Figure 6. ST and nicotine induces Bad phosphorylation enhances interaction with 14-3-3ζ.

SCC4 were treated with (A) ST (20 µg/ml) or (B) with nicotine (10 µM) for different time intervals and immunoprecipitation assays were carried out using whole cell lysates and analyzed by western blotting as described in Materials and Methods. 14-3-3ζ was immunoprecipitated using specific antibody and the bound pBad protein co-precipitated with 14-3-3ζ was determined by western blot analysis. Reverse immunoprecipitation assays were carried out in which pBad was immunoprecipitated, followed by western blotting analysis of 14-3-3ζ using a specific antibody against this protein. (C) Figure shows the hypothetical model for inhibition of ST / nicotine induced activation of PI3K/Akt pathway by GS. Our results showed treatment with ST and /or nicotine activates Akt (phosphorylated at Ser-473 and Ser-309) resulting in phosphorylation of its downstream targets Raf, GSK3β, and pS6, whereas GS treatment inhibits activation of Akt pathway. Interestingly, pre-treatment of head and neck cancer cells with GS inhibits activation of Akt and its downstream targets (Raf, GSK3β, and pS6) on exposure to ST / nicotine. GS pre-treatment also releases Bad from inhibitory action of 14-3-3ζ, thereby activating intrinsic pathway of apoptosis. Thus, our results demonstrated GS as a potential therapeutic agent for ST-induced head and neck carcinogenesis.