Inhibitory Effects Of 3,3’-diindolylmethane On Migration And Invasion In Human Hepatocellular Carcinoma Cells:the Role Of Calcium Sensing Receptor

Objective: The difference of CaSR expressions in various tissues and organs is closely to tumors,which makes it plays a role in promoting or inhibiting the development of various types of tumors.However,its role in hepatocellular carcinoma(HCC)remains unclear.3,3-diindolylmethane(DIM)is a vital active product derived from cruciferous vegetables.DIM has prominent anti-hepatoma effects,but its specific mechanism remains unclear.In this study,we revealed the expression of CaSR and its influence on cell behavior of HCC cell lines.Meanwhile,we clarified the role and molecular mechanism of CaSR in the effects of proliferation,migration and invasion inhibited by DIM on human HepG2 and SMMC-7721 HCC cell lines,so as to provide theoretical basis for application of CaSR in molecular diagnosis and DIM in the clinical treatment of HCC.Methods 1.The expression of CaSR protein in two HCC cells HepG2 and SMMC-7721 were detected by Western blot and immunofluorescence.NPS-2143,a CaSR specific inhibitor and CaSR si RNA were used to observe the effects of inhibition of CaSR on the proliferation,migration and invasion ability on two HCC cell lines from the pharmacological and genetic levels.Meanwhile,ERK and p-ERK protein expression levels were detected to explore the role of ERK signaling pathway on the abovementioned effects.2.After treatment with DIM on HepG2 and SMMC-7721 cells,the cell proliferation level was detected by MTT,so as to choose an appropriate treatment time and concentration.Transwell assay was applied to detect cell migration and invasion ability after treatment with DIM.The expression of proliferating cell nuclear antigen(PCNA)and proteins closely related to migration and invasion,such as matrix metalloproteinases(MMP-2,MMP-9)was assayed by western blot.3.Western blot and q RT-PCR were applied to observe the changes of CaSR and CaSR m RNA after treatment with DIM in HepG2 and SMMC-7721 cells,also the ERK and p-ERK protein expressions were assayed.4.Gd Cl3,a CaSR specific agonist and ERK specific inhibitor U0126 were used in combination with DIM to explore the influences of activation of CaSR or inhibition of ERK phosphorylation on cell proliferation,migration and invasion ability,and thus to investigate the relationship between CaSR and ERK on inhibitory effects induced by DIM in HCC cells.Results 1.Western blot and immunofluorescence indicated the CaSR protein expression level in HepG2 and SMMC-7721 cells and existed in the cell membrane and cell nucleus.After treatment with CaSR inhibitor NPS-2143 and CaSR si RNA,the proliferation,migration and invasion ability of HCC cells were significantly suppressed and the p-ERK level was decreased while the total ERK protein level remained unchanged.2.DIM significantly suppressed the proliferation in a time-dosedependent manner,and also DIM significantly inhibited the migration and invasion ability in a concentration dependent manner in HepG2 and SMMC-7721 cells.Western blot showed that the PCNA,MMP-2 and MMP-9 protein expressions decreased gradually.3.Western blot and q RT-PCT showed that DIM decreased the expression of CaSR protein and m RNA in a dose-dependent manner.Meanwhile,DIM remarkedly decreased p-ERK protein expression while the total ERK protein level remained unchanged.4.After activation of CaSR by Gd Cl3,the decreased proliferation,migration and invasion ability of HCC cells induced by DIM was restored,and also the reduced p-ERK level was alleviated.The combination of ERK inhibitor U0126 with DIM further decreased the cell proliferation,migration and invasion ability of HCC cells.Conclusion Downregulation of CaSR expressions significantly inhibited cell proliferation,migration and invasion by regulating ERK signaling pathway in HepG2 and SMMC-7721 cell lines.DIM inhibited proliferation,migration and invasion ability of HepG2 and SMMC-7721 cells in a concentration-dependent manner.The inhibitory effects of DIM were mainly carried out through the CaSR/ERK signaling pathway,suggesting that CaSR was a potential target of DIM’s anti-HCC effects,which was mainly achieved by inhibiting ERK signaling pathway.