vitro and in vivo tumor xenografts, which was associated with their inhibitory effects on the cyclooxygenase-2 expression and production of its prostaglandin metabolite, PGE2 (12). In contrast, a significant inhibition of cell proliferation and induction of apoptosis in normal human bronchial epithelial cells after GSPs treatment under identical conditions was not observed [11,12]. In spite of anti-carcinogenic effects of GSPs on NSCLC cells, a precise mechanism of the inhibitory effect on the NSCLC cell growth and apoptosis by GSPs is not well understood. In the present communication, we conducted a comprehensive investi- gation on the mechanism responsible for the inhibition of lung cancer cell proliferation and apoptosis using A549 and H1299 cell lines as an in vitro cell culture model and in vivo tumor xenograft model. To study the in vivo effect of GSPs on tumor xenograft growth, GSPs was given to mice by oral gavage 5 days/week. We report that GSPs-induced apoptotic cell death of NSCLC cells is mediated through modulations in the expression levels of pro- and anti-apoptotic proteins, loss of mitochondrial membrane potential and caspase-3 activation pathways. GSPs also checked the deregulated cell cycle progression and associated regulatory proteins in NSCLC cells. Thus our studies provide insight into the mechanism by which GSPs induce apoptosis in these cells. Additionally, our results provide a convincing rationale for the pharmacological activity of GSPs against human non-small cell lung cancer cells. Materials and Methods Reagents, chemicals and antibodies The GSPs used in this study were obtained from Kikkoman Corporation (Noda, Japan). The JC-1 Mitochondrial Membrane Potential Detection Kit, and Anti-OxPhos Complex IV subunit IV (Cox IV) were purchased from Molecular Probes, Inc. (Eugene, OR). Cell Death Detection ELISA Kits were obtained from Roche Diagnostic Corporation (Indianapolis, IN). The primary antibod- ies were purchased as follows: antibodies for Bcl-2, Bcl-xl, Bax, caspase-9, cleaved caspase-9 and -3, anti-poly(ADP-ribose) polymerase (PARP) and b-actin were purchased from Cell Signaling Technology (Beverly, MA); antibodies for cytochrome c, Smac/DIABLO, Cyclin D1, Cyclin D2, Cyclin E, Cdk2, Cdk4, Cdk6, Cip1/p21, Kip1/p27 and the secondary antibodies, horseradish peroxidase-linked antimouse immunoglobulin G and anti-rabbit immunoglobulin G were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Caspase-3-specific inhibitor (z-DEVD-fmk) was purchased from Calbiochem (San Diego, CA). ApoTarget Kit specific to caspase-3 activity assay was obtained from BioSource International, Inc. (San Diego, CA). Ham’s F-12, RPMI 1640, penicillin, streptomycin and trypsin/EDTA were obtained from Cellgro (Herndon, VA). The enhanced chemilu- minescence western blotting detection reagents were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Cell culture and cell lines Human non-small cell lung carcinoma cell lines, A549 and H1299, and normal human bronchial epithelial cells were purchased from the American Type Culture Collection (Manassas, VA). The A549 and H1299 cell lines were cultured as monolayers in Ham’s F-12 and RPMI 1640 culture medium, respectively, supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 100 mg/mL penicillin, and 100 mg/mL streptomycin and maintained in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37uC. The GSPs were dissolved in a small amount of dimethylsulfoxide [DMSO, maximum concentration, 0.1% (v/v)], which was then added to Proanthocyanidins Inhibit Growth of Lung Cancer complete cell culture medium prior to addition to subconfluent cells (60–70% confluent). Cells treated with vehicle only (DMSO, 0.1% in media) served as control. Analysis of apoptotic cell death by ELISA Cells were treated with GSPs for 48 h and thereafter harvested. GSPs-induced apoptosis was determined using the Cell Death Detection ELISA Kit (Roche Diagnostics, Palo Alto, CA), which quantifies cytoplasmic histone-associated DNA fragments in the form of mononucleosomes or oligonucleosomes, following the manufacturer’s instructions. Caspase-3 activity assay The activity of caspase-3 in cell lysates was measured using the colorimetric protease assay ApoTarget Kit (BioSource Interna- tional, Inc., CA) following the manufacturer’s protocol. Briefly, A549 or H1299 cells were treated with GSPs (20, 40, 60 and 80 mg/mL) for 48 h. Thereafter, the cells were harvested using short trypsinization and cell lysates prepared following the manufacturer’s protocol. Samples of the cell lysates (100 mg protein per sample) were mixed with reaction buffer and 200 mmol/L substrate (DEVD-pNA for caspase-3) and incubated for 3 h at 37uC in the dark. The absorbance was then measured at 405 nm and the sample readings calculated by subtracting the absorbance of blank samples. DNA cell cycle analysis Sub-confluent cells (50–60%) were treated with varying concentrations of GSPs in complete medium for 48 h. The cells were then harvested, washed with cold PBS, and processed for cell cycle analysis, as detailed previously [13,14]. Briefly, the cells (16105) were re-suspended in 50 mL cold PBS to which 450 mL cold methanol was added and the cells were then incubated for 1 h at 4uC. The cells were centrifuged at 1,100 rpm for 5 minutes; the pellet was washed with cold PBS, re-suspended in 500 mL PBS, and incubated with 5 mL RNase (20 mg/mL final concentration) for 30 minutes. The cells were incubated with propidium iodide (50 mg/mL) on ice for 1 h in the dark. The cell cycle distribution of the cells was then determined using FACSCalibur instrument (BD Biosciences, San Jose, CA) equipped with CellQuest 3.3 software in the Fluorescence-activated Cell Sorting (FACS) machine at the Core Facility of the UAB Comprehensive Cancer Center. Immunoprecipitation and western blot analysis Following treatment of A549 and H1299 cells with GSPs, the cells were harvested, washed with cold PBS and lysed with ice-cold lysis buffer supplemented with protease inhibitors, as detailed previously [12,13]. For immunoblotting of cytochrome c, Smac/ DIABLO and Cox IV, mitochondrial and cytosolic fractions were prepared from the cells and the proteins were resolved on 10% Tris-Glycine gels and transferred onto a nitrocellulose membrane. After blocking the non-specific binding sites, the membrane was incubated with the desired primary antibody at 4uC overnight. The membrane was then incubated with appropriate peroxidase- conjugated secondary antibody and the immunoreactive bands were visualized using the enhanced chemiluminescence reagents. Each membrane was stripped and re-probed with anti-b-actin antibody to ensure equal protein loading. For Cdk inhibitor (Cdki)-Cdk binding assay, A549 cells were treated with vehicle or 60 mg/mL GSPs for 48 h, washed with ice- cold PBS, and whole cell lysates prepared as described previously [13]. Aliquots containing 200 mg of protein were cleared with PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27444