Proanthocyanidins Inhibit Growth of Lung Cancer Figure 4. Effect of GSPs on cell cycle progression of A549 and H1299 cells. Cells were treated either with vehicle (0.1% DMSO in medium) or 20, 40 and 60 mg/mL doses of GSPs in complete medium. After 48 h of treatment, cells were harvested and digested with RNase. Cellular DNA was stained with propidium iodide and flow cytometric analysis was performed to analyze the cell cycle distribution, as detailed in the Materials and Methods. (A–D) Cell cycle distribution in A549 (left panels) and H1299 (right panels) cells with the treatment of various concentrations of GSPs. (E) Data from the cell cycle distribution were summarized and presented as the mean 6 SD of three independent experiments. Statistically significant versus non-GSPs treated control group, "P ,0.05 and *P ,0.01. doi:10.1371/journal.pone.0027444.g004 distribution at each dose of GSPs are also summarized in Panel E, which indicated the significant arrest of A549 and H1299 cells in G0–G1 phase after GSPs treatment. GSPs decrease the expressions of G1 regulatory proteins of Cdks and cyclins in NSCLC cells Studies have shown that Cdks and cyclins play crucial roles in the regulation of cell cycle progression [21], therefore we determined the effect of GSPs on the protein levels of the Cdks and cyclins which are negatively regulated by Cdki (Cip1/p21 and Kip1/p27) during G1 phase cell cycle progression. As shown in Figure 5 (Panel A), treatment of A549 cells with GSPs resulted in a marked decrease in the expression of Cdk2, Cdk4 and Cdk6 in a dose-dependent manner at 48 h after GSPs treatment. A strong reduction in Cdks was observed at the doses of 40–80 mg/mL concentrations of GSPs. Similarly, a marked reduction in the expression of cyclins D1, D2 and E was observed in a dose- dependent manner. Identical effect of GSPs was also found when H1299 cells were treated with GSPs and under identical conditions (Figure 5A, right panels). GSPs increase the expression of Cdki (Cip1/p21 and Kip1/ p27) in NSCLC cells The Cdki regulate the progression of cells in the Go/G1 phase of the cell cycle and induction of Cip1/p21 and Kip1/p27 causes a blockade of the G1 to S transition, thereby resulting in a Go/G1 phase arrest of the cell cycle [22]. The loss of Cdki in human cancers leads to uncontrolled cell proliferation [23]. In this context, our results revealed that treatment of human NSCLC cells, A549 and H1299, with varying concentrations of GSPs (0, 20, 40, 60 and 80 mg/mL) for 48 h resulted in a dose-dependent increase in protein expression of Kip1/p27 and Cip1/p21 (Figure 5 B). The GSPs-induced increase in inhibitory Cdki proteins may have a role in blockade of A549 and H1299 cells in G1 phase. These changes in protein expression were not due to differences in the amount of proteins loaded on the gels as the equivalent protein loading was confirmed by probing stripped blots for b-actin, as shown in Figure 5B. GSPs increase Cdk-Cdki binding in NSCLC cells As GSPs induce the expression of Cdki in both A549 and H1299 cells, and since the induction of the Cdki has been shown to result in an increased interaction with Cdks leading to a decrease in their kinase activity [24], we next determined whether GSPs promote the interaction between Cdki and Cdk. To assess this effect, Cip1/p21 and Kip1/p27 were immunoprecipitated from total cell lysates of A549 and their binding with Cdk2, Cdk4 and Cdk6 was assessed using western blot analysis. As compared to vehicle treated controls, treatment with GSPs was found to enhance the binding of Cdk2, Cdk4 and Cdk6 with Cip1/p21 and Kip1/p27 (Figure 5C). These results suggest that an increased interaction between Cdki with Cdks plays an important regulatory role in the GSPs-induced G1 arrest of cell cycle progression in human non-small cell lung cancer cells, possibly through the decrease in their Cdk kinase activity. Administration of GSPs by oral gavage inhibits the growth of tumor xenografts: GSPs are non-toxic to mice Next, we determined the effect of GSPs on A549 and H1299 xenografts growth in immuno-compromised athymic nude mice. Three different doses of GSPs, i.e., 50, 100 and 200 mg GSPs/kg body weight of mice were used and the effects on the growth of the tumors were monitored. The body weights and consumption of diet and water per animal in each treatment group was recorded on weekly basis. The average body weight of the GSPs-treated and non-GSPs-treated control mice remained identical throughout the duration of the experiment protocol (Figure 6A). The daily consumption of diet or drinking water by the mice in each group was also almost identical (data not shown) and the mice that were treated with GSPs did not exhibit impaired movement and posture or any other sign of physical toxicity. These data suggest that administration of GSPs by oral gavage at the concentrations used in these studies is not associated with visible toxicity in laboratory animals. Periodic measurement of the tumor xenograft volume indicated that the treatment with GSPs, particularly with the doses of 100 and 200 mg GSPs/kg body weight, resulted in reduced growth of both the A549 and H1299 lung cancer xenografts throughout the duration of the experiment. As can be seen in Figure 6B (left panel), the GSPs-induced inhibition of A549 xenograft tumor volume was 51% in mice administered GSPs at a concentration of 100 mg/kg body weight and 75% (P,0.001) in the mice administered GSPs at a concentration of 200 mg/kg body weight. Significant inhibition of the H1299 xenograft tumors also was observed after the treatment of mice with GSPs (Figure 6B, right panel). At the termination of the experiment on the 58th day of the experiment, the wet weight of the tumor/mouse in each treatment group was recorded. As shown in Figure 6C (left panel), the wet weight of the A459 xenograft tumor/mouse was significantly lower in the mice administered GSPs than in the mice that did not receive GSPs. The wet weight of the A549 tumors was 14% lower in mice administered 50 mg GSPs/kg body weight; 45% lower (P,0.001) in mice administered 100 mg GSPs/kg body weight; and 71% lower (P,0.001) in mice administered in 200 mg GSPs/ kg body weight compared with non-GSPs-treated control mice. Administration of GSPs at the same concentrations also resulted in a reduction in the wet weight of the H1299 tumor xenografts (Figure 6C, right panel). The wet weight of the H1299 tumors was 15% lower (not significant) in mice administered 50 mg GSPs/kg body weight; 39% lower (P,0.01) in mice administered 100 mg GSPs/kg body weight; and 62% lower (P,0.001) in mice administered in 200 mg GSPs/kg body weight. GSPs inhibit the growth of A549 and H1299 tumor xenografts through the enhancement of apoptotic cell death of tumor cells To determine if administration of GSPs by gavage inhibits the growth of tumor xenografts by enhancing the apoptosis of the lung tumor cells in vivo, the xenograft tumors were subjected to TUNEL-positive staining. As shown in Figure 7A, the percentages of TUNEL-positive cells were significantly higher (P,0.001) in the PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27444