Effects Of 3,3'-Diindolylmethane On Adhesion, Invasion And Migration Of Human Ovarian Cancer Cells Via Blocking Stat3 Signal Pathway

PART I DIM SUPPRESSES THE ADHESION,INVASION AND MIGRATION OF HUMAN OVARIAN CANCER CELLS THROUGH INHIBITING STAT3 SIGNAL PATHWAYObjective To investigate the effects of 3,3'-diindolymethane (DIM) on the migration and invasion of human ovarian cancer SKOV3 and A2780 cells, to improve the role of signal transducer and activator of transcription 3 (STAT3) pathway in its possible mechanism.Methods The human ovarian cancer SKOV3 and A2780 cells were treated with different concentrations of DIM. The adhesion, migration and invasion of SKOV3, A2780 cells were determined by adhesion assay, wound healing assay, and transwell chamber invasion assay, respectinvely. The expression levels of STAT3, phospho-STAT3 (p-STAT3), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) proteins were detected by western blotting.Results The adhesion of DIM-treated SKOV3 and A2780 cells was inhibited in a dose-dependent manner (P< 0.05). Wound healing assay also showed that the rate of wound closure was significantly reduced in DIM-treated ovarian cancer cells (P< 0.05). The invasion assay showed the numbers of SKOV3 and A2780 cells treated with 50 and 100 ?mol/L DIM invading through the chamber were both lower than that of the control cells [(326±20.15)/fild and (59.33±13.32)/fild vs (537.33±53.15) of A2780, (437±23.15)/fild and (49.33±10.92)/fild vs (926.33±43.15) of SKOV3, P<0.01]. Western blotting analysis demonstrated that the expressions of total STAT3 proteins had no signifiant change, but the ex-pression levels of p-STAT3, VEGF and MMP-2, MMP-9 were down-regulated in SKOV3, A2780 cells after the treatment of DIM (all P< 0.05).Conclusion DIM can effectively inhibit the adhesion, invasion and migration of SKOV3 and A2780 cells. These effects might be related to the inhibition of STAT3 activity, and the down-regulation of VEGF and MMP-2, MMP-9 expressions. DIM might be a preventive and therapeutic agent against human ovarian cancer.PART ? EFFECTS OF STAT3 KNOCKDOWN AND/OR DIM TREATMENT ON THE ADHESION, INVASION AND MIGRATION OF HUMAN OVARIAN CANCER CELLSObjective To investigate the effects of the combination of STAT3 knockdown with 3,3'-diindolymethane (DIM) treatment on the adhesion, invasion and migration of human ovarian cancer cells, and clarify mechanism to provide experimental evidence for targeted cancer gene therapy.Methods 1. pGC-STAT3-shRNA (short hairpin RNA) plasmid was built in the experiment, then used the Roche FuGENE HD transfection reagent to transfect SKOV3 ovarian cancer cells by RNA interference (RNAinterference, RNAi) silencing STAT3 expression, inhibiting JAK/ STAT3 signaling pathway. Efficiency of STAT3 expression plasmid on shRNA silencing was detected by fluorescence quantitative PCR, western blotting and immunofluorescence method in SKOV3 ovarian cancer cells. 2. Silencing STAT3 by shRNA, while combined with 25,50,75 ?mol/L DIM, according to the experiment. We divided into control group, DIM group, negative plasmid group, negative plasmid+DIM group, STAT3-shRNA group, STAT3-shRNA+DIM group. Effects of DIM and STAT3 knockdown were detected by MTT assay, adhesion, migration and invasion assays. The protein level of STAT3, p-STAT3 and their dowmstream proteins (VEGF and MMP-2, MMP-9) were detected by western blot.Results RT-PCR and Western Blot results confirmed that STAT3-shRNA plasmids for gene silencing effects are obvious. In this study, we found that adhesion, migration and invasion of SKOV3 was potently suppressed by STAT3 gene silencing. The proliferation and motility were markedly suppressed by the combination of STAT3 gene silencing and DIM in SKOV3 cells. Western blot showed a drastic decrease in expression of p-STAT3 and down-regulated other oncogenic molecules including of VEGF, MMP-2 and MMP-9 in the combination group.Conclusion the combined effects of DIM and silence JAK/STAT3 signaling pathway increased biological activity against ovarian cancer, Which exhibit a more powerful and stable role in anti-cancer. The combination of STAT3 gene silencing and DIM may be a promising therapeutic strategy for the treatment of advanced ovarian cancer. Thus, this study provides a theoretical basis for ovarian cancer cell signal transduction pathways and optimized combination therapy targeted therapy.