Effect of antioxidants on STAT3, cell survival gene products, apoptosis and chemoresistance in lung cancer cells

Abstract

20067 Background: We had earlier demonstrated that antioxidants (beta carotene, vitamins C and E) enhance the apoptotic effect of paclitaxel and carboplatin in vitro and there was a suggestion that when used as an adjunct to the above chemotherapeutic agents in patients with NSCLC, they might enhance the efficacy of the latter. To elucidate the mechanism underlying the synergistic effect between antioxidants and chemotherapy, in the present study we evaluated the effect of antioxidants on STAT3 activation in NSCLC cells. Since this transcription factor has been shown to regulate multiple oncogenic pathways, including pathways regulating tumor cell survival, we further studied how STAT3 modulation relates to apoptosis and downstream apoptosis regulatory proteins. Methods: H520 cells were treated with antioxidants. STAT3 proteins were assessed by western blotting and their nuclear localization was evaluated by immunocytochemistry. Antiapoptotic proteins (survivin, Bcl-2, Bcl-xl and cyclin D1) were evaluated by Western blotting. To assess the interaction, cells were treated with chemotherapy (Paclitaxel and Carboplatin) with or without antioxidants and the effect was assessed by Live & Dead assay, MTT assay and flowcytometry. Results: Antioxidants inhibited STAT3 phosphorylation in a dose and time dependent manner before the initiation of cell death and depleted the nuclear pool of STAT3. This effect was specific since there was no effect on STAT5 phosphorylation and was reversible since STAT 3 reverted back to its constitutively activated state after the removal of antioxidants. Antioxidants suppressed the expression of antiapoptotic proteins Bcl -2, Bcl-xl, cyclin D1 and survivin. This resulted in the inhibition of proliferation of cells and induction of apoptosis as evident on caspase 3 activation and PARP cleavage. Antioxidants arrested the cells in G0/G1 phase of cell cycle making them more vulnerable to the cytotoxic effects of chemotherapy. This synergy was evident on live and dead assay. Conclusions: Our results demonstrate that antioxidants are potent inhibitors of STAT3 phosphorylation and can have a potential role as an adjunct to chemotherapy in lung cancer. No significant financial relationships to disclose.