Papers - IKEDA Ryuji
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Yoshikawa N., Ehara Y., Yamada Y., Matsusaki Y., Shimoda K., Ikeda R.
Scientific Reports 15 ( 1 ) 3364 2025.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Scientific Reports
Intra-patient variability in immunosuppressive blood drug concentrations is a potential biomarker in managing organ transplant patients. However, the association between the time in therapeutic range of tacrolimus blood concentrations and its efficacy in preventing graft-versus-host disease remains unknown. In this study, we analyzed the relationship between the time in therapeutic range of tacrolimus blood concentrations and its efficacy in acute graft-versus-host disease prophylaxis in patients undergoing allogeneic hematopoietic stem cell transplantation. Eligible patients administered tacrolimus were categorized into two groups based on the grade of acute graft-versus-host disease, and propensity score matching was performed using graft-versus-host disease prophylaxis protocols and days to the disease onset to compare time in therapeutic range. In patients with tacrolimus blood concentration therapeutic range ≥ 10 ng/mL, time in therapeutic range during the first 4 weeks post-transplantation was significantly lower in the Grade II–III than in the Grade 0–I group. Among propensity score matching-extracted patients, the Grade II–III group had significantly lower time in therapeutic range during the first 2 and 4 weeks post-transplantation. Our results suggest that high time in therapeutic range early post-transplantation, particularly within 4 weeks, may avert the severity of acute graft-versus-host disease.
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Yasuda K., Hirano Y., Takeda R., Ikeda R., Ishida Y.
Neuropsychopharmacology Reports 45 ( 1 ) e12506 2025.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Neuropsychopharmacology Reports
Aim: Suvorexant is an orexin receptor antagonist (ORA) for the treatment of insomnia. The antagonistic action of suvorexant on orexin receptors is associated with an increase in rapid eye movement (REM) sleep, which can potentially lead to nightmares depending on the patient's condition. However, the precise risk factors for nightmares among patients taking ORAs, such as suvorexant, have yet to be identified. In this retrospective study, we aimed to identify the risk factors for the development of nightmares in patients treated with suvorexant. Methods: The risk factors were determined by comparing parameters between the nightmare group and the nonnightmare group. This study included 440 patients who received suvorexant at the University of Miyazaki Hospital from April 2014 to January 2021. Results: We found that 9.1% (n = 40) of the patients experienced suvorexant-induced nightmares. There was a significant difference in the median age, which was lower in the nightmare group than in the nonnightmare group (p < 0.01). Furthermore, both multiple logistic regression analysis and Cox proportional hazards regression analysis revealed increased odds ratios for nightmares for individuals aged 20–39 years. Conclusions: This study revealed that elderly patients taking suvorexant had fewer nightmares than nonelderly patients did.
DOI： 10.1002/npr2.12506
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Yamada Y., Ishitsuka Y., Fukaura-Nishizawa M., Kawata T., Ishii A., Shirakawa A., Sakai T., Tanaka M., Kondo Y., Takeo T., Nakagata N., Motoyama K., Higashi T., Arima H., Seki T., Kurauchi Y., Katsuki H., Higaki K., Ikeda R., Matsuo M., Era T., Irie T.
Life Sciences 350 122776 2024.8
Language：English Publishing type：Research paper (scientific journal) Publisher：Life Sciences
Niemann–Pick disease type C (NPC) is a lysosomal lipid storage disorder characterized by progressive neurodegeneration and hepatic dysfunction. A cyclic heptasaccharide, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), is currently under clinical investigation for NPC, but its adverse events remain problematic. We previously identified that a cyclic octasaccharide, 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), also ameliorated NPC manifestations with higher biocompatibility than HP-β-CD. However, preclinical studies describing the associations between the biodistribution and pharmacodynamics of these compounds, which are essential for clinical application, are still lacking. Here, we investigated these properties of HP-γ-CD by measuring its organ biodistribution and therapeutic effect after systemic and central administration. The effect of HP-γ-CD on disturbed cholesterol homeostasis appeared within several hours after exposure and persisted for several days in NPC model cells and mice. Tissue distribution indicated that only a small fraction of subcutaneously administered HP-γ-CD rapidly distributed to peripheral organs and contributed to disease amelioration. We found that a subcutaneous dose of HP-γ-CD negligibly ameliorated neurological characteristics because it has limited penetration of the blood–brain barrier; however, an intracerebroventricular microdose unexpectedly attenuated hepatic dysfunction without the detection of HP-γ-CD in the liver. These results demonstrate that central administration of HP-γ-CD can indirectly attenuate peripheral manifestations of NPC.
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Yoshikawa N., Nagatomo T., Matsusaki Y., Yokota T., Yamada Y., Ikeda R.
Pharmazie 79 ( 6 ) 114 - 117 2024.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Pharmazie
The therapeutic effect of tacrolimus against ulcerative colitis (UC) is correlated with its trough blood concentration. Conventionally, oral tacrolimus for the treatment of UC is initiated under fasting conditions; once the symptoms improve, food intake is resumed. Tacrolimus blood concentration decreases with food intake compared with that under fasting conditions. The aim of this study was to explore the characteristics of patients with UC whose tacrolimus blood concentrations tended to decrease after food initiation. Medical data of 13 patients with UC and treated with tacrolimus were retrospectively obtained. The participant characteristics associated with the changes in tacrolimus blood concentrations after food initiation were analyzed using regression analysis based on the rate of decrease in the concentration/dose (C/D) ratio after food initiation. Single regression analysis showed that the number of days required from tacrolimus initiation to food resumption (P = 0.0071) and individual differences in the increase in tacrolimus blood concentration after administration (P = 0.0247) were significantly associated with the rate of decrease in the C/D ratio after food initiation. Furthermore, multiple regression analysis showed a significant effect of the number of days to food resumption (P = 0.0004) and individual differences in the increase in tacrolimus blood concentration after administration (P = 0.0012). The results suggest that the degree of change in blood tacrolimus concentration after food initiation may be related to the severity of the symptoms and pathology of UC. Early identification of participant characteristics may help control tacrolimus blood concentration fluctuations after food initiation.
DOI： 10.1691/ph.2024.4501
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Ishii Saya, Ozaki Mineo, Takamura Norito, Ogata Kenji, Tokunaga Jin, Ikeda Ryuji
Biological and Pharmaceutical Bulletin 47 ( 1 ) 213 - 220 2024.1
Language：English Publishing type：Research paper (scientific journal) Publisher：The Pharmaceutical Society of Japan
Diclofenac instillation is useful in preventing intraoperative miosis and macular edema caused by postoperative inflammation in cataract surgery; however, optimum efficacy is not attained when the instilled diclofenac strongly binds to albumin in patients’ aqueous humor. Therefore, a method that inhibits diclofenac binding and increases the concentration of its free fraction is needed. We conducted a basic study regarding the effects of inhibitors on the binding of instilled diclofenac to albumin and endogenous substances in aqueous humor. Aqueous humor samples from 16 patients were pooled together for analysis. The free fraction of diclofenac was measured using ultrafiltration methods in various experiments with pooled and mimic aqueous humor. Free fraction of diclofenac, a site II drug, in pooled aqueous humor was 0.363 ± 0.013. The binding of diclofenac in the presence of phenylbutazone (PB), a site I inhibitor, was significantly inhibited (free fraction = 0.496 ± 0.013); however, no significant inhibition by ibuprofen, a site II inhibitor, (free fraction = 0.379 ± 0.004), was observed. The unexpected result was due to free fatty acids (FFAs; palmitic acid (PA)) and L-tryptophan (Trp). The inhibition of diclofenac binding by PB in the mimic aqueous humor containing these endogenous substances revealed significant binding inhibition in the presence of PA and Trp. Diclofenac is strongly rebound from site II to site I in the presence of FFAs and Trp in the aqueous humor because FFAs and Trp induce a conformational change in albumin. Therefore, PB significantly inhibits the binding of diclofenac to albumin.
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Different solubilizing ability of cyclodextrin derivatives for cholesterol in Niemann-Pick disease type C treatment. Reviewed
Yamada Y, Fukaura-Nishizawa M, Nishiyama A, Ishii A, Kawata T, Shirakawa A, Tanaka M, Kondo Y, Takeo T, Nakagata N, Miwa T, Takeda H, Orita Y, Motoyama K, Higashi T, Arima H, Seki T, Kurauchi Y, Katsuki H, Higaki K, Minami K, Yoshikawa N, Ikeda R, Matsuo M, Irie T, Ishitsuka Y
Clinical and translational medicine 13 ( 8 ) e1350 2023.8
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Yasuda K., Takeda R., Ikeda R., Ishida Y.
Psychiatry and Clinical Neurosciences Reports 2 ( 1 ) e76 2023.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Psychiatry and Clinical Neurosciences Reports
Aim: Yokukansan is a Japanese herbal medicine used in psychiatry to treat behavioral and psychological symptoms of dementia and other psychiatric symptoms. However, the glycyrrhizic acid included in this medicine can cause pseudoaldosteronism and hypokalemia. We aimed to identify the risk factors for hypokalemia due to yokukansan. Methods: A retrospective cohort study was conducted on patients previously treated with yokukansan. The risk factors were determined by comparing the hypokalemia group with the non-hypokalemia group for each parameter. Results: This study included 304 patients who received yokukansan treatment between April 2009 and March 2019. We found that 17.4% (n = 53) of the patients experienced yokukansan-induced hypokalemia. Risk factors detected as significantly different between patients with and without yokukansan-associated hypokalemia were low serum potassium concentration before yokukansan administration, dose 7.5 g /day or more, and dementia. Hypokalemia occurred earlier in patients with low albumin, low potassium, and dementia. Conclusion: It is necessary to pay attention to hypokalemia onset when administering yokukansan at 7.5 g or more to patients with low potassium levels and dementia. Our findings suggest that potassium levels must be checked early after yokukansan administration, especially in patients with low albumin, low potassium, and dementia.
DOI： 10.1002/pcn5.76
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Koreeda H., Yoshikawa N., Minami K., Yokota T., Ochiai H., Ikeda R.
Pharmazie 77 ( 11-12 ) 348 - 351 2022.12
Publishing type：Research paper (scientific journal) Publisher：Pharmazie
Infections are the most common cause of mortality in patients with burns and are, therefore, a major challenge in burn treatment. Appropriate infection control measures are required for a good prognosis with respect to wound infections. Infection prevention improves the outcomes of burn patients. We aimed to determine the efficacy of early antimicrobial therapy in patients with severe flame burns. We conducted a single-centre, observational, case-control study to assess the relationship between prognosis and timing of antimicrobial therapy in the treatment of severe burns in emergency department patients from April 2018 to March 2020. The primary outcome was the association between the initiation of antimicrobial use and prognosis in patients with severe burns. Twenty-three participants were included in the study: 14 in the Control group, and nine in the Case group (patients with severe flame burns). Analysis of the association between the number of days from admission to the emergency department to the start of antimicrobial therapy and length of hospital stay showed a significant correlation in the Case group (P = 0.0006) but not in the Control group (P = 0.9630). Furthermore, in the Case group, the number of days from admission to initiation of antimicrobial therapy was significantly shorter in the group with a good skin condition prognosis (median, 4; range, 2-5) than in the group with a poor skin condition prognosis (median, 9; range, 7-14) (P = 0.0256). This study showed that early use of antimicrobials in patients with severe burns leads to improved skin graft outcomes and shorter hospital stays for patients.
DOI： 10.1691/ph.2022.2500
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Yoshikawa N., Takeshima H., Matsusaka K., Yokota T., Shimoda K., Ikeda R.
Pharmazie 77 ( 11-12 ) 335 - 337 2022.12
Publishing type：Research paper (scientific journal) Publisher：Pharmazie
Tacrolimus is a prophylactic for graft-versus-host disease (GVHD), a major cause of mortality in hematopoietic stem cell transplantation (HSCT) patients. Individuals homozygous for the ∗3 mutation in cytochrome P450 3A5-encoding gene (CYP3A5) lack CYP3A5 expression, affecting tacrolimus pharmacokinetics. Here, we investigated the relationship between CYP3A5∗3 polymorphism and GVHD in allogeneic HSCT patients receiving tacrolimus. The patients underwent their first allogeneic HSCT and were administered tacrolimus by continuous infusion. A quenching probe method was used to genotype the CYP3A5 6986A>G (∗3) allele. Based on CYP3A5 polymorphism analysis results, the subjects were divided into wild-type allele-bearing (∗1/∗3) and non-bearing (∗3/∗3) groups. The groups were compared regarding their characteristics at the time of transplantation and the frequency of acute GVHD. The ∗1/∗3 and ∗3/∗3 groups comprised nine and 11subjects, respectively. The frequency of acute GVHD during the observation period was higher in the ∗1/∗3 group than in the ∗3/∗3 group (P = 0.049). There was a significant difference in the cumulative incidence of acute GVHD between the two groups (P = 0.019). When GVHD was observed, the blood tacrolimus concentration was close to the target concentration (10-15 ng/mL). CYP3A5∗3 polymorphism could potentially predict clinical outcomes.
DOI： 10.1691/ph.2022.2435
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Yamada Yusei, Miwa Toru, Nakashima Masaki, Shirakawa Aina, Ishii Akira, Namba Nanami, Kondo Yuki, Takeo Toru, Nakagata Naomi, Motoyama Keiichi, Higashi Taishi, Arima Hidetoshi, Kurauchi Yuki, Seki Takahiro, Katsuki Hiroshi, Okada Yasuyo, Ichikawa Atsushi, Higaki Katsumi, Hayashi Ken, Minami Kentaro, Yoshikawa Naoki, Ikeda Ryuji, Ishikawa Yoshihide, Kajii Tomohito, Tachii Kyoko, Takeda Hiroki, Orita Yorihisa, Matsuo Muneaki, Irie Tetsumi, Ishitsuka Yoichi
155 113698 2022.11
Language：English Publishing type：Research paper (scientific journal)
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Okumura M., Iwakiri T., Yoshikawa N., Nagatomo T., Ayabe T., Tsuneyoshi I., Ikeda R.
International Journal of Molecular Sciences 23 ( 16 ) 2022.8
Language：English Publishing type：Research paper (scientific journal) Publisher：International Journal of Molecular Sciences
Aberrant activation of hepatocyte growth factor (HGF) and its receptor c-Met axis promotes tumor growth. Therefore, many clinical trials have been conducted. A phase 3 trial investigating a monoclonal antibody targeting HGF in combination with fluoropyrimidine-based chemotherapy had to be terminated prematurely; however, the reason behind the failure remains poorly defined. In this study, we investigated the influence of HGF on the antineoplastic effects of 5-fluorouracil (5-FU), a fluoropyrimidine, in HepG2 cells. HGF suppressed the proliferative activity of cells concomitantly treated with 5-FU more robustly as compared to that of cells treated with 5-FU alone, and markedly increased the expression of uridine phosphorylase 1 (UPP1). Intracellular concentration of 5-fluorouridine, an initial anabolite of 5-FU catalyzed by UPP1, was increased by HGF. Interestingly, erlotinib enhanced HGF-induced increase in UPP1 mRNA; in contrast, gefitinib suppressed it. Furthermore, erlotinib suppressed HGF-increased phosphorylation of the epidermal growth factor receptor at the Tyr1173 site involved in downregulation of extracellular signal-regulated kinase (Erk) activation, and enhanced the HGF-increased phosphorylation of Erk. Collectively, these findings suggest that inhibition of the HGF/c-Met axis diminishes the effects of fluoropyrimidine through downregulation of UPP1 expression. Therefore, extreme caution must be exercised in terms of patient safety while offering chemotherapy comprising fluoropyrimidine concomitantly with inhibitors of the HGF/c-Met axis.
DOI： 10.3390/ijms23169108
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Takumi S., Hashimoto K., Tomioka M., Sato M., He W., Komatsu Y., Aoki S., Ikeda R., Shiozaki K., Furukawa T., Komatsu M.
Planta Medica 89 ( 6 ) 616 - 623 2022.4
Language：English Publishing type：Research paper (scientific journal) Publisher：Planta Medica
The hepatotoxin microcystin-LR is a strong inhibitor of serine/threonine protein phosphatase (PP) 1 and PP2A. The onset of its cytotoxicity depends on its selective uptake via the hepatocyte uptake transporters, organic anion transporting polypeptide (OATP) 1B1 and OATP1B3. Understanding and preventing the cytotoxicity of microcystin-LR is crucial to maintain human health. This chemoprevention study demonstrates that the herbal plant extract of iwajisha (20 μg/mL) reduced microcystin-LR cytotoxicity in OATP1B3-expressing cells by approximately six times. In addition, 20 μM acteoside, which is one of the major compounds in iwajisha, reduced microcystin-LR cytotoxicity by approximately 7.4 times. Acteoside could also reduce the cytotoxicity of other compounds, such as okadaic acid and nodularin, which are both substrates of OATP1B3 and inhibitors of PP1/PP2A. To investigate the mechanism by which the cytotoxicity of microcystin-LR is attenuated by acteosides, microcystin-LR and microcystin-LR-binding proteins in cells were examined after microcystin-LR and acteosides were co-exposed. Thus, acteoside noncompetitively inhibited microcystin-LR uptake by OATP1B3-expressing cells. Furthermore, acteoside inhibited the intracellular interaction of microcystin-LR with its binding protein(s), including the 22 kDa protein. Furthermore, using immunoblot analysis, acteoside induced the phosphorylation of extracellular signal-regulated kinase (ERK), which is one of the survival signaling molecules. These results suggest that acteoside reduces microcystin-LR cytotoxicity through several mechanisms, including the inhibition of microcystin-LR uptake via OATP1B3, and decreased interaction between microcystin-LR and its binding protein(s), and that ERK signaling activation contributes to the attenuation effect of acteoside against microcystin-LR cytotoxicity.
DOI： 10.1055/a-1978-8768
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Tsuruda T., Sato Y., Tomita M., Tanaka H., Hatakeyama K., Otsu M., Kawano A., Nagatomo K., Yoshikawa N., Ikeda R., Asada Y., Kaikita K.
Frontiers in Cardiovascular Medicine 9 833649 2022.4
Language：English Publishing type：Research paper (scientific journal) Publisher：Frontiers in Cardiovascular Medicine
Background: Cardiac troponin-T (TNNT2) is exclusively present in cardiac muscle. Measurement of TNNT2 is used for diagnosing acute coronary syndrome. However, its expression may not be limited in myocardium. This study aimed at evaluating the expression of TNNT2 in neoplastic tissues. Methods and Results: We used paraffin-embedded blocks of 68 patients with lung cancer (age, 68 ± 11 years old; early-stage, 33; advance-stage, 35) at Miyazaki University Hospital, Japan between January 1, 2017, and March 31, 2019. We stained the slide sections with primary monoclonal antibody against TNNT2 protein, and assessed the frequency of positive staining, and its association with pathological severity. In addition, we examined whether TNNT2 gene is detected in lung cancer tissues of four patients using reverse transcription-polymerase chain reaction. Immunoreactivity for TNNT2 protein was present in the cytoplasm and nucleus of lung cancer cells. The frequency was 37% (25 of 68) in all patients and was irrespective of histologic type (six of 13, squamous cell carcinoma; 18 of 50, adenocarcinoma; 0 of 4, neuroendocrine cell carcinoma; 1 of 1, large cell carcinoma). The prevalence increased with pathological staging [9% (3 of 33) at early-stage (Stage 0–I); 63% (22 of 35) at advance-stage (Stage II–IV and recurrence)]. In addition, frequency of positive staining for TNNT2 increased with pleural (χ2 = 5.877, P = 0.015) and vascular (χ2 = 2.449, P = 0.118) invasions but decreased with lymphatic invasion (χ2 = 3.288, P = 0.070) in specimens performed surgical resection. Furthermore, TNNT2 mRNA was detected in the resected squamous cell carcinoma and adenocarcinoma tissues. Conclusions: Our data suggest the aberrant expression of TNNT2 in lung cancer and its prevalence increases with pathological severity.
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Takeshima H., Yoshikawa N., Akizuki K., Hidaka T., Shimoda K., Ikeda R.
Journal of Clinical Pharmacy and Therapeutics 47 ( 2 ) 260 - 262 2022.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Clinical Pharmacy and Therapeutics
What is known and objective: Cyclosporine A (CyA) causes intrahepatic biliary stasis via inhibition of bile acid excretion through the bile salt export pump. We report a case of a patient in whom ursodeoxycholic acid (UDCA) markedly promoted the absorption of microemulsion-formulated CyA. Case summary: The patient was a 22-year-old Japanese man diagnosed with stage 3 aplastic anaemia. He was treated with CyA, and 2 h post-dose (C2) was increased by UDCA. What is new and conclusion: A remarkable interaction was observed between CyA and UDCA. This is a valuable finding for improving the treatment strategies for haematological disorders.
DOI： 10.1111/jcpt.13496
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池田 龍二
ジェネリック研究 16 22 - 33 2022
Publishing type：Research paper (scientific journal)
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Association between CYP3A5*3 polymorphism and graft-versus-host disease in allogeneic hematopoietic stem cell transplant patients receiving tacrolimus Reviewed
吉川 直樹, 池田 龍二
Die Pharmazie 77 35 - 337 2022
Publishing type：Research paper (scientific journal)
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Urata S., Yoshikawa N., Saito K., Tazaki T., Ohno R., Takeshima H., Ikeda R.
Journal of Clinical Pharmacy and Therapeutics 46 ( 6 ) 1796 - 1799 2021.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Clinical Pharmacy and Therapeutics
What is known and objective: Methotrexate (MTX) is an important agent for the treatment of primary central nervous system lymphomas (PCNSL) but needs to be given in big doses by intravenous infusions to achieve therapeutic concentrations in the cerebrospinal fluid. However, co-administration with many drugs may delay the excretion of MTX which may cause serious adverse effects if the serum concentration exceeds 0.1 µmol/L 72 h after an intravenous infusion. Case summary: A 67-year-old Japanese female with PCNSL was treated with high-dose MTX-based chemotherapy. The serum MTX concentration 72 h post-infusion was 0.153 µmol/L when she was taking levofloxacin (LVFX) but <0.1 µmol/L 72 h after 4 subsequent infusions when she was not taking LVFX. She was given many other drugs but the timing of the short course of LVFX and the fact that ciprofloxacin also delays MTX excretion suggests that LVFX was the cause. What is new and conclusion: Co-administration of LVFX may delay the excretion of MTX. Therefore, serum concentrations of MTX should be monitored to help prevent and improve the management of potentially serious MTX drug-drug interaction.
DOI： 10.1111/jcpt.13425
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Nagata T., Minami K., Yamamoto M., Hiraki T., Idogawa M., Fujimoto K., Kageyama S., Tabata K., Kawahara K., Ueda K., Ikeda R., Kato Y., Komatsu M., Tanimoto A., Furukawa T., Sato M.
International Journal of Molecular Sciences 22 ( 21 ) 2021.11
Language：English Publishing type：Research paper (scientific journal) Publisher：International Journal of Molecular Sciences
Lung cancer constitutes a threat to human health. BHLHE41 plays important roles in circadian rhythm and cell differentiation as a negative regulatory transcription factor. This study investigates the role of BHLHE41 in lung cancer progression. We analyzed BHLHE41 function via in silico and immunohistochemical studies of 177 surgically resected non-small cell lung cancer (NSCLC) samples and 18 early lung squamous cell carcinoma (LUSC) cases. We also examined doxycycline (DOX)-inducible BHLHE41-expressing A549 and H2030 adenocarcinoma cells. BHLHE41 expression was higher in normal lung than in lung adenocarcinoma (LUAD) tissues and was associated with better prognosis for the overall survival (OS) of patients. In total, 15 of 132 LUAD tissues expressed BHLHE41 in normal lung epithelial cells. Staining was mainly observed in adenocarcinoma in situ and the lepidic growth part of invasive cancer tissue. BHLHE41 expression constituted a favorable prognostic factor for OS (p = 0.049) and cause-specific survival (p = 0.042) in patients with LUAD. During early LUSC, 7 of 18 cases expressed BHLHE41, and this expression was inversely correlated with the depth of invasion. DOX suppressed cell proliferation and increased the autophagy protein LC3, while chloroquine enhanced LC3 accumulation and suppressed cell death. In a xenograft model, DOX suppressed tumor growth. Our results indicate that BHLHE41 expression prevents early lung tumor malignant progression by inducing autophagic cell death in NSCLC.
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Yoshikawa N., Yamada A., Yokota T., Yamada Y., Kinoshita M., Moritake H., Ikeda R.
Cancers 13 ( 18 ) 2021.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Cancers
Intrathecal administration of anticancer drugs is an effective dosage strategy, but the elimination of intraventricular drugs is not uniform in all patients. For safety, a system to evaluate local pharmacokinetics in the ventricles after administration is desired. In this study, we developed a simple and reproducible method to measure topotecan concentration in the cerebrospinal fluid (CSF) and confirmed its clinical applicability. High-performance liquid chromatography (HPLC) analysis was performed using a C18 column to measure the total topotecan concentration in the CSF. Clinical CSF samples were obtained from a 1-year old child with poor CSF absorption and stagnation. The patient received topotecan via an intraventricular subcutaneous reservoir. The HPLC method complied with the validation criteria. The lower limit of quantitation of this method was 0.04 µM. Using the developed method, we could determine the difference in topotecan CSF concentrations at 24 and 48 h after administration. The patient’s topotecan elimination rate was extremely low, and signs of adverse effects were observed at high CSF concentration of topotecan. The developed method could detect the delay in topotecan elimination after intrathecal injection. The findings of this study are valuable for the development of personalized treatments for the intrathecal administration of anticancer drugs.
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High-trough plasma concentration of afatinib is associated with dose reduction Reviewed
Takahashi T., Terazono H., Suetsugu T., Sugawara H., Arima J., Nitta M., Tanabe T., Okutsu K., Ikeda R., Mizuno K., Inoue H., Takeda Y.
Cancers 13 ( 14 ) 2021.7
Language：English Publishing type：Research paper (scientific journal) Publisher：Cancers
Afatinib is used to treat non-small-cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutation as a second-generation EGFR-tyrosine kinase inhibitor (TKI). Early prediction of adverse effects based on the pharmacokinetics of afatinib enables support for quality of life (QOL) in patients with no change in efficacy. We examined the pharmacokinetic relationship between trough plasma concentration and adverse effects and evaluated the utility of measuring the trough plasma concentration of afatinib as the first EGFR-TKI treatment for NSCLC in a prospective multicenter study. Twenty-four patients treated with afatinib were enrolled in this study. All blood samples were collected at the trough point, and plasma concentrations were measured using high-performance liquid chromatography–tandem mass spectrometry. Logistic regression analysis for the dose reduction of afatinib was performed, and the receiver operating characteristic (ROC) curve was plotted. Although all patients started afatinib at 40 mg/day, plasma concentrations were variable, and mean and median trough plasma concentrations were 32.9 ng/mL and 32.5 ng/mL in this study, respectively. Minimum and maximum trough plasma concentrations were 10.4 ng/mL and 72.7 ng/mL, respectively. This variability was speculated to involve personal parameters such as laboratory data. However, no patient characteristics or laboratory data examined correlated with the trough plasma concentration of afatinib, except albumin. Albumin showed a weak correlation with plasma concentration (r = 0.60, p = 0.009). The trough plasma concentration of afatinib was significantly associated with the dose reduction of afatinib (p = 0.047). The area under the ROC curve (AUC) for the trough plasma concentration of afatinib was 0.81. The cut-off value was 21.4 ng/mL. The sensitivity and specificity of the cut-off as a risk factor were 0.80 and 0.75. In summary, the trough plasma concentration of afatinib was associated with continued or reduced dosage because of the onset of several adverse effects, and a threshold was seen. Adverse effects not only lower QOL but also hinder continued treatment. Measuring plasma concentrations of afatinib appears valuable to predict adverse effects and continue effective therapy.
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Promotion of Generic Drugs through the Medical SPD System and Efficacy of Drug Cost Reduction Reviewed
Sekiya Hiroshi, Ogata Yutaka, Kajiwara Takahiro, Fukunaga Yoko, Moriki Toyosaka, Ikeda Ryuji
Japanese Journal of Drug Informatics 23 ( 1 ) 38 - 46 2021.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japanese Society of Drug Informatics
DOI： 10.11256/jjdi.23.38
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Impact of green tea intake on the pharmacokinetics of celiprolol in healthy subjects Reviewed
Sonoda J., Ogata K., Yoshikawa N., Sato K., Ikeda R., Shimodozono Y.
International Journal of Clinical Pharmacology and Therapeutics 59 ( 3 ) 198 - 201 2021.3
Publishing type：Research paper (scientific journal) Publisher：International Journal of Clinical Pharmacology and Therapeutics
Objective: To assess the effect of green tea intake on the pharmacokinetics of the β-blocker celiprolol. Materials and method: In an open-label crossover study, 3 healthy subjects were given water or a green tea beverage daily for 3 days. On day 4, each subject received a single oral dose of 200 mg celiprolol with water or green tea. Serum and urinary concentrations of celiprolol were measured for up to 24 hours. Results: Green tea intake decreased the area under the serum concentration-time curve and urinary excretion of celiprolol by 98.6 and 98.0%, respectively. Conclusion: Green tea intake might have a negative impact on the clinical effectiveness of celiprolol.
DOI： 10.5414/CP203795
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Yoshikawa N., Yamada A., Yokota T., Moritake H., Hirabara Y., Ikeda R.
Journal of Clinical Laboratory Analysis 35 ( 3 ) e23661 2021.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Clinical Laboratory Analysis
Background: The concentration of MTX in blood is often measured quickly and easily by immunoassays. Thus, immunoassays may facilitate the easy determination of the concentration of MTX in the cerebrospinal fluid (CSF). In this study, we measured methotrexate (MTX) concentrations in the CSF using a high-performance liquid chromatography (HPLC) method intended for analyzing CSF matrices and a chemiluminescence immunoassay (CLIA) method intended for assessing serum and plasma matrices and verified the differences in the results of the two methods. Methods: HPLC analysis for MTX in the CSF was performed using a Prominence UFLC system with a C18 column. The HPLC method was validated in accordance with the 2018 FDA guideline. The CLIA method was performed using an ARCHITECT i1000SR system intended for serum and plasma matrices. A total of 47 CSF samples (14 clinical and 33 spiked specimens) were analyzed using the two methods. Results: The HPLC method passed the validation criteria. The concentration of MTX in the same sample, determined using the HPLC and CLIA methods, differed proportionally; the percent difference in the concentrations averaged −23.0% (95% confidence interval: −36.9% to −9.1%) as revealed by the Bland-Altman plot. The relationship between the measured values, evaluated using the Passing-Bablok regression, was as follows: HPLC = 1.205 × CLIA – 0.024. Conclusion: The equation deduced in this study can be used to correct the concentration of MTX measured using the CLIA method.
DOI： 10.1002/jcla.23661
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Tsuruda T., Yoshikawa N., Kai M., Yamaguchi M., Toida R., Kodama T., Kajihara K., Kawabata T., Nakamura T., Sakata K., Hatakeyama K., Gi T., Asada Y., Tono T., Kitamura K., Ikeda R.
Internal Medicine 60 ( 3 ) 423 - 429 2021.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Internal Medicine
We herein report the cytokine expression at different stages for three patients who developed cardiac complications after immune checkpoint inhibitor (ICI) therapy. Case 1 with biopsy-proven myocarditis showed increased levels of interleukin (IL)-8, monocyte chemotactic and activating factor, and granulocyte macrophage colony-stimulating factor (GM-CSF) when he developed Takotsubo cardiomyopathy. Case 2 with subclinical myocarditis showed predominant activation of IL-8 during the progressive clinical course. Case 3 with cytokine-releasing syndrome showed substantial activations of IL-6, IL-8, GM-CSF, and interferon-γ. Our data suggest the development of unique cytokine activation in individual patients with cardiac complications after ICI therapy.
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Yoshikawa N., Takeshima H., Sekine M., Akizuki K., Hidaka T., Shimoda K., Ikeda R.
Pharmaceuticals 14 ( 4 ) 2021
Language：English Publishing type：Research paper (scientific journal) Publisher：Pharmaceuticals
A polymorphism in the gene encoding the metabolic enzyme cytochrome P450 family 3 subfamily A member 5 (CYP3A5) is a particularly influential factor in the use of tacrolimus in Japanese patients. Those who are homozygotic for the *3 mutation lack CYP3A5 activity, which results in substantial individual differences in tacrolimus metabolism. The aim of this study was to analyze the relationship between individual differences in tacrolimus blood concentration changes and CYP3A5 polymorphisms in allogeneic hematopoietic stem cell transplantation recipients during the period of increasing blood concentration of the drug following treatment onset. This was a prospective observational cohort study, involving 20 patients administered tacrolimus by continuous infusion. The subjects were divided into the *1/*3 and *3/*3 groups based on CYP3A5 polymorphism analysis. The tacrolimus blood concentration/dose (C/D) ratio increased from day 1 and was largely stable on day 5, and a significant difference was observed between the *1/*3 and *3/*3 groups in the time course of the C/D ratio during this period (p < 0.05). This study reveals the effects om on continuous changes in tacrolimus blood concentration.
DOI： 10.3390/ph14040353
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Retrospective analysis of risk factors for liposomal amphotericin B-associated nephrotoxicity Reviewed
Yokota T., Yoshikawa N., Arimori K., Ikeda R.
Pharmazie 75 ( 11 ) 599 - 601 2020.11
Language：English Publishing type：Research paper (scientific journal) Publisher：Pharmazie
In this study, we examined patients who received liposomal amphotericin B (L-AMB) to determine the risk factors associated with nephrotoxicity before and during L-AMB treatment. In this retrospective, single-center, observational cohort study, we examined 37 patients who received L-AMB treatment between April 2018 and December 2019. Nephrotoxicity was observed in 11 (29.7%) patients. We focused on the baseline albumin level and body surface area (BSA) before L-AMB treatment. Univariate analysis showed that the BSA and baseline albumin levels in patients with nephrotoxicity were significantly higher than those in patients without nephrotoxicity. Moreover, univariate analysis showed that albumin supplementation was significantly associated with the frequency of nephrotoxicity during L-AMB treatment. Multiple logistic regression analysis revealed the following independent risk factors for nephrotoxicity before or during L-AMB treatment: baseline albumin level (odds ratio [OR] = 16.000; 95% CI 1.480-172.000; P = 0.022) and albumin supplementation (OR = 40.800; 95% CI 2.210-753.000; P = 0.013). In conclusion, we identified baseline albumin level and albumin supplementation as novel risk factors for L-AMB-induced nephrotoxicity.
DOI： 10.1691/ph.2020.0731
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Role of FK506 Binding Protein on Tacrolimus Distribution in Red Blood Cells Reviewed
Yoshikawa N., Yokota T., Matsuo A., Matsumoto N., Iwakiri T., Ikeda R.
Pharmaceutical Research 37 ( 7 ) 143 2020.7
Language：English Publishing type：Research paper (scientific journal) Publisher：Pharmaceutical Research
Purpose: Tacrolimus is distributed mainly in red blood cells (RBCs) after transfer into blood. This study aimed to evaluate the effect of FK506-binding proteins (FKBPs) on RBC distribution of tacrolimus in a physiological environment. Methods: Human RBCs were isolated from fresh blood samples from healthy volunteers. The effect of FKBPs on each process of the RBC distribution of tacrolimus was evaluated in vitro. Effect of intracellular FKBPs was assessed by inhibition experiment with rapamycin, which competitively inhibits the binding of tacrolimus to FKBPs. Effect of extracellular FKBPs was examined by pre-exposure of RBCs to FKBP and preincubation of tacrolimus with FKBP. Results: Pretreatment with rapamycin significantly reduced the rate of tacrolimus distribution in RBCs in a concentration-dependent manner. Pre-exposure of RBCs to FKBP12 followed by exposure to tacrolimus significantly decreased tacrolimus distribution in RBCs in a concentration-dependent manner. In addition, preincubation of tacrolimus with FKBP12 significantly reduced the rate of tacrolimus distribution in RBCs. Conclusions: FKBP played an important role in the distribution of tacrolimus in RBCs. The effect of intracellular and extracellular FKBPs on RBC distribution of tacrolimus in circulating blood was substantial. FKBP was shown as a potential biomarker for predicting the pharmacokinetics and pharmacodynamics of tacrolimus.
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Retrospective analysis of the correlation between tacrolimus concentrations measured in whole blood and variations of blood cell counts in patients undergoing allogeneic haematopoietic stem cell transplantation. Reviewed
Yoshikawa N, Urata S, Yasuda K, Sekiya H, Hirabara Y, Okumura M, Ikeda R.
Eur J Hosp Pharm 2020.3
Language：English Publishing type：Research paper (scientific journal)
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Retrospective analysis of the correlation between tacrolimus concentrations measured in whole blood and variations of blood cell counts in patients undergoing allogeneic haematopoietic stem cell transplantation. Reviewed
Yoshikawa N, Urata S, Yasuda K, Sekiya H, Hirabara Y, Okumura M, Ikeda R
European journal of hospital pharmacy : science and practice 27 ( e1 ) e7 - e11 2020.3
Language：English Publishing type：Research paper (scientific journal)
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バイオシミラー導入への取り組みと薬剤費削減効果 Reviewed
池田 龍二
ジェネリック研究 14 25 - 33 2020
Publishing type：Research paper (scientific journal)
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Anti-proliferative Activities of Some Bivalent Symmetrical 5-Substituted Hydantoin Derivatives towards Human Brain Glioma U251 Cells (U251) and Human Carcinoma Cells (KB3-1). Reviewed
Furutachi M, Ota K, Fujisaki F, Ikeda R, Yoshikawa N, Yokota T, Takeda Y, Yokomizo K, Zhou JR, Kashige N, Miake F, Sumoto K.
Biol Pharm Bull. 2019.11
Language：English Publishing type：Research paper (scientific journal)
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医薬品SPDを活用したタスク・シフティングの検討 Reviewed
関屋裕史、友宗直樹、緒方豊、梶原隆広、福永洋子、森木豊栄、長友隆雄、岩田円夏、萩原櫻子、池田 龍二
九州薬学会雑誌 2019.11
Language：Japanese Publishing type：Research paper (scientific journal)
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当院の持参薬鑑別時におけるお薬手帳の利用状況とお薬手帳の記載内容調査 Reviewed
外山智章、畑中真理、田﨑智也、竹島秀美、平原康寿、池田 龍二
九州薬学会雑誌 2019.11
Language：Japanese Publishing type：Research paper (scientific journal)
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電子カルテシステムのベンダー変更を契機としたがん薬物療法レジメンの見直しと医療安全に向けた取り組み Reviewed
浦田修平、大野梨絵、千阪智美、北畑恵理子、髙橋沙季、是枝秀彦、森山徳文、平原康寿、池田 龍二
九州薬学会雑誌 2019.11
Language：Japanese Publishing type：Research paper (scientific journal)
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宮崎県病院施設における薬薬連携の現状調査 Reviewed
田﨑智也、平原康寿、本田憲一、三代朗子、杉田昌子、永友由里子、高橋雅也、津曲恭一、帖佐康弘、寺町文宏、長友隆雄、外山智章、山口博行、奥村学、佐藤智加子、池田 龍二
九州薬学会雑誌 2019.11
Language：Japanese Publishing type：Research paper (scientific journal)
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5-Aza-2-deoxycytidine Enhances the Sensitivity of 5-Fluorouracil by Demethylation of the Thymidine Phosphorylase Promoter. Reviewed
Nishizawa Y, Ikeda R, Yamamoto M, Kawahara K, Shinsato Y, Minami K, Nitta M, Terazono H, Akiyama SI, Furukawa T, Takeda Y.
Anticancer Res 2019.8
Language：English Publishing type：Research paper (scientific journal)
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Anti-proliferative Activities towards Human Brain Glioma U251 Cells and Human Carcinoma Cells (KB3-1) of Some Twin-Drug Type Bivalent C2-Symmetrical Phenylboronic Acid Derivatives. Reviewed
Furutachi M, Gondo T, Ikeda R, Yoshikawa N, Yokota T, Takeda Y, Yokomizo K, Zhou JR, Kashige N, Miake F, Sumoto K.
Biol Pharm Bull 2019.5
Language：English Publishing type：Research paper (scientific journal)
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Drug-drug interactions among drugs prescribed for nontuberculous mycobacterial infection and epilepsy: A case report. Reviewed
Yoshikawa N, Tazaki T, Hatanaka M, Oda Y, Matsumoto N, Sonoda J, Ikeda R.
Journal of Clinical Pharmacy and Therapeutics 2019.2
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Viva-Eシステムとナノピア®TDMエベロリムス試薬によるエベロリムス血中濃度測定の基礎的評価 Reviewed
吉川 直樹、横田 翼、関屋 裕史、平原 康寿、池田 龍二
日本病院薬剤師会雑誌 2019.1
Language：Japanese Publishing type：Research paper (scientific journal)
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Tabata S., Yamamoto M., Goto H., Hirayama A., Ohishi M., Kuramoto T., Mitsuhashi A., Ikeda R., Haraguchi M., Kawahara K., Shinsato Y., Minami K., Saijo A., Toyoda Y., Hanibuchi M., Nishioka Y., Sone S., Esumi H., Tomita M., Soga T., Furukawa T., Akiyama S.
Scientific Reports 8 ( 1 ) 2018.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Scientific Reports
© 2018 The Author(s). Thymidine phosphorylase (TP) is a rate-limiting enzyme in the thymidine catabolic pathway. TP is identical to platelet-derived endothelial cell growth factor and contributes to tumour angiogenesis. TP induces the generation of reactive oxygen species (ROS) and enhances the expression of oxidative stress-responsive genes, such as interleukin (IL)-8. However, the mechanism underlying ROS induction by TP remains unclear. In the present study, we demonstrated that TP promotes NADPH oxidase-derived ROS signalling in cancer cells. NADPH oxidase inhibition using apocynin or small interfering RNAs (siRNAs) abrogated the induction of IL-8 and ROS in TP-expressing cancer cells. Meanwhile, thymidine catabolism induced by TP increased the levels of NADPH and intermediates of the pentose phosphate pathway (PPP). Both siRNA knockdown of glucose 6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme in PPP, and a G6PD inhibitor, dihydroepiandrosterone, reduced TP-induced ROS production. siRNA downregulation of 2-deoxy-D-ribose 5-phosphate (DR5P) aldolase, which is needed for DR5P to enter glycolysis, also suppressed the induction of NADPH and IL-8 in TP-expressing cells. These results suggested that TP-mediated thymidine catabolism increases the intracellular NADPH level via the PPP, which enhances the production of ROS by NADPH oxidase and activates its downstream signalling.
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クラスI自主回収における医薬品SPDのトレーサビリティ分析の効果 Reviewed
関屋 裕史、緒方 豊、梶原 隆広、福永 洋子、北畑 恵理子、岩切 智美、奥村 学、池田 龍二
九州薬学会雑誌 2018.11
Language：Japanese Publishing type：Research paper (scientific journal)
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神経障害性疼痛を伴った難治性癌性疼痛に対しメサドン導入を行った3症例 Reviewed
畑中真理，平原康寿，吉川直樹，山賀昌治，柴田伸弘，船橋英樹，藤山美穂，西村亜希，三輪真砂子，田﨑智也，岩切智美，奥村学，池田龍二
九州薬学会雑誌 2018.11
Language：Japanese Publishing type：Research paper (scientific journal)
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神経・呼吸器・内分泌・代謝・糖尿病内科病棟における病棟専任薬剤師による能動的介入の調査報告 Reviewed
田﨑 智也、吉川 直樹、平原 康寿、大野 梨絵、関屋 裕史、岩切 智美、奥村 学、池田 龍二
九州薬学会雑誌 2018.11
Language：Japanese Publishing type：Research paper (scientific journal)
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Tomiyama N., Ikeda R., Nishizawa Y., Masuda S., Tajitsu Y., Takeda Y.
Oncology Letters 15 ( 6 ) 9929 - 9933 2018.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Oncology Letters
© 2018, Spandidos Publications. All rights reserved. Cancer stem-like cells (CSCs), which possess the ability to self-renewal and are multipotent, are regarded as the cause of tumor formation, recurrence, metastasis and drug resistance. It is necessary to understand the properties of CSCs in order to treat them effectively. It has been previously reported that S100 family proteins, which carry calcium-binding EF-hand motifs and are associated with tumorigenic processes, serve crucial roles in maintaining cancer stem-like properties. S100A16 is upregulated in various types of cancer, including bladder, lung and pancreatic. However, the roles of S100A16 in cancer cells, particularly CSCs, are not clear. The present study investigated the roles of S100A16 in CSCs using the sphere formation assay of Yumoto cells, which are a human cervical carcinoma cell line. The mRNA expression levels were evaluated by reverse transcription-polymerase chain reaction and the protein expression levels were detected by western blot analysis. Following the sphere formation of Yumoto cells, the mRNA and protein expression level of Oct4, Nanog and S100A16 were increased compared with the control cells. Following transfection with S100A16 small interfering RNA (siRNA), the mRNA and protein expression of Oct4 and Nanog were decreased and the spheroid size was significantly decreased in the sphere formation of Yumoto cells compared with control siRNA treated cells. There was no change in the p53 mRNA expression level, whereas the p53 protein expression level, which was decreased by the sphere formation, was recovered by S100A16 knockdown. In addition, the protein expression levels of Oct4 and Nanog, which were increased in the sphere formation, were decreased by the proteasome inhibitor lactacystin. No differences were observed in the S100A16 protein expression between the presence or absence of lactacystin. These results suggest that S100A16 serves an important role in the CSCs of human cervical carcinoma and is a positive regulator of Oct4 and Nanog.
DOI： 10.3892/ol.2018.8568
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ダクラタスビル/アスナプレビル投与による肝機能障害発現の危険因子に関する後方視的調査 Reviewed
吉川 直樹, 北畑 和寛, 奥村 学, 岩切 智美, 関屋 裕史, 有森 和彦, 米澤 ゆう子, 有村 保次, 池田 龍二
医療薬学 2018.1
Language：Japanese Publishing type：Research paper (scientific journal)
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Thymidine Catabolism as a Metabolic Strategy for Cancer Survival Reviewed
Tabata S., Yamamoto M., Goto H., Hirayama A., Ohishi M., Kuramoto T., Mitsuhashi A., Ikeda R., Haraguchi M., Kawahara K., Shinsato Y., Minami K., Saijo A., Hanibuchi M., Nishioka Y., Sone S., Esumi H., Tomita M., Soga T., Furukawa T., Akiyama S.
Cell Reports 19 ( 7 ) 1313 - 1321 2017.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Cell Reports
© 2017 The Author(s) Thymidine phosphorylase (TP), a rate-limiting enzyme in thymidine catabolism, plays a pivotal role in tumor progression; however, the mechanisms underlying this role are not fully understood. Here, we found that TP-mediated thymidine catabolism could supply the carbon source in the glycolytic pathway and thus contribute to cell survival under conditions of nutrient deprivation. In TP-expressing cells, thymidine was converted to metabolites, including glucose 6-phosphate, lactate, 5-phospho-α-D-ribose 1-diphosphate, and serine, via the glycolytic pathway both in vitro and in vivo. These thymidine-derived metabolites were required for the survival of cells under low-glucose conditions. Furthermore, activation of thymidine catabolism was observed in human gastric cancer. These findings demonstrate that thymidine can serve as a glycolytic pathway substrate in human cancer cells.
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Desaki R., Sawada G., Okumura H., Ikeda R., Tanabe K., Komatsu H., Mimori K., Mori M., Kita Y., Uchikado Y., Arigami T., Uenosono Y., Owaki T., Ishigami S., Natsugoe S.
Annals of Surgical Oncology 24 ( 2 ) 586 - 593 2017.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Annals of Surgical Oncology
© 2015, Society of Surgical Oncology. Background: Cysteine/histidine-rich 1 (CYHR1) was first discovered in a yeast two-hybrid screen with murine galectin-3, and no previous reports have described a relationship between the CYHR1 gene and human cancer. The current study evaluated the role and significance of CYHR1 in esophageal cancer. Methods: The human esophageal squamous cell carcinoma (ESCC) cell line TE-8 and the CYHR1 knock-down cell line TE-8/small interfering (si)-CYHR1 were used for in vitro and in vivo assays. For clinical study, ESCC tissues (n = 104) were used. Results: Compared with parental cells, TE-8/si-CYHR1 cells had suppressed proliferation and invasion activities. In the in vivo assay, the tumors from TE-8 cells treated with si-CYHR1 had abrogated tumorigenicity. In the clinical study, the expression of CYHR1 mRNA was associated with lymph node metastasis and stage and shown to be an independent prognostic factor. Conclusions: As the findings show, CYHR1 may represent not only a valuable prognostic marker but also a therapeutic target for ESCC patients.
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Incretin as a novel treatment strategy for NAFLD/NASH
Takeda Y., Ikeda R., Kondo T.
Yakugaku Zasshi 136 ( 4 ) 573 - 577 2016.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Yakugaku Zasshi
© 2016 The Pharmaceutical Society of Japan. Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) have been recognized as metabolic disorders characterized by fatty accumulation in the liver without alcohol consumption. The diseases can cause metabolic syndromes, consisting of obesity, diabetes mellitus (DM), dyslipidemia and hypertension. For the treatment of NAFLD/NASH, losing weight by exercise or diet remains the standard treatment, because no effective pharmacological therapy has yet been developed for NAFLD/NASH. Two incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), stimulate glucose-mediated insulin production in pancreatic β cells. Incretin has also been reported to have various extra-pancreatic effects, including the regulation of hepatic glucose production, appetite and satiety, as well as the stimulation of aferent sensory nerves. Therefore, incretin may have potential as a novel therapeutic agent for NAFLD/NASH.
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Minami K., Shinsato Y., Yamamoto M., Takahashi H., Zhang S., Nishizawa Y., Tabata S., Ikeda R., Kawahara K., Tsujikawa K., Chijiiwa K., Yamada K., Akiyama S., Pérez-Torras S., Pastor-Anglada M., Furukawa T., Yasuo T.
Journal of Pharmacological Sciences 127 ( 3 ) 319 - 325 2015.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Pharmacological Sciences
© 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. Gemcitabine is widely used for pancreatic, lung, and bladder cancer. However, drug resistance against gemcitabine is a large obstacle to effective chemotherapy. Nucleoside transporters, nucleoside and nucleotide metabolic enzymes, and efflux transporters have been reported to be involved in gemcitabine resistance. Although most of the resistant factors are supposed to be related to each other, it is unclear how one factor can affect the other one. In this study, we established gemcitabine-resistant pancreatic cancer cell lines. Gemcitabine resistance in these cells is caused by two major processes: a decrease in gemcitabine uptake and overexpression of ribonucleotide reductase large subunit (RRM1). Knockdown of RRM1, but not the overexpression of concentrative nucleoside transporter 1(CNT1), could completely overcome the gemcitabine resistance. RRM1 knockdown in gemcitabine-resistant cells could increase the intracellular accumulation of gemcitabine by increasing the nucleoside transporter expression. Furthermore, a synergistic effect was observed between hydroxyurea, a ribonucleotide reductase (RR) inhibitor, and gemcitabine on thegemcitabine-resistant cells.Hereweindicate that RRisoneofthe most promising targetstoovercome gemcitabine resistance in gemcitabine-resistant cells with dual resistant factors.
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Factors involved in the cisplatin resistance of KCP-4 human epidermoid carcinoma cells
Oiso S., Takayama Y., Nakazaki R., Matsunaga N., Motooka C., Yamamura A., Ikeda R., Nakamura K., Takeda Y., Kariyazono H.
Oncology Reports 31 ( 2 ) 719 - 726 2014.2
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncology Reports
KCP-4 is a cisplatin-resistant cell line established from human epidermoid carcinoma KB-3-1 cells. Although our previous study revealed that one of the mechanisms for cisplatin resistance in KCP-4 cells is the activation of NF-κB, its high resistance is considered to be induced by multiple mechanisms. In the present study, we explored other factors involved in the development of cisplatin resistance in KCP-4 cells. Since it has been reported that an unknown efflux pump exports cisplatin from KCP-4 cells in an ATP-dependent manner, we examined 48 types of ATP-binding cassette proteins as candidate cisplatin efflux transporters. The mRNA expression levels of ABCA1, ABCA3, ABCA7 and ABCB10 in KCP-4 cells were higher when compared to those in KB-3-1 cells. These expression levels in cisplatin-sensitive revertant KCP-4 cells (KCP-4R cells), were reduced in parallel with the sensitivity of these cells to cisplatin and their intracellular accumulation of cisplatin. Next, we investigated the occurrence of mutations in p53 in KCP-4 cells. We found a heterozygous missense mutation at codon 72 (p.Pro72Arg) in p53 of both KCP-4 and KB-3-1 cells, but the protein expression level of p53 in KCP-4 cells was higher when compared to that in KB-3-1. These results suggest that ABCA1, ABCA3, ABCA7 and ABCB10 are candidate genes for the cisplatin efflux transporter that is involved in the cisplatin resistance of KCP-4 cells, and that the mutation at codon 72 of p53 may contribute to the development of cisplatin resistance.
DOI： 10.3892/or.2013.2896
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Sonoda J., Ikeda R., Baba Y., Narumi K., Kawachi A., Tomishige E., Nishihara K., Takeda Y., Yamada K., Sato K., Motoya T.
Experimental and Therapeutic Medicine 8 ( 1 ) 59 - 63 2014.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Experimental and Therapeutic Medicine
Clinical and epidemiological studies have indicated that the consumption of green tea has a number of beneficial effects on health. Epigallocatechin-3-gallate (EGCg), the major polyphenolic compound present in green tea, has received much attention as an active ingredient. Among the numerous promising profiles of EGCg, the present study focused on the anticancer effects. Apoptosis induced by EGCg and subsequent cell growth suppression have been demonstrated in a number of cell culture studies. However, the underlying mechanism of apoptotic cell death remains unclear. Thus, the aim of the present study was to identify the major molecule that mediates proapoptotic cell death by EGCg. The effect of EGCg on cell proliferation and the induction of mRNA that modulates apoptotic cell death was evaluated in the A549 human non-small-cell lung cancer cell line. In addition, morphological changes were assessed by microscopy in A549 cells that had been treated with 100 μM EGCg for 24 h. The MTT assay revealed that cell proliferation was significantly reduced by EGCg in a dose-dependent manner (3-100 μM). The mRNA expression level of B-cell lymphoma-extra large (Bcl-xL) was decreased in A549 cells following 24 h incubation with 100 μM EGCg. Therefore, the results indicated that the inhibition of cell proliferation by EGCg may be achieved via suppressing the expression of the cell death-inhibiting gene, Bcl-xL.
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Ikeda R., Nishizawa Y., Tajitsu Y., Minami K., Mataki H., Masuda S., Furukawa T., Akiyama S., Yamada K., Takeda Y.
Oncology Reports 31 ( 1 ) 197 - 201 2014.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncology Reports
Major vault protein (MVP) is the main constituent of the vault ribonucleoprotein particle and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several types of normal tissues, little is known about its physiological role. In the present study, we identified the crucial MVP promoter elements that regulate MVP expression. An examination of tissue expression profiles revealed that MVP was expressed in the heart, placenta, lung, liver, kidney and pancreas. Elements of the MVP promoter contain binding sites for transcription factors, STAT, p53, Sp1, E-box, GATA, MyoD and Y-box. By deletion analysis, a conserved proximal E-box binding site was demonstrated to be important for human MVP promoter transactivation. Introduction of siRNA against upstream stimulating factor (USF) 1, which is known to bind the E-box binding site, decreased the expression of MVP in SW620 and ACHN cells. Using a chromatin immunoprecipitation (ChIP) assay, USF1 bound the MVP promoter in SW620 cells. These findings suggest that USF1 binding to an E-box element may be critical for basal MVP promoter activation. The results of the present study are useful in understanding the molecular mechanisms regulating MVP gene expression, and may aid in elucidating the physiological functions of MVP.
DOI： 10.3892/or.2013.2818
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Tabata S., Ikeda R., Yamamoto M., Shimaoka S., Mukaida N., Takeda Y., Yamada K., Soga T., Furukawa T., Akiyama S.
Oncotarget 5 ( 21 ) 10473 - 10485 2014.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncotarget
Thymidine phosphorylase (TP) promotes angiogenesis and metastasis, and confers resistance to anticancer agents in some cancer cell types. We previously reported that TP stimulates the expression of interleukin (IL)-8 in human KB cancer cells by an unknown mechanism. A mutation in the nuclear factor (NF)κB binding site of the IL-8 promoter suppressed promoter activity in KB/TP cells that overexpress TP. Specifically inhibiting NFκB by using BY11-7082 also suppressed TP-induced IL-8 promoter activity and IL-8 expression. Moreover, TP overexpression led to the activation of NFκB and an upregulation in the expression of its target genes, and increased phosphorylated IKKa/β protein levels, while promoting IκBa degradation as well as p65 phosphorylation and nuclear localization. The activation of NFκB in KB/TP cells was suppressed by the antioxidants N-acetylcysteine and EUK-8. In addition, in gastric cancer tissue samples, the expression of the NFκB-regulated genes, including IL-8, IL-6, and fibronectin-1 was positively correlated with TP expression. These findings indicate that reactive oxygen species mediated NFκB activation by TP increases the expression of genes that promote angiogenesis and metastasis in gastric cancer.
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Tamai M., Matsushita S., Miyanohara H., Imuta N., Ikeda R., Kawai K., Nishi J., Sakamoto A., Shigihara T., Kanekura T.
Journal of Dermatology 40 ( 12 ) 1020 - 1026 2013.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Dermatology
Methicillin-resistant Staphylococcus aureus (MRSA) has rapidly emerged as a cause of severe and intractable skin infection. At present, there are no effective topical treatments, and infection or colonization by MRSA of the skin raises serious medical problems. We developed an ultrasonic levitation washer that generates silver ions (Ag+) and ozone (O3) to clean and sterilize medical devices. We report the effect of ultrasonic levitation (levitation) with Ag+ and O3 on MRSA in vitro and in vivo. Antimicrobial effect against six MRSA strains of all agr types was examined under three in vitro conditions; cells floating in a water tank, cells infiltrating-, and cells forming a biofilm on an atelocollagen membrane. In the in vivo studies, we assayed the number of MRSA organisms that survived treatment on murine skin ulcers and evaluated the ulcer size. Levitation with Ag + dramatically decreased the survival of MRSA floating in a water tank. Levitation with Ag+ and O3 significantly decreased the viability of MRSA that had infiltrated or formed a biofilm on atelocollagen membranes regardless of the level of biofilm production. In vivo studies showed that the number of MRSA on murine skin ulcers was significantly decreased when 15-min treatment was performed for 7 consecutive days and that the ulcer size was significantly decreased after the seventh treatment course. Levitation with Ag+ and O3 may be a valuable tool for treating MRSA infestation of the skin and for accelerating wound healing. © 2013 Japanese Dermatological Association.
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Tajitsu Y., Ikeda R., Nishizawa Y., Mataki H., Che X., Sumizawa T., Nitta M., Yamaguchi T., Yamamoto M., Tabata S., Akiyama S., Yamada K., Furukawa T., Takeda Y.
International Journal of Molecular Medicine 32 ( 3 ) 703 - 708 2013.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：International Journal of Molecular Medicine
Major vault protein (MVP) is identical to lung resistance-related protein (LRP), which is the major component of vaults. Vaults are considered to play a protective role against xenobiotics and other types of stress. In a previous study, we reported that the expression levels of MVP in SW620 human colon cancer cells were increased in hypertonic culture medium with sucrose. However, the molecular mechanism behind the induction of MVP expression by osmotic stress has not yet been elucidated. Therefore, in the present study, we investigated the mechanism behind the induction of MVP expression by osmotic stress. Under hyperosmotic stress conditions, the ubiquitination of specificity protein 1 (Sp1) decreased, Sp1 protein levels increased, its binding to the MVP promoter was enhanced, and small interfering RNA (siRNA) for Sp1 suppressed the induction of MVP expression. The inhibition of c-jun N-terminal kinase (JNK) by SP600125, a specific JNK inhibitor, decreased the expression of MVP and Sp1 under hyperosmotic conditions. Our data indicate that the stabilization and upregulation of Sp1 protein expression by JNK participate in the inhibition of the ubiquitination and degradation of Sp1, and thus in the induction of MVP expression under hyperosmotic conditions.
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Molecular basis for the regulation of hypoxia-inducible factor-1α levels by 2-deoxy-D-ribose
Ikeda R., Tabata S., Tajitsu Y., Nishizawa Y., Minami K., Furukawa T., Yamamoto M., Shinsato Y., Akiyama S., Yamada K., Takeda Y.
Oncology Reports 30 ( 3 ) 1444 - 1448 2013.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncology Reports
The angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, inhibits the upregulation of hypoxia-inducible factor (HIF) 1α, BNIP3 and caspase-3 induced by hypoxia. In the present study, we investigated the molecular basis for the suppressive effect of 2-deoxy-D-ribose on the upregulation of HIF-1α. 2-Deoxy-D-ribose enhanced the interaction of HIF-1α and the von Hippel-Lindau (VHL) protein under hypoxic conditions. It did not affect the expression of HIF-1α, prolyl hydroxylase (PHD)1/2/3 and VHL mRNA under normoxic or hypoxic conditions, but enhanced the interaction of HIF-1α and PHD2 under hypoxic conditions. 2-Deoxy-D-ribose also increased the amount of hydroxy-HIF-1α in the presence of the proteasome inhibitor MG-132. The expression levels of TP are elevated in many types of malignant solid tumors and, thus, 2-deoxy-D-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis.
DOI： 10.3892/or.2013.2572
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VEGF expression is augmented by hypoxia-induced PGIS in human fibroblasts
Wang J., Ikeda R., Che X., Ooyama A., Yamamoto M., Furukawa T., Hasui K., Zheng C., Tajitsu Y., Oka T., Tabata S., Nishizawa Y., Eizuru Y., Akiyama S.
International Journal of Oncology 43 ( 3 ) 746 - 754 2013.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：International Journal of Oncology
Prostacyclin synthase (PGIS or PTGIS) is an enzyme that catalyses the conversion of prostaglandin H2(PGH2) to prostaglandin I2(PGI2). PGI2promotes cancer growth by activating peroxisome proliferator-activated receptor δ (PPARδ), and increases the expression levels of the pro-angiogenic factor vascular endothelial growth factor (VEGF). We found that the expression of the PGIS gene was enhanced in WI-38, TIG-3-20 and HEL human lung fibroblast cells and two cancer cell lines (NB-1 and G361) under hypoxic conditions. The main localization of PGIS changed from the cytoplasm to the nucleus by hypoxia in WI-38 cells. The induced PGIS had an enzymatic activity since the intracellular level of 6-keto-prostaglandin, a useful marker of PGI2biosynthesis in vivo, was increased with the increasing levels of PGIS. Expression of VEGF was increased in parallel with PGIS induction under hypoxic conditions. PGIS knockdown resulted in the decreased expression of VEGF mRNA. Since VEGF is a known PPARδ target gene, we examined the effects of siRNAs targeting PPARδ on the expression of VEGF under hypoxic conditions. Knockdown of PPARδ suppressed the expression of VEGF under hypoxic conditions in WI-38 cells. These findings suggest that PGIS is induced by hypoxia and regulates the expression of VEGF in fibroblasts. Fibroblasts in the hypoxic area of tumors may have an important role in tumor growth and angiogenesis.
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Autocrine effects of neuromedin b stimulate the proliferation of rat primary osteoblasts
Saito H., Nakamachil T., Inoue K., Ikeda R., Kitamura K., Minamino N., Shioda S., Miyata A.
Journal of Endocrinology 217 ( 2 ) 141 - 150 2013.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Endocrinology
Neuromedin B (NMB) is a mammalian bombesin-like peptide that regulates exocrine/ endocrine secretion, smooth muscle contraction, body temperature, and the proliferation of some cell types. Here, we show that mRNA encoding Nmb and its receptor (Nmbr) are expressed in rat bone tissue. Immunohistochemical analysis demonstrated thatNMB andNMBR colocalize in osteoblasts, epiphyseal chondrocytes, and proliferative chondrocytes of growth plates frommouse hind limbs. Then, we investigated the effect of NMB on the proliferation of rat primary cultured osteoblasts. Proliferation assays and 5-bromo-2′-deoxyuridine incorporation assays demonstrated that NMB augments the cell number and enhances DNA synthesis in osteoblasts. Pretreatment with the NMBR antagonist BIM23127 inhibited NMB-induced cell proliferation and DNA synthesis. Western blot analysis showed that NMB activates ERK1/2 MAPK signaling in osteoblasts. Pretreatment with the MAPK/ERK kinase inhibitor U0126 attenuatedNMB-induced cell proliferation and DNA synthesis.We also investigated the effects ofmolecules that contribute to osteoblast proliferation and differentiation onNmb expression in osteoblasts. Real-time PCR analysis demonstrated that 17b-estradiol (E2) and transforming growth factor b1 increase and decrease Nmb mRNA expression levels respectively. Finally, proliferation assays revealed that the NMBR antagonist BIM23127 suppresses E2-induced osteoblast proliferation. These results suggest that NMB/NMBR signaling plays an autocrine or paracrine role in osteoblast proliferation and contributes to the regulation of bone formation. © 2013 Society for Endocrinology.
DOI： 10.1530/JOE-12-0488
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Shinsato Y., Furukawa T., Yunoue S., Yonezawa H., Minami K., Nishizawa Y., Ikeda R., Kawahara K., Yamamoto M., Hirano H., Tokimura H., Arita K.
Oncotarget 4 ( 12 ) 2261 - 2270 2013.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncotarget
Although there is a relationship between DNA repair deficiency and temozolomide (TMZ) resistance in glioblastoma (GBM), it remains unclear which molecule is associated with GBM recurrence. We isolated three TMZ-resistant human GBM cell lines and examined the expression of O6-methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) components. We used immunohistochemical analysis to compare MutL homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2) and MGMT expression in primary and recurrent GBM specimens obtained from GBM patients during TMZ treatment. We found a reduction in MLH1 expression and a subsequent reduction in PMS2 protein levels in TMZ-resistant cells. Furthermore, MLH1 or PMS2 knockdown confered TMZ resistance. In recurrent GBM tumours, the expression of MLH1 and PMS2 was reduced when compared to primary tumours.
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Matsushita S., Ikeda R., Fukushige T., Tajitsu Y., Gunshin K., Okumura H., Ushiyama M., Akiyama S., Kawai K., Takeda Y., Yamada K., Kanekura T.
Journal of Dermatological Science 68 ( 1 ) 19 - 24 2012.10
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Dermatological Science
Background The treatment of melanoma, an aggressive, chemo-resistant skin cancer characterized by rapid metastasis and a poor prognosis, requires the development of innovative therapies with improved efficacy. The p53R2 gene that encodes the ribonucleotide reductase small subunit 2 homologue is induced by several stress signals including DNA-damaging agents that activate p53. The p53R2 gene product increases the deoxynucleotide triphosphate pool in the nucleus; this facilitates DNA repair and synthesis.Objective We examined the expression of p53R2 in melanoma and evaluated whether p53R2 is involved in the growth and proliferation of melanoma cells.Methods We examined the clinicopathological significance of p53R2 in melanoma. To investigate the role of p53R2 in melanoma we used KHm5 and KHm6 melanoma cells that express p53R2, and p53R2-targeting small interfering (si) RNA.Results p53R2 expression was detected immunohistochemically in 56 of 78 patients (71.8%). The expression of p53R2 was significantly correlated with the depth of invasion and the tumor stage. p53R2-targeting siRNA successfully knocked down p53R2 and significantly inhibited the growth of KHm5 and 6 cells. Moreover, The degree of KHm5 and 6 cell growth inhibition was greater in the presence of both p53R2-targeting siRNA and nimustine (ACNU) than with ACNU alone, suggesting that p53R2 silencing enhanced the chemosensitivity of KHm5 and 6 cells to ACNU.Conclusions We propose p53R2 as a therapeutic target to enhance the effectiveness of chemotherapy in patients with p53R2-positive melanoma. © 2012 Japanese Society for Investigative Dermatology.
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Stability of infliximab in polyvinyl chloride bags
Ikeda R., Vermeulen L., Lau E., Jiang Z., Saha S., Reichelderfer M., Kolesar J.
American Journal of Health-System Pharmacy 69 ( 17 ) 1509 - 1512 2012.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：American Journal of Health-System Pharmacy
Purpose. The stability of prepared infusions of the tumor necrosis factor (TNF)-α agent infliximab after storage for up to two weeks was investigated. Methods. To determine the feasibility of liberalized expiration dating of infliximab (current recommendations call for the infusion of prepared doses within three hours), the stability of diluted infliximab stored in polyvinyl chloride (PVC) bags at 4°C for up to 14 days was evaluated. A known quantity of TNF-a was combined with infliximab test samples in PVC bags for one hour; immediately after the reaction period and after 7 and 14 days of storage, the residual amount of TNF-α (an indirect measure of the drug's biological activity) was analyzed via a validated enzyme-linked immunosorbent assay (ELISA). Results. The mean ± S.D. amount of TNF-α consumed by infliximab was calculated to be 24.5 ± 5.6 pg/mL at baseline, 29.0 ± 4.4 pg/mL at 7 days, and 24.8 ± 17.3 pg/ mL at 14 days. At all evaluated time points, ELISA results indicated that 19-24% of the original TNF-α had been consumed by infliximab (mean ± S.D. consumption: 19.6% ± 4.5% at baseline, 23.2% ± 3.5% at 7 days, and 19.8% ± 13.8% at 14 days). Conclusion. Infliximab, when prepared at a concentration of 400 μg/mL in 0.9% sodium chloride injection, incurred no loss of biological activity when stored for up to 14 days at 4°C in PVC bags. Changing in- fliximab preparation practices may improve clinic efficiency by reducing patient dissatisfaction with long wait times for infusions and avoiding costly waste. Copyright © 2012, American Society of Health-System Pharmacists, Inc. All rights reserved.
DOI： 10.2146/ajhp100116
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Tabata S., Ikeda R., Yamamoto M., Furukawa T., Kuramoto T., Takeda Y., Yamada K., Haraguchi M., Nishioka Y., Sone S., Akiyama S.
Oncology Reports 28 ( 3 ) 895 - 902 2012.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncology Reports
Thymidine phosphorylase (TP) is an angiogenic factor that plays a pivotal role in tumor angiogenesis. Various kinds of solid tumors express TP and high TP activity is correlated with microvessel density. We have previously reported that TP enhances interleukin-8 (IL-8) expression in KB human epidermoid carcinoma cells. In this study, TP was shown to be involved in enhanced expression of IL-8 in EJ human bladder cancer cells and Yumoto human cervical cancer cells as well as KB human epidermoid carcinoma cells. The enzymatic activity of TP was required for the enhanced expression of IL-8. A degradation product of thymidine was implicated in the enhanced expression of IL-8. TP augmented reactive oxygen species (ROS) generation in KB and Yumoto cells, and the enzymatic activity of TP was again required for the generation of ROS. An antioxidant, N-acetylcysteine (NAC), attenuated the generation of ROS and IL-8 mRNA expression in KB and Yumoto cells, and H2O2increased IL-8 mRNA expression in Yumoto cells, suggesting that ROS generated by TP caused the increased expression of IL-8 mRNA. Since TP also reduced cellular glutathione levels and transcription of γ-GCS in KB cells, the TP-induced augmentation of ROS may be partially attributed to the decreased glutathione. Our findings suggest that thymidine-derived sugars enhanced ROS generation and consequently increased IL-8 expression.
DOI： 10.3892/or.2012.1887
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Neuromedin B stimulates proliferation of mouse chondrogenic cell line ATDC5
Saito H., Ikeda R., Inoue K., Nagata S., Kitamura K., Minamino N., Kangawa K., Miyata A.
Peptides 36 ( 2 ) 299 - 302 2012.8
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Peptides
Neuromedin B (NMB), which was originally isolated from porcine spinal cord, is a mammalian bombesin-related peptide that exerts various physiological effects. Previously, we observed expression of NMB in rib cartilage from chicken. Here, we report the initial attempt to elucidate the role of NMB in cartilage. We used RT-PCR to measure the expression of NMB and its receptor (NMB-R) in mouse chondrogenic cell line ATDC5. During chondrogenic differentiation of ATDC5 cells, NMB mRNA transiently increased on day 4 and then decreased on day 14, whereas NMB-R mRNA decreased on days 7 and 14. We also characterized immunoreactive NMB in ATDC5 culture medium using a combination of specific radioimmunoassay (RIA) and reverse phase-high performance liquid chromatography (RP-HPLC). Furthermore, using the WST-8 assay, we demonstrated that NMB significantly induced ATDC5 proliferation; this was inhibited by NMB-R antagonist, BIM 23127. These results implicate that NMB is involved in cartilage development, either in an autocrine or paracrine manner. Crown Copyright © 2012 Published by Elsevier Inc. All rights reserved.
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Oiso S., Ikeda R., Nakamura K., Takeda Y., Akiyama S., Kariyazono H.
Oncology Reports 28 ( 1 ) 27 - 32 2012.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncology Reports
cis-Diamminedichloroplatinum II (cisplatin) is one of the most potent antitumor agents for the treatment of various types of cancer. In spite of its therapeutic usefulness, the intrinsic resistance acquired under continuous treatment limits its benefit in cancer therapy. KCP-4, a cisplatin-resistant cell line, was derived from human epidermoid carcinoma KB-3-1 cells. Since the accumulation of cisplatin in KCP-4 cells is markedly reduced by the presence of an efflux pump, this pump is thought to be related to cisplatin resistance of the KCP-4 cells. However, given that KCP-4 cells are tremendously resistant to cisplatin compared with KB-3-1 cells, it is possible that another mechanism exists. The aim of this study was to investigate whether the activation of nuclear factor-kappa B (NF-κB) contributes to the cisplatin resistance of KCP-4 cells. We used the level of translocated NF-κB into the nucleus, determined by immunoblot analysis, as the indicator of NF-κB activation. The activation level of NF-κB was higher in KCP-4 cells than in KB-3-1 cells. KCP-4 cells were treated with a combination of cisplatin and curcumin, an inhibitor of NF-κB activation, and the cell viabilities were subsequently determined by the MTT assay using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide. In the presence of 10 μmol/l curcumin, we found that the sensitivity of KCP-4 cells to 100 and 300 μmol/l cisplatin was augmented. Additionally, curcumin reduced the activation levels of NF-κB in KCP-4 cells, and suppressed the expression levels of Bcl-2, Bcl-xL and survivin, which are apoptosis-related proteins regulated by NF-κB. Our results suggest that the high cisplatin resistance of KCP-4 cells compared with KB-3-1 cells results from multiple mechanisms other than increased cisplatin efflux, including the activation of NF-κB.
DOI： 10.3892/or.2012.1801
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Miyawaki A., Hijioka H., Ikeda R., Ishida T., Nozoe E., Nakamura N.
Oncology Letters 3 ( 5 ) 995 - 1001 2012.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncology Letters
We introduced concurrent neoadjuvant chemoradiotherapy (CCRT) with S-1, an oral fluoropyrimidine, as treatment for oral squamous cell carcinoma (OSCC) from October 2005. The clinical usefulness and medical safety of CCRT with S-1 (S-1 group) for OSCC were analyzed and compared with CCRT using super-selective intra-arterial infusion (AI group). The subjects in the S-1 group underwent external irradiation, at a total dose of 30 Gy, with S-1 chemotherapy. The AI group received cisplatin (CDDP) or carboplatin (CBDCA) combined with daily radiotherapy at a total dose of 40 Gy. The histological effects and disease-specific survival rates were almost equivalent in the S-1 and AI groups. Adverse events were less frequent in the S-1 group, while hematological toxicity, including anemia, thrombopenia and pharyngeal edema, was observed in the AI group. The results of this study indicate that CCRT combined with S-1 is a more effective and safer treatment for OSCC than AI.
DOI： 10.3892/ol.2012.606
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Effect of 5-fluorouracil treatment on SN-38 absorption from intestine in rats
Shibayama Y., Iwashita Y., Yoshikawa Y., Kondo T., Ikeda R., Takeda Y., Osada T., Sugawara M., Yamada K., Iseki K.
Biological and Pharmaceutical Bulletin 34 ( 9 ) 1418 - 1425 2011.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biological and Pharmaceutical Bulletin
5-Fluorouracil (5-FU)-based chemotherapies with irinotecan have been applied for the treatment of cancers, and a common dose-limiting toxicity is neutropenia and diarrhea. In this study, we investigated the effect of 5-FU treatment on expression levels of drug transporters for SN-38 transportation and SN-38 absorption from the intestine following 5-FU treatment. Expression levels of several drug transporters and nuclear receptors in rats after 5-FU treatment were evaluated. SN-38 absorption from the intestine was evaluated by SN-38 concentration levels in serum following SN-38 injection into the intestine of 5-FU treated rats. The levels of renal multidrug resistance protein 2 (Mrp2) on day 4 after treatment (400 mg/kg) showed significant upregulation, 359.2±33.2% (mean±S.E.) of control. Mrp2 levels in the intestine were downregulated to 26.2±8.4% of control. 5-FU treatment (400 mg/kg) also significantly downregurated expression levels of P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) to 41.2±14.7%, 15.7±4.3% of control, respectively. To evaluate SN-38 absorption from the intestine, SN-38 was loaded in to the intestine on day 4 after 5-FU treatment. Pretreatment with 5-FU significantly increased SN-38 concentration in the blood 30, 60 and 90 min after SN-38 administration. The area under the curve for SN-38 in the 5-FU group was significantly higher than in vehicle groups. 5-FU treatment decreased expression levels of P-glycoprotein and Bcrp in intestine. The present study suggests that combination chemotherapy of 5-FU with irinotecan (CPT-11) may elevate SN-38 absorption from intestine. © 2011 Pharmaceutical Society of Japan.
DOI： 10.1248/bpb.34.1418
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Multidrug resistance protein 2 implicates anticancer drug-resistance to sorafenib
Shibayama Y., Nakano K., Maeda H., Taguchi M., Ikeda R., Sugawara M., Iseki K., Takeda Y., Yamada K.
Biological and Pharmaceutical Bulletin 34 ( 3 ) 433 - 435 2011.3
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biological and Pharmaceutical Bulletin
Sorafenib and sunitinib is a small molecule inhibitor of certain receptor tyrosine kinases, and have improved outcomes for patients with advanced renal cell carcinoma. Inhibitory concentration of 50% cell growth of sorafenib significantly rose to 6.4-fold in a multidrug resistance protein 2 (MRP2) transfected cell line versus control cell line. The concentration of sorafenib was significantly decreased to 74% of control cells after 3 h treatment. In contrast, a tyrosine kinase inhibitor sunitinib did not show alteration of inhibitory concentration of 50% cell growth and accumulation into the cells of MRP2 transfected cells. The present study suggest that sorafenib is a substrate for MRP2, suggesting that MRP2 may implicate drug resistance to sorafenib. © 2011 Pharmaceutical Society of Japan.
DOI： 10.1248/bpb.34.433
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Isolation and characterization of gemcitabine-resistant human non-small cell lung cancer A549 cells
Ikeda R., Vermeulen L., Lau E., Jiang Z., Sachidanandam K., Yamada K., Kolesar J.
International Journal of Oncology 38 ( 2 ) 513 - 519 2011.2
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：International Journal of Oncology
Gemcitabine is an effective chemotherapy against non-small cell lung cancer (NSCLC). However, resistance to gemcitabine reduces its efficacy. We have isolated gemcitabineresistant human non-small cell lung cancer A549 cells, termed A549/GR cells. A549/GR cells were resistant to gemcitabine as well as paclitaxel and docetaxel but not carboplatin and irinotecan. The expression level of multidrug resistance protein 7 (MRP7) in A549/GR cells was higher than that in A549 cells, and the inhibitor of MRP7 by cepharanthine increased the sensitivity to gemcitabine in A549/GR cells. These findings indicate that cepharanthine reversed gemcitabine resistance. To determine predictive molecular markers of gemcitabine resistance for more effective treatment of these tumors, we performed PCR array. We identified that CDKN1A/p21, CYP3A5, microsomal epoxide hyrolase 1 (EPHX1) and ABCC6 (MRP6) were up-regulated >5-fold in A549/GR cells. Gemcitabine also induced the expression of p21 and CYP3A5 in A549 cells. A better understanding of the characterization and mechanism of the resistance to gemcitabine in A549/GR cells may help identify agents that reverse clinical gemcitabine resistance in NSCLC.
DOI： 10.3892/ijo.2010.866
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Ushiyama M., Ikeda R., Yoshida M., Mori K., Kangawa K., Sugawara H., Inoue K., Yamada K., Miyata A.
Journal of Molecular Neuroscience 42 ( 3 ) 341 - 348 2010.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Molecular Neuroscience
Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides performing a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA region encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. To characterize the molecular forms of alternative splicing variants of PAC1, we examined the binding affinity and activation of two major second messenger pathways (cAMP production and changes in [Ca2+]i) by PACAP-27. Activation of cAMP was influenced by the variant in both of the EC1 domain and IC3 loops. In the N form in the EC1 domain, the suppressive effect of the HOP1 form in the IC3 loop was enhanced. Regarding the intracellular calcium mobilization assay, the rank order of the potency of PACAP-27 for the different PAC1 isoforms was S/HOP1∈>>∈N/R∈∈S/ R∈>>∈N/HOP1. In particular, PACAP-27 exhibited remarkable potency of calcium mobilization in the S/HOP1-expressing cells at sub-picomolar concentrations even though the affinities of PACAP-27 to the four PAC1 isoforms were not significantly different. This suggests the specific functions of PACAP-27 due to PACAP-27 preferring PAC1 activation, and leads in clarification of the pleiotoropic function of PACAP. © 2010 Springer Science+Business Media, LLC.
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Ikeda R., Vermeulen L., Lau E., Jiang Z., Pomplun M., Kolesar J.
Molecular Medicine Reports 3 ( 6 ) 1031 - 1034 2010.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Molecular Medicine Reports
Irinotecan (CTP-11) is a topoisomerase I inhibitor used in the treatment of colorectal cancer and non-small cell lung cancer (NSCLC). Despite an initial response to therapy, resistance to irinotecan reduces its efficacy. We isolated irinotecan-resistant human NSCLC A549 cells, termed A549/CTP-11R cells. A549/CTP-11R cells were resistant to irinotecan, as well as paclitaxel, gemcitabine and carboplatin. Curcumin, a nuclear factor-κB (NF-κB) inhibitor, increased the sensitivity to irinotecan of A549/CTP-11R cells. The expression level of Bcl-XL and X-linked inhibitor of apoptosis protein, target genes of NF-κB, in A549/CTP-11R cells was higher than that in A549 cells. Our result suggests that the addition of curcumin to irinotecan reverses irinotecan resistance in NSCLC.
DOI： 10.3892/mmr.2010.366
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Hirashima M., Tsuda K., Hamada T., Okamura H., Furukawa T., Akiyama S., Tajitsu Y., Ikeda R., Komatsu M., Doe M., Morimoto Y., Shiro M., Van Soest R., Takemura K., Iwagawa T.
Journal of Natural Products 73 ( 9 ) 1512 - 1518 2010.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Natural Products
Seven new isomalabaricane derivatives, rhabdastins A-G (1-7), and a new monocyclic triterpene glycoside, rhabdastoside A (8), have been isolated from the methanol extract of the sponge Rhabdastrella globostellata, collected at Amami-oshima, Japan. Three of them were isolated as their corresponding methyl esters, rhabdastins A-D (1-3). Their structures were determined on the basis of spectroscopic and X-ray diffraction analyses. The isolated compounds were evaluated for their cytotoxicity against the proliferation of promyelocytic leukemia HL-60 cells. Compounds 4, 5, 7, and 11, possessing a cyclopentane side chain, exhibited weak activity, with IC50values of 21, 29, 44, and 11 μM, respectively, while compounds 1, 2, and 3, with a 2-substituted- propanoate side chain, were inactive at 100 μM. In addition, the mechanism of cytotoxicity of compounds 4 and 5 was investigated. © 2010 The American Chemical Society and American Society of Pharmacognosy.
DOI： 10.1021/np100302a
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Ikeda R., Vermeulen L., Jiang Z., Lau E., Kolesar J.
Experimental and Therapeutic Medicine 1 ( 5 ) 853 - 857 2010.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Experimental and Therapeutic Medicine
Vascular endothelial growth factor (VEGF) plays an important role in the process of angiogenesis in many types of cancer, including non-small cell lung cancer (NSCLC), and angiogenesis inhibitors and standard chemotherapy exhibit synergy though an unknown mechanism. We therefore hypothesized that cytotoxic chemotherapy influences VEGF production and analyzed VEGF production in an NSCLCA549 cell line after treatment with standard chemotherapy. Paclitaxel inhibited the production of VEGF in A549 cells, while cisplatin and erlotinib did not. Paclitaxel and gemcitabine inhibited deferoxamine (DFX) (known to mimic hypoxia)-induced VEGF production in A549 cells. Erlotinib also inhibited DFX-induced VEGF production in A549 cells slightly, while cisplatin did not. We subsequently examined the effect of the interaction between paclitaxel or gemcitabine and VEGF protein. Paclitaxel and gemcitabine did not directly affect the binding of VEGF. Since VEGF is known as one of the HIF-1 target genes, we examined the effect of paclitaxel and gemcitabine on HIF-1α levels induced by DFXin A549 cells. Paclitaxel and gemcitabine inhibited DFX-induced HIF-1α in A549 cells. These findings may be useful for future treatment schedules, including anti-cancer agents in NSCLC.
DOI： 10.3892/etm.2010.130
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P53 plays an important role in cell fate determination after exposure to Microcystin-LR
Takumi S., Komatsu M., Furukawa T., Ikeda R., Sumizawa T., Akenaga H., Maeda Y., Aoyama K., Arizono K., Ando S., Takeuchi T.
Environmental Health Perspectives 118 ( 9 ) 1292 - 1298 2010.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Environmental Health Perspectives
Background: Microcystin-LR, a cyclic heptapeptide, possesses the ability to inhibit the serine/threonine protein phosphatases PP1 and PP2A and, consequently, exhibits acute hepatocytotoxicity. Moreover, microcystin-LR induces cellular proliferation, resulting in tumor-promoting activity in hepatocytes. However, mechanisms that regulate the balance between cell death and proliferation after microcystin-LR treatment remain unclear. Objective: We examined the contribution of the transcription factor p53, as well as that of the hepatic uptake transporter for microcystin-LR, organic anion transporting polypeptide 1B3 (OATP1B3), to the cellular response to microcystin-LR exposure. Methods: We analyzed intracellular signaling responses to microcystin-LR by immunoblotting and real-time reverse-transcriptase polymerase chain reaction techniques using HEK293 human embryonic kidney cells stably transfected with SLCO1B3 (HEK293-OATP1B3). In addition, we analyzed the effect of attenuation of p53 function, via the p53 inhibitor pifithrin-α, and knockdown of p53 mRNA on the cytotoxicity of microcystin-LR using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Microcystin-LR induced the phosphorylation and accumulation of p53 in HEK293-OATP1B3 cells, which resulted in up-regulation of the expression of p53 transcript targets, including p21 and seven in absentia homolog 1 (siah-1). In addition, microcystin-LR activated Akt signaling through the phosphorylation of Akt and glycogen synthase kinase 3β. Although Akt signaling was activated, the accumulation of p53 led cells to apoptosis after treatment with 50 nM microcystin-LR for 24 hr. Both pharmacological inhibition of transcription factor activity of p53 by pifithrin-α and knockdown of p53 with small hairpin RNA attenuated the susceptibility of HEK293-OATP1B3 cells to microcystin-LR. Conclusions: This study demonstrates the importance of p53 in the regulation of cell fate after exposure to microcystin-LR. Our results suggest that, under conditions of p53 inactivation (including p53 mutation), chronic exposure to low doses of microcystin-LR may lead to cell proliferation through activation of Akt signaling. Results of this study may contribute to the development of chemoprevention and chemotherapeutic approaches to microcystin-LR poisoning.
DOI： 10.1289/ehp.1001899
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Miyawaki A., Ikeda R., Hijioka H., Ishida T., Ushiyama M., Nozoe E., Nakamura N.
Oncology Reports 23 ( 5 ) 1205 - 1212 2010.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncology Reports
The aim of this study was to analyze the relationship between the maximum standardized uptake value (SUVmax) of 18F-fluoro-2-deoxyglucose-positron emission tomography (FDG-PET) and the effects of neoadjuvant chemoradiotherapy in oral squamous cell carcinoma (OSCC), and to identify the possible biological background of this association. Thirty-seven patients with OSCC, who underwent preoperative FDG-PET followed by cancer treatment with neoadjuvant chemoradiotherapy, were enrolled in this study. The various histological effects following neoadjuvant chemoradiotherapy were compared to the SUVmax in the primary OSCC. These effects were also compared to the immunohistochemical staining score of hypoxia-inducible factor-1α (HIF-1α), glucose membrane transporter (GLUT)-1 and vascular endothelial growth factor (VEGF) in the biopsy specimen. Furthermore, we analyzed the chemosensitivity of KB-3-1 cells to cisplatin under hypoxic conditions using the MTT assay. A negative correlation was observed between the SUVmax and the histological effects following neoadjuvant chemoradiotherapy (p<0.01). The SUVmax was also correlated with the staining score of HIF-1α (p<0.03), but not with GLUT-1 and VEGF. The mean staining score of HIF-1α in the highly effective group was 2.7±1.1, which was significantly lower than that (3.7±0.9) of the poorly effective group (p<0.05). The cell chemosensitivity assay revealed chemoresistant effects under a hypoxic condition in OSCC. In conclusion, the SUVmax is correlated with the effectiveness of neoadjuvant chemoradiotherapy in OSCC. Our clinical and experimental analyses further suggest a possible association of the upregulation of HIF-1α with chemoradiosensitivity in SCC cells.
DOI： 10.3892/or-00000751
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Matsushita S., Ikeda R., Nishizawa Y., Che X., Furukawa T., Miyadera K., Tabata S., Ushiyama M., Tajitsu Y., Yamamoto M., Takeda Y., Minami K., Mataki H., Kanzaki T., Yamada K., Kanekura T., Akiyama S.
International Journal of Oncology 36 ( 5 ) 1193 - 1200 2010.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：International Journal of Oncology
Thymidine phosphorylase (TP) is an enzyme involved in reversible conversion of thymidine to thymine. TP is identical to an angiogenic factor, pletelet-derived endothelial cell growth factor (PD-ECGF) and the expression levels of TP in a variety of malignant tumors were higher than the adjacent non-neoplastic tissues. To investigate the molecular basis for the effect of TP on the metabolic process and the anticancer effect of 5-fluorouracil (5-FU), human gastric carcinoma AZ521 cells and epidermoid carcinoma KB cells were transfected with TP cDNA, and AZ521/TP and KB/ TP were cloned. AZ521/TP and KB/TP cells overexpressed TP and were more sensitive to 5-FU than the counterpart parental cells. TPI, a newly synthesized inhibitor for TP (Ki=2.36x10-9M), decreased the sensitivity to 5-FU of the TP expressing cells but not of the parental cells. 5-Formyltetrahydrofolate (leucovorin; LV) stabilized the complex of thymidylate synthase (TS) and 5-fluoro-deoxyuridine-monophosphate (FdUMP), increased the sensitivity to 5-FU of TP expressing AZ521 cells, but not of the parental cells. The levels of FdUMP in TP expressing cells were significantly higher than in parental cells and TPI considerably decreased FdUMP to the level comparable to that in the parental cells. 5-FU increased the expression of early growth response protein-1 (Egr-1) and an angiogenesis inhibitor, thrombospondin-1 (TSP-1), in KB/TP cells but only slightly in KB/CV cells, if any. TPI attenuated the induction of Egr-1 and TSP-1 mRNA by 5-FU, while LV increased the expression of Egr-1 and TSP-1 mRNA in KB/TP cells. These findings demonstrate that the TP has a principal role in the production of FdUMP and the enhanced responses to 5-FU by leucovorin in TP-overexpressing KB and AZ521 cells, and FdUMP but not FUTP is implicated in the induction of Egr-1 and TSP-1 in KB cells.
DOI： 10.3892/ijo-00000602
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Iwashita K., Ikeda R., Takeda Y., Sumizawa T., Furukawa T., Yamaguchi T., Akiyama S., Yamada K.
Cancer Science 101 ( 4 ) 920 - 926 2010.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Science
Vaults are evolutionarily highly conserved ribonucleoprotein (RNP) particles with a hollow barrel-like structure. Although roles in multidrug resistance and innate immunity have been suggested, the physiological function of vaults remains unclear. Major vault protein (MVP), the main component of the vault particle, has been reported to be induced by hypoxia. However, there are no reports about the effect of vaults on cellular responses to hypoxia. We thus examined whether vaults are implicated in cellular responses to hypoxia. In this study, we focused on hypoxia-inducible factor-1α (HIF-1α), which is a master regulator of hypoxic responses, and found that: (i) MVP knockdown by RNA interference increases HIF-1α protein levels induced by hypoxia and hypoxia mimetics; (ii) MVP knockdown does not affect HIF-1α mRNA levels, but decreases the ubiquitination and degradation of HIF-1α protein; and (iii) vaults form complexes with HIF-1α, PHD2, and pVHL. Taken together, these results suggest that vaults function as scaffolds in HIF-1α degradation pathway and promote the ubiquitination and degradation of HIF-1α. © 2010 Japanese Cancer Association.
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Adverse events of superselective intra-arterial infusion chemotherapy in patients with oral cancer
Ushiyama M., Ikeda R., Yamaguchi H., Miyawaki A., Nitta T., Yamaguchi T., Tazitsu Y., Nishizawa Y., Shimodouzono Y., Hijioka H., Furukawa T., Akiyama S., Nakamura N., Takeda Y., Yamada K.
Journal of Applied Therapeutic Research 7 ( 2 ) 58 - 64 2009.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Applied Therapeutic Research
Superselective intra-arterial infusion chemotherapy (SIC) for oral cancer is an attractive method to achieve a high concentration of anti-cancer agents at the site of the local lesion. Although the therapy is clinically effective, the adverse events remain unclear. We studied thirteen patients with oral cancer who were treated either with a combined docetaxel/cisplatin regimen or carboplatin regimen as SIC. The dosages of docetaxel and cisplatin were based on body surface area (60mg/m2and 50-70 mg/m2, respectively). In contrast, the dosage of carboplatin was determined using the Calvert formula which is based on body weight, height, renal function, age and gender. It was found that the combined docetaxel/cisplatin regimen reduced the leukocytes count to 3,000/mm3or less. However, no patients treated with the carboplatin regimen exhibited signs of hematological toxicity. These results suggest that patients receiving SIC with the docetaxel/cisplatin regimen need more attention than those on the carboplatin regimen. As a result, we have developed a work sheet that will be useful for monitoring adverse events and reconfirming the dosage of anti-cancer agents. Predicting the onset and early detection of adverse events enables early intervention by medical staff leading to safer and more effective cancer chemotherapy.
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Contactin-associated protein (Caspr) 2 interacts with carboxypeptidase e in the CNS
Oiso S., Takeda Y., Futagawa T., Miura T., Kuchiiwa S., Nishida K., Ikeda R., Kariyazono H., Watanabe K., Yamada K.
Journal of Neurochemistry 109 ( 1 ) 158 - 167 2009.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Neurochemistry
To identify proteins interacting with the intracellular domain of the neural cell adhesion molecule contactin-associated protein 2 (Caspr2), yeast two-hybrid screening was performed. We identified carboxypeptidase E (CPE) as a Caspr2-interacting candidate protein. Glutathione S-transferase pull-down and immunoprecipitation analyses indicated that Caspr2 was associated with CPE in vitro and in vivo. Both Caspr2 and CPE were expressed predominantly in the CNS. Immunohistochemical analyses revealed that both Caspr2- and CPE-like immunoreactivities were found to co-localize in the apical dendrites and cell bodies of rat cortical neurons. In subcellular localization analysis, Caspr2- and CPE-like immunoreactivities were co-migrated in the fractions of Golgi/ER. Additionally, in COS-7 cells co-transfected with CPE and Caspr2 cDNAs, Caspr2- and CPE-immunoreactivities were co-localized in both Golgi and membrane, whereas it was only observed in Golgi of either COS-7 cell transfected with CPE or Caspr2 cDNA alone. It is known that the membrane-bound form of CPE functions as a sorting receptor of prohormones in the trans-Golgi network. Taken together, our data suggest that CPE may be a key molecule to regulate Caspr2 trafficking to the cell membrane. © 2009 International Society for Neurochemistry.
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Ayukawa O., Nakamura K., Kariyazono H., Ikeda R., Arima J., Shinkawa T., Iwase H., Sakata R., Yamada K.
Blood Coagulation and Fibrinolysis 20 ( 3 ) 176 - 184 2009.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Blood Coagulation and Fibrinolysis
To investigate platelet responsiveness during cold storage of whole blood, we examined platelet aggregation, expression of CD40 ligand (CD40L) on platelets, the plasma levels of soluble form of CD40L (sCD40L) as well as platelet-leukocyte aggregates. Flow cytometry analysis was performed to investigate platelet-leukocyte aggregate formation using antibodies against CD42b and CD45 and platelet activation using antibodies against P-selectin and PAC-1. Blood samples were collected from healthy volunteers, patients with cardiovascular diseases, or both. In the healthy volunteers' blood samples stored at 4°C for 6 h, platelet aggregation in response to 1 μmol/l ADP was enhanced, and released levels of soluble form of P-selectin and thromboxane B2 in response to 1 μmol/l ADP markedly increased. In the samples stored at 4°C for 6 h but not stimulated by any agonists, CD40L expression on the platelets was increased, and plasma levels of sCD40L were also elevated. Under the same condition, the increase in simultaneous expression of CD45 and CD42b was observed. In patients with cardiovascular diseases, the platelet aggregability, coexpression of P-selectin and PAC-1, expression of CD40L on platelets and both CD45-bound and CD42b-bound subsets were all comparable to those of healthy volunteers, samples stored at 4°C for 6 h. Plasma levels of sCD40L in patients were higher than those in healthy volunteers' control. Taken together, storage of whole blood at 4°C for 6 h caused platelet activation comparable to that of patients with cardiovascular diseases, and enhanced platelet activity in such patients may be involved in increased risk for thromboembolic events. © 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins.
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Ushiyama M., Ikeda R., Nitta T., Tazitsu Y., Miyawaki A., Nishizawa Y., Yamaguchi T., Yamaguchi H., Akatsuka C., Shimodouzono Y., Ushinohama K., Sugawara H., Sugihara K., Nakamura N., Takeda Y., Yamada K.
Cancer Therapy 7 ( ISSUE A ) 277 - 281 2009.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Therapy
An important factor in caring for oral cancer patients receiving chemotherapy and/or radiation therapy is palliation of oral mucositis and acute oral pain due to oral mucositis. One effective treatment is the Azunol Gargle, which contains sodium gualenate hydrate (GAS-Na) with or without lidocaine hydrochloride, and is prepared in four forms: Azunol Saline Gargle (AS, saline solution containing 0.006% GAS-Na), Azunol Lidocaine Saline Gargle (ALS, AS with lidocaine), Azunol Water Gargle (AW, aqueous solution containing 0.006% GAS-Na) and Azunol Lidocaine Water Gargle (ALW, AW with lidocaine). However, the four Azunol Gargles are expected to improve the quality of life of patients with oral mucositis, little is known about the stability of AW and ALW. Therefore, we examined stability of those solutions to the light and the temperature as an index of residual ratio of GAS-Na. As a result, AW and ALW were stable for seven days at room temperature if shielded from light, and at 4°C under lighting conditions similar to those at nursing stations. These results provide useful information regarding the management of oral mucositis in oral cancer patients.
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Ushiyama M., Ikeda R., Nitta T., Tazitsu Y., Miyawaki A., Yamaguchi T., Shimodouzono Y., Ushinohama K., Matsui R., Sugihara K., Nakamura N., Yamada K.
Oral Therapeutics and Pharmacology 27 ( 3 ) 143 - 150 2008.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oral Therapeutics and Pharmacology
In our hospital, hospital preparations of Azunol Saline Gargle (AS, saline solution containing 0.006% sodium gualenate hydrate), and Azunol Lidocaine Saline Gargle (ALS, AS with lidocaine) are used as a treatment for oral cancer with oral mucositis. However, little is known about the stability and microbiological safety of AS and ALS. In this study, the stability and microbiological safety of AS and ALS were assessed as the pH and the percent of sodium gualenate hydrate remaining in both preparations after exposure to various light and temperature conditions and the colony formation, respectively. As a result, we found that under fluorescent lamp lighting, AS and ALS were stable for 7 days at 4°C compared with 25°C or room temperature. Furthermore, by light shielding, they were stable for at least 14 days at 4°C. Bacterial contamination of AS was prevented by preserving at 4°C for 14 days. We have demonstrated the stability and microbiological safety of AS and ALS and established an appropriate preservation method. This study provides useful information regarding the management of oral mucositis in oral cancer patients.
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Copper transport systems are involved in multidrug resistance and drug transport
Furukawa T., Komatsu M., Ikeda R., Tsujikawa K., Akiyama S.
Current Medicinal Chemistry 15 ( 30 ) 3268 - 3278 2008.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Current Medicinal Chemistry
Copper is an essential trace element and several copper containing proteins are indispensable for such processes as oxidative respiration, neural development and collagen remodeling. Copper metabolism is precisely regulated by several transporters and chaperone proteins. Copper Transport Protein 1 (CTR1) selectively uptakes copper into cells. Subsequently three chaperone proteins, HAH1 (human atx1 homologue 1), Cox17p and CCS (copper chaperone for superoxide dismutase) transport copper to the Golgi apparatus, mitochondria and copper/zinc superoxide dismutase respectively. Defects in the copper transporters ATP7A and ATP7B are responsible for Menkes disease and Wilson's disease respectively. These proteins transport copper via HAH1 to the Golgi apparatus to deliver copper to cuproenzymes. They also prevent cellular damage from an excess accumulation of copper by mediating the efflux of copper from the cell. There is increasing evidence that copper transport mechanisms may play a role in drug resistance. We, and others, found that ATP7A and ATP7B are involved in drug resistance against the anti-tumor drug cis-diamminedichloroplatinum (II) (CDDP). A relationship between the expression of ATP7A or ATP7B in tumors and CDDP resistance is supported by clinical studies. In addition, the copper uptake transporter CTR1 has also been reported to play a role in CDDP sensitivity. Furthermore, we have recently found that the effect of ATP7A on drug resistance is not limited to CDDP. Using an ex vivo drug sensitivity assay, the histoculture drug response assay (HDRA), the expression of ATP7A in human surgically resected colon cancer cells correlated with sensitivity to 7-ethyl-10-hydroxy-camptothecin (SN-38). ATP7Aoverexpressing cells are resistant to many anticancer drugs including SN-38, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11), vincristine, paclitaxel, etoposide, doxorubicin (Dox), and mitoxantron. The mechanism by which ATP7A and copper metabolism modulate drug transport appears to involve modulation of drug cellular localization via modulation of the vesicle transport system. In ATP7A overexpressing cells, Dox accumulates in the Golgi apparatus. In contrast, in the parental cells, Dox is localized in the nuclei, where the target molecules of Dox, topoisomerase II and DNA, are found. Disruption of the intracellular vesicle transport system with monensin, a Na+/H+ ionophore, induced the relocalization of Dox from the Golgi apparatus to the nuclei in the ATP7A overexpressing cells. These data suggested that ATP7A-related drug transport is dependent on the vesicle transport system. Thus copper transport systems play important roles in drug transport as well as in copper metabolism. Components of copper metabolism are therefore likely to include target molecules for the modulation of drug potency of not only anti-cancer agents but also of other drugs. © 2008 Bentham Science Publishers Ltd.
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Zhao H., Ooyama A., Yamamoto M., Ikeda R., Haraguchi M., Tabata S., Furukawa T., Che X., Iwashita K., Oka T., Fukushima M., Nakagawa M., Ono M., Kuwano M., Akiyama S.
Cancer Letters 270 ( 1 ) 156 - 163 2008.10
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Letters
5-FU is commonly used for treatment of various solid tumors including colon carcinoma. We have previously demonstrated that Egr-1 induced by 5-FU enhanced TSP-1 expression in human colon cancer KM12C cells. In this study, a Genechip analysis of KM12C cells treated with 5-FU revealed down-regulation of 924 genes and up-regulation of 460 genes. The decreased expression of c-Myc mRNA and phosphorylated c-Myc were detected and confirmed by RT-PCR and immunoblotting. Since 5-FU induced the expression of TSP-1, we examined the effect of c-Myc on the TSP-1 promoter. Deletion of the TSP-1 promoter region in which binding sites for c-Myc reside had no effect on the TSP-1 promoter activity induced by 5-FU. Meanwhile, 5-FU dose-dependently decreased the expression of miR-17-92 cluster. These findings suggest that 5-FU decreased the expression of c-Myc and consequently miR-17-92 cluster and increased the expression of TSP-1 mRNA. © 2008 Elsevier Ireland Ltd. All rights reserved.
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Ikeda R., Iwashita K., Sumizawa T., Beppu S., Tabata S., Tajitsu Y., Shimamoto Y., Yoshida K., Furukawa T., Che X., Yamaguchi T., Ushiyama M., Miyawaki A., Takeda Y., Yamamoto M., Zhao H., Shibayama Y., Yamada K., Akiyama S.
Experimental Cell Research 314 ( 16 ) 3017 - 3026 2008.10
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Experimental Cell Research
The major vault protein (MVP) is the major constituent of the vault particle, the largest ribonuclear protein complex described to date and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several normal tissues, little is known about its physiological role. MVP played a protective role against some xenobiotics and other stresses. We thus investigated the effect of osmotic stress on MVP expression by treating human colon cancer SW620 cells with sucrose or NaCl. The expression level of both MVP protein and MVP mRNA was increased by the osmostress. Sucrose or sodium chloride could also enhance MVP promoter activity. Inhibition of p38 MAPK in SW620 cells by SB203580 inhibited the expression of MVP under hyperosmotic stress. These findings suggested that osmotic stress up-regulated the MVP expression through p38 MAPK pathway. Down-regulation of MVP expression by MVP interfering RNA (RNAi) in SW620 cells increased the sensitivity of the cells to hyperosmotic stress and enhanced apoptosis. Furthermore, MVP RNAi prevented the osmotic stress-induced, time-dependent increase in phosphorylated Akt. These findings suggest that the PI3K/Akt pathway might be implicated in the cytoprotective effect of MVP. Our data demonstrate that exposure of cells to hyperosmotic stress induces MVP that might play an important role in the protection of the cells from the adverse effects of osmotic stress. © 2008 Elsevier Inc. All rights reserved.
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Molecular basis for the induction of an angiogenesis inhibitor, thrombospondin-1, by 5-fluorouracil
Zhao H., Ooyama A., Yamamoto M., Ikeda R., Haraguchi M., Tabata S., Furukawa T., Che X., Zhang S., Oka T., Fukushima M., Nakagawa M., Ono M., Kuwano M., Akiyama S.
Cancer Research 68 ( 17 ) 7035 - 7041 2008.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Research
5-Fluorouracil (5-FU) is one of the most commonly used anticancer drugs in chemotherapy against various solid tumors. 5-FU dose-dependently increased the expression levels of intrinsic antiangiogenic factor thrombospondin-1 (TSP-1) in human colon carcinoma KM12C cells and human breast cancer MCF7 cells. We investigated the molecular basis for the induction of TSP-1 by 5-FU in KM12C cells. Promoter assays showed that the region with the Egr-1 binding site is critical for the induction of TSP-1 promoter activity by 5-FU. The binding of Egr-1 to the TSP-1 promoter was increased in KM12C cells treated with 5-FU. Immunofluorescence staining revealed that 5-FU significantly increased the level of Egr-1 in the nuclei of KM12C cells. The suppression of Egr-1 expression by small interfering RNA decreased the expression level of TSP-1. Furthermore, 5-FU induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27). Blockade of the p38 MAPK pathway by SB203580 remarkably inhibited the phosphorylation of HSP27 induced by 5-FU and decreased the induction of Egr-1 and TSP-1 by 5-FU in KM12C cells. These findings suggest that the p38 MAPK pathway plays a crucial role in the induction of Egr-1 by 5-FU and that induced Egr-1 augments TSP-1 promoter activity, with the subsequent production of TSP-1 mRNA and protein. ©2008 American Association for Cancer Research.
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Thymidine phosphorylase inhibits the expression of proapoptotic protein BNIP3
Ikeda R., Tajitsu Y., Iwashita K., Che X., Yoshida K., Ushiyama M., Furukawa T., Komatsu M., Yamaguchi T., Shibayama Y., Yamamoto M., Zhao H., Arima J., Takeda Y., Akiyama S., Yamada K.
Biochemical and Biophysical Research Communications 370 ( 2 ) 220 - 224 2008.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biochemical and Biophysical Research Communications
An angiogenic factor, thymidine phosphorylase (TP), confers resistance to apoptosis induced by hypoxia. We investigated the molecular basis for the suppressive effect of TP on hypoxia-induced apoptosis using Jurkat cells transfected with TP cDNA, Jurkat/TP, and a mock transfectant, Jurkat/CV. TP and 2-deoxy-d-ribose, a degradation product of thymidine generated by TP enzymatic activity, suppressed hypoxia-induced apoptosis. They also inhibited the upregulation of hypoxia-inducible factor (HIF) 1α and the proapoptotic factor, BNIP3, and caspase 3 activation induced by hypoxia. Introduction of siRNA against BNIP3 in Jurkat cells decreased the proportion of apoptotic cells under hypoxic condition. These findings suggest that the suppression of BNIP3 expression by TP prevents, at least in part, hypoxia-induced apoptosis. Expression levels of TP are elevated in many malignant solid tumors and thus 2-deoxy-d-ribose generated by TP in these tumors might play an important role in tumor progression by preventing hypoxia-induced apoptosis. © 2008 Elsevier Inc. All rights reserved.
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Saigo K., Yoshida K., Ikeda R., Sakamoto Y., Murakami Y., Urashima T., Asano T., Kenmochi T., Inoue I.
Human Mutation 29 ( 5 ) 703 - 708 2008.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Human Mutation
Integration of hepatitis B virus (HBV) DNA into host DNA is detected in about 90% of HBV-related hepatocellular carcinoma (HCC), but the preferential sites of the viral integration etiologically relevant to oncogenesis have been controversial. By using an adaptor-ligation/suppression-PCR, we identified four integrations into the myeloid/lymphoid or mixed-lineage leukemia 4 (MLL4) gene from 10 HCC patients with positive HBV surface antigen (HBsAg). Determination of the cellular-virus DNA junction demonstrated that various lengths of the virus were integrated within 300 bp of intron 3 flanked by the Alu element of MLL4. Chimeric hepatitis B virus X gene (HBx)/MLL4 transcripts and the HBx fusion proteins were detected. DNA microarray revealed that HBx/MLL4 fusion proteins suppressed unique genes in HepG2 cells. Finally, chromosomal translocations of intron 3 of MLL4 to the specific region of chromosome 17p11.2 in 22 out of 32 HCC patients were observed, showing that the intron 3 region of MLL4 gene would be a target of translocation breakpoint. In conclusion, the present data suggest that the translocation breakpoint of MLL4 gene is one of the preferential targets for HBV DNA integration into the MLL4 gene and the HBV DNA integration may be involved in liver oncogenesis. © 2008 Wiley-Liss, Inc.
DOI： 10.1002/humu.20701
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Yokomakura N., Natsugoe S., Okumura H., Ikeda R., Uchikado Y., Mataki Y., Takatori H., Matsumoto M., Owaki T., Ishigami S., Aikou T.
Oncology Reports 18 ( 3 ) 561 - 567 2007.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncology Reports
Chemoradiation therapy (CRT), a combination of X-ray irradiation and anticancer agents as a radiosensitizer, has been found to be an effective treatment for esophageal cancer and has been linked to p53 genetics. The p53 gene family regulates cell-cycle arrest, apoptosis and DNA damage repair. A recently identified ribonucleotide reductase, p53R2, is directly regulated by p53 in the supply of nucleotides for repairing damaged DNA. In the present study, we investigated the improvement in radiosensitivity of human esophageal squamous cell carcinoma (ESCC) cell lines using p53R2 small interfering RNA (siRNA). p53R2 expression in ESCC cells (TE-8) with or without transfection of p53R2 siRNA was examined by Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). The radiosensitivity of TE-8 cells was also measured by cell survival assay. In addition, we investigated the relationship between the expression of p53R2 mRNA in the biopsy specimens of untreated primary tumors and the efficacy of CRT, using RT-PCR. The expression of p53R2 was amplified after X-ray irradiation (14 Gy) and diminished after X-ray irradiation following the transfection of p53R2 siRNA in TE-8 cells. The radiosensitivity of the TE-8 cells significantly improved following the transfection of p53R2 siRNA. In the clinical study, a significantly lower p53R2 mRNA expression was detected in the effective response cases. We demonstrated that p53R2 is associated with the radiosensitivity of ESCC cell lines, and that p53R2 expression is reduced after X-ray irradiation following the transfection of p53R2 siRNA. This protocol could potentially improve the efficacy of radiation therapy.
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Gene expression changes during the chondrogenic differentiation of human mesenchymal stem cells
Ikeda R., Tsukaara S., Yoshida K., Inoue I.
Journal of Biological Sciences 7 ( 5 ) 729 - 736 2007.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Biological Sciences
To gain more insight into the molecular mechanisms of chondrogenic differentiation induced by transforming growth factor (TGF)-β1 and insulin-like growth factor (IGF)-I and insulin, we performed cDNA microarray experiments during the chondrogenic differentiation of human mesenchymal stem cells (hMSCs), which provide an excellent in vitro model system for chondrogenesis. Our repeated cDNA microarray analyses identified the up regulation of 23 transcripts and the down regulation of 25 transcripts after 14 days of chondrogenic induction. We found that many of the up regulated and down regulated genes belonged to overlapping gene categories; specifically, 44 and 40% of the up regulated and down regulated genes were associated with the extracellular matrix and metabolic pathways, respectively. The expression of the identified genes was confirmed by RT-PCR. These analyses suggest that the transcriptional control induced by TGF-β1, IGF-I and insulin signaling during the chondrogenic differentiation of hMSCs is mainly targeted to genes belonging to specialized gene categories. © 2007 Asian Network for Scientific Information.
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Ushiyama M., Ikeda R., Sugawara H., Yoshida M., Mori K., Kangawa K., Inoue K., Yamada K., Miyata A.
Molecular Pharmacology 72 ( 1 ) 103 - 111 2007.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Molecular Pharmacology
Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, performs a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA regions encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. The effects on down-stream signaling produced by combinations of alternative splicing events in the EC1 domain and IC3 loop have not yet been clarified. In this study, we have used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine the tissue distributions of four PAC1 isoforms in mice. We then established cell lines constitutively expressing each of the PAC1 isoforms and characterized the binding properties of each isoform to PACAP-38, vasoactive intestinal polypeptide (VIP), and the PAC1-specific agonist maxadilan, as well as the resulting effects on two major intracellular signaling pathways: cAMP production and changes in the intracellular calcium concentration. The results demonstrate that the variants of the IC3 loop affect the binding affinity of the ligands for the receptor, whereas the variants of the EC1 domain primarily affect the intracellular signaling downstream of PAC1. Accordingly, this study indicates that the combination of alternative splicing events in the EC1 domain and the IC3 loop create a variety of PAC1 isoforms, which in turn may contribute to the functional pleiotropism of PACAP. This study not only contributes to the understanding of the multiple functions of PACAP but also helps to elucidate the relationship between the structures and functions of G-protein-coupled receptors. Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics.
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Molecular basis for the inhibition of anticancer agents-induced apoptosis by thymidine phosphorylase
Ikeda R.
Yakugaku Zasshi 127 ( 7 ) 1097 - 1102 2007.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Yakugaku Zasshi
An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, partially prevented hypoxia-induced apoptosis. TP was expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. Jurkat cells transfected with TP cDNA (Jurkat/TP) and mutant TP cDNA (Jurkat/TPMu) expressed high levels of TP, while Jurkat/CV cells which were transfected with a control vector did not express TP. A high TP enzyme activity was detected in Jurkat/TP cells, but not in Jurkat/CV and Jurkat/TPMu cells. Sensitivities to cisplatin of these cells were determined by MTT assay. IC50 values for cisplatin of Jurkat/CV, Jurkat/TP, and Jurkat/TPMu cells were 4.50, 14.08, 13.40 μM, respectively. Jurkat/TP and Jurkat/TPMu cells were about three times more resistant to cisplatin than Jurkat/CV cells. TP inhibited activation of caspase 3, 9 and mitochondrial cytochrome c release induced by cisplatin. These findings suggest a mechanism by which TP confers the resistance to cisplatin-induced apoptosis. Moreover, mutant TP that has no enzymatic activity also suppressed the cisplatin-induced apoptosis. These suggest that TP molecules have cytoprotective functions against cytotoxic agents. © 2007 The Pharmaceutical Society of Japan.
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Effect of pre-treatment with St John's Wort on nephrotoxicity of cisplatin in rats
Shibayama Y., Kawachi A., Onimaru S., Tokunaga J., Ikeda R., Nishida K., Kuchiiwa S., Nakagawa S., Takamura N., Motoya T., Takeda Y., Yamada K.
Life Sciences 81 ( 2 ) 103 - 108 2007.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Life Sciences
A herbal health care supplement, St John's Wort (SJW, Hypericum perforatum) has become widely used in the treatment of depression, and is known to interact with therapeutic drugs. Here we report a preventive effect of SJW on cisplatin nephrotoxicity in rats. Rats were given SJW (400 mg/kg/day, p.o.) for 10 consecutive days, and were injected with cisplatin (5 mg/kg, i.v.) on the day after the final SJW treatment. Cisplatin treatment increased the serum creatinine level, which is an index of nephrotoxicity, to 1.51 ± 0.22 mg/dl (mean ± SE) from 0.28 ± 0.05 mg/dl (control) on day 5 after the cisplatin injection. This increase fell significantly to 0.86 ± 0.13 mg/dl by pre-treatment with SJW. Cisplatin-induced histological abnormality of the kidney was blocked by pre-treatment with SJW. When SJW was administered for 10 days, the amounts of renal metallothionein (MT) and hepatic multidrug resistance protein 2 (Mrp2) were increased to 164.8 ± 13.0% and 220.8 ± 39.3% (mean ± SE) of controls, respectively. GSH levels in the kidney and liver were not changed. Total and free cisplatin concentration in serum was not influenced by SJW treatment. In conclusion, the results suggest that pre-treatment with SJW may diminish cisplatin nephrotoxicity. © 2007 Elsevier Inc. All rights reserved.
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Komatsu M., Furukawa T., Ikeda R., Takumi S., Nong Q., Aoyama K., Akiyama S., Keppler D., Takeuchi T.
Toxicological Sciences 97 ( 2 ) 407 - 416 2007.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Toxicological Sciences
The serine/threonine protein phosphatase (PP) 2A inhibitor, microcystin-LR, selectively induces liver damage and promotes hepatocarcinogenesis. It is thought that microcystin-LR affects hepatocellular viability mainly through inhibition of PP2A, partially through PP1, and, in addition, by generation of reactive oxygen species (ROS). However, the molecular basis of the selective liver damage and the balance between cell death and survival remained unclear. We analyzed the cytotoxicity of low doses of microcystin-LR using HEK293 cells stably expressing the human hepatocyte uptake transporters, organic anion transporting polypeptide (OATP)1B1 (HEK293-OATP1B1 cells) and OATP1B3 (HEK293-OATP1B3 cells). HEK293-OATP1B1 (IC50 6.6nM) and HEK293-OATP1B3 cells (IC50 6.5nM) were equally very sensitive to microcystin-LR. In contrast, control-vector-transfected (HEK293-CV) cells were resistant to microcystin-LR. Using HEK293-OATP1B3 cells, the cytotoxicity was attenuated by substrates and inhibitors of OATP1B3, including bromosulfophthalein, rifampicin, and cyclosporin A. Microcystin-LR was transported into HEK293-OATP1B3 cells with 1.2mM Km value, and its uptake was inhibited by above substances. Accumulation of microcystin-LR in the HEK293-OATP1B1 and HEK293-OATP1B3 cells was increased in a dose-dependent manner but not in HEK293-CV cells. Cellular serine/threonine PP activity of HEK293-OATP1B3 cells was decreased by microcystin-LR but not in HEK293-CV cells. Apoptotic changes were observed after incubation of the HEK293-OATP1B3 cells with microcystin-LR. We found by FACS analysis that microcystin-LR induced apoptosis but not necrosis in HEK293-OATP1B3 cells. Microcystin-LR activated several mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK, and p38 through inhibition of PP2A. In addition, the cytotoxicity of microcystin-LR was attenuated by the inhibitors of MAPK pathways, including U0126, SP600125, and SB203580. The ROS scavenger N-acetyl-L-cysteine partially attenuated the cytotoxicity of microcystin-LR. Thus, the present study demonstrates that microcystin-LR induces apoptosis through activation of multiple MAPK pathways subsequent to its selective uptake via OATP1B1 and OATP1B3 and followed by inhibition of PP2A, in addition to the ROS generation which might contribute to apoptosis. © The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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Owatari S., Akune S., Komatsu M., Ikeda R., Firth S., Che X., Yamamoto M., Tsujikawa K., Kitazono M., Ishizawa T., Takeuchi T., Aikou T., Mercer J., Akiyama S., Furukawa T.
Cancer Research 67 ( 10 ) 4860 - 4868 2007.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Research
We and others have shown that the copper transporters ATP7A and ATP7B play a role in cellular resistance to cis-diaminedichloroplatinum (II) (CDDP). In this study, we found that ATP7A transfection of Chinese hamster ovary cells (CHO-K1) and fibroblasts isolated from Menkes disease patients enhanced resistance not only to CDDP but also to various anticancer drugs, such as vincristine, paclitaxel, 7-ethyl-10-hydroxy-camptothecin (SN-38), etoposide, doxorubicin, mitoxantron, and 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11). ATP7A preferentially localized doxorubicin fluorescence to the Golgi apparatus in contrast to the more intense nuclear staining of doxorubicin in the parental cells. Brefeldin A partially and monensin completely altered the distribution of doxorubicin to the nuclei in the ATP7A-expressing cells. ATP7A expression also enhanced the efflux rates of doxorubicin and SN-38 from cells and increased the uptake of SN-38 in membrane vesicles. These findings strongly suggested that ATP7A confers multidrug resistance to the cells by compartmentalizing drugs in the Golgi apparatus and by enhancing efflux of these drugs, and the trans-Golgi network has an important role of ATP7A-related drug resistance. ATP7A was expressed in 8 of 34 (23.5%) clinical colon cancer specimens but not in the adjacent normal epithelium. Using the histoculture drug response assay that is useful for the prediction of drug sensitivity of clinical cancers, ATP7A-expressing colon cancer cells were significantly more resistant to SN-38 than ATP7A-negative cells. Thus, ATP7A confers resistance to various anticancer agents on cancer cells and might be a good index of drug resistance in clinical colon cancers. ©2007 American Association for Cancer Research.
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Ikeda R., Yoshida K., Inoue I.
Journal of Cellular Biochemistry 101 ( 1 ) 147 - 154 2007.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Cellular Biochemistry
Bone morphogenetic protein 2 (BMP2) is a key factor in the regulation of osteoblastic differentiation; however, its downstream mediators are not fully understood. Previously, we identified and characterized transcription factor promyelocytic leukemia zinc finger protein (PLZF), composed of an N-terminal BTB/POZ and C-terminal zinc finger motifs, as an upstream factor of CBFA1 (Runx2/core-binding factor 1). PLZF was induced in an osteoblastic differentiation medium, but was not induced by BMP2. Here, we report the identification of transcription factor fanconi anemia zinc finger protein (FAZF), which is closely related to PLZF. FAZF was induced by BMP2 in human mesenchymal stem cells (hMSCs). In addition to the full-length FAZF, we also identified alternatively spliced mRNAs in which the C-terminal zinc finger motifs were deleted (designated BTB/POZ-only FAZF). Both the full-length and BTB/POZ-only FAZF mRNAs were equally expressed in BMP2-treated hMSCs. The full-length FAZF was exclusively detected in the nucleus, whereas the BTB/POZ-only FAZF protein was localized in the cytoplasm of the transfected cells. The full-length FAZF, but not the BTB/ POZ-only FAZF, increased the expression of osteoblastic differentiation markers, including CBFA1, collagen 1A1, osteocalcin, and alkaline phosphatase in C2C12 cells. In conclusion, both FAZF and PLZF differentially participate in the regulation of osteoblastic differentiation via the BMP2 and CBFA1 signaling pathways, respectively. © 2006 Wiley-Liss, Inc.
DOI： 10.1002/jcb.21165
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Hayashi R., Goto Y., Ikeda R., Yokoyama K., Yoshida K.
Journal of Biological Chemistry 281 ( 47 ) 35633 - 35648 2006.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Biological Chemistry
The TRIP-Br1/p34 SEI-1 family proteins participate in cell cycle progression by coactivating E2F1- or p53-dependent transcriptional activation. Here, we report the identification of human CDCA4 (also know as SEI-3/Hepp) as a novel target gene of transcription factor E2F and as a repressor of E2F-dependent transcriptional activation. Analysis of CDCA4 promoter constructs showed that an E2F-responsive sequence in the vicinity of the transcription initiation site is necessary for the E2F1-4-induced activation of CDCA4 gene transcription. Chromatin immunoprecipitation analysis demonstrated that E2F1 and E2F4 bound to an E2F-responsive sequence of the human CDCA4 gene. Like TRIP-Br1/p34 SEI-1 and TRIP-Br2 (SEI-2), the transactivation domain of CDCA4 was mapped within C-terminal acidic region 175-241. The transactivation function of the CDCA4 protein was inhibited by E2F1-4 and DP2, but not by E2F5-8. Inhibition of CDCA4 transactivation activity by E2F1 partially interfered with retinoblastoma protein overexpression. Conversely, CDCA4 suppressed E2F1-3-induced reporter activity. CDCA4 (but not acidic region-deleted CDCA4) suppressed E2F1-regulated gene promoter activity. These findings suggest that the CDCA4 protein functions as a suppressor at the E2F-responsive promoter. Small interfering RNA-mediated knockdown of CDCA4 expression in cancer cells resulted in up-regulation of cell growth rates and DNA synthesis. The CDCA4 protein was detected in several human cells and was induced as cells entered the G 1/S phase of the cell cycle. Taken together, our results suggest that CDCA4 participates in the regulation of cell proliferation, mainly through the E2F/retinoblastoma protein pathway. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
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Shibayama Y., Ushinohama K., Ikeda R., Yoshikawa Y., Motoya T., Takeda Y., Yamada K.
Cancer Science 97 ( 11 ) 1260 - 1266 2006.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Science
The ATP binding cassette (ABC) transporters, multidrug resistance protein 2 (Mrp2; Abcc2) and breast cancer resistance protein (Bcrp; Abcg2), and organic anion transporters (Oats) mediate excretion of methotrexate (MTX) and many other drugs. However, it is not known whether MTX treatment leads to any changes in the expression of these transporters. We examined the effect of MTX treatment on expression of Mrp2, Bcrp and Oats in rats. MTX was single injected intraperitoneally at doses of 10, 50 and 150 mg/kg, and then Western blot analysis was performed. The levels of Mrp2, Oat1 and Oat2 on day 1 after the treatment showed no significant change. Four days after injection of 150 mg/kg MTX, the Mrp2 levels in the liver and ileum, but not in the kidney, were markedly down-regulated to 20 ± 3.6% and 8.9 ± 3.8% (mean ± SEM) of controls, respectively. Renal Oat1 and Oat3 were also down-regulated to 56.4 ± 4.3% (Oat1) and 54.3 ± 5.5% (Oat3) of controls. These effects of MTX were almost recovered by leucovorin which rescues normal cells from MTX toxicity. MTX treatment also decreased mRNA levels of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) to 65.5 ± 17.9% and 59.6 ± 14.5% of controls in the liver, respectively. MTX treatment has no apparent effect on expression levels of Bcrp, cytochrome P450 2B6 and 3A1. In conclusion, these data indicate that MTX treatment down-regulates expression levels of Mrp2, Oat1 and Oat3, and its effects are recovered by leucovorin. © 2006 Japanese Cancer Association.
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Tsukahara S., Ikeda R., Goto S., Yoshida K., Mitsumori R., Sakamoto Y., Tajima A., Yokoyama T., Toh S., Furukawa K., Inoue I.
Biochemical Journal 398 ( 3 ) 595 - 603 2006.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biochemical Journal
To better understand the molecular pathogenesis of OPLL (ossification of the posterior longitudinal ligament) of the spine, an ectopic bone formation disease, we performed cDNA microarray analysis on cultured ligament cells from OPLL patients. We found that TSG-6 (tumour necrosis factor α-stimulated gene-6) is down-regulated during osteoblastic differentiation. Adenovirus vector-mediated overexpression of TSG-6 inhibited osteoblastic differentiation of human mesenchymal stem cells induced by BMP (bone morphogenetic protein)-2 or OS (osteogenic differentiation medium). TSG-6 suppressed phosphorylation and nuclear accumulation of Smad 1/5 induced by BMP-2, probably by inhibiting binding of the ligand to the receptor, since interaction between TSG-6 and BMP-2 was observed in vitro. TSG-6 has two functional domains, a Link domain (a hyaluronan binding domain) and a CUB domain implicated in protein interaction. The inhibitory effect on osteoblastic differentiation was completely lost with exogenously added Link domain-truncated TSG-6, while partial inhibition was retained by the CUB domain-truncated protein. In addition, the inhibitory action of TSG-6 and the in vitro interaction of TSG-6 with BMP-2 were abolished by the addition of hyaluronan. Thus, TSG-6, identified as a down-regulated gene during osteoblastic differentiation, suppresses osteoblastic differentiation induced by both BMP-2 and OS and is a plausible target for therapeutic intervention in OPLL. © 2006 Biochemical Society.
DOI： 10.1042/BJ20060027
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Ikeda R., Yoshida K., Ushiyama M., Yamaguchi T., Iwashita K., Futagawa T., Shibayama Y., Oiso S., Takeda Y., Kariyazono H., Furukawa T., Nakamura K., Akiyama S., Inoue I., Yamada K.
Biological and Pharmaceutical Bulletin 29 ( 9 ) 1815 - 1819 2006.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biological and Pharmaceutical Bulletin
Myoblasts respond to growth factor deprivation either by diffentiation into multinucleated myotubes or by undergoing apoptosis. The induction of apoptosis and differentiation in myogenic lineage may use overlapping cellular mechanisms. Here we demonstrate that the expression of the small heat shock protein αB-crystallin as well as MyoD and myogenin is induced during myogenic differentiation in C2C12 cells, and these inductions occur at an early stage in the differentiation in vitro. To investigate the effect of αB-crystallin on myogenic differentiation and apoptosis, C2C12 cells were infected with adenovirus vector bearing full-length αB-crystallin cDNA. Overexpression of αB-crystallin in C2C12 cells suppressed differentiation-induced apoptosis and activation of caspase 3, and also decreased the expression of MyoD and myogenin during myogenic differentiation of C2C12 cells induced by the differentiation medium. Our findings suggest that stress such as growth factor deprivation plays an important role in triggering apoptosis associated with myogenic differentiation and αB-crystallin suppressed the differentiation, apoptosis and caspase 3 activity. © 2006 Pharmaceutical Society of Japan.
DOI： 10.1248/bpb.29.1815
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Che X., Zheng C., Owatari S., Mutoh M., Gotanda T., Jeung H., Furukawa T., Ikeda R., Yamamoto M., Haraguchi M., Arima N., Akiyama S.
Blood 107 ( 12 ) 4880 - 4887 2006.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Blood
Patients with acute- or lymphoma-type adult T-cell leukemia (ATL) have a poor outcome because of the intrinsic drug resistance to chemotherapy. Protection from apoptosis is a common feature involved in multidrug-resistance of ATL. IAP (inhibitor of apoptosis) family proteins inhibit apoptosis induced by a variety of stimuli. In this study, we investigated the expression of IAP family members (survivin, cIAP1, cIAP2, and XIAP) in the primary leukemic cells from patients with ATL. We found that survivin was overexpressed in ATL, especially in acute-type ATL. Sodium arsenite was shown to down-regulate the expression of survivin at both the protein and RNA levels in a time- and dose-dependent manner, thus inhibiting cell growth, inducing apoptosis, and enhancing the caspase-3 activity in ATL cells. Nuclear factor-κB (NF-κB) enhances the transcriptional activity of survivin. Sodium arsenite suppressed the constitutive NF-κB activation by preventing the IκB-α degradation and the nuclear translocation of NF-κB. These findings suggest that survivin is an important antiapoptotic molecule that confers drug resistance on ATL cells. Sodium arsenite was shown to down-regulate the expression of survivin through the NF-κB pathway, thus inhibiting cell growth and promoting apoptosis of ATL cells. © 2006 by The American Society of Hematology.
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2-Deoxy-D-ribose inhibits hypoxia-induced apoptosis by suppressing the phosphorylation of p38 MAPK
Ikeda R., Che X., Ushiyama M., Yamaguchi T., Okumura H., Nakajima Y., Takeda Y., Shibayama Y., Furukawa T., Yamamoto M., Haraguchi M., Sumizawa T., Yamada K., Akiyama S.
Biochemical and Biophysical Research Communications 342 ( 1 ) 280 - 285 2006.3
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biochemical and Biophysical Research Communications
An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, partially prevented hypoxia-induced apoptosis. 2-Deoxy-D-ribose inhibited hypoxia-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not c-jun NH2-terminal kinase/stress-activated protein kinase in human leukemia HL-60 cells. 2-Deoxy-D-ribose also suppressed the levels of Bax attached to mitochondria under hypoxic conditions. SB203580, a specific inhibitor of the p38 MAPK, suppressed the hypoxia-induced apoptosis of HL-60 cells. These findings suggest that one of the molecular bases for resistance to hypoxia-induced apoptosis conferred by 2-deoxy-D-ribose is the inhibition of the p38 signaling pathway. The expression levels of TP are elevated in many malignant solid tumors and thus the 2-deoxy-D-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis. © 2006 Elsevier Inc. All rights reserved.
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Onimaru S., Nakamura K., Kariyazono H., Ikeda R., Ueno T., Fukumoto Y., Yabuki A., Sakata R., Yamada K.
Heart and Vessels 21 ( 2 ) 108 - 115 2006.3
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Heart and Vessels
We evaluated the effects of edaravone, a hydroxyl radical scavenging agent, on the production of tumor necrosis factor-α (TNF-α) in myocardium, and the release of TNF-α and P-selectin from myocardium after ischemia-reperfusion injury in isolated Langendorff-perfused rat hearts. Cardiodynamic function at stable points during perfusion and 5, 15, 30, and 60min after the initiation of reperfusion was evaluated by left ventricular developed pressure, rate of increase in left ventricular pressure and rate of decrease in ventricular pressure, coronary flow, and heart rate. At 60min after the initiation of reperfusion, myocardial infarct size was estimated microscopically using triphenyltetrazolium chloride staining, and expression of TNF-α in myocardium was detected by Western blot and immunohistochemistry. At the same time points as the measurement of cardiodynamic function, TNF-α and the soluble form of P-selectin in coronary effluent were measured by enzyme immunoassay. At all time points during reperfusion, edaravone markedly improved cardiodynamic function and reduced myocardial infarct size in comparison to the control. In myocardium in the control, TNF-α was detected in the endothelial cells and other cells bearing some resemblance to interstitial cells and monocyte cells. Edaravone suppressed this cytokine expression in the corresponding sites. P-selectin as well as TNF-α was found in the coronary effluent of the control, and edaravone significantly decreased soluble P-selectin levels in comparison to the control (P < 0.01). Edaravone might have protective effects on cardiac function through reduction of infarct size via decrease of production of TNF-α in myocardium induced by ischemia-reperfusion injury and through reduction of the release of adhesion molecules such as P-selectin from vascular endothelial cells. © Springer-Verlag Tokyo 2006.
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Hayashi R., Goto Y., Haga A., Kobayashi D., Ikeda R., Yoshida K.
Gene 367 ( 1-2 ) 126 - 134 2006.2
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Gene
Minichromosome maintenance protein (MCM) is composed of six structurally related subunits (MCM2-7) and is essential for eukaryotic DNA replication initiation and early stage of elongation process. Recently human and Xenopus MCM8 was identified as a novel member of MCM protein. Here we characterized MCM8 orthologous genes by using bioinformatics. Human MCM8 showed approximately 90%, 90%, 93%, and 79% total-amino acid identity with mouse, rat, dog, and chicken MCM8, respectively. Human, mouse, rat, dog, and chicken MCM8 gene, consisting of 19, 18, 17, 18, and 18 exons, was mapped to 20p12.3-13, 2F3, 3q36, 24, and 3, respectively. We identified transcription factor E2F-binding motifs in the vicinity of the transcription start site among MCM8 orthologous genes. The mammalian but not chicken E2F-binding motif was accompanied by NF-Y binding motif. MCM8 mRNA was upregulated by E2E1 in human culture cells. Chromatin immunoprecipitation (ChIP) demonstrated the direct association of E2F1 and NF-Y with human MCM8 promoter. The promoter activities of human, rat, and chicken MCM8 were demonstrated to be E2F1-dependent. Analysis of human MCM8 promoter constructs showed that an E2F-binding motif in the vicinity of the transcription initiation site is necessary for the transcriptional activation. We also showed that the transcription of human MCM8 is activated by transcription factors E2F1-4, but not by factors E2F5-8.
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Cepharanthine potently enhances the sensitivity of anticancer agents in K562 cells
Ikeda R., Che X., Yamaguchi T., Ushiyama M., Zheng C., Okumura H., Takeda Y., Shibayama Y., Nakamura K., Jeung H., Furukawa T., Sumizawa T., Haraguchi M., Akiyama S., Yamada K.
Cancer Science 96 ( 6 ) 372 - 376 2005.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Science
A major impediment to cancer treatment is the development of resistance by the tumor. P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) are involved in multidrug resistance. In addition to the extrusion of chemotherapeutic agents through these transporters, it has been reported that there are differences in the intracellular distribution of chemotherapeutic agents between drug resistant cells and sensitive cells. Cepharanthine is a plant alkaloid that effectively reverses resistance to anticancer agents. It has been previously shown that cepharanthine is an effective agent for the reversal of resistance in P-gp-overexpressing cells. Cepharanthine has also been reported to have numerous pharmacological effects besides the inhibition of P-gp. It has also been found that cepharanthine enhanced sensitivity to doxorubicin (ADM) and vincristine (VCR), and enhanced apoptosis induced by ADM and VCR of P-gp negative K562 cells. Cepharanthine changed the distribution of ADM from cytoplasmic vesicles to nucleoplasm in K562 cells by inhibiting the acidification of cytoplasmic organelles. Cepharanthine in combination with ADM should be useful for treating patients with tumors. © Japanese Cancer Association.
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Ikeda R., Yoshida K., Tsukahara S., Sakamoto Y., Tanaka H., Furukawa K., Inoue I.
Journal of Biological Chemistry 280 ( 9 ) 8523 - 8530 2005.3
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Biological Chemistry
Ossification of the posterior longitudinal ligament of the spine (OPLL) is the leading cause of myelopathy in Japan and is diagnosed by ectopic bone formation in the paravertebral ligament. OPLL is a systemic high bone mass disease with a strong genetic background. To detect genes relevant to the pathogenesis of OPLL, we performed a cDNA microarray analysis of systematic gene expression profiles during the osteoblastic differentiation of ligament cells from OPLL patients (OPLL cells), patients with a disorder called ossification of yellow ligament (OYL), and non-OPLL controls together with human mesenchymal stem cells (hMSCs) after stimulating them with osteogenic differentiation medium (OS). Twenty-four genes were up-regulated during osteoblastic differentiation in OPLL cells. Zinc finger protein 145 (promyelotic leukemia zinc finger or PLZF) was one of the highly expressed genes during osteoblastic differentiation in all the cells examined. We investigated the roles of PLZF in the regulation of osteoblastic differentiation of hMSCs and C2C12 cells. Small interfering RNA-mediated gene silencing of PLZF resulted in a reduction in the expression of osteoblast-specific genes such as the alkaline phosphatase, collagen 1A1 (Col1a1), Runx2/core-binding factor 1 (Cbfa1), and osteocalcin genes, even in the presence of OS in hMSCs. The expression of PLZF was unaffected by the addition of bone morphogenetic protein 2 (BMP-2), and the expression of BMP-2 was not affected by PLZF in hMSCs. In C2C12 cells, overexpression of PLZF increased the expression of Cbfa1 and Col1a1; on the other hand, the overexpression of CBFA1 did not affect the expression of Plzf. These findings indicate that PLZF plays important roles in early osteoblastic differentiation as an upstream regulator of CBFA1 and thereby might participate in promoting the ossification of spinal ligament cells in OPLL patients. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
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Effects of acute and chronic cyclosporin treatment on digoxin pharmacokinetics
Shibayama Y., Kawachi A., Ikeda R., Motoya T., Yamada K.
Journal of Applied Therapeutic Research 4 ( 4 ) 38 - 45 2004.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Applied Therapeutic Research
Administration of the immunosuppressant cyclosporin A (CsA) increases digoxin (DIG) blood levels through inhibition of P-glycoprotein (P-gp). On the other hand, chronic treatment with CsA has been reported to induce overexpression of P-gp. Therefore, chronic treatment with CsA may change the pharmacokinetic profile of DIG. We report here the experimental results of acute and chronic CsA treatment on the pharmacokinetics of DIG. Rats were treated with CsA once daily for 10 consecutive days. Acute and chronic treatment with CsA was found to increase the area under the blood concentration-time curve for DIG (AUC, acute: 42 ± 3 to 195 ± 30, chronic: 52 ± 2 to 206 ± 30 μg·h/l) and peak blood DIG levels (Cmax, acute: 8.8 ± 0.5 to 23.7 ± 5.4, chronic: 7.2 ± 0.6 to 20.6 ± 2.4 ng/ml). Overexpression of the renal P-gp induced about a 1.4-fold difference between control and chronic treatment with CsA. From the results, the inhibition of P-gp by CsA appears to be a potent determinant on the pharmacokinetic profile of DIG in cases of concomitant administration. Thus, caution is required when using CyA and DIG concomitantly to avoid a potential interaction.
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Shibayama Y., Ikeda R., Motoya T., Yamada K.
Food and Chemical Toxicology 42 ( 6 ) 995 - 1002 2004.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Food and Chemical Toxicology
St. John's Wort (Hypericum perforatum, SJW) has been used as a herbal medicine for the treatment of depression in oral doses of 900-1050 mg/day in humans. However, the ingestion of SJW was reported to cause interactions with drugs. In the present study, we examined the effects of SJW treatment on the induction of drug transporters and enzymes in rats. An immunoblot analysis was performed to quantify the expression of the transporters and enzymes. SJW was given at a dose of 400 mg/kg/day, since it was reported that 400 mg/kg/day is antidepressant effective dose in rats. When SJW was administered for 10 days, the amounts of multidrug resistance protein 2 (MRP2), glutathione S-transferase-P (GST-P) and cytochrome P450 1A2 (CYP1A2) in the liver were increased to 304%, 252% and 357% of controls, respectively, although the amounts of P-glycoprotein and multidrug resistance protein 1 were not changed. Under the same conditions, an increase of MRP2 in the kidney was not observed. The increase in the levels of each protein was maximal at 10 days after SJW treatment and lasted for at least 30 consecutive days. These results suggest that SJW induces hepatic MRP2, GST-P and CYP1A2 overexpressions, and thus, it could affect drug metabolism, conjugation and disposition. © 2004 Elsevier Ltd. All rights reserved.
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Inhibition of Metastasis of Tumor Cells Overexpressing Thymidine Phosphorylase by 2-Deoxy-L-Ribose
Nakajima Y., Gotanda T., Uchimiya H., Furukawa T., Haraguchi M., Ikeda R., Sumizawa T., Yoshida H., Akiyama S.
Cancer Research 64 ( 5 ) 1794 - 1801 2004.3
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Research
Thymidine phosphorylase (TP) catalyzes the reversible conversion of thymidine to thymine, thereby generating 2-deoxy-D-ribose-1-phosphate, which upon dephosphorylation forms 2-deoxy-D-ribose (D-dRib), a degradation product of thymidine. We have previously shown that D-dRib promotes angiogenesis and chemotaxis of endothelial cells and also confers resistance to hypoxia-induced apoptosis in some cancer cell lines. 2-Deoxy-L-ribose (L-dRib), a stereoisomer of D-dRib, can inhibit D-dRib anti-apoptotic effects and suppressed the growth of KB cells overexpressing TP (KB/TP cells) transplanted into nude mice. In this study, we examined the ability of L-dRib to suppress metastasis of KB/TP cells using two different models of metastasis. The antimetastatic effect of L-dRib was first investigated in a liver-metastasis model in nude mice inoculated with KB/TP cells. Oral administration of L-dRib for 28 days at a dose of 20 mg/kg/day significantly reduced the number of metastatic nodules in the liver and suppressed angiogenesis and enhanced apoptosis in KB/TP metastatic nodules. Next, we compared the ability of L-dRib and tegafur alone or in combination to decrease the number of metastatic nodules in organs in the abdominal cavity in nude mice receiving s.c. of KB/TP cells into their backs. L-dRib (20 mg/kg/day) was significantly (P < 0.05) more efficient than tegafur (100 mg/kg/day) in decreasing the number of metastatic nodules in organs in the abdominal cavity. By in vitro invasion assay, L-dRib also reduced the number of invading KB/TP cells. L-dRib anti-invasive activity may be mediated by its ability to suppress the enhancing effect of TP and D-dRib on both mRNA and protein expression of vascular endothelial growth factor and interleukin-8 in cultured KB cells. These findings suggest that L-dRib may be useful in a clinical setting for the suppression of metastasis of tumor cells expressing TP.
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Thymidine phosphorylase inhibits apoptosis induced by anticancer agents
Ikeda R., Furukawa T., Sumizawa T., Haraguchi M., Oiso S., Inoue I., Yamada K., Akiyama S.
Folia Pharmacologica Japonica 122 ( SUPPL. 1 ) 2003.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Folia Pharmacologica Japonica
An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, partially prevented hypoxia-induced apoptoisis. TP was expressed at higher levels in tumor tissuses compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected wild-type or mutant (L148R) TP cDNA. TP inhibits a number of steps in the cisplatin-induced apoptotic pathway, activation of caspase 3, 9 and mitochondrial cytochrome c release. These findings suggest a mechanism by which TP confers the resistance to apoptosis by cisplatin. Moreover, mutant TP that has no enzymatic activity also suppressed cisplatin-induced apoptosis. These findings indicate that TP has cytoprotective functions against cytotoxic agents which are independent of its enzymatic activity.
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Sata T., Ishida K., Motoya T., Nakano R., Honda K., Nakao S., Yamashita K., Iwashita Y., Ikeda R., Yamada K.
Journal of Applied Therapeutic Research 4 ( 2 ) 40 - 45 2003.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Applied Therapeutic Research
The usefulness of drug information leaflets with pictures for elderly patient education to facilitate understanding of their medicines was examined. Sixty inpatients, more than 65 years old, were divided into three groups of 20 patients each. Each group was provided drug information such as trade name, efficacy, administration procedure and cautions for use by one of the following three procedures: i) oral explanation; ii) oral explanation and provision of information written on the package; iii) oral explanation and provision of an information leaflet with pictures. Understanding by patients of their medicines was assessed by an interview with a pharmacist. The patients given drug information leaflets with pictures received significantly (p<0.05) higher understanding scores in all of the information items examined than those in the other groups. After withdrawal of education for 6 months, the patients who had kept the leaflet showed significantly (p<0.05) higher understanding scores than those who had lost it. The score in the latter patient group after the beginning of the second period of education, however, improved faster than that after the first. These results indicate that the use of drug information leaflets with pictures is an effective way to improve the understanding by elderly patients of their medicines.
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Aspirin induces hepatoma-derived cell apoptosis via a hydrogen peroxide-dependent pathway
Tuvdendorj D., Oketani M., Ikeda R., Kohara K., Komorizono Y., Ishibashi K., Munkhtuvshin N., Arima T.
Hepatology Research 26 ( 1 ) 47 - 54 2003.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Hepatology Research
Here we studied whether aspirin (ASA) has any influence on viability of human hepatoma-derived SKHep-1 cells and whether hydrogen peroxide (H2O2) has any relation with this effect. ASA inhibited SKHep-1 cell proliferation dose- and time-dependently. Intracellular H2O2increased as early as 15 min after ASA supplementation. Cellular apoptosis correlated with an increase in intracellular H2O2level. Moreover, in the presence of a catalase inhibitor-aminotriazol, ASA showed more apoptotic effect on SKHep-1 cells with increasing intracellular H2O2level. In conclusion, the present results shows that ASA induced SKHep-1 cell apoptosis has a relation with an early increase in intracellular H2O2level and catalase inhibitor synergizes to induce this process. © 2003 Elsevier Science B.V. All rights reserved.
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Thymidine phosphorylase inhibits apoptosis induced by cisplatin
Ikeda R., Furukawa T., Mitsuo R., Noguchi T., Kitazono M., Okumura H., Sumizawa T., Haraguchi M., Che X., Uchimiya H., Nakajima Y., Ren X., Oiso S., Inoue I., Yamada K., Akiyama S.
Biochemical and Biophysical Research Communications 301 ( 2 ) 358 - 363 2003.2
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biochemical and Biophysical Research Communications
An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP, partially prevents hypoxia-induced apoptosis. TP is expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. TP inhibited a number of steps in the cisplatin-induced apoptotic pathway, activation of caspases 3 and 9 and mitochondrial cytochrome c release. These findings suggest a mechanism by which TP confers resistance to apoptosis induced by cisplatin. Moreover, mutant TP that has no enzymatic activity also suppressed cisplatin-induced apoptosis. These findings indicate that TP has cytoprotective functions against cytotoxic agents which are independent of its enzymatic activity. © 2003 Elsevier Science (USA). All rights reserved.
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Che X., Nakajima Y., Sumizawa T., Ikeda R., Ren X., Zheng C., Mukai M., Furukawa T., Haraguchi M., Gao H., Sugimoto Y., Akiyama S.
Cancer Letters 187 ( 1-2 ) 111 - 119 2002.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Letters
A newly synthesized 1,4-benzothiazipine derivate, 4-[3-(4-benzylpiperidin-1-yl) propionyl]-7-methoxy-2,3,4,5-tetrahydro-1, 4-benzothiazepine monohydrochloride (JTV-519) was examined for its ability to reverse P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) mediated multidrug resistance (MDR) in K562/MDR and KB/MRP cells, respectively. JTV-519 at 3μM reversed the resistance of K562/MDR cells to vincristine (VCR), taxol, etoposide (VP16), adriamycin (ADM) and actinomycin D and at 0.5 or 1μM reversed their resistance to STI571. JTV-519 at 10μM enhanced the accumulation of ADM in K562/MDR cells to the level in parental K562 cells and inhibited the efflux of ADM from K562/MDR cells. Photoaffinity labeling of P-gp with3H-azidopine was almost completely inhibited by 500μM JTV-519. JTV-519 at 3μM also partially reversed the resistance of KB/MRP cells to VCR and at 500μM partially inhibited the photoaffinity labeling of MRP1 with125I-II-azidophenyl agosterol A (125I-azidoAG-A). These results suggest that JTV-519 reversed the resistance to the anti-cancer agents in P-gp and MRP1 overexpressing multidrug-resistant cells by directly binding to P-gp and MRP1, and competitively inhibiting transport of the anti-cancer agents. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
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Ren X., Furukawa T., Aoki S., Sumizawa T., Haraguchi M., Nakajima Y., Ikeda R., Kobayashi M., Akiyama S.
Biochemistry 41 ( 48 ) 14132 - 14140 2002.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biochemistry
MRP1 is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. Our recent study demonstrated that GSH is required for the labeling of MRP1932-1531 with a photoanalogue of agosterol A (AG-A) and suggested that GSH interacts with the L0 region of MRP1. In this study, we further characterized the GSH-dependent binding site of azido AG-A on MRP1. Coexpression of the N- and C-terminal halves of MRP1 (residues 1-1222, TM1-16, and 1223-1531, TM17, respectively) in Sf21 insect cells reconstituted a functional drug transporter with a Km for LTC4 (97 nM) similar to that of intact MRP1. In membrane vesicles from those cells, GSH-dependent photolabeling of the MRP1 fragment (1-1222) required the coexpression of the C-terminal MRP1 fragment (1223-1531). An MRP1 fragment extending from residue 1 to 1295 however could be photolabeled by azido AG-A in a GSH-dependent manner. These data indicate that amino acids 1223-1295 of MRP1 are required for AG-A binding to MRP1 in a GSH-dependent manner. However, cross-linking of the photolabel to MRP1 occurs at a more upstream site. An arginine residue at position 1249 of MRP1 was shown to be important for the GSH-dependent binding of AG-A to MRP1. Mutation of this arginine to alanine (R1249A) resulted in a decreased level of GSH-dependent azido AG-A photolabeling of MRP1. Furthermore, this mutant attenuated MRP1 function by decreasing the level of LTC4 substrate transport and impairing resistance to the drug vincristine (VCR). In summary, this study demonstrates that a region of MRP1 (amino acids 1223-1295), which includes TM helix 17, is required for azido AG-A binding to MRP1 in a GSH-dependent manner. A GSH-dependent drug binding site may exist in this region. Furthermore, our findings suggest that the charged amino acid Arg1249 proximal to the C-terminus of TM helix 17 is indispensable for MRP1-substrate interaction and the function of MRP1.
DOI： 10.1021/bi026443s
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Suppression of thymidine phosphorylase-mediated angiogenesis and tumor growth by 2-deoxy-L-ribose
Uchimiya H., Furukawa T., Okamoto M., Nakajima Y., Matsushita S., Ikeda R., Gotanda T., Haraguchi M., Sumizawa T., Ono M., Kuwano M., Kanzaki T., Akiyama S.
Cancer Research 62 ( 10 ) 2834 - 2839 2002.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Research
Thymidine phosphorylase (TP), an enzyme involved in the reversible conversion of thymidine to thymine, is identical to an angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF). Both TP and one of the TP-degradation products of thymidine 2-deoxy-D-ribose (dRib) display endothelial cell chemotactic activity in vitro and angiogenic activity in vivo. Recently, we demonstrated that 2-deoxy-L-ribose (IRib) could abolish the inhibitory effect of dRib on hypoxia-induced apoptosis. This suggested that IRib may be a useful inhibitor of dRib and thereby of TP functions. Therefore, we investigated the ability of IRib to inhibit the range of biological activities of TP and dRib. IRib suppressed both dRib-induced endothelial cell migration in a chemotaxis assay and endothelial tube formation induced by dRib in a collagen gel. IRib could also suppress the biological effects of TP in vivo assays of angiogenesis and tumor growth. Thus, in a corneal assay of angiogenesis, IRib inhibited angiogenesis induced by the implantation of recombinant TP. In a dorsal air sac assay of angiogenesis, IRib inhibited angiogenesis induced by the implantation of KB cells overexpressing TP (KB/TP). In a tumor growth assay, IRib treatment considerably decreased the growth rate of KB/TP cells xenografted into nude mice and also resulted in an increase in the proportion of apoptotic cells in KB/TP tumors. These findings demonstrate that TP and dRib play an Important role in angiogenesis and tumor growth, and that these effects can be inhibited by IRib. Thus, IRib is a potentially useful agent for the suppression of TP-dependent anglogenesis and tumor growth.
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Molecular basis for the inhibition of hypoxia-induced apoptosis by 2-deoxy-D-ribose
Ikeda R., Furukawa T., Kitazono M., Ishitsuka K., Okumura H., Tani A., Sumizawa T., Haraguchi M., Komatsu M., Uchimiya H., Ren X., Motoya T., Yamada K., Akiyama S.
Biochemical and Biophysical Research Communications 291 ( 4 ) 806 - 812 2002.3
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biochemical and Biophysical Research Communications
An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity partially prevented hypoxia-induced apoptosis. 2-Deoxy-D-ribose inhibits a number of components of the caspase-mediated hypoxia-induced apoptotic pathway. It inhibits hypoxia-induced caspase 3 activation, mitochondrial cytochrome c release, downregulation of Bcl-2 and Bcl-xL, upregulation of hypoxia-inducible factor (HIF)-1α, and loss of mitochondrial transmembrane potential in human leukemia HL-60 cell line. These findings suggest a molecular mechanism by which 2-deoxy-D-ribose confers the resistance to apoptosis. Thus 2-deoxy-D-ribose-modulated suppression of HIF-1α expression could prevent the hypoxia-induced decrease of the anti-apoptotic Bcl-2 and Bcl-xLon the mitochondria. 2-Deoxy-L-ribose and its analogs may enhance apoptosis and suppress the growth of tumors by competitively inhibiting the activities of 2-deoxy-D-ribose and thus these analogs show promise for anti-tumor therapy. © 2002 Elsevier Science Ltd. All rights reserved.
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Ishitsuka K., Ikeda R., Utsunomiya A., Uozumi K., Hanada S., Suzuki S., Takeuchi S., Takatsuka Y., Takeshita T., Ohno N., Arima T.
Leukemia and Lymphoma 43 ( 5 ) 1107 - 1114 2002.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Leukemia and Lymphoma
Arsenic trioxide (As2O3) has been reported to induce apoptosis in human T-cell leukemia virus type-I (HTLV-I) infected T-cell lines and fresh adult T-cell leukemia (ATL) cells and to induce G1phase accumulation in HTLV-I infected T-cell lines. The present study aimed to clarify the pathway of AS2O3-induced apoptosis in HTLV-I infected T-cell lines, MT-1 and MT-2, and fresh ATL cells separated from peripheral blood of patients with acute or chronic type ATL. Cells were treated up to 72h at clinically tolerable concentrations of As2O3(1-2 μmol/l) shown to be safe in patients with acute promyelocytic leukemia (APL). Activation of caspases 3, 8, and 9, loss of mitochondrial transmembrane potential and cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) were observed during As2O3treatment. Furthermore, prior exposure to a broad-spectrum caspase inhibitor blocked AS2O3-induced apoptosis but not G1phase accumulation. While pre-treatment with a CD95 receptor-blocking antibody (Ab) or a TNF-α neutralizing Ab did not show such inhibitions in these cells. In conclusion, As2O3induces apoptosis in HTLV-I infected T-cell lines and fresh ATL cells through CD95 or TNF-α receptor independent caspase activation.
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Mori S., Takao S., Ikeda R., Noma H., Mataki Y., Wang X., Akiyama S., Aikou T.
Biochemical and Biophysical Research Communications 295 ( 2 ) 300 - 305 2002.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Biochemical and Biophysical Research Communications
Thymidine phosphorylase (TP) has chemotactic and angiogenic activities resulting from its enzymatic activity in vitro, and it also promotes tumor growth and inhibits apoptosis in vivo. Recently, we have reported that TP plays an important role in Fas-induced apoptosis. Caspase-8 cleavage, subsequent cytochrome c release, and caspase-3 cleavage were prevented in KB cells transfected with a TP cDNA (KB/TP cells). In this study, treatment with thymidine phosphorylase inhibitor (TPI) or thymidine did not affect cell survival of KB/TP cells during Fas-induced apoptosis. Moreover, treatment with thymine or 2-deoxy-D-ribose (degradation products of thymidine generated by TP) also did not affect cell survival of control transfectant (KB/CV) cells during Fas-induced apoptosis. These findings indicate that TP suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity. © 2002 Elsevier Science Ltd. All rights reserved.
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Role of thymidine phosphorylase in Fas-induced apoptosis.
Mori S., Takao S., Ikeda R., Noma H., Mataki Y., Wang X., Akiyama S., Aiko T.
Human cell : official journal of Human Cell Research Society 14 ( 4 ) 323 - 330 2001.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Human cell : official journal of Human Cell Research Society
Thymidine phosphorylase (TP) has chemotactic and angiogenic activity in vitro, and it promotes tumor growth and inhibits apoptosis in vivo. It plays a key role in the invasiveness and metastasis of TP-expressing solid tumors. KB/TP cells transfected with a TP cDNA have been shown to be resistant to hypoxia-induced apoptosis, suggesting that TP has effects on tumor growth and cell death independent of its effects on angiogenesis. However, the mechanisms of cell death inhibition by TP are unknown. In the present study, we demonstrate that caspase-8 is cleaved in control transfectant KB cells early on during Fas-induced apoptosis. Caspase-8 activation leads to the loss of mitochondrial membrane potential, followed by the release of cytochrome c, the activation of caspase-3, and apoptosis. In contrast, Fas-induced caspase-8 cleavage is inhibited in KB/TP cells, which lead to inhibition of the downstream apoptotic cascade and inhibition of apoptosis. These findings indicate that TP plays an important role in intracellular apoptotic signal transduction in the Fas-induced apoptotic pathway. Therefore, inhibition of TP may suppress the progression of TP-overexpressing solid tumors by inducing apoptosis.
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Reversal of LRP-associated drug resistance in colon carcinoma SW-620 cells
Kitazono M., Okumura H., Ikeda R., Sumizawa T., Furukawa T., Nagayama S., Seto K., Aikou T., Akiyama S.
International Journal of Cancer 91 ( 1 ) 126 - 131 2001.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：International Journal of Cancer
Resistance to multiple drugs is mediated by lung resistance-related protein (LRP) as well as P-glycoprotein (P-gp) and multidrug resistance protein (MRP). The levels of expression of LRP mRNA and LRP in a human colon carcinoma cell line, SW-620, were increased by the differentiation-inducing agent, sodium butyrate (NaB). Treatment of SW-620 cells with NaB for 2 weeks conferred resistance to adriamycin (ADM) and VP-16. The resistance was almost completely reversed by PAK-104P, a pyridine analog, but not by cepharanthine. ADM accumulated mainly in the nuclei of SW-620 cells not treated with NaB and in the cytoplasm of SW-620 cells treated with NaB. When the NaB-treated SW-620 cells were incubated with ADM in the presence of PAK-104P, the accumulation of ADM in nuclei was substantially increased. Isolated nuclei from untreated cells accumulated more ADM than nuclei from NaB-treated cells. Efflux of ADM from the nuclei isolated from NaB-treated cells was enhanced. PAK-104P and an antibody against LRP increased the accumulation of ADM in the isolated nuclei from NaB-treated cells, and inhibited the enhanced efflux of ADM from the nuclei. These findings suggest that at leastin part, PAK-104P reverses LRP-mediated drug resistance by inhibiting the efflux of ADM from nuclei. PAK-104P may be useful for reversing MDR in tumors that overexpress LRP. (C) 2001 Wiley-Liss, Inc.
DOI： 10.1002/1097-0215(20010101)91:1<126::AID-IJC1018>3.0.CO;2-8
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Ikeda R., Nagao T., Okabe H., Nakano Y., Matsunaga H., Katano M., Mori M.
Chemical and Pharmaceutical Bulletin 46 ( 5 ) 871 - 874 1998.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Chemical and Pharmaceutical Bulletin
The MeOH extract of the root and the ground part of Anthriscus sylvestris HOFFM. showed a high inhibitory activity against MK-1, HeLa, and B16F10 cell growth in vitro. The activity was found only in the CHCl3- soluble fractions. From the CHCl3-soluble fraction of the root, falcarindiol, 1-(3'-methoxy-4',5'-methylenedioxyphenyl)-1ζ- methoxy-2- propene, elemicin, and nemerosin were newly isolated in addition to deoxypodophyllotoxin (anthricin), anthriscusin, (-)-deoxypodorhizone, and anthriscinol methyl ether which were reported earlier as constituents of the root of this plant. From the CHCl3-soluble fraction of the ground part, deoxypodophyllotoxin, (-)-deoxypodorhizone, nemerosin, and falcarindiol were isolated. In vitro antiproliferative activities of the isolates against MK- 1, HeLa, and B16F10 cells are reported.
DOI： 10.1248/cpb.46.871
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Ikeda R., Nagao T., Okabe H., Nakano Y., Matsunaga H., Katano M., Mori M.
Chemical and Pharmaceutical Bulletin 46 ( 5 ) 875 - 878 1998.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Chemical and Pharmaceutical Bulletin
The constituents in the fruit of Anthriscus sylvestris HOFFM. were investigated, and four lignans [deoxypodophyllotoxin, morelensin, (-)- deoxypodorhizone, and (-)-hinokinin], one phenylpropanoid [l-(3',4'- dimethoxyphenyl)-1ζ-hydroxy-2-propene], two phenylpropanoid esters [3',4'- dimethoxycinnamyl (z)-2-angeloyloxymethyl-2-butenoate and 3',4'- dimethoxycinnamyl (Z)-2-tigloyloxymethyl-2-butenoate], and one polyacetylenic compound (falcarindiol) were isolated. Their antiproliferative activity against MK-1, HeLa and B16F10 cell lines is reported.
DOI： 10.1248/cpb.46.875