LY294002通过PI3K/Akt/Mtor对CD133+CD44+结直肠癌干细胞自我更新、增殖能力的影响

目的:探讨LY294002对CD133+CD44+结直肠癌干细胞自我更新、增殖能力的影响及其机制.方法: 通过流式细胞术从人结直肠癌HCT116细胞中分选CD133+CD44+的肿瘤干细胞(CSCs),不同浓度(10、20、30 μmol/L)LY294002作用CSCs,对照组不使用药物.成球实验及有限浓度稀释法检测细胞自我更新能力,细胞活性计数法及平板克隆实验检测增殖能力,流式细胞术检测细胞周期,实时定量PCR及流式细胞术检测PI3K/Akt/mTOR信号通路基因Akt、磷酸化Akt、诱骗受体3(DcR3)、B淋巴细胞瘤-2(Bc--2)、哺乳动物雷帕霉素靶蛋白(mTOR)及细胞周期蛋白D1(Cyclin D1)表达变化.结果: 与对照组相比,30 μmol/L LY294002作用实验组成球能力明显削弱(P＜0. 01),CSCs比例明显下降(P＜0. 05),增殖能力明显削弱(P＜0. 01),G0G1期细胞周期受到阻滞(P＜0. 01);磷酸化Akt表达降低(P＜0. 01),DcR3、Bcl-2、mTOR及Cyclin D1表达量下调(P＜0. 01).结论: LY294002可能通过PI3K/Akt/mTOR通路导致结直肠癌CSCs周期阻滞、抑制其自我更新、增殖能力.

Effects of LY294002 on self-renewal and proliferation of CD133+CD44+colorectal cancer stem cells by PI3K/Akt/mTOR

Objective:To investigate the effect of LY294002 on the self-renewal and proliferation of CD133+CD44+ colorectal cancer stem cells (CSCs) and its mechanism. Methods:CSCs were isolated from HCT116 human colorectal cancer cell line using flow cytometry methods. LY294002 (10,20,30 μmol/L),an inhibitor of PI3K/Akt/mTOR signaling pathway,was used in the experimental groups,LY294002 was not added to the control group. The self-renewal was detected by sphere-forming assay and limitting dilution as-say (LDA),respectively. The proliferation ability was detected by cell counting kit (CCK-8) and colony formation assay,respectively. Moreover,the cell cycle was detected by flow cytometry (FCM). The mRNA and protein expression of PI3K/Akt/mTOR signaling path-way downstream genes such as phosphorylated Akt,DcR3,Bcl-2,mTOR and Cyclin D1 were tested by real-time PCR (qRT-PCR) and FCM,respectively. Results:30 μmol/L LY294002 could inhibit the self-renewal and proliferation of CSCs ( P ＜0. 01). It also de-creased the fraction of CSCs as compared to the control group (P＜0. 05),the ability to proliferate obviously weakened (P＜0. 01). Moreover,30 μmol/L LY294002 resulted in CSCs G0G1 arrest (P＜0. 01). Mechanismly,we found the expression of phosphorylated Akt protein decreased,which accorded with the decreased expression of DcR3,Bcl-2,mTOR and Cyclin D1 (P＜0. 01). Conclusion:LY294002 can inhibit the self-renewal,cell cycle and proliferation of CSCs via inhibiting PI3K/Akt/mTOR signaling pathway.