益母草碱调节Fas/FasL信号通路对乳腺癌细胞恶性生物行为的影响

Impacts of Leonurine on the malignant biological behaviors of breast cancer cells by regulating the Fas/FasL signaling pathway

目的探讨益母草碱(Leonurine,Leo)对乳腺癌细胞的增殖、迁移、凋亡、细胞周期进展以及上皮间质转化的影响及作用机制。方法体外培养4T1乳腺癌细胞,将细胞分为对照组(Control组)、益母草碱低、中、高浓度组(Leo-L组、Leo-M组、Leo-H组)、益母草碱高浓度+转染短发夹RNA慢病毒阴性对照组(Leo-H+sh-NC组),益母草碱高浓度+转染Fas短发夹RNA慢病毒组(Leo-H+sh-Fas组)。使用不同浓度的益母草碱(10~160 mmol/L)处理细胞,CCK-8法检测细胞的存活率,筛选最佳实验浓度;通过Transwell小室法评价细胞的迁移能力;流式细胞术检测细胞凋亡率及细胞周期进展;Western blot检测N-cadherin、Vimentin、Fas、FasL表达,建立荷瘤小鼠模型评价益母草碱对乳腺癌移植瘤生长的影响。结果使用浓度为10~160 mmol/L的益母草碱处理细胞12 h,4T1细胞存活率显著降低(P<0.05),半数抑制浓度(IC50值)为(53.61±1.729)mmol/L,选择10、20、30 mmol/L为后续实验浓度;与Control组相比,Leo-L组、Leo-M组、Leo-H组4T1细胞的增殖活力、迁移率、G2/M期细胞比例及S期细胞比例、N-cadherin、Vimentin表达显著降低(P<0.05),细胞凋亡率、G0/G1期细胞比例、Fas、FasL表达显著升高(P<0.05);与Leo-H+sh-NC组相比,Leo-H+sh-Fas组4T1细胞的增殖活力、迁移率、G2/M期细胞比例及S期细胞比例、N-cadherin、Vimentin表达显著升高(P<0.05),细胞凋亡率、G0/G1期细胞比例、Fas、FasL表达显著降低(P<0.05);益母草碱可以抑制乳腺癌移植瘤的生长(P<0.05)。结论益母草碱可以抑制乳腺癌细胞恶性生物行为,其作用机制可能与激活Fas/FasL信号通路有关。

Objective To investigate the impacts of Leonurine(Leo)on the proliferation,migration,apoptosis,cell cycle progression and epithelial mesenchymal transformation(EMT)of breast cancer cells and its mechanism.Methods 4T1 breast cancer cells were cultured in vitro.The cells were grouped into control group,low-dose Leonurine group(Leo-L),medium-dose Leonurine group(Leo-M),high-dose Leonurine group(Leo-H),high-dose Leonurine+transfection of short hairpin RNA lentivirus negative control group(Leo-H+sh NC group),and high-dose Leonurine+transfection of Fas short hairpin RNA lentivirus group(Leo-H+sh Fas group).The cells were treated with different concentrations of Leonurine(10-160 mmol/L).CCK-8 assay was applied to detect the cell survival and to screen the optimal experimental concentration.Transwell assay was applied to evaluate the migration ability of cells.Flow cytometry was applied to detect cell apoptosis rate and cell cycle progression.Western blot was applied to detect the protein expressions of N-cadherin,Vimentin,fatty acid synthase(Fas)and fatty acid synthase ligand(FasL).A tumor-bearing mouse model was established to evaluate the effect of Leonurine on the growth of breast cancer xenografts.Results Treatment of Leonurine at the concentration of 10-160mmol/L for 12h significantly reduced the survival rate of 4T1 cells(P<0.05).The inhibitory concentration 50(IC50)value was(53.61±1.729)mmol/L,and the concentrations of 10,20,and 30mmol/L were selected for the subsequent experiments.Compared with those of the Control group,cells in the Leo-L,Leo-M and Leo-H groups presented significantly lower proliferative rate,migratory rate,cell ratio in the G2/M and S phase,and protein expressions of N-cadherin and Vimentin,but significantly higher apoptotic rate,cell ratio in the G0/G1 phase and protein expressions of Fas and FasL(P<0.05).Compared with those of Leo-H+sh-NC group,4T1 cells in the Leo-H+sh-Fas group presented significantly higher proliferative rate,migratory rate,cell ratio in the G2/M and S phase,and protein expressions of N-cadherin and Vimentin,but significantly lower apoptotic rate,cell ratio in the G0/G1 phase and protein expressions of Fas and FasL(P<0.05).Leonurine could inhibit the growth of breast cancer xenografts(P<0.05).Conclusion Leonurine can inhibit the malignant behaviors of breast cancer cells by activating the Fas/FasL signaling pathway.