Grape Proanthocyanidins Induce Apoptosis by Loss of Mitochondrial Membrane Potential of Human Non-Small Cell Lung Cancer Cells In Vitro and In Vivo Tripti Singh 2 , Som D. Sharma 2 , Santosh K. Katiyar1,2,3,4* 1 Birmingham Veterans Affairs Medical Center, Birmingham, Alabama, United States of America, 2 Department of Dermatology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 3 Nutrition Obesity Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 4 Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America Abstract Lung cancer remains the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) represents approximately 80% of total lung cancer cases. The use of non-toxic dietary phytochemicals can be considered as a chemotherapeutic strategy for the management of the NSCLC. Here, we report that grape seed proanthocyanidins (GSPs) induce apoptosis of NSCLC cells, A549 and H1299, in vitro which is mediated through increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase (PARP). Pre-treatment of A549 and H1299 cells with the caspase-3 inhibitor (z-DEVD-fmk) significantly blocked the GSPs-induced apoptosis of these cells confirmed that GSPs-induced apoptosis is mediated through activation of caspases-3. Treatments of A549 and H1299 cells with GSPs resulted in an increase in G1 arrest. G0/G1 phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk), cyclin-dependent kinase inhibitors (Cdki) and cyclins. Our western blot analyses showed that GSPs-induced G1 cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), and a simultaneous decrease in the levels of Cdk2, Cdk4, Cdk6 and cyclins. Further, administration of 50, 100 or 200 mg GSPs/kg body weight of mice by oral gavage (5 d/week) markedly inhibited the growth of s.c. A549 and H1299 lung tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death, increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3 in tumor xenograft cells. Based on the data obtained in animal study, human equivalent dose of GSPs was calculated, which seems affordable and attainable. Together, these results suggest that GSPs may represent a potential therapeutic agent for the non-small cell lung cancer. Citation: Singh T, Sharma SD, Katiyar SK (2011) Grape Proanthocyanidins Induce Apoptosis by Loss of Mitochondrial Membrane Potential of Human Non-Small Cell Lung Cancer Cells In Vitro and In Vivo. PLoS ONE 6(11): e27444. doi:10.1371/journal.pone.0027444 Editor: Surinder K. Batra, University of Nebraska Medical Center, United States of America Received August 6, 2011; Accepted October 17, 2011; Published November 8, 2011 Copyright: 2011 Singh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funds from the Veterans Administration Merit Review Award (SKK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: skatiyar@uab.edu Introduction Lung cancer remains the leading cause of cancer related deaths in the United States and worldwide [1]. One of every three cancer- related deaths is attributable to lung cancer, and the dismal 5-year survival rate of approximately 14% has shown no improvement over the past three decades [2,3]. Small-cell lung cancer and non- small-cell lung cancer (NSCLC) account for 90% of all lung cancers. NSCLC represents approximately 80% of all types of lung cancer and includes squamous cell carcinomas, adenocarci- nomas, and large-cell carcinomas [4,5]. Although a combination of chemotherapy and radiation therapy can improve survival of the patients, most patients die of disease progression, often resulting from acquired or intrinsic resistance to chemotherapeutic drugs [6]. Therefore, the exploration and development of more effective therapeutic agents and therapies that can target the molecules associated with tumor growth and apoptosis resistance will lead to improved outcomes in patients with lung cancer. Natural plant products offer promising new options for the development of more effective chemotherapeutic strategies for cancers of various organs. Grape seed proanthocyanidins (GSPs) are promising phytochemicals that have anti-inflammatory [7] and anti-oxidant properties [8–10], and appear to exhibit minimal toxicity in laboratory animals [9,10]. GSPs are readily extracted from grape-seeds, a by-product of grape juice and wine industries, and are a mixture of several polyphenolic components, which constitute dimers, trimers, tetramers, and oligomers/polymers of monomeric catechins and/or (-)-epicatechins, as described previ- ously [9,10]. We believe that at least some of the constituents present in GSPs act synergistically and may provide better chemotherapeutic effects than a single constituent. Previously, we have shown that dietary supplementation of GSPs with AIN76A control diet resulted in a dose-dependent inhibition of the growth of A549 and H1299 NSCLC tumor xenograft in athymic nude mice, and the growth inhibitory effect of GSPs on the NSCLC xenograft tumors was associated with the enhancement of the levels of insulin-like growth factor binding protein-3 and anti-angiogenic effects in the tumor microenviron- ments (11). In another study, we also have reported that GSPs inhibit the proliferation and induce apoptosis of NSCLC cells in PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27444