LCL161联合Z-VAD-FMK对人乳腺癌细胞MCF-7和MDA-MB-231增殖的影响

目的:探讨第二个线粒体衍生的半胱氨酸蛋白酶激动剂小分子(Smac)类似物LCL161联合天冬氨酸蛋白水解酶(caspase)抑制剂Z-VAD-FMK诱导细胞发生坏死性凋亡对人乳腺癌细胞MCF-7和MDA-MB-231增殖的影响。方法:体外培养MCF-7、MDA-MB-231细胞株。噻唑蓝(MTT)法检测2种细胞株空白对...

Effect of LCL161 combined with Z-VAD-FMK on the induction of necroptosis in breast cancer cells on the proliferation of MCF-7 and MDA-MB-231 human breast cancer cells

Objective To investigate the effect of a second mitochondria-derived cysteine protease agonist small molecule (Smac) analog, LCL161, combined with aspartate protein hydrolase (caspase) inhibitor Z-VAD-FMK to induce necroptosis on the proliferation of human breast cancer cells MCF-7 and MDA-MB-231.Methods MCF-7 and MDA-MB-231 cell lines were cultured in vitro, and the survival rates of the blank control, Z-VAD-FMK (10 mmol/L), LCL161 (1 μmol/L) and LCL161+Z-VAD-FMK groups of the two cell lines were examined by MTT, and the apoptotic submicrostructures were observed by fluoroscopic electron microscopy. dec-1 After Nec-1 pretreatment, the survival rates of the blank control, Nec-1 (20 mmol/L), LCL161 (1 μmol/L) and LCL161+Nec-1 groups were detected by MTT assay. Annexin V-FITC/PI double staining method to detect cell apoptosis in blank control group, LCL161 group, Z-VAD-FMK group, LCL161+Nec-1 group and LCL161+Z-VAD+Nec-1 group. Nude mice tumorigenesis assay was performed to observe the volume and quality of transplanted MCF-7 cell lines in vivo in the blank control group, LCL161 group and LCL161+Z-VAD-FMK group.Results (1) The results of MTT assay showed that the survival rates of MDA-MB-231 and MCF-7 cell lines in the LCL161+Z-VAD-FMK group were lower than those in all other groups, and the difference were statistically significant (all P values＜0.01). The cells in the LCL161+Z-VAD-FMK group were observed by fluoroscopic electron microscopy to have characteristic morphological changes of apoptosis and necrosis together. (2) After Nec-1 pretreatment, the results of MTT assay showed that the survival rate of MDA-MB-231 and MCF-7 cells in the LCL161 group and the Nec-1+LCL161 group was significantly reduced compared with the blank control group and the Nec-1 group, which showed that the cell survival rate was low, and the differences were statistically significant (all P values＜0.05), but the difference between the LCL-161 and Nec-1+LCL161 groups were not statistically significant (all P values＞0.05). (3) The results of Annexin V-FITC/PI double-staining method showed that the apoptosis rate of both cell lines was significantly higher in the LCL161+Z-VAD-FMK group and LCL161 group compared with other groups, and the LCL161+Z-VAD-FMK group was also higher than the LCL161 group, and the differences were statistically significant (all P values＜0.01). (4) Fifteen nude mice were successfully established as MCF-7 cell nude mice tumorigenic model, and the tumor volume and mass of LCL161 and LCL161+Z-VAD-FMK groups were significantly lower than those of the blank control group at day 20 after drug administration, and the differences were statistically significant (all P values＜0.05). There was no significant difference in tumor volume and mass between the LCL161 group and the LCL161+Z-VAD-FMK groups(all P values＞0.05).Conclusions In vitro experiments verified that LCL161+Z-VAD-FMK can inhibit tumor proliferation by inducing necroptosis in breast cancer cells, but the in vivo effect on estrogen receptor-positive breast cancer needs further verification.