左卡尼汀联合多西他赛对GLC-82细胞增殖与凋亡影响研究

Effects of combination treatment with L-carnitine and docetaxel on proliferation and apoptosis in GLC-82 cells

目的左卡尼汀(levocarnitine,L-CNT)在改善由化疗药物引起的心脏毒性和周围神经毒性方面有一定的作用。本实验旨在研究联合应用时,L-CNT对多西他赛(docetaxel,DTX)抑制非小细胞肺癌(non-small cell lung cancer,NSCLC)GLC-82细胞增殖作用的影响,初步探讨其作用机制。方法 MTT法检测不同浓度L-CNT(0.625、1.25、2.5、5、10、20、40和80μg/mL)和DTX(0.01、0.1、1、5、10、20、40和80μg/mL)分别对GLC-82细胞增殖作用的影响,倒置显微镜观察其形态的变化。以终浓度为1.25、20、80μg/mL L-CNT分别和DTX(9.301μg/mL)联合,同时处理GLC-82细胞24h,MTT法检测其对细胞增殖作用的影响,流式细胞术检测细胞凋亡的变化,蛋白质印迹法检测Bcl-2、Bax和ANT1蛋白表达的情况。结果与阴性对照组相比,L-CNT(1.25、20和80μg/mL)对GLC-82细胞作用24h后,促进增殖率分别为(14.52±0.73)%、(27.03±1.21)%和(19.77±0.65)%,F=42.538,P=0.001;48h后分别为(11.37±0.13)%、(19.56±1.01)%和(14.89±0.51)%,F=52.119,P=0.001。与DTX(27.319μg/mL)单药组相比,L-CNT(1.25、20和80μg/mL)、DTX联合用药组的细胞增殖率由(49.49±2.52)%增加至(52.64±2.43)%、(59.44±3.20)%和(53.15±2.37)%,F=7.412,P=0.011。与DTX(9.301μg/mL)单药组相比,L-CNT(1.25、20和80μg/mL)、DTX联合用药组GLC-82细胞密度增大,细胞凋亡率由(24.67±1.95)%分别降低至(21.90±1.98)%、(14.38±2.35)%和(22.42±1.43)%,F=15.697,P=0.001。L-CNT、DTX联合作用后,抑凋亡蛋白Bcl-2表达增多,促凋亡蛋白Bax表达减少,同时ANT1蛋白表达降低。结论 L-CNT和DTX联合应用对GLC-82细胞增殖与凋亡有一定的影响,主要通过下调Bax和上调Bcl-2蛋白的表达以及影响线粒体膜通透性转换孔的开放和关闭,减弱DTX对细胞凋亡的诱导作用。

OBJECTIVE L-CNT has certain effects in improving the cardiac toxicity and peripheral nerve toxicity aspects caused by chemotherapy drugs. This study aimed to investigate the proliferation and cell apoptosis effects of com- bination treatment L-CNT and DTX on NSCLC GLC-82 cells, and to explore its mechanism. METHODS GLC-82 cells were treated with different concentrations of L-CNT（0. 625, 1. 25, 2. 5, 5, 10, 20, 40, 80 μg/mL） and DTX（0.01, 0.1, 1, 5, 10, 20, 40, 80 μg/mL） in vitro. The inhibitory rates of cells were measured by MTT assay. Morphological changes were observed with inverted microscope. GLC-82 cells were treated with the combination treatment of L-CNT （1.25, 20, 80 μg/mL） and DTX（9. 301 μg/mL） for 24 h, and then the proliferation inhibitory rates were detected by MTT assay. The cell apoptosis was analyzed by Annexin V-FITC/PI double staining. Western blot was used to analyzed the expression of Bcl-2, Bax and adenine nucleotide translocatorl（ANT1）. RESULTS After 24 h and 48 h, compared with the control group, the proliferation rates of L-CNT （1. 25, 20, 80 μg/mL） were （14. 52 ± 0. 73） %, （27. 03 ± 1.21）%,（19.77±0.65）%, F=42. 538, P=0. 001 and （11.37±0. 13）%,（19.56± 1.01）M, （14. 89±0. 51）%, F= 52. 119, P=0. 001, respectively. Compared with DTX（27. 319 μg/mL）, the cell proliferation rate of L-CNT（1.25, 20, 80 μg/mL） and DTX combined group increased from（49.49±2.52）% to （52.64±2.43）%, （59. 44±3. 20）% and （53.15±2.37） %, F=7. 412, P=0. 011. Compared with the DTX（9. 301 μg/mL）group, cell density of the combination group was increased, Flow cytometry showed that the apoptosis rates were reduced to （21. 90± 1.98）%, （14. 38±2.35）% and （22.42±1.43）% from （24. 67±1. 95）%, F=15.697, P=0.001. The combination group could increase the expression of Bcl-2 and decrease the expression of Bax, at the same time, the expression of ANT1 was also decreased. CONCLUSIONS Treatment with L-CNT and DTX has impacts on the proliferation of GLC-82 cells. The possible mecha- nism of action is achieved by regulating the expression of Bcl-2 protein family and regulating the opening and closing of mitochondrial membrane permeability transition pore.