LCL161联合多西他赛对人乳腺癌细胞株MCF-7增殖、凋亡的影响及机制

Effects of LCL161 combined with docetaxel on proliferation and apoptosis of human breast cancer cell line MCF-7 and its mechanism

目的观察LCL161联合多西他赛对人乳腺癌细胞株MCF-7增殖及凋亡的影响,并探讨其作用机制。方法 CCK-8法测算1～5μmol/L浓度的LCL161作用于MCF-7细胞24 h后的细胞存活率,选取对细胞增殖无明显抑制作用、细胞毒性较小的2μmol/L浓度的LCL161用于后续实验。将MCF-7细胞分为4组,LCL161组加入2μmol/L的LCL161,多西他赛组加入0. 6 mg/L的多西他赛,LCL161联用多西他赛组加入0. 6 mg/L的多西他赛和2μmol/L的LCL161,对照组加入不含药物的培养液。采用CCK-8法测算各组细胞存活率,采用细胞集落克隆实验检测各组细胞集落克隆能力;采用Hoechst33258染色法观察各组细胞晚期凋亡情况,采用流式细胞术测算各组细胞凋亡率,采用Western blotting法检测各组细胞凋亡抑制蛋白c IAP1、c IAP2及程序性坏死蛋白RIP1。结果LCL161组、多西他赛组、LCL161联用多西他赛组、对照组的细胞存活率分别为86. 84%±3. 83%、81. 29%±6. 71%、46. 91%±4. 76%和92. 77%±2. 18%,集落数目分别为(117±14)、(95±9)、(61±12)、(132±7)个,LCL161组与对照组相比,P> 0. 05;其余组间两两相比,P均<0. 05。LCL161组细胞膜通透性增加,染色质固缩,部分细胞发生了核碎裂;多西他赛组和对照组的细胞核均匀淡染,细胞核形态呈椭圆形,细胞膜相对完整; LCL161联用多西他赛组细胞数量明显减少,细胞核碎裂,部分发生了核解体。LCL161组、多西他赛组、LCL161联用多西他赛组、对照组细胞凋亡率分别为15. 16%±2. 26%、21. 42%±1. 17%、34. 38%±3. 27%、5. 18%±0. 89%,LCL161组与对照组相比,P> 0. 05;其余组间两两相比,P均<0. 05。LCL161组、多西他赛组、LCL161联用多西他赛组、对照组c IAP1蛋白的相对表达量分别为0. 41±0. 09、0. 29±0. 11、0. 11±0. 04、0. 62±0. 17,c IAP2蛋白的相对表达量分别为0. 86±0. 11、0. 77±0. 15、0. 51±0. 12、0. 98±0. 01,LCL161组与对照组相比,P> 0. 05;其余组间两两相比,P均<0. 05。LCL161组、多西他赛组、LCL161联用多西他赛组、对照组RIP1蛋白的相对表达量分别为1. 97±0. 22、1. 84±0. 19、3. 17±0. 26、1. 00±0. 07,LCL161组与多西他赛组相比,P> 0. 05;其余组间两两相比,P均<0. 05。结论LCL161联合多西他赛可抑制乳腺癌细胞的增殖,促进其凋亡; LCL161联合多西他赛可通过下调凋亡抑制蛋白、上调程序性坏死蛋白的表达,诱导乳腺癌细胞的凋亡及程序性坏死来发挥抗肿瘤作用。

Objective To detect the effects of LCL161 combined with docetaxel on the proliferation and apoptosis of human breast cancer MCF-7 cells and to explore its mechanism. Methods CCK-8( Cell counting kit-8) was used to detect the proliferation inhibition effect of various concentrations of LCL161( 1-5 μmol/L) on the human breast cancer MCF-7 cells. MCF-7 cells were divided into four groups: the LCL161 group which was added with 2 μmol/L LCL161,docetaxel group with 0. 6 mg/L docetaxel,LCL161 combined with docetaxel group with 0. 6 mg/L docetaxel and 2 μmol/L LCL161,and the control group with a drug-free medium. CCK-8 was used to detect the cell viability. The colony cloning ability of each group was detected by cell colony cloning assay. The apoptosis of each group was observed by Hoechst33258 staining.The apoptosis rate of each group was measured by flow cytometry. Western blotting was used to detect the changes of apoptosis-related protein cellular inhibitor of apoptosis protein 1/2( c IAP1/2) and necroptosis protein RIP1. Results The relative cell viability rates of the LCL161 group,docetaxel group,LCL161 combined with docetaxel group,and control group were 86. 84%± 3. 83%,81. 29%± 6. 71%,46. 91%± 4. 76%,and 92. 77%± 2. 18%,and the number of colonies was117 ± 14,95 ± 9,61 ± 12,and 132 ± 7,respectively;no significant difference was found between the LCL161 group and the control group( P > 0. 05),and significant difference was found between the rest groups( all P < 0. 05). In the LCL161 group,the cell membrane permeability increased,with chromatin condensation and some existed nuclear fragmentation.The nuclei of the docetaxel group and control group were lightly stained,the nucleus morphology was elliptical,and the cell membrane was relatively intact. The number of cells in the LCL161 combined with docetaxel group was significantly decreased,with fragmented nucleus,and some nuclear disintegration. The apoptosis rates of the LCL161 group,docetaxel group,LCL161 combined with docetaxel group,and control group were 15. 16%± 2. 26%,21. 42%± 1. 17%,34. 38%± 3. 27%,and 5. 18%± 0. 89%,respectively;no significant difference was found between the LCL161 group and the control group( P > 0. 05),and significant difference was found between the rest groups( all P < 0. 05). The relative expression levels of c IAP1 protein in the LCL161 group,docetaxel group,LCL161 combined with docetaxel group,and control group were 0. 41 ± 0. 09,0. 29 ± 0. 11,0. 11 ± 0. 04,and 0. 62 ± 0. 17. The relative expression levels of c IAP2 protein in the LCL161 group,docetaxel group,LCL161 combined with docetaxel group,and control group were 0. 86 ± 0. 11,0. 77± 0. 15,0. 51 ± 0. 12,and 0. 98 ± 0. 01,respectively;no significant difference was found between the LCL161 group and the control group( P > 0. 05),and significant difference was found between the rest groups( all P < 0. 05). The relative expression levels of RIP1 protein in the LCL161 group,docetaxel group,LCL161 combined with docetaxel group and control group were 1. 97 ± 0. 22,1. 84 ± 0. 19,3. 17 ± 0. 26,and 1. 00 ± 0. 07;no significant difference was found between the LCL161 group and the control group( P > 0. 05),and significant difference was found between the rest groups( all P <0. 05). Conclusion LCL161 combined with docetaxel can inhibit the proliferation and promote apoptosis of breast cancer cells;LCL161 combined with docetaxel can induce the apoptosis of breast cancer cells by down-regulating apoptosis inhibitory protein and up-regulating the expression of necroptosis protein to exert its anti-tumor effect.