盐霉素通过Na^(+),K^(+)-ATP酶诱导脂肪干细胞和肝癌干细胞胀亡的作用机制研究

Mechanism of salinomycin-induced oncosis in adipose-derived stem cells and hepatocellular carcinoma stem cells via Na^(+),K^(+)-ATPase

目的观察脂肪干细胞(ASCs)和肝癌干细胞(HCCSCs)对盐霉素处理的反应,探讨Na^(+),K^(+)-ATP酶在盐霉素选择性杀伤ASCs和HCCSCs中的作用。方法采用CCK-8、Transwell实验分析盐霉素对ASCs及HCCSCs的细胞毒性反应,显微镜下观察细胞形态变化,采用Na^(+),K^(+)-ATP酶检测试剂盒测定细胞内Na^(+)和K^(+)离子浓度,通过ATP检测试剂盒测定细胞内ATP浓度。采用Western blot法分析Na^(+),K^(+)-ATP酶各亚基及细胞上皮间质转化(EMT)相关蛋白的表达水平。通过上述方法验证盐霉素通过调控Na^(+),K^(+)-ATP酶诱导ASCs和HCCSCs胀亡(细胞肿胀、坏死)的作用机制。结果盐霉素选择性诱导ASCs和HCCSCs胀亡。在胀亡的ASCs和HCCSCs中,细胞内ATP浓度显著下降,伴随Na^(+),K^(+)-ATP酶活性丧失和细胞内Na^(+)滞留。所有类型的Na^(+),K^(+)-ATP酶亚基在ASCs中均有表达,而在盐霉素诱导分化的ASCs中仅表达α1和β1亚基。β1亚基的丧失是阿霉素诱导肝癌Huh-7细胞EMT的关键事件,相反β1亚基的过表达可以抑制阿霉素诱导的Huh-7细胞EMT。结论Na^(+),K^(+)-ATP酶的活性差异是盐霉素对干细胞产生选择性细胞毒性的原因,且β1亚基是肝癌EMT过程中一个重要因素。Na^(+),K^(+)-ATP酶亚基对ASCs和HCCSCs的干性及癌症干细胞样的EMT过程起关键作用。

Objective Salinomycin,an ionophore antibiotic,has been reported to selectively kill cancer stem cells.However,the underlying mechanism remains unclear.This study investigated the responses of adipose-derived stem cells(ASCs)and hepatocellular carcinoma stem cells(HCCSCs)to salinomycin treatment and explored the underlying mechanisms.Methods The cytotoxic effects of salinomycin on ASCs and HCCSCs were analyzed using CCK-8 and Transwell assays,while morphological changes of cells were observed under a microscope.Intracellular Na^(+)and K^(+)ion concentrations were measured using a Na^(+),K^(+)-ATPase assay kit,and ATP levels were determined with an ATP assay kit.Additionally,the expression levels of Na^(+),K^(+)-ATPase subunits and cell epithelial-mesenchymal transition(EMT)related proteins were examined using Western blot analysis.These methods were employed to investigate the mechanism by which salinomycin induces oncosis(cellular swelling and necrosis)in ASCs and HCCSCs through the regulation of Na^(+),K^(+)-ATPase.Results This study observed that salinomycin selectively induced cellular and organelle swelling in ASCs and HCCSCs,morphologically defined as oncosis.In oncotic ASCs and HCCSCs,intracellular ATP concentration was significantly reduced,accompanied by a loss of Na^(+),K^(+)-ATPase activity and retention of intracellular Na^(+).All types of Na^(+),K^(+)-ATPase subunits were expressed in ASCs,whereas only theα1 andβ1 subunits were expressed in salinomycin-induced differentiated ASCs.Additionally,this study found that the loss ofβ1 subunits was a key event in doxorubicin-induced EMT in Huh-7 cells,and overexpression ofβ1 subunits could inhibit doxorubicin-induced EMT in Huh-7 cells.Conclusion The study demonstrates that the difference in Na^(+),K^(+)-ATPase activity determined by subunits is the reason for the selective cytotoxicity of salinomycin on stem cells,and that theβ1 subunit serves as a key marker in the HCC EMT process.These results indicate that Na^(+),K^(+)-ATPase subunits play a key role in the stemness of ASCs and HCCSCs,as well as in the cancer stem cell-like EMT process.