Resveratrol inhibits TRAF6/PTCH/SMO signal and regulates prostate cancer progression

Abstract

Prostate cancer (PC) is one of the most common types of cancers among men, referring to the uncontrolled growth of the prostate gland. It is increasingly recognized that the interaction of the glioma-associated oncogene (GLI) pathway and androgen receptor affects PC progression. Nevertheless, the effects of resveratrol on PC progression via Hedgehog (HH) signaling remain unexplored. In this study, the castration-sensitive and castration-resistant xenograft models in mice are systematically established using two different PC cell lines (LNCaP and PC-3). Further, the Western blotting, immunohistochemistry, MTT, Transwell, and RT-qPCR analyses are performed to verify the mechanistic views of resveratrol on PC and HH signals in vitro and in vivo. Resveratrol showed epithelial-mesenchymal transition (EMT) progression, inhibiting the tumor size and expression levels of vimentin, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP) 7, as well as upregulating the expression profiles the E-cadherin and Annexin 2. Moreover, resveratrol inhibited the hedgehog (HH) signals and tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) levels exhibiting the therapeutic action on castration-sensitive and castration-resistant PC cell lines. In summary, the overexpression of TRAF6 enhanced the viability and EMT progression of cancer cells. The resveratrol could alleviate the TRAF6 effect and regulate the HH signal to affect PC progression.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-022-00544-0.

Keywords: Hedgehog; Prostate cancer; Resveratrol; TRAF6.

Fig. 1

Resveratrol inhibits tumor growth in vivo. A Bright-field images show the formation of PC-3 and LNCaP cells-based xenografts in nude mice. B Resveratrol reduces the tumor volume induced by LPS. C Resveratrol alleviates the tumor weight induced by LPS. D The TUNEL staining shows the effect of resveratrol on the apoptosis of PC-3-induced tumors. E The TUNEL staining presents the effect of resveratrol on the apoptosis of LNCaP-induced tumors. F The WB analysis presents the effect of resveratrol on the relative expressions of Bax, Bcl-2, and p53 proteins in PC-3-induced tumors and their corresponding quantified levels. G The WB analysis indicates the effect of resveratrol on the relative expression of Bax, Bcl-2, and p53 proteins in LNCaP-induced tumors and their corresponding quantified levels (^# refers to comparison with control group, ^# indicates P < 0.05 and ^## represents P < 0.01; * refers to comparison with LPS group, * indicates P < 0.05 and ** represents P < 0.01; n.s. no significant)

Fig. 2

Resveratrol inhibits EMT progression and HH signal in vitro. A Representative microscopic images of PC-3 cells show the EMT morphological changes induced by LPS treatment. B Resveratrol enhances the expression of E-cadherin. C Resveratrol decreases the vimentin level. D The immunohistochemical staining presents the relative expressions of E-cadherin and vimentin. E The WB analysis shows the relative expressions of the various proteins of EMT, i.e., E-cadherin, Vimentin, VEGF, MMP-7, and Annexin 2, compared to GAPDH. F Resveratrol inhibits the HH signal in vivo (^# refers to comparison with control group, ^# indicates P < 0.05, ^## represents P < 0.01; * refers to comparison with LPS group, * indicates P < 0.05, ** represents P < 0.01; n.s. means no significant)

Fig. 3

Resveratrol alleviates EMT by targeting TRAF6 and regulates HH signal. A MTT assay results show the cell viability at the different concentrations of resveratrol. B RT-PCR analysis shows the TRAF6 expression after transfection; C TRAF6 overexpression enhances the cell viability. D Resveratrol downregulates the increase of cell invasion induced by TRAF6 overexpression. E The WB analysis determines relative levels of factors in the HH signal pathway. F RT-PCR determines the relative expressions of various EMT factors (# refers to comparison with control group, ^# indicates P < 0.05 and ^## represents P < 0.01; * refers to comparison with Ov-ex group, * indicates P < 0.05 and ** represents P < 0.01; n.s. means no significant)