| Background and objectivesHepatocellular carcinoma(HCC)has poor prognosis,which is due to the advanced stage by the time it is diagnosed,high recurrence rates and metastasis.Therefore,the potential agents for the treatment of HCC with fewer side effects are urgently needed in clinical.Accordingly,new therapeutic strategies focus on inhibiting growth of existing tumors and preventing cancer cells invasion,metastasis and angiogenesis.Metformin,a well-tolerated anti-diabetic drug,has been reported to reduce the risk of various cancers including HCC.Curcumin,a turmeric polyphenol extracted from rhizome of Curcuma longa Linn.,with pleiotropic pharmacological effects,good tolerance and low toxicity,has been found to have anti-tumor effects in numerous cancers,including HCC.A growing number of in vitro and in vivo studies have suggested that combination of dietary phytochemical may be far more effective for enhancing cytotoxic activity and inhibiting the growth of cancer cells compared to individual compounds.In the present study,we investigated the synergistic anti-tumor effects of metformin and curcumin on the proliferation,apoptosis,invasion,metastasis and angiogenesis of HepG2 cells in vitro and in vivo.Methods and resultsMethods:Anti-proliferative activity of metformin and curcumin on hepatocellular carcinoma HepG2 cells was evaluated by MTT assay,CCK-8 assay,colony formation assay and HCC xenografts mouse model.Hoechst 33342 staining and Annexin V-FITC/PI double-staining were employed to detect the apoptosis of HepG2 cells after treatment of metformin and curcumin.The mitochondrial transmembrane potential(??m)was measured to detect the mitochondrial integrity using JC-1 staining and the cell cycle arrest of HepG2 cells was measure by PI staining.The inhibitory effect of metformin and curcumin on migration and invasion of HepG2 cells was examined using wound scratch assay and Transwell assay.Gelatin zymography and Western blot were performed to evaluate the expression of MMP9 and MMP2.Wound scratch assay and capillary tube formation assay were carried out to examine the anti-angiogenic effect of metformin and curcumin,using HUVECs.The expression of all proteins was detected by Western blot method.Results:Metformin enhanced the anti-proliferative and anti-tumor effects of curcumin on HepG2 cells in vitro and in vivo without obvious side effects.Combination treatment of metformin and curcumin evoked mitochondrial dysfunction-related apoptosis through activation of p53,which resulted in the increase of the ratio of Bax/Bcl-2 and the level of cleaved PARP in HepG2 cells.Combined treatment of metformin and curcumin inhibited the migration and invasion of HepG2 cells via decreasing the level of MMP2 and MMP9.Metformin combined with curcumin suppressed the migration and capillary tube formation of HUVECs,resulting in inhibition of tumor angiogenesis.Co-treatment of metformin and curcumin inhibited PTEN/PI3K/Akt/mTOR signaling pathway,suppressed the transportation of NF-?B and prevented VEGF expression by blocking its transcription mediated by STAT3 in HepG2 cells to exert inhibitory effect on tumour cell proliferation,metastasis and angiogenesis,which confirmed in HepG2 xenografts.ConclusionIn conclusion,combination treatment of metfromin and curcumin synergistically inhibited the development and progression of liver cancer cells in vitro and in vivo.Co-treatment of metfromin and curcumin could not only induce tumor cells into apoptosis through the activation of mitochondria pathways,but also suppress the invasion,metastasis and angiogenesis abilities of HepG2 cells by downregulating the expression of MMP2/9 and the level of angiogenesis-relative factors,including VEGF,VEGFR2.What is more important,these effects of metformin and curcumin should act through up-regulating PTEN,P53 and suppressing PI3K/Akt/mTOR/NF-?B and EGFR/STAT3 signal transduction pathways.The combination treatment of metformin and curcumin would provide a promising treatment option in HCC. |
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