Manjinder Kaur

Anticancer and cancer chemopreventive potential of grape seed extract and other grape-based products.

With emerging trends in the incidence of cancer of various organ sites, additional approaches are needed to control human malignancies. Intervention or prevention of cancer by dietary constituents, a strategy defined as chemoprevention, holds great promise in our conquest to control cancer, because it can be implemented on a broader population base with less economic burden. Consistent with this, several epidemiological studies have shown that populations that consume diets rich in fruits and vegetables have an overall lower cancer incidence. Based on these encouraging observations, research efforts from across the globe have focused on identifying, characterizing, and providing scientific basis to the efficacy of various phytonutrients in an effort to develop effective strategy to control various human malignancies. Cancer induction, growth, and progression are multi-step events and numerous studies have demonstrated that various dietary agents interfere with these stages of cancer, thus blocking malignancy. Fruits and vegetables represent untapped reservoir of various nutritive and nonnutritive phytochemicals with potential cancer chemopreventive activity. Grapes and grape-based products are one such class of dietary products that have shown cancer chemopreventive potential and are also known to improve overall human health. This review focuses on recent advancements in cancer chemopreventive and anticancer efficacy of grape seed extract and other grape-based products. Overall, completed studies from various scientific groups conclude that both grapes and grape-based products are excellent sources of various anticancer agents and their regular consumption should thus be beneficial to the general population.

Grape seed extract inhibits in vitro and in vivo growth of human colorectal carcinoma cells.

Purpose: Accumulating evidences suggest the beneficial effects of fruit-and-vegetable consumption in lowering the risk of various cancers, including colorectal cancer. Herein, we investigated the in vitro and in vivo anticancer effects and associated mechanisms of grape seed extract (GSE), a rich source of proanthocyanidins, against colorectal cancer. Experimental Design: Effects of GSE were examined on human colorectal cancer HT29 and LoVo cells in culture for proliferation, cell cycle progression, and apoptosis. The in vivo effect of oral GSE was examined on HT29 tumor xenograft growth in athymic nude mice. Xenografts were analyzed by immunohistochemistry for proliferation and apoptosis. The molecular changes associated with the biological effects of GSE were analyzed by Western blot analysis. Results: GSE (25-100 μg/mL) causes a significant dose- and time-dependent inhibition of cell growth with concomitant increase in cell death. GSE induced G1 phase cell cycle arrest along with a marked increase in Cip1/p21 protein level and a decrease in G1 phase–associated cyclins and cyclin-dependent kinases. GSE-induced cell death was apoptotic and accompanied by caspase-3 activation. GSE feeding to mice at 200 mg/kg dose showed time-dependent inhibition of tumor growth without any toxicity and accounted for 44% decrease in tumor volume per mouse after 8 weeks of treatment. GSE inhibited cell proliferation but increased apoptotic cell death in tumors. GSE-treated tumors also showed enhanced Cip1/p21 protein levels and poly(ADP-ribose) polymerase cleavage. Conclusions: GSE may be an effective chemopreventive agent against colorectal cancer, and that growth inhibitory and apoptotic effects of GSE against colorectal cancer could be mediated via an up-regulation of Cip1/p21.

Gallic Acid, an Active Constituent of Grape Seed Extract, Exhibits Anti-proliferative, Pro-apoptotic and Anti-tumorigenic Effects Against Prostate Carcinoma Xenograft Growth in Nude Mice

PurposeGallic acid, a natural agent present in a wide-range of fruits and vegetables, has been of potential interest as an anti-cancer agent; herein, we evaluated its efficacy in androgen-independent DU145 and androgen-dependent-22Rv1 human prostate cancer (PCa) cells.Materials and MethodsCell viability was determined by MTT and apoptosis by Annexin V-PI assays. In vivo anti-cancer efficacy was assessed by DU145 and 22Rv1 xenograft growth in nude mice given normal drinking water or one supplemented with 0.3% or 1% (w/v) gallic acid. PCNA, TUNEL and CD31 immunostaining was performed in tumor tissues for in vivo anti-proliferative, apoptotic and anti-angiogenic effects of gallic acid.ResultsGallic acid decreased cell viability in a dose-dependent manner in both DU145 and 22Rv1 cells largely via apoptosis induction. In tumor studies, gallic acid feeding inhibited the growth of DU145 and 22Rv1 PCa xenografts in nude mice. Immunohistochemical analysis revealed significant inhibition of tumor cell proliferation, induction of apoptosis, and reduction of microvessel density in tumor xenografts from gallic acid-fed mice as compared to controls in both DU145 and 22Rv1 models.ConclusionTaken together, our findings show the anti-PCa efficacy of gallic acid and provide a rationale for additional studies with this naturally-occurring agent for its efficacy against PCa.

p21 and p27 induction by silibinin is essential for its cell cycle arrest effect in prostate carcinoma cells

Recent studies have shown that silibinin induces p21/Cip1 and p27/Kip1 and G1 arrest in different prostate cancer cells irrespective of p53 status; however, biological significance and mechanism of such induction have not been studied. Here, using two different prostate cancer cell lines DU145 and 22Rv1, representing androgen-independent and androgen-dependent stages of malignancy, first we investigated the importance of p21 and p27 induction in silibinin-mediated G1 arrest. Silencing p21 and p27 individually by RNA interference showed marked reversal in G1 arrest; however, their simultaneous ablation showed additional reversal of G1 arrest in 22Rv1 but not DU145 cells. These results suggest that whereas relative importance of these molecules might be cell line specific, their induction by silibinin is essential for its G1 arrest effect. Next, studies were done to examine mechanisms of their induction where cycloheximide-chase experiments showed that silibinin increases p21 and p27 protein half-life. This effect was accompanied by strong reduction in Skp2 level and its binding with p21 and p27 together with strong decrease in phosphorylated Thr187 p27 without considerable change in proteasomal activity, suggesting a posttranslational mechanism. Skp2 role was further elucidated using Skp2-small interfering RNA–transfected cells, where decreased G1 arrest and attenuated Cip/Kip induction were observed with silibinin treatment. Further, silibinin caused a marked increase in p21 and p27 mRNA levels together with an increase in their promoter activity, also indicating a transcriptional mechanism. Together, our results for the first time identify a central role of p21 and p27 induction and their regulatory mechanism in silibinin-mediated cell cycle arrest. [Mol Cancer Ther 2007;6(10):2696–707]

Grape seed extract induces cell cycle arrest and apoptosis in human colon carcinoma cells.

One approach to control colorectal cancer (CRC) is its preventive intervention by dietary agents or those consumed as supplements. However, because most of these products are often consumed by patients as an complementary and alternative medicine practice, a scientific base such as efficacy, mechanism, and standardized preparation needs to be developed. Grape seed extract (GSE) is one such supplement widely consumed by humans for its several health benefits. We reported recently that GSE inhibits CRC cell HT29 growth in culture and nude mice xenograft. Because GSE is available commercially through different vendors, here we assessed whether GSE from 2 different manufacturers produces comparable biological effects in a panel of human CRC cell lines. Our results show that irrespective of source, GSE strongly inhibits LoVo, HT29, and SW480 cell growth, with a G1 arrest in LoVo and HT29 cells but an S and/or G2/M arrest in SW480 cell cycle progression. GSE also induced Cip/p21 levels in all 3 cell lines. Furthermore, an induction of apoptosis was observed in all 3 cell lines by GSE. Taken together, our findings suggest that GSE could be an effective CAM agent against CRC possibly due to its strong growth inhibitory and apoptosis-inducing effects.

Grape seed extract induces anoikis and caspase-mediated apoptosis in human prostate carcinoma LNCaP cells: possible role of ataxia telangiectasia mutated–p53 activation

Prostate cancer is the second leading cancer diagnosed in elderly males in the Western world. Epidemiologic studies suggest that dietary modifications could be an effective approach in reducing various cancers, including prostate cancer, and accordingly cancer-preventive efficacy of dietary nutrients has gained increased attention in recent years. We have recently shown that grape seed extract (GSE) inhibits growth and induces apoptotic death of advanced human prostate cancer DU145 cells in culture and xenograft. Because prostate cancer is initially an androgen-dependent malignancy, here we used LNCaP human prostate cancer cells as a model to assess GSE efficacy and associated mechanisms. GSE treatment of cells led to their detachment within 12 hours, as occurs in anoikis, and caused a significant decrease in live cells mostly due to their apoptotic death. GSE-induced anoikis and apoptosis were accompanied by a strong decrease in focal adhesion kinase levels, but an increase in caspase-3, caspase-9, and poly(ADP-ribose) polymerase cleavage; however, GSE caused both caspase-dependent and caspase-independent apoptosis as evidenced by cytochrome c and apoptosis-inducing factor release into cytosol. Additional studies revealed that GSE causes DNA damage–induced activation of ataxia telangiectasia mutated kinase and Chk2, as well as p53 Ser15 phosphorylation and its translocation to mitochondria, suggesting this to be an additional mechanism for apoptosis induction. GSE-induced apoptosis, cell growth inhibition, and cell death were attenuated by pretreatment with N-acetylcysteine and involved reactive oxygen species generation. Together, these results show GSE effects in LNCaP cells and suggest additional in vivo efficacy studies in prostate cancer animal models. [Mol Cancer Ther 2006;5(5):1265–74]

Chemoprevention of Intestinal Tumorigenesis in APCmin/+ Mice by Silibinin

Chemoprevention is a practical and translational approach to reduce the risk of various cancers including colorectal cancer (CRC), which is a major cause of cancer-related deaths in the United States. Accordingly, here we assessed chemopreventive efficacy and associated mechanisms of long-term silibinin feeding on spontaneous intestinal tumorigenesis in the APC(min/+) mice model. Six-week-old APC(min/+) mice were p.o. fed with vehicle control (0.5% carboxymethyl cellulose and 0.025% Tween 20 in distilled water) or 750 mg silibinin/kg body weight in vehicle for 5 d/wk for 13 weeks and then sacrificed. Silibinin feeding strongly prevented intestinal tumorigenesis in terms of polyp formation in proximal, middle, and distal portions of small intestine by 27% (P < 0.001), 34% (P < 0.001), and 49% (P < 0.001), respectively. In colon, we observed 55% (P < 0.01) reduction in number of polyps by silibinin treatment. In size distribution analysis, silibinin showed significant decrease in large-size polyps (>3 mm) by 66% (P < 0.01) and 88% (P < 0.001) in middle and distal portions of small intestine, respectively. More importantly, silibinin caused a complete suppression in >3 mm sized polyps and 92% reduction in >2 to 3 mm sized polyps in colon. Molecular analyses of polyps suggested that silibinin exerts its chemopreventive efficacy by inhibiting cell proliferation, inflammation, and angiogenesis; inducing apoptosis; decreasing beta-catenin levels and transcriptional activity; and modulating the expression profile of cytokines. These results show for the first time the efficacy and associated mechanisms of long-term p.o. silibinin feeding against spontaneous intestinal tumorigenesis in the APC(min/+) mice model, suggesting its chemopreventive potential against intestinal cancers including CRC.

Silibinin suppresses growth and induces apoptotic death of human colorectal carcinoma LoVo cells in culture and tumor xenograft

Colorectal cancer is one of the leading causes of cancer-related morbidity and mortality. The use of nontoxic phytochemicals in the prevention and intervention of colorectal cancer has been suggested as an alternative to chemotherapy. Here we assessed the anticancer efficacy of silibinin against advanced colorectal cancer LoVo cells both in vitro and in vivo. Our results showed that silibinin treatment strongly inhibits the growth of LoVo cells (P < 0.05-0.001) and induces apoptotic death (P < 0.01-0.001), which was associated with increased levels of cleaved caspases (3 and 9) and cleaved poly(ADP-ribose) polymerase. Additionally, silibinin caused a strong cell cycle arrest at G1 phase and a slight but significant G2-M-phase arrest at highest concentration (P < 0.01-0.001). Molecular analyses for cell cycle regulators showed that silibinin decreases the level of cyclins (D1, D3, A and B1) and cyclin-dependent kinases (1, 2, 4, and 6) and increases the level of cyclin-dependent kinase inhibitors (p21 and p27). Consistent with these results, silibinin treatment also decreased the phosphorylation of retinoblastoma protein at Ser780, Ser795, and Ser807/Ser811 sites without significantly affecting its total level. In animal studies, oral administration of silibinin for 6 weeks (at 100 and 200 mg/kg/d for 5 days/wk) significantly inhibited the growth of LoVo xenograft (P < 0.001) in athymic nude mice without any apparent toxicity. Analyses of xenograft tissue showed that silibinin treatment inhibits proliferation and increases apoptosis along with a strong increase in p27 levels but a decrease in retinoblastoma phosphorylation. Together, these results suggest the potential use of silibinin against advanced human colorectal cancer. [Mol Cancer Ther 2009;8(8):2366–74]

Grape seed extract upregulates p21 (Cip1) through redox-mediated activation of ERK1/2 and posttranscriptional regulation leading to cell cycle arrest in colon carcinoma HT29 cells.

Abnormalities in cell cycle progression provide unlimited replicative potential to cancer cells, and therefore targeting of key cell cycle regulators could be a sound cancer chemopreventive strategy. Earlier, we found that grape seed extract (GSE) increases Cip/p21 protein level and inhibits growth and induces apoptosis in human colon carcinoma HT29 cells both in vitro and in vivo. However, the mechanism of GSE‐induced p21 upregulation and its role in biological efficacy of GSE are not known, which were investigated here. GSE treatment of HT29 cells resulted in a strong dose‐ and time‐dependent phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), consistent with p21 induction. The inhibition of sustained ERK1/2 activation by GSE using pharmacological inhibitors abrogated GSE‐induced p21 upregulation. Furthermore, pretreatment of cells with N‐acetylcysteine inhibited GSE‐induced ERK1/2 phosphorylation as well as p21 upregulation, suggesting the involvement of GSE‐induced oxidative stress as an upstream event. Consistent with this, GSE also decreased intracellular level of reduced glutathione. Next, we determined whether GSE‐induced signaling regulates p21 expression at transcriptional and/or translational levels. GSE was found to increase the stability of p21 message with resultant increase in p21 protein level, but it did not alter the protein stability to a great extent. Importantly, knock‐down of p21 abrogated GSE‐induced G1 arrest suggesting that p21 induction by GSE is essential for its G1 arrest effect. Together, our results for the first time identify a central role of p21 induction and associated mechanism in GSE‐induced cell cycle arrest in HT29 cells. Mol. Carcinog.

The marine alkaloid naamidine A promotes caspase-dependent apoptosis in tumor cells

Apoptosis is important for normal development and removal of damaged cells. Evasion of apoptosis by cancer cells is one of the key characteristics of many tumor types. Thus, discovering agents that promote apoptosis in tumor cells could have great therapeutic value. Marine natural products have demonstrated great potential as anticancer agents, and the proapoptotic activity of some of these products is emerging as a potentially useful property for cancer treatments. Using a tumor xenograft assay in rodents, we previously found that the marine alkaloid naamidine A is a potent antitumor agent. In this study, we further characterize the mechanism of action of naamidine A. In cultured tumor cells, we find that naamidine A induces cell death, which is accompanied with annexin V staining, disruption of the mitochondrial membrane potential, and cleavage and activation of caspases 3, 8, and 9, all of which are hallmarks of apoptosis. Furthermore, naamidine A-induced cell death is caspase dependent. We also find that under conditions where naamidine A inhibits tumor xenograft growth, it induces activation of caspase 3, suggesting that apoptosis is part of its antitumorigenic activity in vivo. Apoptosis is not dependent on extracellular signal-regulated kinase 1/2, previously characterized molecular targets of naamidine A, nor does it require functional p53. Our studies support the continued study of naamidine A and its target(s) for the potential development of better clinical treatments for cancer.