Empirical Study Of Methyl Jasmonate Anti-hepatocellular Carcinoma Effect And Its Mechanism

Objective:To observe the inhibitory effect of Methyl jasmonate on hepatocellular carcinoma cell in vitro, to detect the affection of AMPK and mTOR signaling pathway, and to explore its potential mechanisms.Method:Human hepatocellular carcinoma cell lines HepG2^PLC/PRF/5、Hep3B、HepG2.2.15、Mahlavuand SK-Hep1were cultured and treated with Methyl jasmonate at different concentration and at different time points. The effect of Methyl jasmonate on hepatocellular carcinoma cell lines proliferation was studied by means of SRB,the cell cycle and mitochondrial membrane potential were detected with flow eytometry; RT-PCR and western blot were used to detect the expressions of AMPK and mTOR signaling pathway related gene at mRNA and protein levels. Morphological change of HepG2after exposure to Methyl jasmonate by scanning electron microscope.Determination of TSC1-TSC2association by Immunoprecipitation assay.Results:The rank order of Methyl jasmonate potency against HCCs is HepG2> HepG2.2.15> Mahlavu> PLC/PRF/5> SK-Hep1> Hep3B. Methyl jasmonate completely abolished cell-cycle progression released from double-thymidine-block synchronization and caused a subsequent apoptosis. The data were supported by down-regulation and reduced nuclear translocation of G1-regulator proteins, including cyclin D1, cyclin E, Cdk4and Cdk2. Further analysis showed that the mRNA expressions of the G1-regulator proteins were not modified by Methyl jasmonate, indicating an inhibition of translational but not transcriptional levels.Methyl jasmonate induced the assembly of tuberous sclerosis complex (TSC)-1/TSC2, leading to the blockade of cellular protein synthesis through inhibition of protein phosphory lation including mTOR (Ser2448), p70S6K (Thr421/Ser424and Thr389) and4E-BP1(Thr37/Thr46and Thr70). Furthermore, the AMPK activity was elevated by Methyl jasmonate. Compound C, a selective AMPK inhibitor, significantly reversed Methyl jasmonate-mediated effects suggesting the crucial role of AMPK. Besides, the loss of mitochondrial membrane potential and depletion of mitochondrial content indicated the mitochondrial stress caused by Methyl jasmonate.Conclusion:Methyl jasmonate induces anticancer signaling cascades in a sequential manner. The exposure of cells to Methyl jasmonate induces mitochondrial stress and activation of AMPK that further induces the loss of mitochondrial membrane potentia and activates TSC1/TSC2association. Consequently, the mTOR mediated translational pathways are blocked, leading to G1arrest of the cell-cycle and subsequent cell death. Ⅱ Effects of Methyl jasmonate on invasive, angiogenic ability of hepatocellular carcinoma cellObjective:To study the effects of Methyl jasmonate on invasive, angiogenic ability of hepatocellular carcinoma cell lines HepG2.Method:Evaluation of cell migration by scratch wound healing assay. Analysis of the expression of phosphoERKl/2, phosphoJNK1/2, phosphop38, RhoB, NF-κB, IκB,Semi Quantitative RT-PCR and Real time RT-PCR analysis of MMPs/TIMPs, Rho and VEGF-A inhibition by MJ. The cells were transfected with pRL-null, MMP-2/MMP-9luciferase reporter plasmids and treated with and without MJ/PMA. Luciferase reporter assays were performed by Dual Luciferase Reporter Assay System. The relative luminescence units were normalized for the pRL-Null activity/protein content. Gelatinolytic activity was analyzed by using Innozyme Gelatinase MMP-2/9Activity Assay Kit. HUVECs were plated on the Matrigel and cultured in DMEM with and without MJ for24h. The enclosed network of complete tubes/incomplete structures were observed and photographed under light microscope. Results:A relatively non toxic concentration of MJ suppressed the PMA induced migration and invasion of tumor cells. On analysis for the molecules involved in the migration/invasion, we found MJ downregulated mRNA expression and promoter/gelatinolytic activity of Matrix Metalloproteinase (MMP)-2/9, as well as the RhoB expression at gene and protein level. It was also a strong inhibitor of Vascular Endothelial Growth Factor (VEGF) expression, promoter activity and endothelial cell migration/invasion and in vitro angiogenesis. MJ suppressed the nuclear translocation and DNA binding of NF-κB, which is an important transcription factor for controlling MMP-2/9and VEGF gene expression.Conclusion:MJ effectively inhibited key regulatory molecules involved in cell migration such as MMP-2/9and RhoB expression, at both the mRNA and protein level. It was a strong inhibitor of VEGF-A gene expression and promoter activity. All these effects of MJ could be due to inhibition of nuclear translocation and DNA binding of NF-κB. Inhibitory effect of MJ on crucial angiogenic events such as endothelial cell proliferation, migration/invasion and in vitro tube formation was also shown. Ⅲ The enhancing effect of Methyl jasmonate on the sensitivity of Hepatocellular carcinoma cell line HepG2to cisplatinObjective:To investigate the association of Methyl jasmonate use with the sensitivity of Hepatocellular carcinoma cell line HepG2to cisplatin.Methods:To establish a model of Hepatocellular carcinoma in nude mice. The expression of Ki-67、CD34、VEGF、3-NT、HO-1and NQO-1in tumor, liver and kidney tissues were detected by Immunohistochemical technique.Apoptosis were detected by TUNEL. The content of serum BUN, creatinine, AST and ALT in the model of Hepatocellular carcinoma were detected by ELISA. MDA and SOD were detected after Preparation of kidney and liver homogenates. The proliferation of HepG2after exposure to MJ and/or DDP were detected by MTT. the cell cycle and apoptosis were detected with flow eytometry. The expression of HO-1and NQO-1in HepG2were studied by West Blot.Results:Oral administration of Methyl jasmonate significantly inhibited tumor growth and promoted the anti-neoplastic efficacy of cisplatin in mice inoculated with HepG2liver cancer cells. In addition, Methyl jasmonate administration remarkably inhibited cisplatin-induced nephrotoxicity, hepatotoxicity and oxidative stress.In cell-based experiments, Methyl jasmonate inhibited cisplatin-induced cytotoxicity in LLC-RK1kidney and NCTC1469liver cells but not in HepG2liver cancer cells and significantly decreased cisplatin-induced intracellular ROS levels in these cells. In normal cells with cytoplasmically localized Nrf2and negligible levels of HO-1and NQO-1, Methyl jasmonate substantially decreased cisplatin-induced elevation in HO-1/NQO-1levels and inhibited cisplatin-induced translocation of Nrf2into the nucleus. In chemoresistant cancer cells with high levels of HO-1/NQO-1and nuclear Nrf2, both basal and cisplatin-induced levels of HO-1/NQO-1and nuclear Nrf2were decreased by Methyl jasmonate treatment, thereby enhancing the susceptibility of cancer cells to cisplatin.Conclusion:Methyl jasmonate exhibits antitumor activity, increases the therapeutic efficacy of cisplatin and protects the kidney and liver against cisplatin-induced tissue damage. These beneficial effects of Methyl jasmonate may be attributable to its ability to scavenge ROS and reduce Nrf2-mediated HO-1/NQO-1expression and appear to be linked to the differential basal levels of HO-1/NQO-1and Nrf2localization in normal and cancer cells.