二甲双胍通过PP2A/AKT通路诱导结直肠癌细胞衰老

Metformin induces senescence of colorectal cancer cells through PP2A/AKT pathway

目的明确二甲双胍(1,1-二甲基双胍盐酸盐,Met)是否通过诱导细胞衰老抑制结直肠癌细胞增殖,并初步探讨其诱导细胞衰老的机制。方法以不同浓度Met(0、2.5、5.0、10.0、20.0、40.0 mmol/L)处理结直肠癌LOVO、SW480细胞,采用CCK-8检测细胞活力,流式细胞仪检测细胞凋亡和周期。根据凋亡结果将实验分为对照组、5.0 mmol/L Met组(n=3),采用EdU和克隆形成实验检测细胞增殖,衰老相关β-半乳糖苷酶(SA-β-gal)染色检测细胞衰老代谢,ELASA检测蛋白磷酸酶2A(protein phosphatase 2A,PP2A)酶活性,Western blot检测蛋白表达。用PP2A抑制剂LB-100联合Met处理细胞并分为对照组、Met组、Met+LB-100组、LB-100组(n=3),再次用CCK-8和克隆形成实验检测细胞增殖,SA-β-ga1染色检测细胞衰老情况。结果Met以浓度和时间依赖方式显著抑制结直肠癌细胞LOVO、SW480增殖(P<0.05);低浓度(≤5.0 mmol/L)Met作用下,细胞无明显凋亡,但有明显增殖抑制和G0/G1期阻滞;细胞呈大而扁平、空泡化增多的典型衰老样态,衰老标志SA-β-gal阳性细胞明显增加[LOVO:对照组(5.43±1.37)%,Met组(17.56±1.89)%,P<0.001;SW480:对照组(8.49±2.12)%,Met组(20.02±3.68)%,P<0.001];此时,PP2A蛋白表达无改变,但磷酸化水平显著降低,PP2A酶活性则显著升高(P<0.05),其下游AKT蛋白磷酸化水平降低,衰老相关蛋白P53、P21等表达明显增加。PP2A抑制剂LB-100联合Met处理后,可明显逆转Met对细胞增殖的抑制,并延缓Met诱导的细胞衰老[LOVO:Met组(10.55±1.86)%,Met+LB-100组(3.48±0.85)%,P<0.001;SW480:Met组(20.07±4.89)%,Met+LB-100组(8.61±1.68)%,P<0.001]。结论低浓度Met可通过PP2A/AKT通路诱导细胞衰老从而抑制结直肠癌细胞增殖。

Objective To determine whether metformin(Met) inhibits the proliferation of colorectal cancer cells by inducing cell senescence, and to preliminarily explore the underlying mechanism. Methods LOVO and SW480 colorectal cancer cells were treated with different concentrations of Met(0, 2.5, 5.0, 10.0, 20.0 and 40.0 mmol/L). The cell viability was detected by CCK-8 assay, and apoptosis and cell cycle were detected by flow cytometry. According to the results of apoptosis, differences between control group(0 mmol/L) and 5.0 mmol/L Met group(n=3) were compared in following experiments. Cell proliferation was detected by CCK-8 assay, EdU assay, and clone formation assay, senescence metabolism was detected by senescence-associated β-galactosidase(SA-β-gal) staining, protein phosphatase 2 A(PP2 A) enzyme activity was measured with ELASA, and protein expression was detected with Western blotting. PP2 A inhibitor LB-100 was used to treat the cells alone or combined with Met, and then the cells were divided into control group, Met group, Met+LB-100 group, and LB-100 group(n=3). Above experiments were performed again.Results Met treatment significantly inhibited LOVO and SW480 cells proliferation in a concentration-and time-dependent manner(P<0.05). Under the treatment of low concentration(≤5.0 mmol/L) of Met, the cells presented no obvious apoptosis, but were obviously inhibited for proliferation and arrested at G0/G1 phase. The cells displayed a typical senescence-like morphology of large, flat and vacuolated, and the number of SA-β-gal positive cells was increased significantly [LOVO: control group(5.43±1.37)% vs Met group(17.56±1.89)%, P<0.001;SW480: control group(8.49±2.12)% vs Met group(20.02±3.68) %, P<0.001]. For PP2 A, total protein expression showed no change, but phosphorylation level decreased obviously and PP2 A activity increased statistically(P<0.05), in the meantime, the phosphorylation level of downstream AKT protein decreased and senescence-related proteins p53 and P21 increased significantly. Treatment by PP2 A inhibitor LB-100 combined with Met significantly reversed the inhibition of Met on cell proliferation and delayed Met-induced cell senescence [LOVO: Met group(10.55±1.86)% vs Met+LB-100 group(3.48±0.85)%, P<0.001;SW480: Met group(20.07±4.89)% vs Met+LB-100 group(8.61±1.68)%, P<0.001]. Conclusion Low concentration of Met can induce cell senescence through PP2 A/AKT pathway and then inhibit the proliferation of colorectal cancer cells.