Wang Zhuo, Goulet Robert, Stanton Katie J, Sadaria Miral, Nakshatri Harikrishna
Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Anticancer Res. 2005 May-Jun;25(3c):2367-79.
Intrinsic or acquired resistance to chemotherapy is a major clinical problem leading to the fatality of patients with advanced and metastatic breast cancer. The overexpression of anti-apoptotic genes is believed to play a role in the resistance to chemotherapy. In the present study, we tested the sensitivity of MCF-7 breast cancer cells overexpressing anti-apoptotic genes TRAF-1, c-FLIP, Bcl-xL, clAP-2 or Mn-SOD to paclitaxel and docetaxel.
MTT and trypan blue dye exclusion assays were performed to examine the sensitivity of different cell lines to docetaxel and paclitaxel. Cell cycle analysis and carboxyfluorescein FLICA assay were employed to determine whether defects in the cell cycle arrest or apoptotic pathway are responsible for the resistance of cells overexpressing Bcl-xL or c-FLIP. Caspase 8 and 9 activities were measured in cells overexpressing Bcl-xL or c-FLIP exposed to docetaxel and paclitaxel using fluorescent substrate cleavage assay.
MCF-7 cells overexpressing Bcl-xL but not TRAF-1, cIAP-2 or Mn-SOD were less sensitive to both paclitaxel and docetaxel compared to vector-transfected control cells. Resistance of Bcl-xL-overexpressing cells to taxanes correlated with the failure to activate caspase 9. 2-Methoxyantimycin A3, a chemical inhibitor of Bcl-xL, sensitized Bcl-xL-overexpressing cells to paclitaxel and docetaxel, which suggests the drugs that inhibit Bcl-xL activity can be used as sensitizers to taxanes. MCF-7 cells overexpressing c-FLIP were less sensitive to paclitaxel but not to docetaxel. Paclitaxel failed to induce caspase 8 in c-FLIP-overexpressing cells.
Because c-FLIP inhibits the extrinsic pathway of cell death whereas Bcl-xL inhibits the intrinsic pathway of cell death, these results suggest that overexpression of anti-apoptotic genes that inhibit either the extrinsic or intrinsic cell death pathways can reduce sensitivity of cancer cells to paclitaxel, whereas anti-apoptotic genes that inhibit only the intrinsic pathway reduce sensitivity to docetaxel.
对化疗的内在或获得性耐药是导致晚期和转移性乳腺癌患者死亡的一个主要临床问题。抗凋亡基因的过表达被认为在化疗耐药中起作用。在本研究中，我们检测了过表达抗凋亡基因TRAF-1、c-FLIP、Bcl-xL、clAP-2或Mn-SOD的MCF-7乳腺癌细胞对紫杉醇和多西他赛的敏感性。
进行MTT和台盼蓝染料排除试验以检测不同细胞系对多西他赛和紫杉醇的敏感性。采用细胞周期分析和羧基荧光素FLICA分析来确定细胞周期阻滞或凋亡途径的缺陷是否导致过表达Bcl-xL或c-FLIP的细胞产生耐药性。使用荧光底物切割试验测量过表达Bcl-xL或c-FLIP的细胞在暴露于多西他赛和紫杉醇时的半胱天冬酶8和9的活性。
与载体转染的对照细胞相比，过表达Bcl-xL而非TRAF-1、cIAP-2或Mn-SOD的MCF-7细胞对紫杉醇和多西他赛均不敏感。过表达Bcl-xL的细胞对紫杉烷类药物的耐药性与未能激活半胱天冬酶9相关。Bcl-xL的化学抑制剂2-甲氧基抗霉素A3使过表达Bcl-xL的细胞对紫杉醇和多西他赛敏感，这表明抑制Bcl-xL活性的药物可作为紫杉烷类药物的增敏剂。过表达c-FLIP的MCF-7细胞对紫杉醇不敏感，但对多西他赛敏感。紫杉醇未能在过表达c-FLIP的细胞中诱导半胱天冬酶8。
由于c-FLIP抑制细胞死亡的外源性途径，而Bcl-xL抑制细胞死亡的内源性途径，这些结果表明，抑制外源性或内源性细胞死亡途径的抗凋亡基因的过表达可降低癌细胞对紫杉醇的敏感性，而仅抑制内源性途径的抗凋亡基因可降低对多西他赛的敏感性。
