Proanthocyanidins Inhibit Growth of Lung Cancer Figure 2. GSPs disrupt mitochondrial membrane potential in non-small cell lung cancer cells. (A & B) Immunoblotting for cytochrome c and Smac/DIABLO using cytosolic and mitochondrial fractions prepared from A549 and H1299 cells following treatment with indicated concentrations of GSPs for 48 h. The blots were stripped and re-probed with anti-COX IV antibody to ensure equal mitochondrial protein loading as well as to rule out cross-contamination of mitochondrial and cytosolic fractions. (C) Treatment of A549 and H1299 cells with GSPs induces loss of mitochondrial membrane potential. Cells were treated with indicated doses of GSPs for 48 h. JC-1 dye-stained cell were analyzed by flow cytometry, as described in Materials and Methods. Data are representative of two separate experiments with identical observations. doi:10.1371/journal.pone.0027444.g002 GSPs-induced apoptosis of A549 and H1299 cells was affected by the addition of the caspase-3-specific inhibitor (z-DEVD-fmk). A549 and H1299 cells had been treated with GSPs with or without z-DEVD-fmk (60 mmol/L) for 48 h. Cells were harvested and cell death was assayed using Cell Death Detection ELISA kit following the instructions of the manufacturer. As shown in Figure 3C (left panel), treatment of A549 cells with GSPs at the concentrations of 60 and 80 mg/mL resulted in 31% and 49% apoptosis or cell death respectively compared to 6.5% in non-GSPs-treated control cells. The treatment of A549 cells with GSPs (60 or 80 mg/mL) in the presence of z-DEVD-fmk resulted in only 14% and 17% apoptosis, respectively, which clearly indicated that the presence of caspase-3-specific inhibitor significantly blocked GSPs-induced apoptotic cells death (P,0.001) in A549 cells. Similar results were found when H1299 cells were treated with GSPs in the presence or absence of z-DEVD-fmk, as shown in Figure 3C (right panel). These results indicate that GSPs-induced apoptosis of both A549 and H1299 human lung cancer cells is associated with the activation of caspase-3. GSPs induce G1 phase cell cycle arrest in NSCLC cells Based on the preliminary studies where we observed a strong growth inhibitory effect of GSPs on NSCLC cells, we then determined the possible mechanism of anti-proliferative activity of GSPs which may lead to apoptotic cell death. For this purpose the effect of GSPs on cell cycle progression in A549 and H1299 cells PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27444