Proanthocyanidins Inhibit Growth of Lung Cancer Figure 3. Induction of apoptosis in lung cancer cells by GSPs is mediated through caspase-3 activation. (A) Treatment of A549 and H1299 cells with GSPs reduces the level of caspase-9 while increases the activation/cleavage of caspase-9, caspase-3 and PARP in a dose-dependent manner. Cells were treated with varying concentrations of GSPs (0, 20, 40, 60 and 80 mg/mL) for 48 h, thereafter cells were harvested, cell lysates prepared and subjected to western blot analysis to detect the levels of caspase-9, cleaved caspase-9, caspase-3 and PARP. A representative blot is shown from three independent experiments with similar results. (B) The caspase-3 activity in cell lysates from the samples of Panel A was measured using a colorimetric protease assay (ApoTarget Kit). GSPs treatment to A549 and H1299 cells increases the activity of caspase-3 dose-dependently. Significant difference versus non-GSPs treatment group, "P,0.01 and *P,0.001. (C) The effect of GSPs (60 and 80 mg/mL) on apoptosis of A549 and H1299 cells was determined after 48 h in the absence or presence of 60 mmol/L of the caspase-3 inhibitor (z-DEVD-fmk). The content of apoptotic cells was determined using cell death detection ELISA kit, as detailed in Materials and Methods. The cells treated with z-DEVD-fmk blocked the GSPs- induced apoptosis in A549 and H1299 cells. The percentage of apoptotic cells in different treatment groups was summarized and data are presented as mean 6 SD from two repeated experiments. Significant inhibition by caspase-3 inhibitor versus GSPs alone treated cells, *P ,0.001. CI = caspase-3 inhibitor, C = control, without any treatment. doi:10.1371/journal.pone.0027444.g003 was determined following the treatment with GSPs for 48 h. As summarized in Figure 4, (Panels A–D), treatment of A549 cells with GSPs resulted in a significant higher number of cells in the G1 phase at all the concentrations used: 20 mg/mL (58.3%, P,0.05), 40 mg/mL (67.9%, P,0.001) and 60 mg/mL (75.5%, P,0.001) compared to the non-GSPs treated control (52.7%). As indicated in Figure 4 (Left panels A–D), the dose-dependent effect of GSPs on G1 arrest in A549 cells was largely at the expense of cells in S phase with a minimal change in G2-M cell population compared with the non-GSPs-treated control cells (Panel A). Similar effect of GSPs on G1 phase arrest was also found in H1299 cells (Figure 4, right panels A–D). The results of cell cycle PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27444