Farrukh Afaq

Targeting Multiple Signaling Pathways by Green Tea Polyphenol (−)-Epigallocatechin-3-Gallate

Cell signaling pathways, responsible for maintaining a balance between cell proliferation and death, have emerged as rational targets for the management of cancer. Emerging data amassed from various laboratories around the world suggests that green tea, particularly its major polyphenolic constituent (-)-epigallocatechin-3-gallate (EGCG), possesses remarkable cancer chemopreventive and therapeutic potential against various cancer sites in animal tumor bioassay systems and in some human epidemiologic studies. EGCG has been shown to modulate multiple signal transduction pathways in a fashion that controls the unwanted proliferation of cells, thereby imparting strong cancer chemopreventive as well as therapeutic effects. This review discusses the modulations of important signaling events by EGCG and their implications in cancer management.

Anthocyanin- and hydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NF-κB pathways and inhibits skin tumorigenesis in CD-1 mice

Chemoprevention has come of age as an effective cancer control modality; however, the search for novel agent(s) for the armamentarium of cancer chemoprevention continues. We argue that agents capable of intervening at more than one critical pathway in the carcinogenesis process will have greater advantage over other single‐target agents. Pomegranate fruit extract (PFE) derived from the tree Punica granatum possesses strong antioxidant and antiinflammatory properties. Pomegranate fruit was extracted with acetone and analyzed based on matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and found to contain anthocyanins, ellagitannins and hydrolyzable tannins. We evaluated whether PFE possesses antitumor‐promoting effects. We first determined the effect of topical application of PFE to CD‐1 mice against 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced conventional markers and other novel markers of skin tumor promotion. We found that topical application of PFE (2 mg/mouse) 30 min prior to TPA (3.2 nmole/mouse) application on mouse skin afforded significant inhibition, in a time‐dependent manner, against TPA‐mediated increase in skin edema and hyperplasia, epidermal ornithine decarboxylase (ODC) activity and protein expression of ODC and cyclooxygenase‐2. We also found that topical application of PFE resulted in inhibition of TPA‐induced phosphorylation of ERK1/2, p38 and JNK1/2, as well as activation of NF‐κB and IKKα and phosphorylation and degradation of IκBα. We next assessed the effect of skin application of PFE on TPA‐induced skin tumor promotion in 7,12‐dimethylbenz(a)anthracene‐initiated CD‐1 mouse. The animals pretreated with PFE showed substantially reduced tumor incidence and lower tumor body burden when assessed as total number of tumors per group, percent of mice with tumors and number of tumors per animal as compared to animals that did not receive PFE. In TPA‐treated group, 100% of the mice developed tumors at 16 weeks on test, whereas at this time in PFE‐treated group, only 30% mice exhibited tumors. Skin application of PFE prior to TPA application also resulted in a significant delay in latency period from 9 to 14 weeks and afforded protection when tumor data were considered in terms of tumor incidence and tumor multiplicity. The results of our study provide clear evidence that PFE possesses antiskin‐tumor‐promoting effects in CD‐1 mouse. Because PFE is capable of inhibiting conventional as well as novel biomarkers of TPA‐induced tumor promotion, it may possess chemopreventive activity in a wide range of tumor models. Thus, an in‐depth study to define active agent(s) in PFE capable of affording antitumor‐promoting effect is warranted.

Involvement of nuclear factor-kappa B, Bax and Bcl-2 in induction of cell cycle arrest and apoptosis by apigenin in human prostate carcinoma cells

Apigenin, a common dietary flavonoid abundantly present in fruits and vegetables, may have the potential for prevention and therapy for prostate cancer. Here, we report for the first time that apigenin inhibits the growth of androgen-responsive human prostate carcinoma LNCaP cells and provide molecular understanding of this effect. The cell growth inhibition achieved by apigenin treatment resulted in a significant decrease in AR protein expression along with a decrease in intracellular and secreted forms of PSA. These effects were also observed in DHT-stimulated cells. Further, apigenin treatment of LNCaP cells resulted in G1 arrest in cell cycle progression which was associated with a marked decrease in the protein expression of cyclin D1, D2 and E and their activating partner cdk2, 4 and 6 with concomitant induction of WAF1/p21 and KIP1/p27. The induction of WAF1/p21 appears to be transcriptionally upregulated and is p53 dependent. In addition, apigenin inhibited the hyperphosphorylation of the pRb protein in these cells. Apigenin treatment also resulted in induction of apoptosis as determined by DNA fragmentation, PARP cleavage, fluorescence microscopy and flow cytometry. These effects were found to correlate with a shift in Bax/Bcl-2 ratio more towards apoptosis. Apigenin treatment also resulted in down-modulation of the constitutive expression of NF-κB/p65. Taken together, these findings suggest that apigenin has strong potential for development as an agent for prevention against prostate cancer.

Botanical antioxidants in the prevention of photocarcinogenesis and photoaging

Abstract:  Exposure of the skin to ultraviolet (UV) radiation, particularly its UV‐B component (280–320 nm), from the sun results in erythema, edema, hyperplasia, hyperpigmentation, sunburn cells, immunosuppression, photoaging, and skin cancer. Amongst these various adverse effects of UV‐B radiation, skin cancer and photoaging are of great concern. More recent changes in lifestyle have led to a significant increase in the amount of UV‐B radiation people receive leading to a surge in the incidence of skin cancer and photoaging. As these trends are likely to continue in the foreseeable future, the adverse effect of UV‐B has become a major human health concern. Therefore, development of novel strategies to reduce the occurrence of skin cancer and delay the process of photoaging are highly desirable goals. One approach to reduce their occurrence is through photochemoprevention, which we define as the use of agents capable of ameliorating the adverse effects of UV‐B on the skin. Photochemoprevention via use of botanical antioxidants, present in the common diet of human have gained considerable attention as photochemopreventive agents for human use. Many such agents have also found a place in skin care products. This review will focus on the effects of selected botanical antioxidants in the prevention of photocarcinogenesis and photoaging.

Inhibition of ultraviolet B-mediated activation of nuclear factor κB in normal human epidermal keratinocytes by green tea Constituent (-)-epigallocatechin-3-gallate

Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, possesses significant anti-inflammatory and cancer chemopreventive properties. Studies have shown the photochemopreventive effects of green tea and EGCG in cell culture, animal models, and human skin. The molecular mechanism(s) of photochemopreventive effects of EGCG are incompletely understood. We recently showed that EGCG treatment of the normal human epidermal keratinocytes (NHEK) inhibits ultraviolet (UV)B-mediated activation of the mitogen-activated protein kinase (MAPK) pathway. In this study, we evaluated the effect of EGCG on UVB-mediated modulation of the nuclear factor kappa B (NF-κB) pathway, which is known to play a critical role in a variety of physiological functions and is involved in inflammation and development of cancer. Immunoblot analysis demonstrated that the treatment of NHEK with EGCG (10–40 μM) for 24 h resulted in a significant inhibition of UVB (40 mJ/cm2)-mediated degradation and phosphorylation of IκBα and activation of IKKα, in a dose-dependent manner. UVB-mediated degradation and phosphorylation of IκBα and activation of IKKα was also observed in a time-dependent protocol (15 and 30 min, 1, 2, 3, 6, 12 h post-UVB exposure). Employing immunoblot analysis, enzyme-linked immunosorbent assay, and gel shift assay, we demonstrate that EGCG treatment of the cells resulted in a significant dose- and time-dependent inhibition of UVB-mediated activation and nuclear translocation of a NF-κB/p65. Our data suggest that EGCG protects against the adverse effects of UV radiation via modulations in NF-κB pathway, and provide a molecular basis for the photochemopreventive effect of EGCG.

Lupeol modulates NF-κB and PI3K/Akt pathways and inhibits skin cancer in CD-1 mice

Chemoprevention has become an effective cancer control modality; however, the search for novel agent(s) for the armamentarium of cancer chemoprevention continues. We argue that agents capable for inhibition of promotion stage of tumorigenesis with the ability to intervene at several critical pathways in the tumorigenesis process will have greater advantage over other single-target agents. Lupeol, a triterpene, is the principal constituent of common fruit plants such as olive, mango, fig and medicinal herbs that have been used to treat skin aliments. Lupeol has been reported to possess a wide range of medicinal properties that include strong antioxidant, antimutagenic, anti-inflammatory and antiarthritic effects. In the present study, we show that Lupeol possesses antitumor-promoting effects in a mouse skin tumorigenesis model. We first determined the effect of topical application of Lupeol to CD-1 mouse against 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced conventional markers and other novel markers of skin tumor promotion. We found that topical application of Lupeol (1–2 mg/mouse) 30 min prior to TPA (3.2 nmol/mouse) application onto the skin of CD-1 mice afforded significant inhibition, in a time- and dose-dependent manner, against TPA-mediated increase in (i) skin edema and hyperplasia, (ii) epidermal ornithine decarboxylase (ODC) activity, and (iii) protein expression of ODC, cyclo-oxygenase-2 and nitric oxide synthase. As of the role of nuclear factor kappa B (NF-κB) and phosphatidyl inositol 3-kinase (PI3K)/Akt signaling in tumor promotion, we next determined the effect of topical application of Lupeol to mouse skin against these signaling pathways. We found that Lupeol treatment to mouse skin resulted in the inhibition of TPA-induced (i) activation of PI3K, (ii) phosphorylation of Akt at Thr308, (iii) activation of NF-κB and IKKα, and (iv) degradation and phosphorylation of IκBα. The animals pretreated with Lupeol showed significantly reduced tumor incidence, lower tumor body burden and a significant delay in the latency period for tumor appearance. At the termination of the experiment at 28 weeks, 100% of the animals in TPA-treated group exhibited seven to eight tumors/mouse, whereas only 53% of the mice receiving Lupeol prior to TPA treatment exhibited one to three tumors/mouse. These results for the first time provide evidence that Lupeol possesses antiskin tumor-promoting effects in CD-1 mouse and inhibits conventional as well as novel biomarkers of tumor promotion. We suggest that Lupeol is an attractive antitumor-promoting agent that must be evaluated in tumor models other than skin carcinogenesis.

Cannabinoid Receptor as a Novel Target for the Treatment of Prostate Cancer

Cannabinoids, the active components of Cannabis sativa Linnaeus (marijuana) and their derivatives have received renewed interest in recent years due to their diverse pharmacologic activities such as cell growth inhibition, anti-inflammatory effects and tumor regression. Here we show that expression levels of both cannabinoid receptors, CB1 and CB2, are significantly higher in CA-human papillomavirus-10 (virally transformed cells derived from adenocarcinoma of human prostate tissue), and other human prostate cells LNCaP, DUI45, PC3, and CWR22Rnu1 than in human prostate epithelial and PZ-HPV-7 (virally transformed cells derived from normal human prostate tissue) cells. WIN-55,212-2 (mixed CB1/CB2 agonist) treatment with androgen-responsive LNCaP cells resulted in a dose- (1-10 micromol/L) and time-dependent (24-48 hours) inhibition of cell growth, blocking of CB1 and CB2 receptors by their antagonists SR141716 (CB1) and SR144528 (CB2) significantly prevented this effect. Extending this observation, we found that WIN-55,212-2 treatment with LNCaP resulted in a dose- (1-10 micromol/L) and time-dependent (24-72 hours) induction of apoptosis (a), decrease in protein and mRNA expression of androgen receptor (b), decrease in intracellular protein and mRNA expression of prostate-specific antigen (c), decrease in secreted prostate-specific antigen levels (d), and decrease in protein expression of proliferation cell nuclear antigen and vascular endothelial growth factor (e). Our results suggest that WIN-55,212-2 or other non-habit-forming cannabinoid receptor agonists could be developed as novel therapeutic agents for the treatment of prostate cancer.

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.

Photochemoprevention of skin cancer by botanical agents

Photochemoprevention has become an important armamentarium in the fight against ultraviolet radiation (UVR)‐induced damage to the skin. Among many UVR‐induced damages, skin cancer is of the greatest concern as its rates have been steadily increasing in recent years and the same trend is expected to continue in the future. Ultraviolet radiation increases oxidative stress in skin cells by causing excessive generation of reactive oxygen species (ROS), leading to cancer initiation and promotion. Antioxidants have the capability to quench these ROS and much recent work shows that some of these can inhibit many UVR‐induced signal transduction pathways. Thus, identifying nontoxic strong antioxidants – capable of preventing UVR‐induced skin cancer – has become an important area of research. The use of botanical antioxidants in skin care products is growing in popularity. A wide range of such agents has been shown to prevent skin cancer in animal model systems. New agents are constantly being investigated; however, only a few have been tested for their efficacy in humans. Animal model and cell culture studies have clarified that antioxidants act by several mechanisms at various stages of skin carcinogenesis. This review focuses on skin cancer photochemopreventive effects of selected botanical antioxidants.

Essential role of caspases in epigallocatechin-3-gallate-mediated inhibition of nuclear factor kappaB and induction of apoptosis

Green tea constituent (−) epigallocatechin-3-gallate (EGCG) has shown remarkable cancer-preventive and some cancer-therapeutic effects. This is partially because of its ability to induce apoptosis in cancer cells without affecting normal cells. Previous studies from our laboratory have shown the involvement of NF-κB pathway in EGCG-mediated cell-cycle deregulation and apoptosis of human epidermoid carcinoma A431 cells. Here we show the essential role of caspases in EGCG-mediated inhibition of NF-κB and its subsequent apoptosis. Treatment of A431 cells with EGCG (10–40 μg/ml) resulted in dose-dependent inhibition of NF-κB/p65, induction of DNA breaks, cleavage of poly(ADP-ribose) polymerase (PARP) and morphological changes consistent with apoptosis. EGCG treatment of cells also resulted in significant activation of caspases, as shown by the dose- and time-dependent increase in DEVDase activity, and protein expression of caspase-3, -8 and -9. EGCG-mediated caspase activation induces proteolytic cleavage of NF-κB/p65 subunit, leading to the loss of transactivation domains, and driving the cells towards apoptosis. EGCG-mediated induction of apoptosis was significantly blocked by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-FMK), and moderately blocked by the specific caspase-3 inhibitor Z-DEVD-FMK. Further, pretreatment of cells with Z-VAD-FMK was found to suppress the cleavage of NF-κB/p65 subunit, thereby increasing nuclear translocation, DNA binding and transcriptional activity, thus protecting the cells from EGCG-induced apoptosis. Taken together, these studies for the first time demonstrate that EGCG-mediated activation of caspases is critical, at least in part, for inhibition of NF-κB and subsequent apoptosis.