INVESTIGATION OF MECHANISMS OF CDDP-INDUCED APOPTOSIS IN THE CDDP-RESISTANT A431 CELL LINE

CDDP抗性A431细胞系中CDDP诱导凋亡机制的研究

• 批准号: 10671880
• 负责人: TAKEBAYASHI Toshiaki
• 金额: $ 1.98万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671880/
• 关键词: Cisplatin (CDDP) resistance; squamous cell carcinoma; A431; apoptosis; Bcl-2; CPP32; ICE; CED3; シスプラチン(CDDP)耐性; p53; p21; WAF-1; Cip1; Bax; Fas

Cisplatin, cis-diamminedichloroplatinum (II) (CDDP) is one of the most important anticancer agents, producing initial good responses in various tumors. However, the resistance to this drug often develops in various tumors, and the additional administration declines its chemotherapeutic efficacy in the treatment of the cancer patient. Despite of this clinically important issue, the precise mechanisms of acquisition of resistance to this drug is still uncertain. We established the CDDP-resistant subline A431/CDDP2 from human epidermoid carcinoma cell line A431. These resistant sublines were constituted by mutagenic induction with mutagen (A431/CDDP2). A431/CDDP2 have developed 2.7 times more resistance to CDDP than the original A431 cell in terms of ICィイD250ィエD2. The CDDP-resistant subline showed a cross-resistance to CDDP analogue, Carboplatin (CBDCA) , but no cross-resistance to other chemotherapeutic drugs such as Adriamycin (ADR) and 5-Fluorouracil (5-FU)> This CDDP-resistant subline … More were transplanted into nude mice to demonstrate the resistance to CDDP treatment in vivo. The established CDDP-resistant sublines may be used in further trials to improve the understanding of the mechanisms of resistance to CDDP.Recent reports have demonstrated that chemotherapy can induce apoptosis in some cancer cells indicating that apoptosis may play a very important role in cancer therapy. Therefore, to investigate whether modulation of apoptosis influences CDDP resistance. The DNA gel electrophoresis, and ELISA of CDDP-resistant cell showed a reduced apoptosis when compared with the A431 cells treated with CDDP. We determined the p53, Bcl-2, Bax and CPP32 protein levels by western blotting. This analysis demonstrated a marked increase in Bcl-2 protein levels and reduction of CPP32 protein levels in CDDP-resistant cells. Our results indicate that reduction of apoptosis was one of the CDDP resistant mechanisms and that reduced apoptosis in CDDP-resistant cells was influenced by Bcl-2 and CPP32 protein.Recent studies have revealed that apoptosis induced by CDDP could be mediated by the activation of CPP32, one of ICE/CED-3 family protease. The parent A431 cells (A431/P) and the A431/CDDP2 were exposed to CDDP with or without ICE/CED-3 family protease inhibitors (Z-Asp-CHィイD22ィエD2-DCB). In the A431/P, the cytotoxity of CDDP was clearly reduced by Z-Asp-CHィイD22ィエD2-DCB compared with the A431/CDDP2. Furthermore, quantitative analysis of DNA fragmentation revealed that Z-Asp-CHィイD22ィエD2-DCB surely inhibited the DNA fragmentation induced by CDDP in A431/Pcells, but A431/CDDP2 were not inhibited the DNA fragmentation. We determined the CPP32 protein level by Western Blotting. This analysis demonstrated a marked reduction of CPP32 protein levels in A431/P treated with Z-Asp-CHィイD22ィエD2-DCB. In the A431/CDDP2, this protein levels were no change with or without Z-Asp-CHィイD22ィエD2-DCB. These findings suggest that CPP32 may mediate apoptosis induced by CDDP, and its induction could represent a novel approach for the effective treatment of malignant tumors. Less

顺铂，顺式 - 二氨基氯铂（II）（CDDP）是最重要的抗癌药之一，在各种肿瘤中产生最初的良好反应。然而，对这种药物的耐药性通常在各种肿瘤中发展，而额外的给药降低了其在癌症患者治疗方面的化学治疗效率。尽管存在这个临床上重要的问题，但对这种药物的抗药性的确切机制仍然不确定。我们从人表皮类癌细胞系A431建立了耐CDDP的subline A431/CDDP2。这些耐药的子宫素是通过诱变剂（A431/CDDP2）配置的。就ICII D250 D2而言，A431/CDDP2对CDDP的阻力比原始A431单元增强了2.7倍。耐CDDP的subline表现出与CDDP类似物Carboplatin（CBDCA）的交叉抗性，但没有其他化学治疗药物（例如Adramycin（ADR）（ADR）和5-氟尿嘧啶（5-FU）（5-FU）和5-FU（5-FU）> CDDP-CDDP抗性的Subline cod to to to to tonp todp todp todp to n ude nudp the n ude nude nude nude nude nude nude nude nude nude nude nude nude nude nude nude nude nude nude，体内。已建立的耐CDP次额定线可以在进一步的试验中使用，以提高对CDDP耐药性机制的理解。过时的报告表明，化学疗法可以在某些癌细胞中诱导凋亡，表明细胞凋亡可能在癌症治疗中起着非常重要的作用。因此，研究凋亡的调节是否会影响CDDP的耐药性。与用CDDP处理的A431细胞相比，CDDP耐药细胞的DNA凝胶电泳和ELISA的凋亡降低。我们通过蛋白质印迹确定了p53，bcl-2，bax和cpp32蛋白水平。该分析表明，Bcl-2蛋白水平显着升高和CPP32耐药细胞中CPP32蛋白水平的降低。我们的结果表明，凋亡的减少是CDDP耐药机制之一，CDDP耐药细胞中凋亡的降低受BCl-2和CPP32蛋白的影响。进行的研究表明，CDDP诱导的凋亡可以通过CPP32的激活来介导CDDP，一种CPP32的激活，一种CPP32。母体A431细胞（A431/p）和A431/CDDP2暴露于具有或没有ICE/CED-3家族蛋白酶抑制剂（Z-ASP-CHII D22-DCB）的CDDP。在A431/P中，与A431/CDDP2相比，Z-ASP-CHII D22-DCB明显降低了CDDP的细胞毒性。此外，对DNA碎片化的定量分析表明，Z-ASP-CHII D22-DCB无疑抑制了CDDP在A431/PCELL中诱导的DNA片段化，但是A431/CDDP2并未抑制DNA损伤。我们通过蛋白质印迹确定了CPP32蛋白水平。该分析表明，用Z-ASP-CHII D22-DCB处理的A431/P中CPP32蛋白水平的显着降低。在A431/CDDP2中，如果有或没有Z-ASP-CHII D22-DCB，该蛋白水平没有变化。这些发现表明，CPP32可能介导CDDP诱导的凋亡，其诱导可能代表了一种有效治疗恶性肿瘤的新方法。较少的

项目成果

Hiroshi Mese, Akira Sasaki, Rafael E. Alcalde, Shuko Nakayama, Tomohiro Matsumura: "Establishment and Characterization of Cisplatin-Resistant Human Epidermoid Carcinoma Cell Line, A431 Cell"Chemotherapy. 44(6). 414-420 (1998)

Hiroshi Mese、Akira Sasaki、Rafael E. Alcalde、Shuko Nakayama、Tomohiro Matsumura：“顺铂耐药人表皮样癌细胞系 A431 细胞的建立和表征”化疗。

Hiroshi Mese, Akira Sasaki et al.: "Regulation of Apoptosis Reduction in the Cisplatin-Resistant A431 Cell Line by Bcl-2 and CPP32"Chemotherapy. 46・1. 69-76 (2000)

Hiroshi Mese、Akira Sasaki 等：“Bcl-2 和 CPP32 对顺铂耐药 A431 细胞系的凋亡减少的调节”化疗 46・1。

Hiroshi Mese, Akira, Sasaki et al.: "Regulation of Apoptosis Reduction in the Cisplatin-Resistant A431 Cell Line by Bcl-2 and CPP32"Chemotherapy. 46・1. 69-76 (2000)

Hiroshi Mese、Akira、Sasaki 等：“Bcl-2 和 CPP32 对顺铂耐药 A431 细胞系中细胞凋亡的减少”化疗 46・1。