Santosh K. Katiyar

Berberine, a natural product, induces G1-phase cell cycle arrest and caspase-3-dependent apoptosis in human prostate carcinoma cells

Berberine, a naturally occurring isoquinoline alkaloid, has been shown to possess anti-inflammatory and antitumor properties in some in vitro systems. Here, we report that in vitro treatment of androgen-insensitive (DU145 and PC-3) and androgen-sensitive (LNCaP) prostate cancer cells with berberine inhibited cell proliferation and induced cell death in a dose-dependent (10–100 μmol/L) and time-dependent (24–72 hours) manner. Treatment of nonneoplastic human prostate epithelial cells (PWR-1E) with berberine under identical conditions did not significantly affect their viability. The berberine-induced inhibition of proliferation of DU145, PC-3, and LNCaP cells was associated with G1-phase arrest, which in DU145 cells was associated with inhibition of expression of cyclins D1, D2, and E and cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 proteins, increased expression of the Cdk inhibitory proteins (Cip1/p21 and Kip1/p27), and enhanced binding of Cdk inhibitors to Cdk. Berberine also significantly (P < 0.05–0.001) enhanced apoptosis of DU145 and LNCaP cells with induction of a higher ratio of Bax/Bcl-2 proteins, disruption of mitochondrial membrane potential, and activation of caspase-9, caspase-3, and poly(ADP-ribose) polymerase. Pretreatment with the pan-caspase inhibitor z-VAD-fmk partially, but significantly, blocked the berberine-induced apoptosis, as also confirmed by the comet assay analysis of DNA fragmentation, suggesting that berberine-induced apoptosis of human prostate cancer cells is mediated primarily through the caspase-dependent pathway. The effectiveness of berberine in checking the growth of androgen-insensitive, as well as androgen-sensitive, prostate cancer cells without affecting the growth of normal prostate epithelial cells indicates that it may be a promising candidate for prostate cancer therapy. [Mol Cancer Ther 2006;5(2):296–308]

Polyphenolic Antioxidant (-)-Epigallocatechin-3-Gallate from Green Tea Reduces UVB-lnduced Inflammatory Responses and Infiltration of Leukocytes in Human Skin

Abstract— Identification of natural products capable of affording protection against UVB radiation‐induced inflammatory responses and generation of oxidative stress may have important human health implications. The UVB exposure‐induced skin injury and oxidative stress has been associated with a variety of skin disease conditions including photoaging, inflammation and cancer. Tea is a popular beverage consumed worldwide. In several mouse skin models, topical application as well as oral consumption of green tea has been shown to afford protection against chemical and UVB‐induced carcinogenesis and inflammatory responses. In the present study, we investigated in human skin, whether topical application of (‐)epigallocatechin‐3‐gallate (EGCG), the major polyphenolic constituent in green tea, inhibits UVB‐induced infiltration of leukocytes (macrophage/neutrophils), a potential source of generation of reactive oxygen species (ROS), and generation of prostaglandin (PG) metabolites. Human subjects were UVB irradiated on sun‐protected skin to four times their minimal erythema dosage (MED) and skin biopsies or keratomes were obtained either 24 h or 48 h later. We found that topical application of EGCG (3 mg/2.5 cm2) before UVB (4 MED) exposure to human skin significantly blocked UVB‐induced infiltration of leukocytes and reduced myeloperoxidase activity. These infiltrating leukocytes are considered to be the major source of generation of ROS. In the same set of experiments we found that topical application of EGCG before UVB exposure decreased UVB‐induced erythema. In additional experiments, we found that microsomes from EGCG pretreated human skin and exposed to UVB, compared to UVB exposure alone, produced significantly reduced PG metabolites, particularly PGE2. The PG metabolites play a critical role in free radical generation and skin tumor promotion in multistage skin carcinogenesis. Careful microscopic examination of skin sections, stained with hematoxylin and eosin, under higher magnification (x400) also revealed that EGCG pretreated and UVB exposed human skin contained fewer dead cells in the epidermis with comparison to nonpretreated UVB‐exposed skin. Taken together, our data demonstrate that EGCG has the potential to block the UVB‐induced infiltration of leukocytes and the subsequent generation of ROS in human skin. This may explain the possible mechanism involved in anti‐inflammatory effects of green tea. We suggest that EGCG may be useful as a topical agent for protection against UVB‐induced ROS‐associated inflammatory dermatoses, photoaging and photocarcinogenesis. Further studies are warranted in this direction.

Green tea polyphenol (−)-epigallocatechin-3-gallate treatment to mouse skin prevents UVB-induced infiltration of leukocytes, depletion of antigen-presenting cells, and oxidative stress

Ultraviolet (UV) radiation‐induced infiltrating leukocytes, depletionof antigen‐presenting cells, and oxidative stress in the skin play animportant role in the induction of immune suppression andphotocarcinogenesis. Earlier we have shown that topical application ofpolyphenols from green tea or its major chemopreventive constituent(−)‐epigallocatechin‐3‐gallate (EGCG) prevents UV‐B‐inducedimmunosuppression in mice. To define the mechanism of prevention, wefound that topical application of EGCG (3 mg/mouse/3 cm2 ofskin area) to C3H/HeN mice before a single dose of UV‐B (90mJ/cm2) exposure inhibited UV‐B‐induced infiltration ofleukocytes, specifically the CD11b+ cell type, and myeloperoxidaseactivity, a marker of tissue infiltration of leukocytes. EGCG treatmentwas also found to prevent UV‐B‐induced depletion in the number ofantigen‐presenting cells when immunohistochemically detected as classII MHC+ Ia+ cells. UV‐B‐induced infiltrating cell production of H2O2 and nitric oxide (NO) was determined as amarker of oxidative stress. We found that pretreatment of EGCGdecreased the number of UV‐B‐induced increases inH2O2‐producing cells and inducible nitric oxidesynthase‐expressing cells and the production of H2O2 and NO in both epidermis and dermis at aUV‐B‐irradiated site. Together, these data suggest that prevention of UV‐B‐induced infiltrating leukocytes, antigen‐presenting cells, andoxidative stress by EGCG treatment of mouse skin may be associated withthe prevention of UV‐B‐induced immunosuppression andphotocarcinogenesis.

Multi-targeted prevention and therapy of cancer by proanthocyanidins

In recent years, a considerable emphasis has been focused on the importance of the naturally available botanicals that can be consumed in an individuals everyday diet and that can also be useful as a chemopreventive or chemotherapeutic agent for certain diseases, including cancers. A wide variety of botanicals, mostly dietary flavonoids or polyphenolic substances, have been reported to possess substantial anti-carcinogenic and antimutagenic activities because of their antioxidant and anti-inflammatory properties. Proanthocyanidins are considered as one of them, and are abundantly available in various parts of the plants, such as fruits, berries, bark and seeds. Their modes of action were evaluated through a number of in vitro and in vivo studies which showed their potential role as anti-carcinogenic agent. We summarize and highlight the latest developments on anti-carcinogenic activities of proanthocyanidins from different sources, specifically from grape seeds, and their molecular targets, such as NF-kappaB, mitogen-activated protein kinases, PI3K/Akt, caspases, cytokines, angiogenesis and cell cycle regulatory proteins and other check points, etc. Although the bioavailability and metabolism data on proanthocyanidins is still largely unavailable, certain reports indicate that at least monomers and smaller oligomeric procyanidins are absorbed in the gut. The modulation of various molecular targets by proanthocyanidins in vitro and in vivo tumor models suggests their importance, contribution and mechanism of action to the prevention of cancers of different organs.

(-)-Epigallocatechin-3-gallate reactivates silenced tumor suppressor genes, Cip1/p21 and p16INK4a, by reducing DNA methylation and increasing histones acetylation in human skin cancer cells.

The anti-skin carcinogenic effects of green tea catechins have been studied extensively in vitro and in vivo models but the precise epigenetic molecular mechanisms are still unclear. Accumulating data suggest that dietary phytochemicals may alter cancer risk by modifications of epigenetic processes in the cells. The present study was designed to investigate whether tea catechins, particularly (-)-epigallocatechin-3-gallate (EGCG), would modify epigenetic events to regulate DNA methylation-silenced tumor suppressor genes in skin cancer cells. DNA methylation, histone modifications and tumor suppressor gene expressions were studied in detail using human epidermoid carcinoma A431 cells as an in vitro model after EGCG treatment using cytostaining, western blotting, dot blot analysis, real-time polymerase chain reaction and enzymatic activity assays. Our study shows that EGCG treatment decreased global DNA methylation levels in A431 cells in a dose-dependent manner. EGCG decreased the levels of 5-methylcytosine, DNA methyltransferase (DNMT) activity, messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b. EGCG decreased histone deacetylase activity and increased levels of acetylated lysine 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysine 5, 12 and 16 on histone H4 but decreased levels of methylated H3-Lys 9. Additionally, EGCG treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, p16INK4a and Cip1/p21. Together, our study provides new insight into the epigenetic mechanism of action of EGCG that may contribute to the chemoprevention of skin cancer and may have important implications for epigenetic therapy.

Tea antioxidants in cancer chemoprevention

In recent years, the concept of cancer chemoprevention has matured greatly. Significant reversal or suppression of premalignancy in several sites by chemopreventive agents appears achievable. This article summarizes experimental data on chemopreventive effects of tea polyphenols in different tumor bioassay systems. Tea (Camellia sinensis) is cultivated in about 30 countries, and is the most widely consumed beverage in the world. Three main commercial tea varieties—green, black, and oolong—are usually consumed, but most experimental studies demonstrating the antimutagenic and anticarcinogenic effects of tea have been conducted with water extract of green tea, or a polyphenolic fraction isolated from green tea (GTP). The majority of these studies have been conducted in a mouse skin tumor model system where tea is fed either as water extract through drinking water, or as purified GTP. GTP has been shown to exhibit antimutagenic activity in vitro, and inhibit carcinogen‐ as well as UV‐induced skin carcinogenesis in vivo. Tea consumption has also been shown to afford protection against chemical carcinogen‐induced stomach, lung, esophagus, duodenum, pancreas, liver, breast, and colon carcinogenesis in specific bioassay models. Several epicatechin derivatives (polyphenols) present in green tea have been shown to possess anticarcinogenic activity; the most active is (−)‐epigallocatechin‐3‐gallate, which is also the major constituent of GTP. The mechanisms of teas broad cancer chemopreventive effects are not completely understood. Several theories have been put forward, including inhibition of UV‐ and tumor promoter‐induced ornithine decarboxylase, cyclo‐oxygenase, and lipoxygenase activities, antioxidant and free radical scavenging activity; enhancement of antioxidant (glutathione peroxidase, catalase, and quinone reductase) and phase II (glutathione‐S‐transferase) enzyme activities; inhibition of lipid peroxidation, and anti‐inflammatory activity. These properties of tea polyphenols make them effective chemopreventive agents against the initiation, promotion, and progression stages of multistage carcinogenesis. J. Cell. Biochem. Suppl. 27:59–67. Published 1998 Wiley‐Liss, Inc.

Cyclooxygenase-2 Expression in Murine and Human Nonmelanoma Skin Cancers: Implications for Therapeutic Approaches¶

Abstract Inflammatory stimuli result in the production of cutaneous eicosanoids, which are known to contribute to the process of tumor promotion. Cyclooxygenase (COX), the rate-limiting enzyme for the production of prostaglandins (PG) from arachidonic acid, exists in at least two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and plays various physiological roles, whereas increased COX-2 expression is known to occur in several types of epithelial neoplasms. Enhanced PG synthesis is a potential contributing factor in UVB-induced nonmelanoma skin cancers (NMSC). Increased COX-2 staining occurs in murine skin neoplasms after chronic exposure to carcinogenic doses of UVB. In this study, immunohistochemical and Western blot analyses were employed to assess longitudinally COX-2 expression in a standard mouse UVB complete carcinogenesis protocol and in human basal cell carcinomas (BCC) and squamous cell carcinomas (SCC). During UVB irradiation of mice, COX-2 expression consistently increased in the hyperplastic skin, the benign papillomas and the SCC. COX-2 expression was also increased in human actinic keratoses, SCC and BCC as well as in murine SCC and BCC. The pattern of COX-2 expression was quite variable, occurring in a patchy distribution in some lesions with staining confined mainly to suprabasal cell layers. In general, COX-2 expression progressively became more extensive in benign papillomas and well-differentiated murine SCC. The staining was predominantly cytoplasmic and perinuclear in some focal areas in tissue stroma around both murine and human tumors. Western blot analysis confirmed negative COX-2 expression in normal skin, whereas acute UVB exposure resulted in increased enzyme expression, which continued to increase in developing papillomas and SCC. Because of the evidence indicating a pathogenic role for eicosanoids in murine and human skin neoplasms, we performed studies to assess the anti-inflammatory and anticarcinogenic effects of green tea extracts, which are potent antioxidants. Acute exposure of the human skin to UVB (minimum erythema dose × 4) caused a transient enhancement of the COX-2 expression, which reverted to baseline within hours; however, in murine skin the expression persisted for several days. Pretreatment with the topically applied green tea extract (1 mg/cm2) largely abrogated the acute COX-2 response to UVB in mice or humans. In summary, enhanced COX-2 expression serves as a marker of epidermal UVB exposure for murine and human NMSC. These results suggest that COX-2 inhibitors could have potent anticarcinogenic effects in UVB-induced skin cancer.

Chemoprevention of photocarcinogenesis by selected dietary botanicals

Epidemiological, clinical and laboratory studies have implicated solar ultraviolet (UV) radiation as a tumor initiator, tumor promoter and complete carcinogen, and their excessive exposure can lead to the development of various skin disorders including melanoma and nonmelanoma skin cancers. Sunscreens are useful, but their protection is not adequate to prevent the risk of UV-induced skin cancer. It may be because of inadequate use, incomplete spectral protection and toxicity. Therefore new chemopreventive methods are necessary to protect the skin from photodamaging effects of solar UV radiation. Chemoprevention refers to the use of agents that can inhibit, reverse or retard the process of skin carcinogenesis. In recent years, considerable interest has been focused on identifying naturally occurring botanicals, specifically dietary, for the prevention of photocarcinogenesis. A wide variety of botanicals, mostly dietary flavonoids or phenolic substances, have been reported to possess substantial anticarcinogenic and antimutagenic activities because of their antioxidant and antiinflammatory properties. This review summarizes chemopreventive effects of some selected botanicals, such as apigenin, curcumin, grape seed proanthocyanidins, resveratrol, silymarin, and green tea polyphenols, against photocarcinogenesis in in vitro and in vivo systems. Attention has also been focused on highlighting the mechanism of chemopreventive action of these dietary botanicals. We suggest that in addition to the use of these botanicals as dietary supplements for the protection of photocarcinogenesis, these botanicals may favorably supplement sunscreens protection and may provide additional antiphotocarcinogenic protection including the protection against other skin disorders caused by solar UV radiation.

PROTECTION AGAINST ULTRAVIOLET-B RADIATION-INDUCED LOCAL and SYSTEMIC SUPPRESSION OF CONTACT HYPERSENSITIVITY and EDEMA RESPONSES IN C3H/HeN MICE BY GREEN TEA POLYPHENOLS

Abstract— Exposure of skin to UV radiation can cause diverse biological effects, including induction of inflammation, alteration in cutaneous immune cells and impairment of contact hypersensitivity (CHS) responses. Our laboratory has demonstrated that oral feeding as well as topical application of a poly‐phenolic fraction isolated from green tea (GTP) affords protection against the carcinogenic effects of UVB (280–320 nm) radiation. In this study, we investigated whether GTP could protect against UVB‐induced immunosuppression and cutaneous inflammatory responses in C3H mice. Immunosuppression was assessed by contact sensitization with 2,4‐dinitrofluorobenzene applied to UVB‐irradiated skin (local suppression) or to a distant site (systemic suppression), while double skin‐fold swelling was used as the measure of UVB‐induced inflammation. Topical application of GTP (1–6 mg/animal), 30 min prior to or 30 min after exposure to a single dose of UVB (2 kj/m2) resulted in significant protection against local (25–90%) and systemic suppression (23–95%) of CHS and inflammation in mouse dorsal skin (70–80%). These protective effects were dependent on the dose of GTP employed; increasing the dose (1–6 mg/animal) resulted in an increased protective effect (25–93%). The protective effects were also dependent on the dose of UVB (2–32 kJ/m2). Among the four major epicatechin derivatives present in GTP, (‐)‐epigallocatechin‐3‐gallate, the major constituent in GTP, was found to be the most effective in affording protection against UVB‐caused CHS and inflammatory responses. Our study suggests that green tea, specifically polyphenols present therein, may be useful against inflammatory dermatoses and immunosuppression caused by solar radiation.

Dietary grape seed proanthocyanidins inhibit UVB-induced oxidative stress and activation of mitogen-activated protein kinases and nuclear factor-κB signaling in in vivo SKH-1 hairless mice

We have shown previously that dietary grape seed proanthocyanidins (GSP) inhibit UVB-induced photocarcinogenesis in mice. As UVB-induced oxidative stress and oxidative stress–mediated signaling has been implicated in photocarcinogenesis, this study was designed to investigate the effect of dietary GSPs on UVB-induced oxidative stress in in vivo SKH-1 hairless mice. Here, we report that provision of dietary GSPs (0.2 and 0.5%, w/w) to mice exposed to either acute UVB irradiation (120 mJ/cm2) or chronic irradiation of UVB inhibited depletion of glutathione peroxidase, catalase, and glutathione, and inhibited UVB-induced H2O2, lipid peroxidation, protein oxidation, and nitric oxide in mouse skin. As UV-induced oxidative stress mediates activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways, we determined the effect of dietary GSPs on these pathways. We observed that dietary GSPs inhibited UVB-induced phosphorylation of extracellular signal-regulated kinase 1/2, c-Jun-NH2-kinase, and p38 proteins of MAPK family, which seems to be mediated through reactivation of MAPK phosphatases. GSPs inhibited UVB-induced activation of NF-κB/p65 through inhibition of degradation of IκBα and activation of IκB kinase α (IKKα). As NF-κB–targeted genes play critical roles in inflammation and cellular proliferation, we assessed the effect of GSPs on proteins encoded by these genes. Dietary GSPs resulted in inhibition of the expression of proliferating cell nuclear antigen, cyclin D1, inducible nitric oxide synthase, and cyclooxygenase-2 in the skin. Collectively, our data show that GSPs have the ability to protect the skin from the adverse effects of UVB radiation via modulation of the MAPK and NF-κB signaling pathways and provide a molecular basis for the photoprotective effects of GSPs in an in vivo animal model. [Mol Cancer Ther 2007;6(3):995–1005]