Proanthocyanidins Inhibit Growth of Lung Cancer are summarized and presented as mean 6SD of 6–7 tumor samples from each group. Statistical significance versus non-GSPs-fed controls, {P,0.01; *P,0.001. doi:10.1371/journal.pone.0027444.g007 to the activation of caspase-3 which is one of the key-mediators of apoptosis [15,35]. GSPs also result in an increase in the release of cytochrome c and Smac/DIABLO in cytosols (Figure 2), activa- tion of caspase-9 and finally an increase in the activation or cleavage of caspase-3 and PARP (Figure 3). The involvement of GSPs-induced increase in activation of caspase-3 and its effect on apoptosis was further confirmed by measuring its activity and induction of apoptotic cell death by cell death detection ELISA. The blockade of GSPs-induced apoptosis in A549 and H1299 cells by the addition of the caspase-3 inhibitor (z-DEVD-fmk) Figure 7. Administration of GSPs by gavage induces apoptosis in tumor xenograft cells. (A) The data on terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL)-positive cells in tumor xenograft tissues from GSPs-treated and GSPs-untreated samples are summarized. (B) GSPs inhibit the expression of anti-apoptotic proteins while increases the expression of pro-apoptotic protein in A549 and H1299 xenograft tissues. (C) GSPs enhance the activation of caspase-9, caspase-3 and PARP proteins in tumor xenografts. Tumor lysates were prepared from the tumors collected at the termination of the experiment and subjected to western blot analysis, as described in Materials and methods. Representative blots are presented from the independent experiments from at least six tumors from six different mice per group with identical observations. (D) Analysis of PCNA- positive cells for proliferation index. Immunohistochemical data in terms of percentage of positive cells (TUNEL-positive or PCNA-positive) Figure 8. Effect of dietary GSPs on G1 phase cell cycle regulatory proteins in xenograft tissues. The A549 tumor xenograft tissues were harvested at the termination of the experiment, and tumor lysates were subjected to the analyses of cell cycle proteins of G1 phase using Western blot analysis, as described in Materials and Methods. Effect of GSPs was determined on the following: (A) the expression of cyclin D1, cyclin D2 and cyclin E, and the expression levels of Cdk2, Cdk4 and Cdk6; and (B) the expression of Cdk inhibitory proteins, Kip1/p27 and Cip1/p21. b-actin was used to verify equal loading of the samples. Representative blots are presented from the independent experiments from at least six tumors from six different mice per group with identical expression levels of proteins. doi:10.1371/journal.pone.0027444.g008 PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e27444