protein A/G-plus agarose beads (Santa Cruz, CA). Cip1/p21 and Kip1/p27 proteins were immunoprecipitated from whole cell lysates using specific antibodies after incubation for 8 h followed by the addition of protein A/G-plus agarose beads (50 mL, Santa Cruz, CA) and continued incubation overnight at 4uC. Immuno- precipitates were washed, and subsequently subjected to western blot analysis using Tris-Glycine gels followed by immunoblotting using Cdk2, Cdk4 and Cdk6 antibodies. Assay for mitochondrial membrane potential The change in the mitochondrial membrane potential in lung cancer cells after treatment with GSPs was determined by flow cytometry using the fluorescent lipophilic cationic probe JC-1 (5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolcarbocyanine iodide) Detection Kit following the instructions of the manufacturer, and as described by us previously [13,14]. JC-1 accumulates selectively within intact mitochondria to form multimer J-aggregates emitting fluorescence light at 590 nm. The monomeric form emits light at 527 nm after excitation at 490 nm. Thus, the color of the dye changes from orange to green, depending on the mitochondrial membrane potential, and can be analyzed by FACS with green fluorescence in channel 1 (FL1) and orange emission in channel 2 (FL2). Animals and tumor xenograft study Female athymic nude mice (4–5 weeks-old) were purchased from National Cancer Institute (Frederick, MD), housed in accordance with the Institutional Animal Care and Use Commit- tee guidelines, and provided with sterilized AIN76A diet and water ad libitum. All mice were maintained under standard conditions of a 12-h dark/12-h light cycle, a temperature of 2462uC, and relative humidity of 50610%. The animal protocol used in this study was approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham, and approved Animal Protocol Number is: 101109267. To determine the in vivo efficacy of GSPs against human lung cancer tumor xenograft growth, exponentially growing A549 and H1299 cells (26106) were mixed at a 1:1 ratio with Matrigel (Becton Dickinson, Bedford, MA), and a 100 mL suspension containing 26106 cells was injected s.c. in the right flank of each mouse. After 24 h, mice were randomly divided in four groups (n = 10). Experimental animals were treated by oral gavage with 50, 100 or 200 mg GSPs/kg body weight/day in 100 mL of PBS five-days a week (Monday through Friday) beginning one day after tumor cell implantation. Control mice received an equal volume of PBS. The experiment was terminated 58 days after tumor cell inoculation. Tumor growth was monitored 2-times per week. Body weight/mouse and diet consumption/mouse were recorded regularly throughout the experiment. Animals were also moni- tored if they become ill or suffering (lack normal grooming and avoidance behaviors) during the entire experiment period. At the termination of the experiment, the whole tumor mass was harvested, weighed, and a part of the tumor was used for the preparation of tumor lysates to analyze the expression levels of different proteins of interest and remaining part was used for immunohistochemical analysis. Immunohistochemical detection of PCNA-positive and TUNEL-positive cells Five-mm thick tumor sections were deparaffinized and rehy- drated in a graded series of alcohols. Following rehydration, an antigen retrieval process was performed by placing the slides in 10 mM sodium citrate buffer, pH 6.0 at 95uC for 20 minutes Proanthocyanidins Inhibit Growth of Lung Cancer followed by 20-minutes cooling. The sections were washed in PBS and non-specific binding sites were blocked with 1% BSA with 2% goat serum in PBS prior to incubation with anti-PCNA antibodies for 2 h at room temperature. After washing, the sections were incubated with biotinylated secondary antibody for 45 min followed by HRP-conjugated streptavidin, washing in PBS, incubation with diaminobenzidine substrate, and counterstaining with hematoxylin. Apoptotic cell death in tumor tissues was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. The numbers of PCNA- positive and TUNEL-positive cells were detected and counted using a light microscope. The results are presented as the number of positive cells x 100/total number of cells. Statistical analysis The results from the in vivo studies are representative of at least 2–3 independent experiments. The statistical significance of difference between control and GSPs-treated groups were calculated by Student’s t test (Sigma Stat 2.03, Jandel Scientific, San Rafael, CA). All quantitative data are shown as mean 6 SD. In tumor xenograft study, the statistical significance of difference between control and GSPs-treated groups was determined by ANOVA followed by Bonferroni t test, and in each case P,0.05 was considered statistically significant. Results NSCLC cells are sensitive to GSPs-induced apoptosis We have shown that treatment of A549 and H1299 NSCLC cells with various concentrations of GSPs inhibits the growth or proliferation potential of these cells in a dose- and time-dependent manner. GSPs also induced apoptotic cell death of these cells in a concentration dependent manner [11,12]. The GSPs-induced apoptosis of A549 and H1299 cells after treatment for 48 h using FACS analysis is summarized in Figure 1A. However, a precise mechanism of anti-apoptotic effects of GSPs against NSCLC cells is not clearly understood. Therefore, the studies were conducted to determine the precise mechanism involved in apoptotic cell death of NSCLC cells by the treatment with GSPs. GSPs reduce the expression of the anti-apoptotic proteins Bcl-xl and Bcl-2 while increasing the expression of pro-apoptotic protein Bax in A549 and H1299 cells The proteins of the Bcl-2 family play a major role in regulation of apoptosis by functioning as promoters (e.g., Bax) or inhibitors (Bcl-2 or Bcl-xl) of cell death [15–17]. Using western blot analysis, we found that treatment of A549 and H1299 cells with GSPs for 48 h resulted in a dose-dependent reduction in the levels of the anti-apoptotic proteins Bcl-xl and Bcl-2, and an increase in the levels of the pro-apoptotic protein, Bax, as compared with the vehicle-treated control cells (Figure 1 B). Thus, treatment of these lung cancer cells with GSPs can alter the protein levels of key members of the Bcl-2 family in a manner that favors an increase in the ratio of Bax: Bcl-2, which may contribute to the susceptibility of cancer cells to GSP-induced apoptosis [15]. GSPs induce loss of mitochondrial membrane potential and a subsequent enhancement of the release of cytochrome c and Smac/DIABLO in NSCLC cells Loss of mitochondrial membrane potential has been linked to the initiation and activation of apoptotic process in cells [15,16]. The release of cytochrome c and Smac/DIABLO from mito- chondria into the cytosol contributes to the activation of caspases PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27444