Abstract 5687: Grape seed extract induces apoptosis via reactive oxygen species-dependent mechanism in human non-small-cell lung cancer cells
Authors: Alpna Tyagi, Subhash Chander Gangar, Komal Raina, Rajesh Agarwal, Chapla Agarwal
Journal: Cancer Research (2010)

Abstract Lung cancer is the most frequently diagnosed malignancy and leading cause of cancer-related deaths worldwide. The 5-year survival rate of 14% has shown little improvement over the last 30 years even with the development of molecularly targeted therapies. Non-small-cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers, suggesting that additional approaches and strategies are needed to control this malignancy. For the last few decades, major emphasis has also been placed on identifying natural dietary and/or non-dietary products for both prevention and intervention of several malignancies including lung cancer. One such naturally occurring polyphenolic product is grape seed extract (GSE); which is especially rich in proanthocyanidins. In recent studies, GSE has shown promising cancer chemo-preventive and anti-cancer effects against various epithelial cancers. Accordingly, here we evaluated the efficacy of GSE against a panel of human NSCLC cell lines, namely A549, H460, H1299 and H322 cells. GSE (50-100 µg/ml) treatment for 24 and 48 hrs significantly suppressed cell proliferation and induced cell death in all the four cell lines studied; however, H460 and H322 cells were more sensitive to GSE treatment even at comparatively lower doses (20-50 µg/ml). Additional studies showed that GSE causes a dose- and a time-dependent apoptotic cell death in A549, H460, H322 and H1299 cells which was confirmed by the Annexin V-PI staining, activation of caspase-3 and −9, and cleavage of PARP. In detailed mechanistic studies, to determine the cause of GSE-induced apoptotic cell death, pre-treatment of cells with N-acetylcysteine (NAC) strongly inhibited GSE-caused apoptotic cell death, suggesting the involvement of reactive oxygen species (ROS). Interestingly, GSE treatment also resulted in a strong dose- and time-dependent phosphorylation of ERK1/2 in these cell lines, which was inhibited by pre-treatment of cells with NAC, further supporting the role of GSE-caused oxidative stress in the activation of ERK1/2. Based on these results, next we assessed whether GSE causes oxidative stress in these NSCLC cell lines. Dihydroethidium staining followed by flow cytometry analysis of cells showed that indeed GSE treatment results in ROS generation. Next we assessed the intracellular reduced glutathione levels, which were decreased by 82% and 60% in A549 and H460 cells, respectively, after GSE treatment. Together, these results confirm that GSE generates ROS causing oxidative stress that leads to apoptotic death in NSCLC cells. Based on these findings, more studies are needed in future to evaluate both anti-cancer and chemopreventive efficacy of GSE in relevant pre-clinical lung cancer models to establish its potential usefulness against human lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5687.