Ozvaran Mustafa K, Cao Xiaobo X, Miller Steven D, Monia Brett A, Hong Waun Ki, Smythe W Roy
Department of Thoracic and Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Mol Cancer Ther. 2004 May;3(5):545-50.
Malignant pleural mesothelioma (MPM) is resistant to both conventional chemotherapy and apoptosis. The bcl-2 family proteins are major determinants of apoptotic homeostasis. MPM lines and tumors routinely overexpress the anti-apoptotic protein BCL-XL. We have previously shown that antisense inhibition of BCL-XL in MPM cells leads to apoptosis. We sought to determine whether antisense oligonucleotides directed at the bcl-xl gene product would augment response to a conventional chemotherapeutic agent in human mesothelioma cell lines.
The human MPM cell lines REN and I-45 were exposed to two bcl-xl antisense oligonucleotides (15999, 16009) and one sense oligonucleotide (113529) construct at varying doses, followed by IC(50) cisplatin. Cellular viability was assessed by a calorimetric assay, and apoptosis was evaluated by Hoechst staining, Annexin V staining, and sub-G(1) fluorescence-activated cell sorter analysis. Western blot analysis of BCL-2 family proteins was performed following single agent and combined treatment. Isobologram mathematical analysis was used to determine whether or not combination therapies were additive or synergistic.
Cell viability was most affected with the 15999 antisense oligonucleotides plus IC(50) cisplatin combination (70% of I-45 and 90% of REN cells killed), and apoptosis was markedly increased with this combination by all measures. Western blot demonstrated 15999 antisense oligonucleotides construct down-regulation of BCL-XL, but no further effect on expression of BCL-2 proteins with cisplatin. Isobologram analysis demonstrated 15999 + cisplatin synergistic effect.
Exposure of human MPM cells to bcl-xl antisense oligonucleotides sensitizes human mesothelioma cells to the conventional chemotherapeutic agent cisplatin. Similar approaches using a combination of molecular and conventional treatment may have clinical utility for this tumor.
恶性胸膜间皮瘤（MPM）对传统化疗和凋亡均具有抗性。bcl-2家族蛋白是凋亡稳态的主要决定因素。MPM细胞系和肿瘤通常过表达抗凋亡蛋白BCL-XL。我们之前已表明，在MPM细胞中反义抑制BCL-XL可导致细胞凋亡。我们试图确定针对bcl-xl基因产物的反义寡核苷酸是否会增强人胸膜间皮瘤细胞系对传统化疗药物的反应。
将人MPM细胞系REN和I-45暴露于两种不同剂量的bcl-xl反义寡核苷酸（15999、16009）和一种正义寡核苷酸（113529）构建体，随后给予半数抑制浓度（IC50）的顺铂。通过比色法测定细胞活力，并用Hoechst染色、膜联蛋白V染色及亚G1期荧光激活细胞分选分析评估细胞凋亡情况。在单药及联合治疗后，对BCL-2家族蛋白进行蛋白质印迹分析。采用等效线图数学分析来确定联合治疗是否具有相加或协同作用。
15999反义寡核苷酸与IC50顺铂联合使用时对细胞活力影响最大（I-45细胞的70%和REN细胞的90%被杀死），且通过所有检测方法均显示该联合用药显著增加了细胞凋亡。蛋白质印迹显示15999反义寡核苷酸构建体下调了BCL-XL，但顺铂对BCL-2蛋白的表达无进一步影响。等效线图分析显示15999与顺铂具有协同作用。
人MPM细胞暴露于bcl-xl反义寡核苷酸可使人间皮瘤细胞对传统化疗药物顺铂敏感。使用分子治疗与传统治疗相结合的类似方法可能对该肿瘤具有临床应用价值。
