Abstract 1003: S-nitrosylation of Bcl-2 negatively affects autophagy in malignantly transformed lung epithelial cells

Abstract

Abstract Purpose The purpose of this study is to identify the key autophagic biomarkers involved in Cr(VI)-induced malignant transformation of non-tumorigenic Beas-2B cells and to investigate the underlying mechanisms. Experimental Methods The non-tumorigenic (Beas-2B) and malignantly transformed (B-Cr) lung epithelial cell lines were used in all experiments. Cells were exposed to hexavalent chromium (Cr (VI)) doses of 20 and 50μM for 6h time points. Separate experiments were conducted using inhibitors for the anti-apoptotic protein Bcl-2 (ABT-737), nitric oxide (AG (Aminoguanidine)), and p38 (SB203580) for 1hr before Cr treatment as detailed above. Cells were transfected with myc-tagged wild-type (WT) Bcl-2 (WT Bcl-2), double-mutant (C158A/C229A) Bcl-2 (DM Bcl-2), or control (pcDNA3) plasmid DNA (5 μg) using Lipofectamine 2000 transfecting agent. S-nitrosylation of Bcl-2 was measured using the S-Nitrosylation Western Blot kit. For autophagy and apoptosis analysis, cell vacuoles and nuclei were stained using the Cyto-ID dye kit and Hoechst dye respectively, and scored by fluorescence microscopy. Results Here we show that Cr(VI)-induced carcinogenesis triggers autophagy in non-tumorigenic human lung epithelial cells, while malignantly-transformed lung cells are resistant to autophagy. We previously reported that chronic exposure of human lung epithelial cells to the carcinogenic hexavalent chromium Cr(VI) caused malignant transformation and Bcl-2 upregulation. In this study we report that nitric oxide (NO), a potent cellular messenger, has a negative effect on autophagy initiation by nitrosylating and stabilizing the Bcl-2 protein, thereby preserving the anti-autophagic Bcl-2-Beclin-1 interaction. We demonstrate that p38 MAPK is also critical in the autophagic response along with LC3 and Beclin-1. Further results also link apoptosis, analyzed by cell staining and measuring caspase-9 protein levels, with the autophagic proteins, suggesting a possible autophagy-apoptosis pathway crosstalk. The effect of Bcl-2 nitrosylation on autophagy was corroborated using a Bcl-2 inhibitor (ABT-737) and by transfecting cells with non-nitrosylable Bcl-2 double mutant (DM) plasmid, in which the two cysteine residues (Cys158 and Cys229) critical for nitrosylation are mutated. Both ABT-737 and transfection with Bcl-2 DM plasmid resulted in decreased S-nitro-cysteine expression and increased autophagy in lung cells. Conclusions We report a novel mechanism involving nitric oxide (NO)-mediated post-translational modification of Bcl-2 protein as being responsible for this resistance to autophagy induction. This Bcl-2 S-nitrosylation was observed to affect the Bcl-2-Beclin-1 interaction which is key for autophagy initiation. This novel regulation of autophagy may be important in cancer development and progression, as well as in carcinogenesis and its prevention. Citation Format: Clayton A. Wright, Anand Krishnan V. Iyer, Yogesh Kulkarni, Neelam Azad. S-nitrosylation of Bcl-2 negatively affects autophagy in malignantly transformed lung epithelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1003. doi:10.1158/1538-7445.AM2015-1003