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

Abstract

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 2A(PP2A) enzyme activity was measured with ELASA, and protein expression was detected with Western blotting. PP2A 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. For PP2A, total protein expression showed no change, but phosphorylation level decreased obviously and PP2A 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 PP2A inhibitor LB-100 combined with Met significantly reversed the inhibition of Met on cell proliferation and delayed Met-induced cell senescence. Conclusion Low concentration of Met can induce cell senescence through PP2A/AKT pathway and then inhibit the proliferation of colorectal cancer cells.

Keywords

• metformin
• colorectal cancer
• cell senescence
• cell proliferation
• pp2a