Molecular Mechanism Of 3,3'-Diindomethane Inducing Apoptosis Of Gastric Cancer Cells Through TRAF2/p38MAPK Signaling Pathway

Objective3,3'-diindolylmethane(DIM)is a kind of bioactive phytochemical derived from cruciferous vegetables.DIM can induce apoptosis of a variety of tumor cells in vivo and in vitro,but its specific molecular mechanism for inducing apoptosis of gastric cancer cells remains unclear.In this study,our main purpose is to clarify that DIM can inhibit gastric cancer cells by promoting apoptosis,elucidate the molecular mechanism of DIM inducing apoptosis of gastric cancer cells.To provide new theoretical basis and therapeutic target for DIM to become a clinical drug for gastric cancer treatment,it also provides a new target for molecular diagnostic strategy of gastric cancer.Methods1.After treatment of BGC-823,SGC-7901 and GES-1 cells with DIM,the cell viability and proliferative activity were detected by MTT,proliferation and cell cycle related proteins,such as anti proliferation protein p21,cyclin dependent kinases 4(CDK4),cyclin D1 were detected by Western blot.2.TUNEL kit was used to measure the level of apoptosis in BGC-823 and SGC-7901 cells,apoptosis related proteins cleaved caspase 3,cleaved poly ADP ribose polymerase(cleaved PARP),Bcl-2 and Bcl-2 associated X protein(Bax)were detected by Western blot.3.The effects of DIM on p38 mitogen-activated protein kinase(p38 MAPK)were observed,after pretreatment of p38 MAPK specific inhibitor SB203580 combined with DIM,to observe the changes of inhibition of p38 MAPK signaling pathway on the above-mentioned proliferation and apoptosis indicators.4.Immunohistochemical analysis of gastric tissues and para-cancer tissues of 15 patients with gastric cancer,and the expression differences of TNF receptor associated factor 2(TRAF2)in gastric cancer tissues and normal tissues were compared.The expression level of TRAF2 in BGC-823,SGC-7901 and GES-1 cells was detected by Western blot.The level of TRAF2 in BGC-823 and SGC-7901 cells was detected by used small interference RNA fragment of TRAF2(TRAF2 siRNA),to observe the effect of TRAF2 reduction on proliferation and apoptosis of gastric cancer cells.5.The changes of TRAF2 under the influence of DIM were observed,after TRAF2 overexpressed plasmid was transfected cells combined with DIM,and the effect of TRAF2 overexpression plasmid on proliferation and apoptosis was observed.Results1.DIM significantly reduced the cell activity of BGC-823 and SGC-7901 cells and showed a significant concentration and time effect relationship,while GES-1 cells were less sensitive to DIM by MTT assay.Western blot analysis showed that the p21 increased significantly,the protein level of CDK4 and cyclin D1 decreased significantly.2.The results of TUNEL assay showed that the apoptotic level of BGC-823 and SGC-7901 cells increased significantly.Western blot detected DIM significantly increased the expression of cleaved caspase 3,cleaved PARP,and Bax in a concentration dependent manner,the ratio of Bax/Bcl-2,the related index of apoptosis,was significantly increased.3.Western blot showed that DIM could activate p38 MAPK signal pathway,while the inhibitor SB203580 was combined with DIM,DIM-induced proliferation inhibition and apoptosis induction of gastric cancer cells were alleviated.4.The expression of TRAF2 in gastric cancer tissues was higher than that in the adjacent tissues,Western blot showed that the protein expression level of TRAF2 in BGC-823 and SGC-7901 cells was significantly higher than normal human gastric mucosa epithelial cells GES-1.After co-treated with TRAF2 siRNA and DIM,it was found that the proliferation level of gastric cancer cells was inhibited,which promoted apoptosis and activated the p38 MAPK signaling pathway.5.Western blot showed that the expression of TRAF2 in gastric cancer cells was significantly reduced by DIM in a concentration dependent manner.The overexpression plasmid of TRAF2 was transfected into gastric cancer cells and then treated with DIM,the activation of p38 MAPK was inhibited,and DIM-induced apoptosis of gastric cancer cells were also alleviated.ConclusionDIM could inhibit cell proliferation and promote cell apoptosis in BGC-823 and SGC-7901 human gastric cancer cells in a concentration dependent manner.Moreover,DIM mainly regulated the TRAF2/p38 MAPK axis to achieve the inhibitory effect on gastric cancer cells.TRAF2 was highly expressed in human gastric cancer tissues and cells,down-regulation ofTRAF2 expression was associated with inhibition of proliferation and induction of apoptosis in gastric cancer cells.In conclusion,TRAF2 was a key regulatory target for DIM to inhibit gastric cancer.The DIM anti-gastric cancer was mainly promotes gastric cancer cell apoptosis through TRAF2/p38 MAPK axis.