Research On The Mechanism Involved In Curcumin-suppressed Cell Proliferation In Human Hepatocarcinoma HepG-2 Cells

Hepatocellular carcinoma(HCC), a malignant tumor arisen from liver, is one of the most prevalent cancers and the main leading cause of cancer mortality worldwide,as well as in China. Recently, people’s healthy daily life has been threatened by its high incidence and mortality. Curcumin is a typical natural ingredient derived from Curcuma longa. Amounts of studies suggest that curcumin has the pharmacological activity in inhibiting tumor cell proliferation and inducing apoptosis. Unfortunately,the precise mechanisms are still poorly understood.To study the effect and mechanism of curcumin on hepatocarcinoma cell proliferation, the cultured HepG-2 cells were treated with different concentrations of curcumin(0-50 μmol/L) for 24 or 48 h as well as 20 μmol/L curcumin for different time periods(0, 12, 24 and 48 h) separately. After that, the effects of curcumin on cell proliferation and apoptosis were analyzed. Semi-quantitative RT-PCR was used to screen the curcumin-upregulating genes in HepG-2 cells and then the expression of selected gene Egr-1 was further verified by qPCR and Western Blot. Besides, the Egr-1 shRNA was designed to construct the mammal lentiviral shRNA expression vector pGreenPuro-Egr-1 and the over-expression vector pEGFP-Egr-1 was also constructed, then the distribution of Egr-1 protein in HepG-2 cells was analyzed by immunofluorescence after exposed to curcumin. Hereafter, the end ogenous Egr-1stably interfered cell line HepG-2/pGreenPuro-Egr-1 and the negative control cell line HepG-2/pGreenPuro-NC were established. After that, the effects of curcumin on cell proliferation and apoptosis of the established cell lines were also analyzed. Besides,the expression level of p53 and p21 were investigated when enforced expression of Egr-1 in HepG-2 cells. It was found HepG-2 cell proliferation was decreased in a dose-dependent manner when treated with different concentrations of curcumin for 24 or 48 h, when treated with 20 μmol/L curcumin, the proliferation was decreased in time-dependent manner. And the results of Hoechst 33258 staining indicated that curcumin induced HepG-2 cell apoptosis also in dose- and time-dependent manner.The result of semi-quantitative RT-PCR suggested curcumin changed some genes’ mRNA expression level in HepG-2 cells and the results of semi-quantitative RT-PCR,qPCR and Western Blot showed that curcumin induced Egr-1 gene mRNA and protein expression level in HepG-2 cells. The immunofluorescence suggested curcumin induced transient accumulation of Egr-1 protein in the nuclei, which indicated that the location of Egr-1 to the nuclei. The stable cell line that could express the Egr-1shRNA was successfully constructed that verified by the method of qPCR and Western Blot. Interestingly, it was discovered that in HepG-2/pGreenPuro-Egr-1 cells,the suppression effect of curcumin in cell proliferation was attenuated when compared to HepG-2/WT and HepG-2/pGreenPuro-NC cells. But no obvious difference in cell apoptosis was found between the normal cells and the Egr-1 interfered cells. Finally,the results of qPCR and Western Blot showed when enforced expression of Egr-1gene in HepG-2 cells, the expression level of p53 was unchanged but p21 was obviously upregulated both in mRNA and protein level. Collectively, these data suggested curcumin could inhibit HepG-2 cell proliferation and induce cell apoptosis.The upregulation of Egr-1 gene in mRNA and protein level may be involved in the anti-proliferation effect of curcumin in HepG-2 cells and Egr-1 inhibits HepG-2 cell proliferation may through the upregulation of p21, which was independent of p53.Thus, on the one hand, the results of this study indicated the possible molecular mechanism of curcumin in suppressing HepG-2 cell proliferation, which may provided a new alternative target for the design of curcumin analog; on the other hand,those data indicated Egr-1 might be involved a p53-independent regulation of p21 that also providing a new evidence of potential tumor suppressing gene Egr-1.