Transforming growth factor beta-1 promotes migration and invasion of ovarian epithelial carcinoma cells through TRAF6-mediated activation of Notch3 signaling pathway / 中国癌症杂志

ABSTRACT

Background and purpose: Abnormal expression and amplification of transforming growth factor beta 1 (TGF-β1) and Notch3 in ovarian carcinoma tissues are associated with metastasis and low survival rate, respectively. The crosstalk between TGF-β1 and Notch3 signaling pathway promotes invasion and metastasis in various cancers. However, the mechanism is still under debate. Therefore, this study was designed, using in vitro cytological assays, to investigate the effects of TGF-β1 and Notch3 signaling pathway on ovarian cancer cell biological behavior and the potential mechanisms in terms of the crosstalk between TGF-β1 and Notch3 signaling pathway. Methods: Hey A8 and Hey cell lines were used as models in the study. The levels of TGF-β1 in supernatants from culture media were measured by ELISA. Both cell lines were treated with 500 ng/mL TGF-β1 neutralizing antibody (control group), 10 ng/mL TGF-β1, 50 μmol/L DAPT, 10 ng/mL TGF-β1 and 50 μmol/L DAPT, 50 μmol/L tumor necrosis factor receptor-associated factor 6 (TRAF6) peptide inhibitor, 10 ng/mL TGF-β1 and 50 μmol/L TRAF6 peptide inhibitor, respectively. The protein expression levels of TGF-β1 and Notch3 signaling pathway molecules as well as TRAF6 from cell lines with different treatments were detected by Western blot. Cell proliferation, migration and invasion were tested by cell counting kit-8 (CCK-8), scratch and Transwell assays, respectively. Results: The levels of TGF-β1 were timedependently increased in supernatants of culture media from Hey A8 and Hey cell lines. Compared with control group, TGF-β1 treatment increased the expression levels of Notch3-ICD and Hes1, while no obvious change was observed in the group treated with DAPT and TGF-β1. Moreover, TGF-β1 promoted cell proliferation, migration and invasion while DAPT decreased the proliferation, migration and invasion in cell lines treated with TGF-β1. These results indicated that TGF-β1 might promote proliferation, invasion and migration of ovarian epithelial cancer cells through activating the Notch3 signaling pathway. Further study showed that TGF-β1 up-regulated TRAF6 and activated the Notch3 signaling pathway. The activation of the Notch3 signaling pathway by TGF-β1 was inhibited in cells treated with the TRAF6 specific inhibitor. Conclusions: TGF-β1 may promote the proliferation, invasion and migration of ovarian epithelial carcinoma cells through TRAF6-mediated activation of the Notch3 signaling pathway.