卵巣癌の制癌剤抵抗性に関与するアポトーシスシグナルの分子生物学的解析-遺伝子治療の分子標的の確立をめざして-

卵巢癌抗癌药物耐药性中涉及的细胞凋亡信号的分子生物学分析 - 旨在建立基因治疗的分子靶标 -

基本信息

批准号：
11671613
负责人：
MANDAI Masaki
金额：
$ 2.3万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671613/
关键词：
drug resistance apoptosis gene therapy ovarian cancer cisplatin bax adenovirus paclitaxel IGF-1 制癌制抵抗性

项目摘要

The objective of this study is to clarify the association between the intracellular apoptotic signaling and the cytotoxic/therapeutic effect of cisplatin in ovarian cancer. Then, according to the findings obtained, we tried to develop a new strategy to manage cisplatin-resistant ovarian cancer.Firstly, the influence of apoptosis on the effect of cisplatin is examined. Treatment with cisplatin in cisplatin-sensitive ovarian cancer cell line A2780 markedly induced apoptosis, which was detected both by morphological examination as well as quantification of apoptosis using cell death ELISA method or measurement of caspase 3 activity. In contrast, cisplatin in the same dose did not induce significant apoptosis in cisplatin-resistant SK-OV-3 cell line. In addition, an inhibitor of caspase 3, Ac-DEVD-CHO, reduced the cytotoxity of cisplatin, suggesting that the effect of cisplatin is closely linked to the induction of apoptosis in ovarian cancer cells.Secondly, the role of pro-apoptotic Bax p … More rotein in the development of cisplatin-resistance of ovarian cancer was investigated. Immunohistochemical analysis revealed that decreased expression of Bax protein correlated with poor prognosis of patient with ovarian cancer. In vitro, Bax expression was attenuated in cisplatin-resistant ovarian cancers, A2780/cDDP and SK-OV-3, compared with cisplatin sensitive A2780. These findings suggest that the disturbance in pro-apoptotic signaling through Bax play an inportant role in the development of cisplatin-resistance. Accordingly, we tried to induce Bax protein in ovarian cancer cells using recombinant adenovirus which highly express the Bax protein. The pro-apoptotic effect of Bax induction was similarly intensive either in A2780 or A2780/cDDP, while it was minimum in SK-OV-3. Preliminarily, adenovirus-mediated transfer of the Bax gene into human ovarian xenografts showed suppressive effect on tumor growth. Therefore, adenovirus-mediated introduction of Bax may be a useful tool to manage a part of cisplatin-resistant ovarian cancer.Thirdly, the influence of gonadotropins, luteinizing hormone (LH) and human chorionic gonadotropin (hCG), on the apoptosis of ovarian cancer was examined. More than a half of ovarian cancer specimens expressed LH/hCG receptor. hCG treatment markedly reduced cisplatin-induced apoptosis in LH/hCG receptor-positive ovarian cancer cell line, OVCAR-3, and induced insulin-like growth factor 1 (IGF-1 ) expression. IGF-1 also inhibited cisplatin-induced apoptosis and a neutralizing antibody to IGF-1 receptor restored apoptosis in OVCAR-3, suggesting that IGF-1 mediates anti-apoptotic signaling from hCG.Wortmannin, an inhibitor of phosphatidyl inositol 3 kinase (PI3K), blocked the anti-apoptotic effect of IGF-1 to restore the cisplatin-induced apoptosis. This indicates that specific phosphorylation signal is related to cisplatin-sensitivity/resistance of ovarian cancer.In conclusion, both the intrinsic disturbance of intracellular apoptosis signal including Bax and the presence of anti-apoptotic extrinsic factors such as IGF-1 may cause the resistance to chemotherapy in ovarian cancer. Consequently, to regulate apoptosis-related gene and to facilitate apoptosis by the methods including adenovirus-mediated gene therapy may be a hopeful strategy to overcome drug-resistance. Less

本研究的目的是阐明细胞内凋亡信号传导与顺铂在卵巢癌中的细胞毒性/治疗效果之间的关联，然后根据所获得的结果，我们尝试开发一种新的策略来治疗顺铂耐药的卵巢癌。首先，考察了细胞凋亡对顺铂作用的影响，顺铂处理对顺铂敏感的卵巢癌细胞系A2780显着诱导细胞凋亡。通过形态学检查以及使用细胞死亡 ELISA 方法或 caspase 3 活性测量来检测细胞凋亡。此外，相同剂量的顺铂不会诱导顺铂耐药的 SK-OV-3 细胞系显着凋亡。 Caspase 3 抑制剂 Ac-DEVD-CHO 降低了顺铂的细胞毒性，表明顺铂的作用与诱导细胞凋亡密切相关。其次，研究了促凋亡 Bax p 蛋白在卵巢癌顺铂耐药性发展中的作用，免疫组织化学分析表明，Bax 蛋白表达降低与卵巢癌患者的不良预后相关。在体外，Bax 表达在顺铂耐药的卵巢癌、A2780/cDDP 和SK-OV-3 与顺铂敏感的 A2780 相比，这些发现表明 Bax 促凋亡信号传导的干扰在顺铂耐药性的发展中发挥着重要作用。因此，我们尝试使用诱导卵巢癌细胞中的 Bax 蛋白。高度表达 Bax 蛋白的重组腺病毒在 A2780 或 A2780 中的促凋亡作用同样强烈。 A2780/cDDP，而在 SK-OV-3 中最低，腺病毒介导的 Bax 基因转移到人卵巢异种移植物中显示出对肿瘤生长的抑制作用，因此，腺病毒介导的 Bax 导入可能是一种有用的工具。治疗部分顺铂耐药性卵巢癌。三、促性腺激素、黄体生成素（LH）和绒毛膜的影响促性腺激素 (hCG) 对卵巢癌细胞凋亡的影响超过一半的卵巢癌标本表达 LH/hCG 受体，hCG 治疗显着减少了 LH/hCG 受体阳性卵巢癌细胞系 OVCAR- 中顺铂诱导的细胞凋亡。 3、诱导胰岛素样生长因子 1 (IGF-1) 表达也抑制顺铂诱导的细胞凋亡，并且恢复了 IGF-1 受体的中和抗体。 OVCAR-3 中细胞凋亡，表明 IGF-1 介导 hCG 的抗凋亡信号。磷脂酰肌醇 3 激酶 (PI3K) 抑制剂 Wortmannin 阻断 IGF-1 的抗凋亡作用，恢复顺铂诱导的细胞凋亡。这就说明特异性磷酸化信号与卵巢癌顺铂敏感性/耐药性相关。细胞内凋亡信号Bax的紊乱以及IGF-1等抗凋亡外源因子的存在可能导致卵巢癌化疗耐药，通过腺病毒介导等方法调节凋亡相关基因，促进细胞凋亡。基因治疗可能是克服耐药性的一种有希望的策略。