7-Difluoromethyl-5,4’-dimethoxygenistein inhibited the angiogenesis induced by cervical cancer SiHa cells via inhibiting TLR4/VEGF-A axis (217)

Abstract

<b>Objectives:</b> VEGF inhibitors, such as bevacizumab, inhibit angiogenesis and improve the prognosis of patients with recurrent or metastatic cervical cancer. Antiangiogenic drugs reduce the formation of immature and poorly functional blood vessels and induce the normalization of blood vessels. However, in many cases, features of the vascular normalization brought by VEGF inhibitors are quickly replaced by marked vascular regression, which in turn will exacerbate the degree of hypoxia in the tumor microenvironment. In addition, a variety of adverse effects of antiangiogenic drugs have also been found at present. Therefore, it is a very attractive work to extract natural molecules that are non-toxic or low in toxicity from plants as antiangiogenic drugs. 7-Difluoromethyl-5,4'-dimethoxygenistein (DFMG) is a new chemical entity synthesized from the genistein extracted from the natural plant fruit soybean by ourselves. Previously, DFMG has been found to maintain plaque stability in atherosclerosis through antiangiogenesis. This study explored the effect of DFMG on angiogenesis in cervical cancer and its possible mechanism. <b>Methods:</b> Cell proliferation was detected by the CCK-8 experiment. The expressions of TLR4 and VEGF-A were detected by Q-PCR and Western blot. Angiogenesis was detected by tubule formation test and chorioallantoic membrane (CAM) test. TLR4 was overexpressed and silenced on SiHa cells to detect its effect on VEGF-A secretion and angiogenesis. The effects of DFMG on VEGF-A secretion and angiogenesis were detected on TLR4-overexpressed or -silenced SiHa cells. <b>Results:</b> 1) 50 μM DFMG can inhibit the activity of Siha cells, but not HUVE-12 cells. 2) DFMG inhibits the formation of tubules induced by Siha cells, while bevacizumab can inhibit the ability of HUVE-12 cells to form tubules, but DFMG and genistein have no effect on it. 3) DFMG inhibits the expression and secretion of VEGF-A protein in Siha cells. 4) Overexpression of TLR4 in Siha cells can antagonize the angiogenesis-inhibiting effect caused by bevacizumab, and the silent expression of TLR4 can antagonize the angiogenesis-promoting effect caused by VEGF-A. 5) The numbers of tubules in the tubule formation experiment and new blood vessels in CAM in the DFMG-treated Siha cells with TLR4 overexpression was significantly higher than that in the DFMG-treated Siha cells with TLR4 silence. 6) The overexpression of TLR4 could antagonize the effect of DFMG on the secretion and expression of VEGF-A in SiHa cells, while the silent expression of TLR4 could cooperate with DFMG to inhibit the secretion and expression of VEGF-A.Fig. 1 <b>Conclusions:</b> DFMG inhibits the angiogenesis induced by cervical cancer Siha cells which could be regulated by TLR4/VEGF-A signaling pathway. DFMG reduces angiogenesis by inhibiting VEGF-A secretion of SiHa cells, which is not observed in normal cells.