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-

本研究的目的是阐明细胞内凋亡信号与顺铂治疗卵巢癌的细胞毒性/治疗效果之间的关系。然后，根据获得的结果，我们试图开发一种新的策略来管理顺铂耐药卵巢癌。首先，研究细胞凋亡对顺铂治疗效果的影响。顺铂治疗对顺铂敏感的卵巢癌细胞系A2780明显诱导凋亡，通过形态学检查和细胞死亡ELISA法或caspase 3活性测定对凋亡进行定量检测。相反，相同剂量的顺铂在顺铂耐药的SK-OV-3细胞系中未引起明显的细胞凋亡。此外，caspase 3抑制剂Ac-DEVD-CHO降低了顺铂的细胞毒性，提示顺铂的作用与诱导卵巢癌细胞凋亡密切相关。其次，探讨促凋亡baxp . More蛋白在卵巢癌顺铂耐药过程中的作用。免疫组化分析显示Bax蛋白表达降低与卵巢癌患者预后不良相关。体外，与顺铂敏感的A2780相比，Bax在顺铂耐药卵巢癌、A2780/cDDP和SK-OV-3中的表达减弱。这些发现表明，通过Bax干扰促凋亡信号在顺铂耐药的发展中起重要作用。因此，我们尝试利用高表达Bax蛋白的重组腺病毒在卵巢癌细胞中诱导Bax蛋白。Bax诱导的促凋亡作用在A2780和A2780/cDDP中同样强烈，而在SK-OV-3中最低。初步发现腺病毒介导的Bax基因转染人卵巢异种移植物对肿瘤生长有抑制作用。因此，腺病毒介导的Bax导入可能是治疗部分顺铂耐药卵巢癌的有用工具。第三，观察促性腺激素、促黄体生成素（LH）和人绒毛膜促性腺激素（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治疗显著降低顺铂诱导的LH/hCG受体阳性卵巢癌细胞系的OVCAR-3细胞凋亡和诱导的胰岛素样生长因子1 （IGF-1）表达。IGF-1也抑制顺铂诱导的细胞凋亡，IGF-1受体中和抗体恢复OVCAR-3细胞凋亡，提示IGF-1介导hCG的抗凋亡信号传导。Wortmannin是一种磷脂酰肌醇3激酶（PI3K）抑制剂，可阻断IGF-1的抗凋亡作用，恢复顺铂诱导的细胞凋亡。这表明特异性磷酸化信号与卵巢癌顺铂敏感/耐药有关。综上所述，包括Bax在内的细胞内凋亡信号的内在干扰和IGF-1等抗凋亡外源性因子的存在都可能导致卵巢癌化疗耐药。因此，通过腺病毒介导的基因治疗等方法调控细胞凋亡相关基因，促进细胞凋亡可能是克服耐药的一个有希望的策略。少

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