参灵抗癌方经PI3K/AKT通路调控Bim表达逆转胃癌细胞失巢凋亡抵抗的作用及机制

Role and Mechanism of Shenling Kang’ai Formula on Reversing Anoikis Resistance of Gastric Cancer Cells by Regulating Bim Expression via PI3K/AKT Pathway

目的观察参灵抗癌方(SLKAF)逆转胃癌细胞失巢凋亡抵抗作用,并从磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(PI3K/AKT)调控Bcl-2细胞死亡调解子抗体(Bim)表达为切入点探讨其作用机制。方法采用梯度剂量的SLKAF(0,1,2,4,8,16 mg/mL)与SGC-7901胃癌细胞共培养,MTT法检测胃癌细胞活力,GraphPad Prism7.0计算半数抑制浓度(IC50),并以1×IC50、2×IC50分别作为SLKAF干预SGC-7901细胞的低剂量和高剂量。运用RNA干扰技术将Bim-siRNA转染SGC-7901细胞,qPCR技术检测Bim表达,观察对Bim表达的抑制效率;以SLKAF低、高剂量分别与SGC-7901细胞和经Bim-siRNA转染的SGC-7901细胞共培养,分别采用qPCR和Western Blot检测PI3K、AKT的表达,流式细胞技术检测细胞凋亡率,软琼脂克隆形成实验检测细胞克隆形成能力。结果SLKAF对SGC-7901细胞增殖有显著的抑制作用(P<0.01),并呈明显的量—效应关系,IC50为7.626 mg/mL,故确定8、16 mg/mL分别作为低剂量和高剂量。Bim-siRNA转染SGC-7901细胞可抑制Bim表达(P<0.05)。SLKAF高、低剂量均降低SGC-7901细胞PI3K、AKT表达(P<0.05,P<0.01),提高胃癌细胞凋亡率(P<0.05,P<0.01),降低胃癌细胞克隆形成能力(P<0.01);与低剂量比较,高剂量SLKAF作用下的SGC-7901细胞/经Bim-siRNA转染的SGC-7901细胞表达PI3K、AKT均降低(P<0.01),胃癌细胞凋亡率升高(P<0.05),细胞克隆形成数降低(P<0.05);在SLKAF相同剂量下,SGC-7901细胞和经Bim-siRNA转染的SGC-7901细胞表达PI3K、AKT比较,差异无统计学意义(P>0.05),但细胞凋亡率和细胞克隆形成能力差异均有统计学意义(P<0.01)。结论SLKAF经PI3K/AKT调控Bim表达逆转胃癌细胞失巢凋亡抵抗。

Objective To study the role of Shenling Kang’ai Formula(SLKAF)on reversing anoikis resistance of gastric cancer cells,and explore its mechanism based on phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT)signal transduction pathway regulating Bcl-2 interacting mediator of cell death(Bim)expression.Methods The viability of human gastric cancer SGC-7901 cells exposed to various concentrations of SLKAF(0,1,2,4,8 and 16 mg/mL)in vitro was assessed separately by MTT,and the half inhibition concentration(IC50)was calculated by GraphPad Prism7.0 software,and therefore the 1×IC50and 2×IC50 were selected respectively as the low and high doses of for intervening SLKAF on SGC-7901 cells.The SGC-7901 cells were transfected with interfering small RNAs targeting Bim gene(Bim-siRNA)by RNA interference technique,and the inhibition efficiency of Bim expression was detected by real-time fluorescence quantitative PCR(qRT-PCR).The SGC-7901 cells,besides those transfected with Bim-siRNA,were exposed to the low and high doses of SLKAF,respectively.The expression levels of PI3K and AKT were measured separately by qRT-PCR and Western Blot.Apoptosis rate was measured by flow cytometry(FCM).The soft agar colony formation assay was performed to determine the colony-formation ability.Results SLKAF had a significant inhibitory effect on the proliferation of SGC-7901 cells(P<0.01),and there was an obvious dose-effect relationship.IC50 calculated by Graph Pad prism 7.0 software was 7.626 mg/mL,therefore,8 and 16 mg/mL were selected as low-dose and high-dose,respectively.Bim-siRNA transfection significantly inhibited Bim expression in SGC-7901 cells(P<0.05).The low and high doses of SLKAF significantly decreased the expressions of PI3K and AKT in SGC-7901 cells(P<0.05,P<0.01),increased apoptotic rate(P<0.05,P<0.01),and decreased the colony-formation ability of SGC-7901 cells(P<0.01).Compared with the low doses,the expression levels of PI3K and AKT in the SGC-7901 cells/those transfected with Bim-siRNA exposed separately to the high doses of SLKAF were significantly decreased(P<0.01),the apoptotic rate of SGC-7901 cells was significantly increased(P<0.05),and the colony-formation ability of SGC-7901 cells was significantly decreased(P<0.05);moreover,there were no significant differences of the expression levels of PI3K and AKT between the SGC-7901 cells and those transfected with Bim-siRNA at the same dose of SLKAF(P>0.05),while there were significant differences of the apoptosis rate and the colony-formation ability between them(P<0.01).Conclusion SLKAF could reverse anoikis resistance of gastric cancer cells by regulating the expression of Bim via PI3K/AKT signal pathway.