Anti-Cancer Effects Of 3,3'-Diindolylmethane On Human Hepatocellular Carcinoma Cells:The Role Of Cytosolic Ca²⁺ And P38 MAPK

Objective:3,3'-diindolylmethane?DIM?,derived from indole-3-carbinol?I3C?in the brassica species of cruciferous vegetables has anti-hepatoma carcinogenic effects,but its exact underlying mechanism of action remains unclear.Therefore,in this study,we explored the roles of cytosolic free calcium([Ca²⁺]_i)and p38 MAPK in the anti-cancer effects of DIM on human SMMC-7721 and HepG2 hepatocellular carcinoma cells?HCC?,and provided therapeutic targets and in accordance with theories for the development of novel antitumor drugs.Methods:1.Cell proliferation was assessed by CCK-8 and the clonogenic formation assay.Cell apoptosis was examined by Hoechst staining and flow cytometric analysis.PCNA,cleaved-caspase3,cleaved-PARP,Bax,total and phosphorylated p38 MAPK were assayed by western blot.2.Cytolic[Ca²⁺]_i was measured with Fluo-3/AM by fluorescence microscopy.We also detected CaMK,calpain,CaSR protein expression and CaMK mRNA levels.3.Pretreatment HCC with the selective calcium ionphore?A23187?,endoplasmic reticulum calcium pump inhibitor?TG,BHQ?increased[Ca²⁺]_i levels,or using intracellular calcium chelator?BAPTA/AM?,the PLC-IP₃R pathway inhibitor?2-APB,U73122?reduced[Ca²⁺]_i levels,explored the role of[Ca²⁺]_i in DIM-induced proliferation inhibition and apoptosis promotion on hepatoma cells.We pretreated cells with EGTA to observe the role of calcium influx in DIM-induced effects.4.Pretreatment SMMC-7721 and HepG2 with calcium channel blocker?mibefradil?for 0.5 h,we observed the effect of mibefradil on the effects of DIM on hepatoma cells.Results:1.DIM reduced cell viability significantly when comparing with the control group?P<0.05?.The colongenic formation and PCNA were inhibited by DIM significantly?P<0.05?.The Hoechst staining showed the level of apoptosis increased in a concentration-dependent manner.DIM increased the level of cleaved-caspase3,cleaved-PARP,Bax proteins in HCC?P<0.05?.2.DIM activates phosphorylation of p38 MAPK while total p38 MAPK levels remain unchanged.CCK-8 and colongenic formation assay showed that pretreatment with p-p38inhibitor significantly attenuated DIM-induced cell growth inhibition?P<0.05?,and restored PCNA level when comparing with DIM-alone group?P<0.05?.DIM-induced apoptosis was also attenuated among them?P<0.05?.3.The level of[Ca²⁺]_i was increased by fluorescence observation,the expression of CaMK and calpain was significantly increased?P<0.05?,and CaSR was decreased?P<0.05?,but with no change in mRNA level of CaMK.Pretreatment with A23187,TG increased[Ca²⁺]_i and DIM-induced cell proliferation inhibition and apoptosis promotion?P<0.05?.Pretreatment with BAPTA/AM,2-APB reduced[Ca²⁺]_i level,a decrease in p38 MAPK phosphoralytion and cleaved-caspase3 protein level-mediated apoptosis were observed?P<0.05?,compared with the DIM-treated group.DIM-induced apoptosis effect was inhibited by BAPTA/AM,resulting in a decrease in p-p38 MAPK activation and apoptosis in DIM-treated cells,although the proliferation inhibition of DIM is unchanged.Pretreatment with EGTA reduced[Ca²⁺]_i levels indirectly,DIM-induced cell proliferation inhibition and apoptosis promotion were attenuated compared with DIM-treated group?P<0.05?.Both calcium influx and calcium release from the calcium pump are associated with an increase in[Ca²⁺]_i levels induced by DIM.4.Compared with 60?M DIM group,DIM induced-anti cancer effects were significantly increased by pretreatment with mibefradil?P<0.05?.Conclusion:DIM inhibited cell proliferation and induced cell apoptosis in SMMC-7721and HepG2 cells in a concentration-and time-dependent manner.Up-regulation of[Ca²⁺]_i and activation of p38 MAPK were involved in these outcomes.Calcium ionophore enhanced DIM-inducedanti-cancereffectsinhepatocellularcarcinomacells,secondaryto[Ca²⁺]_i-dependent activation of p38 MAPK.Treatment with a combination of DIM and calcium ionophore may offer a new approach to enhance the chemotherapeutic efficacy in liver cancer,regulating Ca²⁺level will become a new promising target for liver cancer treatment.