Abstract 1328: Differential autophagy responses in non-tumorigenic and malignantly transformed lung epithelial cells

Abstract Purpose We have developed an in vitro malignant transformation model for lung epithelial cells using hexavalent chromium (Cr(VI)) compounds which are known human carcinogens associated with lung cancer. The purpose of this study is to identify the key autophagic biomarkers that may be dysregulated in malignantly transformed cells. Experimental Methods The non-tumorigenic (Beas-2B) and malignantly transformed (B-Cr) lung epithelial cell lines were used in all experiments. Cells were plated in 6-well plates and exposed to hexavalent chromium (Cr (VI)) doses of 20, 50, and 100μM for 6 and 24h time points. Separate experiments were conducted using inhibitors for the anti-apoptotic protein Bcl-2, nitric oxide (NO), and p38, with Rapamycin as a positive control. Cells were pre-treated with Rapamycin, ABT, AG, and SB for 1hr before Cr treatment as detailed above. Expression of auophagy-related proteins were analyzed by Western blotting. 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 Microscopy and Western blotting results indicate that the malignant B-Cr cells express lower levels of key autophagic proteins as compared to Beas-2B cells. We also observed fewer vacuoles in B-Cr cells as opposed to Beas-2B cells. To analyze the effect of known autophagic markers like NO, p38, and Bcl-2 in Cr (VI)-induced autophagy, we pre-treated cells as described above with inhibitors to Bcl-2, NO, and p38 proteins. Rapamycin pre-treatment was also used as a positive control for autophagy induction. Microscopy results indicate that the inhibition of Bcl-2 and NO lead to an increase in vacuole formation. Western blotting indicated an overall increase in P-p38 expression, while Beclin-1 and LC3/atg expression levels were decreased. Conclusions We conclude that early autophagic events trigger apoptosis in response to Cr(VI) treatment. However, these events are dysregulated in the malignantly transformed cell line. Results indicate that the key autophagy-related proteins; Beclin-1, LC3, p38, and Bcl-2 all play a key role in autophagy induction in these lung cells. Published data has indicated that cancerous cells that are under stress from medical treatments such as radiation therapy and chemotherapy may initiate the autophagy process in order to evade inevitable cell death that the medical treatment is designed to cause. This study can lead to the manipulation of protein expression to determine how cell death can be specifically targeted to cancerous lung cells thereby preventing them from using autophagy as a protective mechanism. Citation Format: Clayton A. Wright. Differential autophagy responses in non-tumorigenic and malignantly transformed lung epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1328. doi:10.1158/1538-7445.AM2014-1328