confirmed the role of activated caspase-3 in the GSPs-induced apoptosis. It has been recognized that control of cell cycle progression in cancer cells is an effective strategy to inhibit tumor growth [22,36] as the molecular analyses of human cancers have revealed that cell cycle regulators are frequently deregulated in most of the common malignancies [37,38]. Our in vitro data demonstrate that treatment of A549 and H1299 cells with GSPs induces G1 phase arrest of cell cycle progression (Figure 4) indicating that one of the mechanisms by which GSPs inhibit the proliferation of lung cancer cells is inhibition of cell cycle progression. Our studies demonstrate a marked decrease in the expressions of cyclins D1, D2 and E, and Cdk2, Cdk4 and Cdk6 in both A549 and H1299 cells dose- dependently on GSPs treatment suggests the disruption of the uncontrolled cell cycle progression of human NSCLC cells (Figure 5) and that the GSPs-induced G1 arrest is mediated through the up-regulation of cyclin-dependent kinase inhibitory proteins, Cip1/p21 and Kip1/p27, which enhances the formation of heterotrimeric complexes with the G1/S Cdks and cyclins thereby resulting in inhibition of their activity. Uncontrolled cell division depends on the activation of cyclins, which bind to Cdk to induce cell cycle progression towards S phase. Cdk kinase activity is one of the major causes of cancer progression, and their functions are tightly regulated by Cdki, such as, Cip1/p21 and Kip1/p27 proteins. Cip1/p21 is a universal inhibitor of Cdk(s) and Kip1/p27 is commonly up-regulated in response to anti-proliferative signals [39]. The increased expres- sion of G1 cyclins in cancer cells provides them an uncontrolled growth advantage because most of these cells either lack Cdki, harbor nonfunctional Cdki, or Cdki expression is not at a sufficient level to control Cdk-cyclin activity [38,40]. Consistent with these reports, the increased expressions of Cdkis together with decreased expression of cyclins and Cdks on the GSPs treatment to A549 and H1299 cells suggest that GSPs might be effective as a chemotherapeutic agent for the treatment of non-small cell lung cancers. Our in vivo study provides further evidence that administration of GSPs by gavage inhibits the growth of both A549 and H1299 tumor xenografts by inducing cell death and without any apparent sign of toxicity in athymic nude mice (Figures 6 and 7). Further, consistent with the findings in cell culture model, an increase in the levels of pro-apoptotic protein Bax, and cleaved caspase-9, caspase-3 and PARP proteins were also observed in the tumors of GSPs-treated group compared to the control mice which were References 1. American Cancer Society (2011) Cancer facts and figures. Available: http:// www.cancer.org/. Accessed 2011, June 20. 2. Proctor RN (2001) Tobacco and the global lung cancer epidemic. Nat Rev Cancer 1: 82–86. 3. Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, et al. (2005) Cancer statistics. CA Cancer J Clin 55: 10–30. 4. Maziak DE, Markman BR, MacKay JA, Evans WK, Cancer Care Ontario Practice Guidelines Initiative Lung Cancer Disease Site Group (2004) Photodynamic therapy in nonsmall cell lung cancer: a systematic review. Ann Thorac Surg 77: 1484–1491. 5. Hoffman PC, Mauer AM, Vokes EE (2000) Lung cancer. Lancet 355: 479–485. 6. 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It is reasonable to consider whether the chemotherapeutic effect of GSPs examined in an animal model can be translated in human system. For appropriate conversion of the doses of chemopreven- tive or chemotherapeutic agent from animal studies to humans, the body surface area normalization method has been recom- mended [41]. In the present study, maximum dose of GSPs by oral gavage was given 200 mg/kg body weight of the mouse. Based on this information, the human equivalent dose of GSPs was calculated using the following formula: Human equivalent dose ðmg=kgÞ~ Animal doseðmg=kgÞ X Animal Km f actor Human Km f actor (Km factor for mouse = 3; Km factor for adult human = 37). If, the body weight of a normal standard person is considered to be 70 Kg, then 1.13 g GSPs will be required for a healthy person/ day to produce same level of anti-lung carcinogenic effects as observed in mice, which seems reasonable, affordable and attainable. In summary, the novelty of this study lies in the analysis of chemotherapeutic effects of GSPs on additional new molecular targets of NSCLC cells using both in vitro and in vivo models. This detailed and systematic study revealed that GSPs induce apoptosis of human non-small cell lung cancer cells by loss of mitochondrial membrane potential, which was not reported earlier in this model. The present findings provide pre-clinical data suggesting that grape seed proanthocyanidins have the potential to be developed as a pharmacologically safe agent either alone or in combination with other drugs for the treatment of non-small cell lung cancers in humans. Author Contributions Conceived and designed the experiments: SKK TS SDS. Performed the experiments: TS SDS. Analyzed the data: SKK TS SDS. Contributed reagents/materials/analysis tools: SKK. Wrote the paper: SKK TS SDS. 9. 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Mantena SK, Sharma SD, Katiyar SK (2006) Berberine inhibits growth, induces G1 arrest and apoptosis in human epidermoid carcinoma A431 cells by regulating Cdki-Cdk-cyclin cascade, disruption of mitochondrial membrane potential and cleavage of caspase-3 and PARP. Carcinogenesis 27: 2018–2027. 14. Mantena SK, Sharma SD, Katiyar SK (2006) Berberine, a natural product, induces G1-phase cell cycle arrest and caspase-3-dependent apoptosis in human prostate carcinoma cells. Mol Cancer Ther 5: 296–308. PLoS ONE | www.plosone.org 12 November 2011 | Volume 6 | Issue 11 | e27444