厚果崖豆藤提取物抑制NF-κB p65的活化对三阴性乳腺癌细胞增殖、凋亡和转移的影响

Effect of extract of millettia pachycarpa benth on proliferation,apoptosis and metastasis of triple negative breast cancer cells by inhibiting activation of NF-κB p65

目的:本研究探索厚果崖豆藤提取物MIL对MDA-MB-231乳腺癌细胞增殖、凋亡和转移的影响。方法:将MDA-MB-231细胞随机分为4组,分别在4组细胞的血清DMEM培养基中加入0、2、4、8μmol/L的MIL继续培养,用CCK8试剂盒检测处理细胞存活率;EDU染色检测细胞增殖状态;流式细胞术检测细胞凋亡;Transwell实验观察细胞侵袭的情况;RT-PCR检测E-cadherin、N-cadherin、Vimentin的表达;免疫印迹法检测NF-κB p65蛋白表达变化;免疫荧光检测p65的细胞核转位情况。结果:MIL对MDA-MB-231细胞有毒性作用,其毒性作用呈一定的剂量关系(P<0.05)MIL浓度大于16μmol/L时,细胞存活率显著降低(P<0.05);2、4、8μmol/L MIL三个处理组增殖抑制作用较0μmol/L MIL处理组明显(P<0.05);2、4、8μmol/L MIL三个处理组细胞凋亡率较0μmol/L MIL处理组明显升高,但差异无统计学意义;与对照组比较,MIL加药组显著抑制细胞增殖(P<0.05);而对细胞凋亡率无显著影响;4μmol/L和8μmol/L两个浓度的MIL对MDA-MB-231细胞侵袭能力有显著的抑制作用(P<0.05);与0μmol/L MIL处理组对照组比较,2μmol/L、4μmol/L和8μmol/L MIL处理MIL加药组组Vimentin mRNA的表达均显著降低(P<0.05);4μmol/L和8μmol/L MIL两个处理组E-cadherin mRNA的表达均显著升高(P<0.05),N-cadherin mRNA的表达均显著降低(P<0.05);4μmol/L和8μmol/L MIL两个处理组p65磷酸化水平较0μmol/L MIL处理组显著降低(P<0.05)。4μmol/L和8μmol/L两个浓度的MIL明显抑制p65的核转位(P<0.05)。结论:MIL通过抑制NF-κB p65的活化有效抑制MDA-MB-231细胞增殖、侵袭能力并促进其发生凋亡,表明MIL对三阴性乳腺癌有缓解作用。

Objective:To explored the effects of MIL extract from millettia pachycarpa benth on proliferation,apoptosis and metastasis of MDA-MB-231 breast cancer cells.Methods:MDA-MB-231 cells were randomly divided into 4 groups,and 0,2,4 and 8μmol/L MIL were added into the serum DMEM medium of the 4 groups of cells for further culture,and the cell survival rate was detected by CCK8 kit.Cell proliferation status was detected by EDU staining.Apoptosis was detected by flow cytometry.Transwell experiment observed cell invasion.The expression of E-cadherin,N-cadherin and Vimentin were detected by RT-PCR.Changes of protein expression of NF-κB p65 were detected by Western blot.The nuclear translocation of p65 was detected by immunofluorescence.Results:MIL had a toxic effect on MDA-MB-231cells,and its toxic effect showed a dose relationship(P<0.05).When the MIL concentration was greater than 16μmol/L the cell survival rate was significantly decreased.The proliferation inhibition of the 2,4 and 8μmol/L MIL treatment groups was more significant than that of the 0 micron MIL treatment group(P<0.05).Apoptosis rate of the 2,4 and 8μmol/L MIL treatment groups was significantly higher than that of the 0 muon MIL treatment group,but the difference was not statistically significant.Compared with the control group,the MIL treatment group significantly inhibited cell proliferation(P<0.05).There was no significantly effect on apoptosis.MIL of 4 and 8μmol/L significantly inhibited the invasion of MDA-MB-231 cells(P<0.05).The expression of Vimentin mRNA in the 2,4,and 8μmol/L MIL groups was significantly decreased(P<0.05).Compared with the control group,the expression of Vimentin mRNA in the MIL dosing group was significantly decreased(P<0.05).The expression of E-cadherin mRNA was significantly increased(P<0.05)and the expression of N-cadherin mRNA was significantly decreased(P<0.05).The phosphorylation level of p65 in the 4 and 8μmol/L MIL treatment groups was significantly lower than that in the 0 MIL treatment group(P<0.05).Nuclear transposition of p65 was significantly inhibited by MIL at concentrations of 4μmol/L and 8μmol/L(P<0.05).Conclusion:By inhibiting the activation of NF-κB p65,MIL can effectively inhibit the proliferation and invasion of MDA-MB-231 cells and promote their apoptosis,indicating that MIL can alleviate triple-negative breast cancer.