The Role Of Autophagy In The Curcumin-induced Proliferation Inhibition And Apoptosis In SMMC-7721 Hepatocellular Carcinoma Cells

Study Objective Curcumin, the principal polyphenolic curcuminoid, obtained from the turmeric rhizome Curcuma longa L and other Curcuma spp. Previous studies have shown that curcumina can exert a wide range of pharmacological activities, such as neuroprotective action, anti-mutagenic, anti-atherosclerosis and anti-microbial properties. In addition, it has been traditionally used to cure several chronic ailments, such as allergies, neurodegenerative, osteoporosis, diabetes, and neoplastic. Besides, curcumin could play a role as antitumor drug which has become a research focus recently. It has been proven that curcumin can inhibit the proliferation of various types of human cancer cells, such as leukemia, pancreatic and cervical cancers. In view of the present situation that high mortality of most patients with hepatocellular carcinoma of advanced stage and barely effective using anticancer drugs alone. Many scholars, domestic and overseas, have carried out an increasing number of researches involving in the use of curcumin in primary liver cancer. However the relevant underlying mechanism is yet to be completely clear.We used SMMC-7721 cell as the object of this study to research:(1) Effects of curcumin on cell proliferation and apoptosis in human hepatocellular carcinoma cells.(2) To explore the potential mechanism of activation of curcumin in human hepatocellular carcinoma cells with focus on regulation of autophagy.(3) Potential mechanism of autophagy in the curcumin-induced proliferation inhibition and apoptosis in SMMC-7721 hepatocellular carcinoma cells. Our study will be beneficial to exploring the pharmacological characteristic of curcumin in hepatocellular carcinoma and better guiding the further clinicial research.Main methods1. The cells were treated by serially diluted curcumin at different concentrations(2.5, 5, 10, 20, 40mM) for 12, 24 and 48 h, and the effects of curcumin on cell proliferation were evaluated by MTT assay. Apoptosis of SMMC-7721 cells was assessed by Annexin V-FITC and PI apoptosis detection kit.2. To investigate whether curcumin activated autophagy in SMMC-7721 cells, expression levels of autophagosome-associated proteins(LC3-II, Beclin1 and p62) were evaluated by western blot.3. To clarify the activation of autophagy in SMMC-7721 cells after treatment with curcumin, we used immunofluorescence staining with antibodies against LC3(Atg8) protein, which is a key marker of autophagy.4. The cells were pretreated with 3-MA/CQ prior to curcumin treatment for 24 h. The effects of curcumin on cell proliferation were evaluated by MTT assay and apoptosis of SMMC-7721 cells was assessed by Annexin V-FITC and PI apoptosis detection kit.Main results1. Curcumin can exert not only proliferation inhibition in SMMC-7721 cells but also induction of apoptosis in a time- and dose-dependent manner.2. Curcumin increased the expression level of some autophagy-related proteins, such as LC3-II, Beclin-1 and p62. In addition, the expression of p-Akt and p-m TOR were siginificantly downregulated in the curcumin groups compared with the control group.3. Pretreated with CQ induced higher expression level of LC3-II caused by curcumin alone.4. Immunofluorescence data shows that accumulation of LC3 in SMMC-7721 cells after treatment with curcumin for 24 h.5. Pretreatment of 3-MA/CQ enhanced the effect of curcumin-induced cell proliferation inhibition and apoptosis in SMMC-7721 cells.ConclusionCurcumin can inhibit cell proliferation and induce cell apoptosis in SMMC-7721 cells. In addition, curcumin may induce autophagy by downregulating Akt/m TOR signaling pathway in SMMC-7721 hepatocellular carcinoma cells. Pharmacological inhibition of autophagy enhanced cell proliferation inhibition and apoptosis of curcumin in SMMC-7721 cells. These findings may provide theoretical basis for clinical application of curcumin.