咪达唑仑诱导乳腺癌细胞MDA-MB-231凋亡并抑制细胞恶性增殖、干样特性及AKT磷酸化

Midazolam induces breast cancer cell MDA-MB-231 apoptosis and inhibits cell malignant proliferation, stem-like properties and AKT phosphorylation

目的 探讨咪达唑仑对乳腺癌细胞MDA-MB-231增殖、凋亡、干样特性及蛋白激酶B(AKT)的影响。方法 CCK8法检测不用浓度咪达唑仑对乳腺癌细胞MDA-MB-231活力的影响,筛选合适的剂量;克隆形成检测细胞增殖;流式检测细胞凋亡;Western印迹检测凋亡相关蛋白的表达[半胱天冬酶(Caspase)-3、Caspase-9];显微观察干细胞成球;Western印迹检测干细胞标志物八聚体结合转录因子(OCT)4、性别决定区γ框蛋白(SOX)2和AKT的磷酸化;免疫荧光检测AKT的核转位。结果 与对照组相比,75μmol/L咪达唑仑组对MDA-MB-231细胞活力的影响无明显差异,随浓度的增加,细胞活力明显减少。CCK8实验结果,筛选4个剂量组(0、75、150、300μmol/L),进行后续实验;与对照组相比,除75μmol/L剂量组差异无统计学意义外,150、300μmol/L剂量组,细胞凋亡率、凋亡蛋白表达水平明显升高(P<0.05);克隆细胞形成率、干细胞成球数量及直径、OCT4及SOX2表达量、AKT的磷酸化表达均明显降低。结论 咪达唑仑诱导乳腺癌细胞MDA-MB-231凋亡并抑制肿瘤细胞恶性增殖、干样特性,其机制可能是通过下调SOX2、OCT4表达,减少癌细胞内AKT信号通路的调节,从而抑制AKT磷酸化水平及AKT核转位。缓解乳腺癌的发生发展。

Objective To investigate the effects of midazolam on the proliferation, apoptosis, stem-like properties and protein kinase B(AKT) of breast cancer cell MDA-MB-231.Methods CCK8 method was used to detect the effect of different concentrations of midazolam on the viability of breast cancer cells MDA-MB-231, and the appropriate dose was screened;cell proliferation was detected by clone formation;cell apoptosis was detected by flow cytometry;expression of apoptosis-related protein(Caspase-3, Caspase-9) were detected by Western blot;stem cell formation was observed by microscopic;stem cell markers octamer-binding transcription factor(OCT)4, sex-determining γ-frame protein(SOX)2 and phosphorylation of AKT were detected by Western blot;immunofluorescence was used to detect nuclear translocation of AKT.Results Compared with the control group, 75 μmol/L midazolam group had no significantly difference in the effect of MDA-MB-231 cell viability. The cell viability was decreased significantly with the increase of concentration. CCK8 experiment results, four dose groups(0, 75, 150, 300 μmol/L) were screened for follow-up experiments;compared with the control group, except for the 75 μmol/L dose group had no statistical difference, in the 150 and 300 μmol/L dose groups, the apoptosis rate and the expression level of apoptotic proteins were significantly increased(P<0.05);the formation rate of cloned cells, the number and diameter of stem cell spheroids, the expressions of OCT4 and SOX2, and AKT phosphorylation expression were significantly reduced.Conclusions Midazolam could induce breast cancer cell MDA-MB-231 apoptosis and inhibit cell malignant proliferation and stem-like characteristics. The mechanism might be related to down-regulation of the expression of SOX2 and OCT4, reduce the regulation of AKT signaling pathway in cancer cells, inhibition of AKT phosphorylation level and nuclear translocation, alleviating the occurrence and development of breast cancer.