Molecular mechanism of paclitaxel on CDDP resistant ovarian cancer

紫杉醇治疗CDDP耐药卵巢癌的分子机制

• 批准号: 10671523
• 负责人: AOKI Yoichi
• 金额: $ 1.15万
• 依托单位: Niigata 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-10671523/
• 关键词: CDDP resistant; paclitaxel; bcl-2; bcl-2 phosphorylation; ovarian cancer; apoptsis; パクリタキモル; タキソール; 塩酸イリノテカン; 分子生物学

We analyzed molecular mechanisms of paclitaxel action on CDDP resistant ovarian cancer cells on the basis of bcl-2 expression and phosphorylation, and apoptotic cell death.We measured IC50 by MTT assay when treated with CDDP and paclitaxel using ovarian cancer cell lines TYK, Nakajima, and HRA, and their CDDP-resistant lines TYK-R, Nakajima-R, and HRA-R. Two of the 3 CDDP resistant lines turned out to be sensitive to paclitaxel, however, only HRA-R showed resistance to paclitaxel. When treated with CDDP and paclitaxel, DNA fragmentation was observed on agarose gel electrophoresis, revealing apoptotic cell death. We also analyzed mRNA expression of bcl-2 and p53 by RT-PCR, and protein expression by Western blotting. In the 3 CDDP resistant lines, bcl-2 expression was increased, and p53 expression was decreased both in mRNA and protein level. Although we did riot observe the phosphorylation of bcl-2 by the treatment of CDDP, paclitaxel treatment of the parental cell lines clearly induced the phosphorylation of bcl-2. On the other hand, bcl-2 phosphorylation on paclitaxel sensitive TYK-R, and Nakajima-R was shown by the treatment of paclitaxel, however, on paclitaxel resistant HRA-R, even 10 μM of paclitaxel was unable to induce the phosphorylatisn of bcl-2. These data raise the possibility of the mechanism by which paclitaxel treatment induce the phosphorylation of bcl-2 and lead to apoptolic cell death in CDDP resistant cells.

我们从bcl-2的表达、磷酸化和细胞凋亡的角度分析了紫杉醇对CDDP耐药卵巢癌细胞的作用机制，并采用MTT法测定了CDDP和紫杉醇对卵巢癌细胞株TYK、Nakajima和HRA及其耐药株TYK-R、Nakajima-R和HRA-R的IC_（50）。3个CDDP耐药株系中有2个对紫杉醇敏感，而只有HRA-R对紫杉醇耐药。当用CDDP和紫杉醇处理时，在琼脂糖凝胶电泳上观察到DNA片段化，揭示了凋亡性细胞死亡。RT-PCR检测bcl-2和p53基因mRNA表达，Western blotting检测蛋白表达。在3个顺铂抗性株系中，bcl-2表达增强，p53表达减弱。尽管我们没有观察到CDDP处理后bcl-2的磷酸化，但紫杉醇处理亲本细胞系明显诱导了bcl-2的磷酸化。紫杉醇处理对紫杉醇敏感的TYK-R和Nakajima-R细胞后，bcl-2磷酸化，而对紫杉醇耐药的HRA-R细胞，即使10 μM紫杉醇也不能诱导bcl-2磷酸化。这些数据提高了紫杉醇处理诱导bcl-2磷酸化并导致CDDP抗性细胞中的凋亡细胞死亡的机制的可能性。