Zhang Qi, Si Shuhui, Schoen Sue, Chen Jindong, Jin Xun-Bo, Wu Guan
Minimally Invasive Urology Center, Provincial Hospital Affiliated to Shandong University, Jinan 250021, China.
J Exp Clin Cancer Res. 2013 Dec 4;32(1):99. doi: 10.1186/1756-9966-32-99.
Paclitaxel, a widely used chemotherapeutic drug, can induce apoptosis in variety of cancer cells. A previous study has shown preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cell line, UOK257. In this report, we investigate the cellular and molecular mechanism of paclitaxel-induced autophagy and apoptosis in renal cancer cells with and without FLCN expression.
Two pairs of cell lines were used: FLCN siRNA-silenced ACHN cell line (ACHN-5968) and scrambled ACHN cell line (ACHN-sc); FLCN-null UOK257 cell line and UOK257-2 cell line restored with ectopic expression of FLCN. Autophagy was examined by western blot, GFP-LC3, transmission electron microscopy, and MDC assay. Cell viability and apoptosis were detected using MTT assay, DAPI stain and TUNEL assay. After inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, cell viability and apoptosis were measured by MTT assay and TUNEL assay.
After paclitaxel treatment, a dose-dependent decrease in cell viability and increase in apoptosis were observed in FLCN-deficient UOK257 and ACHN-5968 cells compared to their FLCN-expressing counterparts, suggesting that renal cancer cells without FLCN were more sensitive to paclitaxel. Enhanced autophagy was found to be associated with paclitaxel treatment in FLCN-deficient RCC cells. The MAPK pathway was also identified as a key pathway for the activation of autophagy in these kidney cancer cells. Inhibition of phosphorylated ERK with ERK inhibitor U0126 showed a significant decrease in autophagy. Furthermore, after inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, apoptosis induced by paclitaxel was significantly increased in FLCN-deficient UOK257 and ACHN-5968 cells.
Preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cells is associated with enhanced autophagy. Suppression of autophagy further enhances paclitaxel-induced apoptosis in FLCN-deficient renal cancer cells. Our results suggest that paclitaxel combined with an autophagy inhibitor might be a potentially more effective chemotherapeutic approach for FLCN-deficient renal cancer.
紫杉醇是一种广泛使用的化疗药物，可诱导多种癌细胞凋亡。先前的一项研究表明，紫杉醇对缺乏卵泡抑素（FLCN）的肾癌细胞系UOK257具有优先毒性。在本报告中，我们研究了紫杉醇诱导有或无FLCN表达的肾癌细胞自噬和凋亡的细胞及分子机制。
使用了两对细胞系：FLCN siRNA沉默的ACHN细胞系（ACHN-5968）和乱序ACHN细胞系（ACHN-sc）；缺乏FLCN的UOK257细胞系和通过FLCN异位表达恢复的UOK257-2细胞系。通过蛋白质免疫印迹法、绿色荧光蛋白-微管相关蛋白1轻链3（GFP-LC3）、透射电子显微镜和单丹磺酰尸胺（MDC）测定法检测自噬。使用噻唑蓝（MTT）测定法、4',6-二脒基-2-苯基吲哚（DAPI）染色和末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）测定法检测细胞活力和凋亡。在用3-甲基腺嘌呤（3-MA）或Beclin 1小干扰RNA（siRNA）抑制自噬后，通过MTT测定法和TUNEL测定法测量细胞活力和凋亡。
紫杉醇处理后，与表达FLCN的对应细胞相比，在缺乏FLCN的UOK257和ACHN-5968细胞中观察到细胞活力呈剂量依赖性下降且凋亡增加，这表明没有FLCN的肾癌细胞对紫杉醇更敏感。发现增强的自噬与缺乏FLCN的肾透明细胞癌（RCC）细胞中的紫杉醇处理有关。丝裂原活化蛋白激酶（MAPK）途径也被确定为这些肾癌细胞中自噬激活的关键途径。用细胞外信号调节激酶（ERK）抑制剂U0126抑制磷酸化的ERK显示自噬显著减少。此外，在用3-MA或Beclin 1 siRNA抑制自噬后，在缺乏FLCN的UOK257和ACHN-5968细胞中紫杉醇诱导的凋亡显著增加。
紫杉醇对缺乏FLCN的肾癌细胞的优先毒性与自噬增强有关。抑制自噬进一步增强了紫杉醇在缺乏FLCN的肾癌细胞中诱导的凋亡。我们的结果表明，紫杉醇与自噬抑制剂联合使用可能是一种对缺乏FLCN的肾癌潜在更有效的化疗方法。
