The mechanism of metformin inhibiting liver cancer cell proliferation through the REDD1-mTOR signaling pathway

Abstract

Abstract: Objective To investigate the mechanism by which metformin (MET) inhibits the proliferation of liver cancer cells by regulating the regulated in development and DNA damage response 1 (REDD1) - mammalian target of rapamycin (mTOR) signaling pathway. Methods MTT assay was used to detect the effects of different concentrations of MET (0.0, 2.5, 5.0, 10.0 mmol/L) on the proliferation of HepG2 cells. 10.0 mmol/L MET was selected for subsequent experiments and the cells were separated into Control group, MET group, sh-NC group, and sh-REDD1 group. QRT-PCR method was applied to detect the expression of REDD1 mTOR p70s6k and Cyclin D1 mRNA in HepG2 cells in each group. The plate cloning experiment was applied to detect the proliferation of HepG2 cells in each group. Flow cytometry was applied to detect the cell cycle of HepG2 in each group. WB was applied to detect the expression of REDD1, mTOR, p70s6k, and Cyclin D1 proteins in HepG2 cells of each group. Results The proportion of G1 phase cells (81.25%±10.35%), apoptosis rate(42.55%±4.52%), REDD1 mRNA (7.52±0.80) and protein expression (0.86±0.13) in the MET group were higher than those in the Control group 36.58%±8.49%,1.87%±0.64%,1.00±0.33,0.27±0.08, the proportions of S phase(11.19%±2.58%) and G2 phase cells(7.56%±2.12%), number of clones(53.41±9.28 number), mTOR mRNA (0.36±0.08) and protein (0.52±0.15), p70s6k mRNA (0.47±0.14) and protein (0.43±0.09) , and Cyclin D1 mRNA (0.53±0.13) and protein expression (0.48±0.11) were lower than those in the control group (30.87%±4.28%, 32.55%±4.36%, 118.43±12.92 number, 0.99±0.12, 1.17±0.17, 1.00±0.21, 0.94±0.14, 1.01±0.17, 1.05±0.17) (P<0.05). Compared with the MET group and sh-NC group, proportion of G1 phase cells, apoptosis rate, REDD1 mRNA and protein expression in the sh-REDD1 group were lower, the proportions of S phase and G2 phase cells, number of clones, mTOR mRNA and protein, p70s6k mRNA and protein, and Cyclin D1 mRNA and protein expression were higher (P<0.05). Conclusion MET can inhibit the proliferation activity of HepG2 cells, and its possible molecular mechanism is to exert an inhibitory effect on liver cancer cell proliferation through the REDD1-mTOR signaling pathway.

Key words: Metformin, Liver cancer, Regulated in development and DNA damage response 1, Mammalian target of rapamycin, Cell proliferation

WANG Yan, LI Qin, LI Jun-jun, ZHANG Xiao-li, Tan Li-ling. The mechanism of metformin inhibiting liver cancer cell proliferation through the REDD1-mTOR signaling pathway[J]. Chinese Hepatolgy, 2025, 30(12): 1668-1673.

References

[1] Chen H, Xu J, Wang P, et al. Protein phosphatase 2 regulatory subunit B′′Alpha silencing inhibits tumor cell proliferation in liver cancer[J]. Cancer Med, 2019, 8(18):7741-7753.
[2] Zhang K, Zhang M, Luo Z, et al. The dichotomous role of TGF-β in controlling liver cancer cell survival and proliferation[J]. J Genet Genomics, 2020 , 47(9):497-512.
[3] Liu Z, Wang Y, Aizimuaji Z, et al. Elevated FOXA1 expression indicates poor prognosis in liver cancer due to its effects on cell proliferation and metastasis[J]. Dis Markers, 2022, 1:3317315-3317330.
[4] Park D, Lee S, Boo H. Metformin induces lipogenesis and apoptosis in H4IIE hepatocellular carcinoma cells[J]. Dev Reprod, 2023, 27(2):77-89.
[5] Hu L, Zeng Z, Xia Q, et al. Metformin attenuates hepatoma cell proliferation by decreasing glycolytic flux through the HIF-1α/PFKFB3/PFK1 pathway[J]. Life Sci, 2019, 239:116966-16974.
[6] Ferrín G, Guerrero M, Amado V, et al. Activation of mTOR signaling pathway in hepatocellular carcinoma[J]. Int J Mol Sci, 2020, 21(4):1266-1281.
[7] Zhidkova E M, Lylova E S, Grigoreva D D, et al. Nutritional sensor REDD1 in cancer and inflammation: friend or foe[J]. Int J Mol Sci, 2022, 23(17):9686-9698.
[8] Kim T S, Lee M, Park M, et al. Metformin and dichloroacetate suppress proliferation of liver cancer cells by inhibiting mTOR complex 1[J]. Int J Mol Sci, 2021, 22(18):10027-10041.
[9] Cai L, Jin X, Zhang J, et al. Metformin suppresses Nrf2-mediated chemoresistance in hepatocellular carcinoma cells by increasing glycolysis[J]. Aging (Albany NY), 2020, 12(17):17582-17600.
[10] Zhuo H, Miao S, Jin Z, et al. Metformin suppresses hepatocellular carcinoma through regulating alternative splicing of LGR4[J]. J Oncol, 2022, 1:1774095-1774103.
[11] Shen Z, Zhou H, Li A, et al. Metformin inhibits hepatocellular carcinoma development by inducing apoptosis and pyroptosis through regulating FOXO3[J]. Aging (Albany NY), 2021, 13(18):22120-22133.
[12] Hu Z, Zhao Y, Li L, et al. Metformin promotes ferroptosis and sensitivity to sorafenib in hepatocellular carcinoma cells via ATF4/STAT3[J]. Mol Biol Rep, 2023, 50(8):6399-6413.
[13] Maehara O, Ohnishi S, Asano A, et al. Metformin regulates the expression of CD133 through the AMPK-CEBPβ pathway in hepatocellular carcinoma cell lines[J]. Neoplasia, 2019, 21(6):545-556.
[14] Yu X N, Chen H, Liu T T, et al. Targeting the mTOR regulatory network in hepatocellular carcinoma: are we making headway[J]. Biochim Biophys Acta Rev Cancer, 2019, 1871(2):379-391.
[15] Zhang H, Su X, Burley S K, et al. mTOR regulates aerobic glycolysis through NEAT1 and nuclear paraspeckle-mediated mechanism in hepatocellular carcinoma[J]. Theranostics, 2022, 12(7):3518-3533.
[16] Chen M, Fang Y, Liang M, et al. The activation of mTOR signalling modulates DNA methylation by enhancing DNMT1 translation in hepatocellular carcinoma[J]. J Transl Med, 2023, 21(1):276-292.
[17] Feehan R P, Coleman C S, Ebanks S, et al. REDD1 interacts with AIF and regulates mitochondrial reactive oxygen species generation in the keratinocyte response to UVB[J]. Biochem Biophys Res Commun, 2022, 616:56-62.
[18] Chen Z, Lai X, Ding H, et al. ATF4/TXNIP/REDD1/mTOR signaling mediates the antitumor activities of liver X receptor in pancreatic cancers[J]. Cancer Innov, 2022, 1(1):55-69.
[19] Guo M, Li N, Zheng J, et al. Epigenetic regulation of hepatocellular carcinoma progression through the mTOR signaling pathway[J]. Can J Gastroenterol Hepatol, 2021, 2021(1):5596712-5596720.
[20] Yang L, Tian X, Chen X, et al. Upregulation of Rab31 is associated with poor prognosis and promotes colorectal carcinoma proliferation via the mTOR/p70S6K/Cyclin D1 signalling pathway[J]. Life Sci, 2020, 257(1):118126.