Proanthocyanidins Inhibit Growth of Lung Cancer Figure 1. GSPs induce apoptosis in non-small cell lung cancer cells. (A) In vitro treatment of A549 and H1299 cells with GSPs induces apoptosis in a dose-dependent manner. Apoptotic cell death was analyzed using FACS analysis, and total percentage of apoptotic cells in A549 and H1299 cells are summarized as mean 6 SD, n = 3. Significant difference versus non-GSPs-treated controls: *P,0.05; "P,0.01; {P,0.001. (B) Treatment of A549 and H1299 cells with GSPs results in a dose-dependent reduction in the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xl while increasing the expression of the pro-apoptotic protein, Bax, as determined by western blot analysis. Data are representative of three independent experiments with similar results. doi:10.1371/journal.pone.0027444.g001 and subsequently leads to apoptotic cell death. To explore the effects of GSPs on this process in the A549 and H1299 cells, cytosolic and mitochondrial fractions were prepared from cells that had been treated with GSPs for 48 h. The results of western blot analysis revealed that treatment of GSPs resulted in a dose- dependent increase in cytochrome c and Smac/DIABLO release into the cytosol and a corresponding reduction in the levels of cytochrome c and Smac/DIABLO in the mitochondrial fractions in both A549 and H1299 cells (Fig. 2A and 2B), thus suggesting the loss of the mitochondrial membrane potential on GSPs treatment in these cells. The blots were stripped and re-probed with anti-COX IV antibody to rule out the possibility of cross- contamination of mitochondrial and cytosolic fractions. The presence of COX IV was not detected in cytosolic fractions. To further verify that GSPs induce a loss of mitochondrial membrane potential, we used the cationic lipophilic dye, JC-1, which accumulates within mitochondria in a potential-dependent manner. On disruption of the mitochondrial membrane potential, the fluorescence emission of JC-1 dye changes from red to green. Forty-eight h after the addition of the GSPs, the A549 and H1299 cells were harvested, incubated with JC-1 dye and the fluorescence emission analyzed using flow cytometry. As shown in Figure 2C, GSP treatment of A549 and H1299 cells resulted in a dose- dependent increase in the numbers of green-fluorescence-positive cells, as shown in the lower right (LR) quadrant of the FACS histogram. Collectively, these studies suggest that GSPs treatment induce apoptosis of both A549 and H1299 lung carcinoma cells through disruption of the mitochondrial membrane potential. GSPs induce the activation of caspases and PARP in both A549 and H1299 cells The release of the cytochrome c and Smac/DIABLO into cytosol activates procaspase 9 in the apoptosome and leads to active caspase-9 cleavage and cleavage of caspase-3 [18–20]. Activation of caspase-3 subsequently leads to apoptotic cell death through cleavage of a broad spectrum of target proteins including PARP. Therefore, we determined whether induction of apoptosis of A549 and H1299 cells by GSPs is mediated through the activation of procaspase-9, caspase-3 and PARP proteins. Treatment of A549 and H1299 cells with GSPs for 48 h resulted in a dose-dependent reduction in the levels of caspase-9 while increasing the cleavage of caspases-9, caspase-3 and PARP proteins compared with the vehicle-treated cells (Figure 3A). The GSPs-induced activation of caspase-3 in A549 and H1299 cells also was determined using a colorimetric caspase-3 activity assay. Treatment of both A549 and H1299 lung cancer cells with GSPs for 48 h resulted in a significantly higher (p,0.05; p,0.001) caspase-3 activity in a dose-dependent manner than that observed in the vehicle-treated control cells (Figure 3B). Treatment with the caspase-3 inhibitor (z-DEVD-fmk) blocks GSPs-induced apoptosis of both A549 and H1299 cells To further confirm that GSPs-induced activation of caspase-3 is involved in the induction of apoptotic cell death of A549 and H1299 human lung cancer cells, we determined whether the PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27444