Papers - IKEDA Ryuji
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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.