拓扑替康对肺癌细胞生长周期的影响及诱导凋亡的作用机制

目的　研究拓扑替康 (topotecan ,TPT)对人肺癌SPC A 1细胞生长周期的影响及诱导凋亡的作用 ,并探讨Caspase 3及Bcl 2在此过程中的作用。 方法　体外培养肺癌细胞 ,分为对照组 (未处理组 )、TPT(5、10、15和 2 0mg·L-1)组、半胱天冬酶 3 (Caspase 3 )特异性拮抗剂Ac DEVD CHO +TPT组 ,加药后培养 2 4h ,TUNEL法观察肺癌细胞凋亡形态学特征 ;流式细胞仪观察细胞生长周期变化并检测凋亡率和肺癌细胞Bcl 2蛋白的表达。结果　TPT (5mg·L-1)处理细胞 2 4h ,流式细胞仪未检测到凋亡峰 ,随着TPT浓度增大 ,凋亡率显著增高 ,各浓度组间差异具有显著性 (P<0 0 1) ;TUNEL及透射电镜检测到典型的凋亡细胞形态学特征 ,凋亡细胞DNA呈梯状电泳 ;流式细胞仪检测到细胞周期发生改变 ,G1期细胞较对照组增多 (P <0 0 1) ,S期细胞减少 (P <0 0 1) ;TPT(2 0mg·L-1)处理细胞后肺癌细胞凋亡率为 11 9%± 2 6% ,高于对照组细胞 (P <0 0 1) ;Bcl 2蛋白阳性表达细胞率为 7 9%± 1 2 % ,较对照组 (44 7%±7 1% )降低 (P <0 0 1) ;Ac EDVD CHO (5 0 μmol·L-1)和TPT(2 0mg·L-1)共同处理细胞 2 4h其凋亡率为 6 1%±1 8% ,较单纯TPT作用组降低 (P <0 0 1) ;Bcl 2蛋白阳性表达率为 3 3 4%± 5 2 % ,较单纯TPT作用组增高

The effect of topotecan on lung cancer cell cycle and the mechanism of topotecan-induced apoptosis

AIM To study the effect of Topotecan(TPT) on cell cycle and apoptosis of hunman lung cancer SPC A 1 cells and the role of Caspase 3 and bcl 2 involved in TPT induced apoptosis. METHODS Cells were incubated with TPT(5,10,15 and 20 mg·L -1 ) for 24 h. Apoptosis was detected by TUNEL staining, electronic microscope and DNA agarose gel electropheresis. Flow cytometer was used to analyse cell cycle changes and to detect apoptotic rates and bcl 2 expression of cells treated with TPT or TPT and 50 μmol·L -1 Ac DEVD CHO(specific Caspase 3 inhibitor). RESULTS Apoptosis of cells treated with TPT(5 mg·L -1 ) was not detected, but apoptosis rates increased with the increase of TPT concentration and there was significant difference between each concentration group( P <0 01).After treated with TPT(20 mg·L -1 ) for 24 h, typical morphological features of apoptotic cells were detected by TUNEL staining and electronic microscope.DNA ladder was also viewed on agarose gel. Cell cycle changes were detected by flow cytometer with G 1 phase significantly more than control ( P <0 01) and S phase significantly less than control ( P <0 01).Apoptotic rate of cells with TPT(20 mg·L -1 ) was 11 9%±2 6%, higher than control cells ( P <0 01). bcl 2 expression rate of cells with TPT was 7 9%±1 2%,much lower than control cells (44 7%±7 1%, P <0 01). Apoptosis rate of cells with TPT(20 mg·L -1 ) and Ac DEVD CHO(50 μmol·L -1 ) for 24 h was 6 1%±1 8%,lower than cells with TPT only ( P <0 01). bcl 2 expression rate of cells with TPT and Ac DEVD CHO was 33 4%±5 2%, higher than cells with TPT only ( P <0 01). CONCLUSION TPT can act on hunman lung cancer SPC A 1 cells at phase G 1 and effectively induce apoptosis in the way of dose dependent.The possible mechanism was through Caspase 3 activation and its cleavage to inhibit bcl 2.