Proanthocyanidins Inhibit Growth of Lung Cancer Figure 6. Administration of GSPs by gavage inhibits the growth of lung tumor xenografts in mice. Each mouse was s.c. implanted with either 26106 A549 or H1299 cells mixed with Matrigel on the right flank. Twenty-four h later, mice were given either PBS (100 mL) or GSPs (50, 100 or 200 mg/kg body weight in 100 mL PBS) by gavage 5 days/week. (A) Change in body weight of mice during the 8 weeks study was monitored. The body weights of the control and GSPs-fed mice did not differ significantly throughout the experiment protocol. (B) Average tumor volume 6SD/ mouse (mm3) in each group. (C) Tumors were harvested at the termination of the experiment, and the wet weight of the tumor/mouse in grams in each group is reported as mean 6 SD. Statistical significance of difference between control and GSPs-fed groups was analyzed by one-way ANOVA followed by Bonferroni t test. n = 10. Statistical significance vs non-GSPs-fed controls, "P,0.05; {P,0.01; *P,0.005. doi:10.1371/journal.pone.0027444.g006 proliferation and induction of apoptosis, while these effects of GSPs were not observed in normal human bronchial epithelial cells [11,12]. Induction of apoptosis is considered as one of the possible mechanisms of inhibition of cancer development, and many therapeutic agents have been shown to act through the induction of apoptosis in their inhibition or blockade of the carcinogenic process [27–29]. Some evidence indicates that apoptosis may represent a protective mechanism against neoplastic development by eliminating genetically damaged cells or those cells that have improperly been induced to proliferate by factors such as carcinogens [30,31]. Experimental evidence suggests that apoptosis can be mediated by several different pathways and that there are numerous regulatory molecules associated with these pathways. The proteins of the Bcl-2 family include both pro- and anti-apoptotic members that elicit opposing effects on mitochondria [15,32,33]. Enhance- ment of pro-apoptotic Bax over Bcl-2 proteins can enhance the permeability of the mitochondrial membrane, which in turn results in the release of apoptogenic factors. Repression of anti-apoptotic members of this family, including Bcl-2 and Bcl-xl, preserves the integrity of the mitochondria. This blocks the release of soluble inter-membrane factors such as cytochrome c that activate the effectors of apoptosis [34]. In this study, we found that treatment of the NSCLC cells with GSPs up-regulate the pro-apoptotic Bax protein and down-regulate the anti-apoptotic proteins Bcl-2 and Bcl-xl and that this occurs in both A549 and H1299 cells (Figure 1). The alteration in the Bax/Bcl-2 ratio is critical for apoptosis and causes the release of cytochrome c and Smac/DIABLO from mitochondria into the cytosol after the loss of mitochondrial membrane potential [15,16]. Cytosolic cytochrome c can bind to Apaf-1 and activate caspase-9 in the apoptosomes in response to diverse inducers of cell death [17,35]. Activation of caspase-9 leads PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27444