Background: Metastasis remains the primary cause in lung cancer. Epithelial-to-mesenchyme transition (EMT) has been identified as the hallmark for metastasis in NSCLC, and is being explored for drug targeting. Aberrant signaling mechanisms for cytokines such as TGF-β1 and TNFα are known to induce EMT. In this study we demonstrate the effect and mechanisms of WFA inhibition of EMT in NSCLC cell lines.
Objective: The present study was done to determine the effect of WFA on TGF-β1 and TNFα induced EMT, migration and invasion in human NSCLC cells. Furthermore, to determine underlying the mechanisms of WFA inhibition of EMT.
Methods: Two human NSCLC cell lines (H1299 and A549) were cultured in DMEM media. To induce the EMT, cells in serum free media were challenged with TGF-β1 (5 ng/mL) or TNFα (5 ng/mL) alone or in combination. To determine the effects of WFA, cells were either pre- or co-treated with different concentrations of WFA for various time points. Western blot analysis, electrophoretic mobility shift assay (EMSA), RT-PCR and immunofluorescence staining were used to determine effects of WFA on Smad2/3 and NFkB activation. Cell adhesion, migration and invasion assays were used to assess in vitro anti-metastatic activity.
Results: Our findings indicate that the cytokines TGF-β1 and TNFα cooperate to promote the acquisition of EMT phenotype, migration and invasion of NSCLC cells in vitro. There was a marked increase in the expression of EMT transcription factors Snail, Slug, ZEB and EMT markers such as vimentin and N-cadherin. Cells treated with TGF-β1/TNFα had significantly increased migratory and invasive capacities. WFA treatment significantly inhibited the TGF-β1 induced Smad2/3 activation and TNFα-induced translocation of NFkB into the nucleus. We observed a dose and time dependent inhibition of the phosphorylation of Smad2/3 and NFkB in both cell lines. The cell adhesion, migratory and invasive capacities of both H1299 and A549 cells were greatly diminished by WFA in the presence or absence of cytokines. Conclusion: Based on the current finding, it can be concluded that WFA inhibits EMT via negative regulation of Smad2/3 and NFkB signaling in NSCLC cells.
Citation Format: Al Hassan Kyakulaga, Farrukh Aqil, Radha Munagala, Ramesh Gupta. Withaferin A inhibits epithelial-to-mesenchymal transition in non-small lung cell cancer cells via regulation of SMAD and NFkB signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2040.