Arshi Malik

Combined inhibitory effects of green tea polyphenols and selective cyclooxygenase-2 inhibitors on the growth of human prostate cancer cells both in vitro and in vivo.

Purpose: Cyclooxygenase-2 (COX-2) inhibitors hold promise for cancer chemoprevention; however, recent toxicity concerns suggest that new strategies are needed. One approach to overcome this limitation is to use lower doses of COX-2 inhibitors in combination with other established agents with complementary mechanisms. In this study, the effect of (−)epigallocatechin-3-gallate (EGCG), a promising chemopreventive agent from green tea, was tested alone and in combination with specific COX-2 inhibitors on the growth of human prostate cancer cells both in vitro and in vivo. Experimental Design: Human prostate cancer cells LNCaP, PC-3, and CWR22Rν1 were treated with EGCG and NS398 alone and in combination, and their effect on growth and apoptosis was evaluated. In vivo, athymic nude mice implanted with androgen-sensitive CWR22Rν1 cells were given green tea polyphenols (0.1% in drinking water) and celecoxib (5 mg/kg, i.p., daily, 5 days per week), alone and in combination, and their effect on tumor growth was evaluated. Results: Combination of EGCG (10-40 μmol/L) and NS-398 (10 μmol/L) resulted in enhanced (a) cell growth inhibition; (b) apoptosis induction; (c) expression of Bax, pro-caspase-6, and pro-caspase-9, and poly(ADP)ribose polymerase cleavage; (d) inhibition of peroxisome proliferator activated receptor γ; and (e) inhibition of nuclear factor-κB compared with the additive effects of the two agents alone, suggesting a possible synergism. In vivo, combination treatment with green tea polyphenols and celecoxib resulted in enhanced (a) tumor growth inhibition, (b) lowering of prostate-specific antigen levels, (c) lowering of insulin-like growth factor-I levels, and (d) circulating levels of serum insulin-like growth factor binding protein-3 compared with results of single-agent treatment. Conclusions: These data suggest synergistic and/or additive effects of combinatorial chemopreventive agents and underscore the need for rational design of human clinical trials.

Green tea polyphenol EGCG sensitizes human prostate carcinoma LNCaP cells to TRAIL-mediated apoptosis and synergistically inhibits biomarkers associated with angiogenesis and metastasis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising candidate for cancer therapy, however, emergence of drug resistance limits its potential use. Here, we report for the first time that epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea, sensitizes TRAIL-resistant LNCaP cells to TRAIL-mediated apoptosis through modulation of intrinsic and extrinsic apoptotic pathways. When combined with EGCG, Apo2L/TRAIL exhibited enhanced apoptotic activity in LNCaP cells characterized by three major molecular events. First, apoptosis induction was accompanied by the upregulation of poly(ADP-ribose) polymerase cleavage and modulation of pro- and antiapoptotic Bcl2 family of proteins. A synergistic inhibition of inhibitors of apoptosis with concomitant increase in caspase cleavage was observed. Second, pretreatment of cells with EGCG resulted in modulation of death-inducing signaling cascade complex involving DR4/TRAIL R1, Fas-associated death domain and FLICE-inhibitory protein proteins. Last, we observed a synergistic inhibition in the invasion and migration of LNCaP cells. This effect was observed to be mediated through inhibition in the protein expression of vascular endothelial growth factor, uPA and angiopoietin 1 and 2. Further, the activity and protein expression of MMP-2, -3 and -9 and upregulation of TIMP1 in cells treated with a combination of EGCG and TRAIL was observed. These data might have implications for developing new strategies aimed at eliminating prostate cancer cells resistant to TRAIL.

A Dietary Anthocyanidin Delphinidin Induces Apoptosis of Human Prostate Cancer PC3 Cells In vitro and In vivo: Involvement of Nuclear Factor-κB Signaling

Delphinidin, a major anthocyanidin present in many pigmented fruits and vegetables, possesses antioxidant, anti-inflammatory, and antiangiogenic properties. In this study, we provide evidence that it could be developed as a novel agent against human prostate cancer (PCa). We observed that delphinidin treatment to human PCa LNCaP, C4-2, 22Rnu1, and PC3 cells resulted in a dose-dependent inhibition of cell growth without having any substantial effect on normal human prostate epithelial cells. We selected PC3 cells as a test model system because of their highly aggressive proliferative nature. Delphinidin treatment of cells resulted in a dose-dependent induction of apoptosis and arrest of cells in G(2)-M phase. This induction of apoptosis seems to be mediated via activation of caspases because N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluromethylketone significantly reduced apoptosis induced by delphinidin. We also observed that delphinidin treatment of cells resulted in a dose-dependent decrease in (a) phosphorylation of IkappaB kinase gamma (NEMO), (b) phosphorylation of nuclear factor-kappaB (NF-kappaB) inhibitory protein IkappaBalpha, (c) phosphorylation of NF-kappaB/p65 at Ser(536) and NF-kappaB/p50 at Ser(529), (d) NF-kappaB/p65 nuclear translocation, and (e) NF-kappaB DNA binding activity. Delphinidin administration (2 mg, i.p. thrice weekly) to athymic nude mice implanted with PC3 cells resulted in a significant inhibition of tumor growth. Analysis of tumors from delphinidin-treated mice showed significant decrease in the expression of NF-kappaB/p65, Bcl2, Ki67, and PCNA. Taken together, our data suggest that delphinidin could be developed as an agent against human PCa.

Cannabinoid Receptor Agonist-induced Apoptosis of Human Prostate Cancer Cells LNCaP Proceeds through Sustained Activation of ERK1/2 Leading to G1 Cell Cycle Arrest

We have recently shown that the expression levels of both cannabinoid receptors CB1 and CB2 are higher in human prostate cancer cells than in normal prostate epithelial cells, and treatment of LNCaP cells with WIN-55,212-2 (a mixed CB1/CB2 agonist) resulted in inhibition of cell growth and induction of apoptosis (Sarfaraz, S., Afaq, F., Adhami, V. M., and Mukhtar, H. (2005) Cancer Res. 65, 1635-1641). This study was conducted to understand the mechanistic basis of these effects. Treatment of LNCaP cells with WIN-55,212-2 (1-10 μm; 24 h) resulted in: (i) an arrest of the cells in the G0/G1 phase of the cell cycle; (ii) an induction of p53 and p27/KIP1; (iii) down-regulation of cyclins D1, D2, E; (iii) decrease in the expression of cdk-2, -4, and -6; (iv) decrease in protein expression of pRb; (v) down-regulation of E2F (1-4); and (vi) decrease in the protein expression of DP1 and DP2. Similar effects were also observed when androgen-independent PC3 cells were treated with WIN-55,212-2 (5-30 μm). We further observed sustained up-regulation of ERK1/2 and inhibition of PI3k/Akt pathways in WIN-55,212-2-treated cells. Inhibition of ERK1/2 abrogated WIN-55,212-2-indued cell death suggesting that sustained activation of ERK1/2 leads to cell cycle dysregulation and arrest of cells in G0/G1 phase subsequently leading to an induction of apoptosis. Further, WIN-55,212-2 treatment of cells resulted in a dose-dependent increase in Bax/Bcl-2 ratio in such a way that favors apoptosis. The induction of apoptosis proceeded through down-regulation of caspases 3, 6, 7, and 9 and cleavage of poly (ADP-ribose) polymerases. Based on these data we suggest that cannabinoid receptor agonists should be considered as novel agents for the management of prostate cancer.

Pomegranate Fruit Extract Modulates UV‐B–mediated Phosphorylation of Mitogen‐activated Protein Kinases and Activation of Nuclear Factor Kappa B in Normal Human Epidermal Keratinocytes¶

Abstract Excessive exposure of solar ultraviolet (UV) radiation, particularly its UV-B component, to humans causes many adverse effects that include erythema, hyperplasia, hyperpigmentation, immunosuppression, photoaging and skin cancer. In recent years, there is increasing use of botanical agents in skin care products. Pomegranate derived from the tree Punica granatum contains anthocyanins (such as delphinidin, cyanidin and pelargonidin) and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) and possesses strong antioxidant and anti-inflammatory properties. Recently, we have shown that pomegranate fruit extract (PFE) possesses antitumor promoting effects in a mouse model of chemical carcinogenesis. To begin to establish the effect of PFE for humans in this study, we determined its effect on UV-B–induced adverse effects in normal human epidermal keratinocytes (NHEK). We first assessed the effect of PFE on UV-B–mediated phosphorylation of mitogen-activated protein kinases (MAPK) pathway in NHEK. Immunoblot analysis demonstrated that the treatment of NHEK with PFE (10–40 μg/mL) for 24 h before UV-B (40 mJ/cm2) exposure dose dependently inhibited UV-B–mediated phosphorylation of ERKl/2, JNK1/2 and p38 protein. We also observed that PFE (20 μg/mL) inhibited UV-B–mediated phosphorylation of MAPK in a time-dependent manner. Furthermore, in dose- and time-dependent studies, we evaluated the effect of PFE on UV-B–mediated activation of nuclear factor kappa B (NF-κB) pathway. Using Western blot analysis, we found that PFE treatment of NHEK resulted in a dose- and time-dependent inhibition of UV-B–mediated degradation and phosphorylation of IκBα and activation of IKKα. Using immunoblot analysis, enzyme-linked immunosorbent assay and electrophoretic mobility shift assay, we found that PFE treatment to NHEK resulted in a dose- and time-dependent inhibition of UV-B–mediated nuclear translocation and phosphorylation of NF-κB/p65 at Ser536. Taken together, our data shows that PFE protects against the adverse effects of UV-B radiation by inhibiting UV-B–induced modulations of NF-κB and MAPK pathways and provides a molecular basis for the photochemopreventive effects of PFE.

Prostate cancer prevention through pomegranate fruit

Prostate cancer (CaP) is the second leading cause of cancer-related deaths among U.S. males with a similar trend in many Western countries. CaP is an ideal candidate disease for chemoprevention because it is typically diagnosed in men over 50 years of age, and thus even a modest delay in disease progression achieved through pharmacological or nutritional intervention could significantly impact the quality of life of these patients. In this regard we and others have proposed the use of dietary antioxidants as candidate CaP chemopreventive agents. The fruit pomegranate derived from the tree Punica granatum has been shown to possess strong antioxidant and anti-inflammatory properties. In a recent study, we showed that pomegranate fruit extract (PFE), through modulations in the cyclin kinase inhibitor-cyclin-cyclin-dependent kinase machinery, resulted in inhibition of cell growth followed by apoptosis of highly aggressive human prostate carcinoma PC3 cells. These events were associated with alterations in the levels of Bax and Bcl-2 shifting the Bax:Bcl-2 ratio in favor of apoptosis. Further, we showed that oral administration of a human acceptable dose of PFE to athymic nude mice implanted with CWR22R1 cells resulted in significant inhibition of tumor growth with concomitant reduction in secretion of prostate-specific antigen (PSA) in the serum. The outcome of this study could have a direct practical implication and translational relevance to CaP patients, because it suggests that pomegranate consumption may retard CaP progression, which may prolong the survival and quality of life of the patients.

Photochemopreventive Effect of Pomegranate Fruit Extract on UVA-mediated Activation of Cellular Pathways in Normal Human Epidermal Keratinocytes

Abstract UVA is the major portion (90–99%) of solar radiation reaching the surface of the earth and has been described to lead to formation of benign and malignant tumors. UVA-mediated cellular damage occurs primarily through the release of reactive oxygen species and is responsible for immunosuppression, photodermatoses, photoaging and photocarcinogenesis. Pomegranate fruit extract (PFE) possesses strong antioxidant and anti-inflammatory properties. Our recent studies have shown that PFE treatment of normal human epidermal keratinocytes (NHEK) inhibits UVB-mediated activation of MAPK and NF-κB pathways. Signal transducers and activators of transcription 3 (STAT3), Protein Kinase B/AKT and Map Kinases (MAPKs), which are activated by a variety of factors, modulate cell proliferation, apoptosis and other biological activities. The goal of this study was to determine whether PFE affords protection against UVA-mediated activation of STAT3, AKT and extracellular signal–regulated kinase (ERK1/2). Immunoblot analysis demonstrated that 4 J/cm2 of UVA exposure to NHEK led to an increase in phosphorylation of STAT3 at Tyr705, AKT at Ser473 and ERK1/2. Pretreatment of NHEK with PFE (60–100 μg/mL) for 24 h before exposure to UVA resulted in a dose-dependent inhibition of UVA-mediated phosphorylation of STAT3 at Tyr705, AKT at Ser473 and ERK1/2. mTOR, structurally related to PI3K, is involved in the regulation of p70S6K, which in turn phosphorylates the S6 protein of the 40S ribosomal subunit. We found that UVA radiation of NHEK resulted in the phosphorylation of mTOR at Thr2448 and p70S6K at Thr421/Ser424. PFE pretreatment resulted in a dose-dependent inhibition in the phosphorylation of mTOR at Thr2448 and p70S6K at Thr421/Ser424. Our data further demonstrate that PFE pretreatment of NHEK resulted in significant inhibition of UVA exposure–mediated increases in Ki-67 and PCNA. PFE pretreatment of NHEK was found to increase the cell-cycle arrest induced by UVA in the G1 phase of the cell cycle and the expression of Bax and Bad (proapoptotic proteins), with downregulation of Bcl-XL expression (antiapoptotic protein). Our data suggest that PFE is an effective agent for ameliorating UVA-mediated damages by modulating cellular pathways and merits further evaluation as a photochemopreventive agent.

Pomegranate Fruit Juice for Chemoprevention and Chemotherapy of Prostate Cancer

Prostate cancer is the most common invasive malignancy and the second leading cause of cancer-related deaths among U.S. males, with a similar trend in many Western countries. One approach to control this malignancy is its prevention through the use of agents present in diet consumed by humans. Pomegranate from the tree Punica granatum possesses strong antioxidant and antiinflammatory properties. We recently showed that pomegranate fruit extract (PFE) possesses remarkable antitumor-promoting effects in mouse skin. In this study, employing human prostate cancer cells, we evaluated the antiproliferative and proapoptotic properties of PFE. PFE (10-100 microg/ml; 48 h) treatment of highly aggressive human prostate cancer PC3 cells resulted in a dose-dependent inhibition of cell growth/cell viability and induction of apoptosis. Immunoblot analysis revealed that PFE treatment of PC3 cells resulted in (i) induction of Bax and Bak (proapoptotic); (ii) down-regulation of Bcl-X(L) and Bcl-2 (antiapoptotic); (iii) induction of WAF1/p21 and KIP1/p27; (iv) a decrease in cyclins D1, D2, and E; and (v) a decrease in cyclin-dependent kinase (cdk) 2, cdk4, and cdk6 expression. These data establish the involvement of the cyclin kinase inhibitor-cyclin-cdk network during the antiproliferative effects of PFE. Oral administration of PFE (0.1% and 0.2%, wt/vol) to athymic nude mice implanted with androgen-sensitive CWR22Rnu1 cells resulted in a significant inhibition in tumor growth concomitant with a significant decrease in serum prostate-specific antigen levels. We suggest that pomegranate juice may have cancer-chemopreventive as well as cancer-chemotherapeutic effects against prostate cancer in humans.