Figure 5. Effect of GSPs on G1 phase cell cycle regulatory proteins in A549 and H1299 cells. The cells were treated with either vehicle (0.1% DMSO in medium) or GSPs (20, 40, 60 and 80 mg/mL) for 48 h and thereafter harvested, cell lysates prepared and then subjected to SDS-PAGE followed by Western blot analysis, as described in Materials and methods. Effect of GSPs was determined on the following: (A) the expression of cyclin D1, cyclin D2 and cyclin E, and the expression levels of Cdk2, Cdk4 and Cdk6; and (B) the expression of Kip1/p27 and Cip1/p21. b-actin was used to verify equal loading of the samples. Representative blots are shown from three independent experiments with almost identical results. (C) In binding assay, Cip1/p21 and Kip1/p27 were immunoprecipitated using specific antibody from total protein lysates followed by SDS-PAGE and western blot analysis for Cdk2, Cdk4 and Ckd6 as detailed in Materials and methods. IP, immunoprecipitation; IB, immunoblotting. doi:10.1371/journal.pone.0027444.g005 A549 and H1299 xenograft tumors of the GSPs-treated mice as compared to percentages of TUNEL-positive cells in the xenograft tumors in the control mice that did not receive GSPs. The Proanthocyanidins Inhibit Growth of Lung Cancer percentage of TUNEL-positive cells was comparatively higher in the A549 xenograft tumors than the H1299 xenograft tumors. To further determine the mechanisms involved in the GSPs- mediated induction of apoptosis in vivo tumor cells, we examined tumor xenograft samples from each of the treatment groups for the expressions of pro- and anti-apoptotic proteins of the Bcl-2 family using western blot analysis. As shown in Figure 7B, the levels of the anti-apoptotic proteins, Bcl-2 and Bcl-xl, were lower in the A459 and H1299 xenograft tumors from mice treated with GSPs than in the tumors in control mice that did not receive GSPs, whereas the levels of the pro-apoptotic protein, Bax, were higher. The cleavage of caspase-3 and caspase-9 proteins was markedly higher in the tumor from the GSPs-treated mice than the control mice. Similarly, the cleavage of PARP protein was enhanced, as shown in Figure 7C. Additionally, we determined whether GSPs treatment also affects the tumor cell proliferation potential in the A549 and H1299 tumor xenografts. As summarized in Figure 7D, immuno- histochemical detection analysis of PCNA-positive cells in tumor xenograft tissues indicated that the percentage of proliferating cells was significantly lower in both the A549 (P,0.001) and H1299 (P,0.01) tumor xenografts from GSPs-treated mice than the xenograft tumors from the control mice. Administration of GSPs decreases the expressions of G1 regulatory proteins of Cdks and cyclins while increases the levels of Cdk inhibitory proteins in tumor xenograft tissues of NSCLC cells As the inhibitory effect of GSPs on the tumor xenograft growth of A549 and H1299 cells was identical, we selected the tumor tissues of A549 cells for further analyses of cell cycle regulatory proteins. As shown in Figure 8A, treatment of mice with GSPs resulted in a marked decrease in the expression of Cdk2, Cdk4 and Cdk6 compared to the tumors from non-GSPs-treated mice in a dose-dependent manner. Similarly, a marked reduction in the expression of cyclins D1, D2 and E was also observed. As the Cdki regulate the progression of cells in the Go/G1 phase of the cell cycle, we also examined the effect of GSPs on the levels of Cdk inhibitory proteins in tumor xenograft tissues from each treatment group. Western blot analysis revealed that administration of GSPs resulted in a dose-dependent increase in protein expression of Kip1/p27 and Cip1/p21 (Figure 8B). The GSPs-induced increase in the levels of Cdki proteins may have a role in controlling the cell cycle progression and that may have resulted in suppression of tumor growth. Discussion Chemotherapy offers a promising strategy for control of cancer. Several phytochemicals including dietary plant products have been shown to have anti-carcinogenic properties. GSPs represent one such phytochemicals and have been shown to have anti- carcinogenic activity in different organs [10–12,25,26]. We have shown previously that long-term dietary feeding of GSPs supplemented with AIN76A control diet of laboratory animals does not show apparent signs of toxicity [10,11]. In the present study, we further verify that long-term administration of GSPs by oral gavage also does not induce any visible sign of toxicity in mice. Similarly, in vitro studies indicate that GSPs exhibit anti- proliferation or cytotoxic effects against tumor cells, but do not exhibit toxic effects on the growth and viability of normal (non- neoplastic) cells in culture [11,12,25]. Previously we have shown that treatment of both A549 and H1299 human non-small cell lung cancer cells with GSPs resulted in inhibition of cell PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27444