Mancini M, Sedghinasab M, Knowlton K, Tam A, Hockenbery D, Anderson B O
Department of Surgery, University of Washington, Seattle 98195, USA.
Ann Surg Oncol. 1998 Apr-May;5(3):287-95. doi: 10.1007/BF02303787.
Chemotherapeutic agents induce apoptosis in cancer cells. Drugs failing to induce apoptosis are likely to have decreased clinical efficacy. We hypothesize that (1) chemotherapeutic agents induce mitochondrial changes and apoptosis through mechanisms associated with reactive oxidant species production; (2) the anti-apoptotic protein Bcl-2 prevents drug-induced mitochondrial changes, reactive oxygen species (ROS) production, and apoptosis; and (3) the assay of drug-induced mitochondrial changes can reflect drug-specific chemoresistance in a given cancer cell line.
A stable Bcl-2 transfectant of the Bcl-2 negative breast cancer cell line SKBr3 was created (SKBr3/Bcl2-2). Both SKBr3 and SKBr3/Bcl2-2 cells were treated with Herbimycin A (300 ng/mL) or vehicle (1% DMSO). Cell cycle changes were assessed by BRDU staining. Apoptosis was determined by electron microscopy, TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining, and diphenylamine assay of DNA fragmentation. Changes in mitochondrial mass and transmembrane potential (deltapsi(m)) were assessed by flow cytometric assessment of JC-1 fluorescence. Reactive oxygen species production was measured by 2',7'-dichlorodihydrofluorescein diacetate (DCFH) fluorescence.
Both SKBr3 and SKBr3/Bcl2-2 cells show cell cycle arrest after Herbimycin treatment. However, SKBr3 cells, but not SKBr3/Bcl2-2 cells, undergo apoptosis. Herbimycin-treated SKBr3 cells show increased mitochondrial mass (JC-1 green fluorescence), with no corresponding increase in deltapsi(m) (JC-1 red fluorescence). By contrast, Herbimycin-treated SKBr3/Bcl2-2 cells show no change in mitochondrial mass or deltapsi(m). Similarly, drug-treated SKBr3 cells, but not SKBr3/Bcl2-2 cells, demonstrate increased reactive oxygen species (ROS) production concomitant with the development of apoptosis.
SKBr3 cells undergoing apoptosis demonstrate mitochondrial changes associated with ROS production. Bcl-2 transfection prevents these changes because it prevents apoptosis and induces chemoresistance to Herbimycin in SKBr3. Flow cytometric measurement of drug induced mitochondrial changes and ROS production may facilitate in vitro assessment of chemosensitivity or chemoresistance in breast cancer.
化疗药物可诱导癌细胞凋亡。无法诱导凋亡的药物临床疗效可能会降低。我们推测：（1）化疗药物通过与活性氧生成相关的机制诱导线粒体变化和凋亡；（2）抗凋亡蛋白Bcl-2可阻止药物诱导的线粒体变化、活性氧（ROS）生成及凋亡；（3）药物诱导的线粒体变化检测可反映特定癌细胞系中的药物特异性化疗耐药性。
构建了Bcl-2阴性乳腺癌细胞系SKBr3的稳定Bcl-2转染细胞株（SKBr3/Bcl2-2）。用赫曲霉素A（300 ng/mL）或溶剂（1%二甲基亚砜）处理SKBr3和SKBr3/Bcl2-2细胞。通过溴脱氧尿苷（BRDU）染色评估细胞周期变化。通过电子显微镜、末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）染色及DNA片段化的二苯胺检测来确定凋亡。通过流式细胞术评估JC-1荧光来检测线粒体质量和跨膜电位（ΔΨm）的变化。用二氯二氢荧光素二乙酸酯（DCFH）荧光测量活性氧的生成。
赫曲霉素处理后，SKBr3和SKBr3/Bcl2-2细胞均出现细胞周期停滞。然而，SKBr3细胞发生凋亡，而SKBr3/Bcl2-2细胞未发生凋亡。赫曲霉素处理的SKBr3细胞线粒体质量增加（JC-1绿色荧光），但ΔΨm（JC-1红色荧光）无相应增加。相比之下，赫曲霉素处理的SKBr3/Bcl2-2细胞线粒体质量和ΔΨm无变化。同样，药物处理的SKBr3细胞而非SKBr3/Bcl2-2细胞，活性氧（ROS）生成增加并伴随凋亡的发生。
发生凋亡的SKBr3细胞表现出线粒体变化并与ROS生成相关。Bcl-2转染可阻止这些变化，因为它可阻止凋亡并诱导SKBr3对赫曲霉素产生化疗耐药性。通过流式细胞术检测药物诱导的线粒体变化和ROS生成可能有助于体外评估乳腺癌的化疗敏感性或化疗耐药性。
