Curcumin Suppresses NF-κB Pathway To Reverse Cigarette Smoke Extract-induced Human Bladder Epithelial-mesenchymal Transition In Vitro

Objectives Bladder cancer(BC) is a common genitourinary malignant disease worldwide. Epithelial-mesenchymal transition(EMT) is a crucial pathophysiological process in cancer development. Curcumin is a promising chemopreventive agent for several types of cancers. Cigarette smoke(CS) is a crucial factor for bladder cancer and the role of NF-κB signal pathway in the development of CS-associated BC was not fully elicited.Methods SV-HUC-1 cells were used as in vitro CS exposure models. Cell morphology and invasive capacity were examined by healing assays and transwell assays. The protein markers and m RNA expression were analyzed by Western blotting, and quantitative reverse transcriptase–polymerase chain reaction(q RT-PCR). PDTC, a specific NF-κB inhibitor, treated with cells for 7 days. Meanwhile, curcumin was treated with SV-HUC-1 cells for 7 days.Results(1) To study the cell viability of the SV-HUC-1 cells after treatment with CSE, the cells were incubated with CSE(0, 0.05, 0.1, 0.25, 0.5, 1 and 2%) for 7 days and examined by MTT assay. The results showed that the cell viability decreased below 80% when the cells were exposed to 1% or higher CSE concentrations, which proved to be toxic to the SV-HUC-1 cells. Therefore, 0.5% CSE was selected as the maximumconcentration for the following experiments.(2) Treatment of the SV-HUC-1 cells with CSE for 7 days resulted in significant morphological change from a urothelial oblate-shape to a spindle-like mesenchymal form. Additionally, the cells became dispersive and some SV-HUC-1 cells even generated a tail-like change after treatment with CSE. To further examine the effect of CSE on EMT, wound healing assays and transwell assays were carried out to analyze SV-HUC-1 cell migratory and invasive capacities in response to CSE. CSE treatment significantly increased SV-HUC-1 cell migration.(3) Exposure of the SV-HUC-1 cells to CSE resulted in decreased protein expression of the epithelial markers E-cadherin and ZO-1. In contrast, the protein levels of mesenchymal markers Vimentin and N-cadherin were increased, as shown by western blot analyses. Moreover, q RT-PCR analyses revealed similar changes in the m RNA levels of epithelial and mesenchymal markers in the SV-HUC-1 cells exposed to CSE. Immunoflorescent staining also showed that CSE decreased E-cadherin protein expression and increased Vimentin expression in SV-HUC-1 cells(4) CSE–induced EMT is associated with activate NF-κB activation in SV-HUC-1 cells.(5) PDTC downregulated p65 and p50 in the SV-HUC-1 cells. Inhibition of NF-κB decreased CSE-mediated migration and invasion capacities of SV-HUC-1 cells. Furthermore, inhibition of NF-κB by PDTC resulted in upregulation of the protein and m RNA levels of the epithelial markers E-cadherin and ZO-1, as well as downregulation of the mesenchymal markers Vimentin and N-cadherin.(6) Western blot analyses showed that curcumin inhibited CSE-induced NF-κB activation in a dose-dependently manner. Meanwhile, CSE-induced alterations in m RNA and protein expressions of the EMT markers, including decreases of the epithelial markers E-cadherin and ZO-1, and increases of the mesenchymal markers Vimentin and N-cadherin, were effectively attenuated with curcumin treatmentConclusion In summary, exposure to CSE induced EMT in normal human bladderepithelial cells and activated the NF-κB pathway, while the special inhibitor inhibited NF-κB activity and afterwards restrained the EMT triggered by CSE. Meanwhile, we found that curcumin effectively attenuated CSE exposure triggered NF-κB activation and EMT alterations in SV-HUC-1 cells. All of these data provide important information that inhibition of NF-κB pathway may be a therapeutic target for the treatment of early stage bladder cancer and regarding the chemopreventive function of curcumin against CSE-associated bladder cancer.