Basic Helix-loop-helix Transcription Factors DEC1Regulates The8-MOP-induced Apoptosis Of HepG2Cells

The hepatocarcinoma occupies the second rate of cancer in China. At present,there were no special drugs to treat late, midtrimester and recurrent hepatocarcinoma.The Chinese medicine was received great attention for improving clinical symptomsand life quality, prolonging survival times in clinical anti-hepatocarcinoma therapy.8-MOP, a furocoumarine, is an active component of the fruit of Fagavazanthoxyloides Lam. or the herb of Ruta graveolens L., and has been widely used inthe treatment of angina pectoris, vitiligo, psoriasis and cutaneous T-cell lymphoma. Inpresent，it ws found that8-MOP in combination with ultraviolect light were potentmodulators of epidermal cell growth and differentiation.Recently, it wasdemonstrated that its isomeride Bergapten could be a promising anti-cancer agentbased on its cytotoxicity. Nevertheless, the effects of8-MOP on cell proliferation andcytotoxicity in HCC cells have not been previously reported.Differentiated embryonic chondrocyte gene (DEC)1(BHLHE40/Stra13/Sharp2)and DEC2(BHLHE41/Sharp1), basic helix-loop-helix (bHLH) transcription factors,has been reported to be involved in the regulation of apoptosis, cell proliferation, andcircadian rhythms, progression to malignancy, and the response to hypoxia. DEC1ishighly expressed in tumor tissues of breast and colon cancers compared to theirnon-tumor regions. Overexpression and knockdown of DEC1and DEC2, respectively,affected the expression of apoptosis related-factors such as Bcl-2, Fas, Bax, c-Myc,survivin, and vascular endothelial growth factor (VEGF) as well as the amounts ofcleaved PARP and cleaved caspases.However, the roles of DEC1in apoptosisinduced by anti-tumor drugs are still unknown.In this report we investigated the effects and mechanisms of8-MOP,independent on the exposure to UV, on apoptosis in HepG2cells. And then, we foundthe expression of DEC1decreaed significantly after8-MOP treatment in HepG2Cells.Therefore, we examined the effects of DEC1on8-MOP-induced apoptosis in HepG2 cells.Objective: To study the growth inhibitory effect of8-MOP on hepatocellularcarcinoma cells and to determine the involved molecular mechanism．Methods:1.Different concentrations of8-MOP（1~64μM）was used to treat thehepatocarcinoma cells HepG2cell. The cell viabilitiy was assessed by MTT assayand cell morphology was monitored using an inverted light microscope.2. Aftertreated with different concentration of8-MOP（0，3，10，30μM） for48h, apoptosiswas detected by fluorescence microscopy and flow cytometry.3.Western Blottingwas performed to detect the apoptosis related protein expression of p53, survivin,caspase-3，caspase-9, Bax, Bcl-2.4. Treatment with8-MOP（3,10and30μM）for48h, the expression of DEC1were detected by realtime PCR and Western Blotting.5.Apoptosis was detected by fluorescence microscopy and and the lever of apoptosisrelated protein (survivin, caspase-3） was detected by Western Blotting afteroverexpressing DEC1by Transient co-transfection in8-MOP（30μM）treated HepG2cells.Results:1.8-MOP inhibits the viability of HepG2cells.(1) By MTT assay wedemonstrated that the inhibitory effects of8-MO (1~64μM) on HepG2cells wereboth time-and concentration-dependent.(2) Treatment with Different concentrationsof8-MOP（1~64μM）for48h, Cell morphol showsogical assessment showeduntreated HepG2cells grew well with clear skeletons, while cells treated with8-MOPwere distorted and some became round.2.8-MOP induces apoptosis in HepG2cells.Treatment with different concentrations of8-MOP （0，3，10，30μM）for48h:(1)Cells presented morphological features of early apoptosis, such as bright, nuclearcondensation and apoptotic bodies.(2)8-MOP-induced apoptosis was further assayedby AnnexinV/PI staining.The apoptpsis rate were5.1％，5.4％，9.8％and17％，respectively.3. Effects of8-MOP on the expression of apoptotic-related proteins.Following treatment of HepG2cells with8-MOP (0，3，10，30μM), we observedactivation of caspase-3, caspase-8and caspase-9. The expression ratio of Bax/Bcl-2was increased in the treated cells, where Bax was up-regulated and Bcl-2was down-regulated. In addition, we found that the expression of p53, a modulator ofp21WAF1/Cip1and Bax, was up-regulated in8-MOP-treated cells. We also foundthe amount of survivin，a member of the inhibitor of apoptosis (IAP) gene family，was decreased after treatment with8-MOP.4.8-MOP down-regulates the expressionof DEC1.Treatment with8-MOP（0，3,10，30μM） for48h，both the mRNA leveland protein expression of DEC1were significantly decreased in a concentrationdependent manner.5. Overexpression of DEC1in the presence of8-MOP treatmenthas opposite effects on the apoptosis of HepG2cells. Treatment with8-MOP（0，3,10，30μM）for48h:(1)Overexpression of DEC1inhibited the nuclear condensationin these cells.(2)The amounts of pro-caspase-3and survivin were decreased aftertreatment with8-MOP, and the decreased levels were increased by overexpression ofDEC1.Conclusion: we have demonstrated that8-MOP, independently on the exposure toUV, induces apoptosis in HepG2cells and DEC1has anti-apoptotic effects on the8-MOP-induced apoptosis in HepG2cells. It could be concluded that8-MOP appearsto be a promising chemopreventive agent for treating hepatocellular carcinoma, andDEC1may be an potential target for liver cancer therapy.