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

基本信息

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

项目摘要

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，顺式二氯二氨铂（II）（CDDP）是最重要的抗癌药物之一，在多种肿瘤中产生良好的初始反应，但在多种肿瘤中经常出现对该药物的耐药性，并且额外给药会降低其化疗效果。尽管存在这个临床上重要的问题，但对该药物产生耐药性的确切机制仍不确定。我们从 A431 建立了 CDDP 耐药亚系。人表皮样癌细胞系A431。这些耐药亚系是通过诱变剂(A431/CDDP2)诱变形成的，其对CDDP的耐药性比原始A431细胞的IC-D250-D2高2.7倍。耐药亚系表现出对 CDDP 类似物卡铂 (CBDCA) 的交叉耐药性，但没有与其他化疗药物如阿霉素（ADR）和5-氟尿嘧啶（5-FU）的交叉耐药>这种CDDP耐药亚系……更多被移植到裸鼠体内，以证明体内对CDDP治疗的耐药性。亚系可能用于进一步的试验，以提高对CDDP耐药机制的理解。最近的报道表明，化疗可以诱导一些癌细胞凋亡，表明细胞凋亡可能在癌症中发挥非常重要的作用因此，与CDDP处理的A431细胞相比，CDDP抗性细胞的DNA凝胶电泳和ELISA显示细胞凋亡减少，因此我们测定了p53、Bcl-2、Bax和CPP32。该分析表明 CDDP 抗性细胞中 Bcl-2 蛋白水平显着增加，而 CPP32 蛋白水平降低。我们的结果表明细胞凋亡减少是其中之一。 CDDP耐药机制以及CDDP耐药细胞凋亡减少受到Bcl-2和CPP32蛋白的影响。最近的研究表明CDDP诱导的细胞凋亡可能是通过激活ICE/CED-3家族蛋白酶之一的CPP32来介导的。将亲本 A431 细胞 (A431/P) 和 A431/CDDP2 暴露于含或不含 ICE/CED-3 家族蛋白酶抑制剂的 CDDP (Z-Asp-CHD22D2-DCB)，与A431/CDDP2相比，Z-Asp-CHD22D2-DCB明显降低了CDDP的细胞毒性。 -CH2D22-DCB 确实抑制了 CDDP 诱导的 DNA 断裂A431/P 细胞，但 A431/CDDP2 不抑制 DNA 片段化，我们通过蛋白质印迹测定了 CPP32 蛋白水平。该分析表明，用 Z-Asp-CH-D22 ED2- 处理的 A431/P 中 CPP32 蛋白水平显着降低。在A431/CDDP2中，无论有无此蛋白水平均无变化。 Z-Asp-CH22D2-DCB。这些发现表明CPP32可能介导CDDP诱导的细胞凋亡，其诱导可能代表一种有效治疗恶性肿瘤的新方法。