Diallyl trisulfide inhibited tobacco smoke-mediated bladder EMT and cancer stem cell marker expression via the NF-κB pathway in vivo

Abstract

Objective: This study examined the effect of the NF-κB pathway on tobacco smoke-elicited bladder epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) marker expression in vivo. The effect of diallyl trisulfide (DATS) treatment was also examined.

Methods: BALB/c mice were exposed to tobacco smoke and treated with an NF-κB inhibitor and DATS. Western blotting, quantitative real-time PCR, and immunohistochemical staining were used to detect the changes of relevant indices.

Results: Phosphorylated inhibitor of kappa-B kinase alpha/beta expression and p65 and p50 nuclear transcription were increased by tobacco smoke exposure, whereas inhibitor of kappa-B expression was decreased. In addition, tobacco smoke reduced the expression of epithelial markers but increased that of mesenchymal and CSC markers. Our study further demonstrated that tobacco smoke-mediated EMT and CSC marker expression were attenuated by inhibition of the NF-κB pathway. Moreover, DATS reversed tobacco smoke-induced NF-κB pathway activation, EMT, and the acquisition of CSC properties in bladder tissues.

Conclusions: These data suggested that the NF-κB pathway regulated tobacco smoke-induced bladder EMT, CSC marker expression, and the protective effects of DATS.

Keywords: IKKα/β; IκB; NF-κB; Tobacco smoke; bladder cancer; cancer stem cells; diallyl trisulfide; epithelial–mesenchymal transition.

Figure 1.

Tobacco smoke alters the expression of EMT and CSC markers. (a) Tobacco smoke exposure reduced the mRNA levels of epithelial markers and increased the levels of mesenchymal markers. (b) Tobacco smoke exposure induced alterations in the protein expression of EMT markers. (c) Immunohistochemistry illustrated that tobacco smoke decreased E-cadherin expression and increased vimentin expression. (d) Alterations in the mRNA expression of CSC markers induced by tobacco smoke. (e) Alterations in the protein expression of CSC markers induced by tobacco smoke. **P < 0.01, compared with FA. EMT, epithelial–mesenchymal transition; CSC, cancer stem cell; FA, filtered air; TS, tobacco smoke.

Figure 2.

Tobacco smoke exposure increased NF-κB pathway activity. Tobacco smoke effectively elevated the expression of p65, p50, and p-IKKα/β and decreased the expression of IκBα. **P < 0.01, compared with FA. p-IKKα/β, phosphorylated inhibitor of kappa-B kinase alpha/beta; IκBα, inhibitor of kappa-B alpha; FA, filtered air; TS, tobacco smoke N-p50, nuclear p50; N-p65, nuclear p65; C-p50, cytoplasmic p50; C-p65, cytoplasmic p65.

Figure 3.

PDTC inhibited tobacco smoke-induced NF-κB pathway activation. Western blotting revealed that PDTC reversed tobacco smoke-induced changes of p65, p50, p-IKKα/β, and IκB expression. **P < 0.01, compared with FA; ^##P < 0.01, compared with TS+DMSO. PDTC, pyrrolidinedithiocarbamate ammonium; p-IKKα/β, phosphorylated inhibitor of kappa-B kinase alpha/beta; IκBα, inhibitor of kappa-B alpha; FA, filtered air; TS, tobacco smoke; N-p50, nuclear p50; N-p65, nuclear p65.

Figure 4.

NF-κB pathway suppression reversed tobacco smoke-induced changes of EMT and CSC marker expression. (a) Real-time PCR of EMT marker expression. (b) Western blotting of EMT marker expression. (c) Real-time PCR of CSC marker expression. (d) Western blotting of CSC marker expression. **P < 0.01, compared with FA; ^#P < 0.05, ^##P < 0.01, compared with TS+DMSO. EMT, epithelial–mesenchymal transition; CSC, cancer stem cell; FA, filtered air; TS, tobacco smoke; PDTC, pyrrolidinedithiocarbamate ammonium.

Figure 5.

DATS attenuated tobacco smoke-mediated changes of urocystic EMT and CSC marker expression. (a) Real-time PCR of the mRNA levels of EMT markers. (b) Western blotting of EMT marker expression. (c) Real-time PCR of the mRNA levels of CSC markers. (d) Western blotting of CSC marker expression. **P < 0.01, compared with FA; ^#P < 0.05, ^##P < 0.01, compared with TS+DMSO. DATS, diallyl trisulfide; EMT, epithelial–mesenchymal transition; CSC, cancer stem cell; FA, filtered air; TS, tobacco smoke.

Figure 6.

DATS reversed tobacco smoke-induced activation of the NF-κB pathway. DATS attenuated tobacco smoke-stimulated NF-κB pathway activation in the mouse bladder. Western blotting was performed to analyze the expression changes of p65, p50, p-IKKα/β, and IκB. DATS, diallyl trisulfide; EMT, epithelial–mesenchymal transition; CSC, cancer stem cell; FA, filtered air; TS, tobacco smoke; p-IKKα/β, phosphorylated inhibitor of kappa-B kinase alpha/beta; IκBα, inhibitor of kappa-B alpha.