Combinatorial bioactive botanicals re-sensitize tamoxifen treatment in ER-negative breast cancer via epigenetic reactivation of ERα expression.

Conventional cancer prevention has primarily focused on single chemopreventive compounds that may not be sufficiently efficacious. We sought to investigate potential combinatorial effects of epigenetic bioactive botanicals including epigallocatechin-3-gallate (EGCG) in green tea polyphenols (GTPs) and sulforaphane (SFN) in broccoli sprouts (BSp) on neutralizing epigenetic aberrations in estrogen receptor-α (ERα) leading to enhanced anti-hormone therapeutic efficacy in ERα-negative breast cancer. Our results showed that this combinatorial treatment re-sensitized ERα-dependent cellular inhibitory responses to an estrogen antagonist, tamoxifen (TAM), via at least in part, epigenetic reactivation of ERα expression in ERα-negative breast cancer cells. Further in vivo studies revealed the combinatorial diets of GTPs and BSp significantly inhibited breast tumor growth in ERα-negative mouse xenografts, especially when combined with TAM treatment. This novel treatment regimen can lead to remodeling of the chromatin structure by histone modifications and recruitment changes of transcriptional factor complex in the ERα promoter thereby contributing to ERα reactivation and re-sensitized chemotherapeutic efficacy of anti-hormone therapy. Our studies indicate that combinatorial bioactive botanicals from GTPs and BSp are highly effective in inhibiting ERα-negative breast cancer due at least in part to epigenetic reactivation of ERα, which in turn increases TAM-dependent anti-estrogen chemosensitivity in vitro and in vivo.

Epigenetic reactivation of p21CIP1/WAF1 and KLOTHO by a combination of bioactive dietary supplements is partially ERα-dependent in ERα-negative human breast cancer cells.

Available treatment strategies against estrogen receptor (ER)-negative breast cancer patients are limited due to their poor response to hormonal therapy. We have shown previously that the combinations of green tea polyphenols (GTPs), a dietary DNA methyltransferase inhibitor, and sulforaphane (SFN), a dietary histone deacetylase inhibitor, reactivate ERα expression in ERα-negative MDA-MB-231 cells. Here, we investigated the functional significance of ERα reactivation in the reactivation of silenced tumor suppressor genes (TSGs) in ERα-negative human breast cancer cells. We found that the treatment of MDA-MB-231 cells with the combinations of GTPs and SFN leads to the reactivation of silenced TSGs such as p21(CIP1/WAF1) and KLOTHO through active chromatin modifications. Further, GTPs- and SFN-mediated reactivation of TSGs was, at least in part, dependent on ERα reactivation in ERα-negative MDA-MB-231 cells. Collectively, our findings suggest that a novel combination of bioactive dietary supplements could further be explored as an effective therapeutic option against hormonal refractory breast cancer.

Bioactive dietary supplements reactivate ER expression in ER-negative breast cancer cells by active chromatin modifications.

Breast cancer is the most common cancer and the leading cause of cancer death in women. Although tamoxifen therapy is successful for some patients, it does not provide adequate benefit for those who have estrogen receptor (ER)-negative cancers. Therefore, we approached novel treatment strategies by combining two potential bioactive dietary supplements for the reactivation of ERα expression for effective treatment of ERα-negative breast cancer with tamoxifen. Bioactive dietary supplements such as green tea polyphenols (GTPs) and sulforaphane (SFN) inhibit DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), respectively, which are of central importance to cancer prevention. In the present study, we have observed that treatment of ERα-negative breast cancer cells with GTPs and SFN alone or in combination leads to the reactivation of ERα expression. The combination of 20 µg/mL GTPs and 5 µM SFN was found to be the optimal dose of ERα-reactivation at 3 days in MDA-MB-231 cells. The reactivation of ERα expression was consistently correlated with ERα promoter hypomethylation and hyperacetylation. Chromatin immunoprecipitation (ChIP) analysis of the ERα promoter revealed that GTPs and SFN altered the binding of ERα-transcriptional co-repressor complex thereby contributing to ERα-reactivation. In addition, treatment with tamoxifen in combination with GTPs and SFN significantly increased both cell death and inhibition of cellular proliferation in MDA-MB-231 cells in comparison to treatment with tamoxifen alone. Collectively, our findings suggest that a novel combination of bioactive-HDAC inhibitors with bioactive-demethylating agents is a promising strategy for the effective treatment of hormonal refractory breast cancer with available anti-estrogens.

Synergistic epigenetic reactivation of estrogen receptor-α (ERα) by combined green tea polyphenol and histone deacetylase inhibitor in ERα-negative breast cancer cells.

BACKGROUND: The status of estrogen receptor-α (ERα) is critical to the clinical prognosis and therapeutic approach in breast cancer. ERα-negative breast cancer is clinically aggressive and has a poor prognosis because of the lack of hormone target-directed therapies. Previous studies have shown that epigenetic regulation plays a major role in ERα silencing in human breast cancer cells. Dietary green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), is believed to be an anticancer agent in part through its regulation of epigenetic processes. RESULTS: In our current studies, we found that EGCG can reactivate ERα expression in ERα-negative MDA-MB-231 breast cancer cells. Combination studies using EGCG with the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), revealed a synergistic effect of reactivation of ERα expression in ERα-negative breast cancer cells. Reactivation of ERα expression by EGCG and TSA treatment was found to sensitize ERα-dependent cellular responses to activator 17ß-estradiol (E2) and antagonist tamoxifen in ERα-negative breast cancer cells. We also found that EGCG can lead to remodeling of the chromatin structure of the ERα promoter by altering histone acetylation and methylation status thereby resulting in ERα reactivation. A decreased binding of the transcription repressor complex, Rb/p130-E2F4/5-HDAC1-SUV39H1-DNMT1, in the regulatory region of the ERα promoter also contributes to ERα transcriptional activation through treatment with EGCG and/or TSA. CONCLUSIONS: Collectively, these studies show that green tea EGCG can restore ERα expression by regulating epigenetic mechanisms, and this effect is enhanced when combined with an HDAC inhibitor. This study will facilitate more effective uses of combination approaches in breast cancer therapy and will help to explore more effective chemotherapeutic strategies toward hormone-resistant breast cancer.

Green tea polyphenols prevent UV-induced immunosuppression by rapid repair of DNA damage and enhancement of nucleotide excision repair genes.

UV radiation-induced immunosuppression has been implicated in the development of skin cancers. Green tea polyphenols (GTP) in drinking water prevent photocarcinogenesis in the skin of mice. We studied whether GTPs in drinking water (0.1-0.5%, w/v) prevent UV-induced immunosuppression and (if so) potential mechanisms of this effect in mice. GTPs (0.2% and 0.5%, w/v) reduced UV-induced suppression of contact hypersensitivity (CHS) in response to a contact sensitizer in local (58-62% reductions; P < 0.001) and systemic (51-55% reductions; P < 0.005) models of CHS. Compared with untreated mice, GTP-treated mice (0.2%, w/v) had a reduced number of cyclobutane pyrimidine dimer-positive (CPD(+)) cells (59%; P < 0.001) in the skin, showing faster repair of UV-induced DNA damage, and had a reduced (2-fold) migration of CPD(+) cells from the skin to draining lymph nodes, which was associated with elevated levels of nucleotide excision repair (NER) genes. GTPs did not prevent UV-induced immunosuppression in NER-deficient mice but significantly prevented it in NER-proficient mice (P < 0.001); immunohistochemical analysis of CPD(+) cells indicated that GTPs reduced the numbers of UV-induced CPD(+) cells in NER-proficient mice (P < 0.001) but not in NER-deficient mice. Southwestern dot-blot analysis revealed that GTPs repaired UV-induced CPDs in xeroderma pigmentosum complementation group A (XPA)-proficient cells of a healthy person but did not in XPA-deficient cells obtained from XPA patients, indicating that a NER mechanism is involved in DNA repair. This study is the first to show a novel NER mechanism by which drinking GTPs prevents UV-induced immunosuppression and that inhibiting UV-induced immunosuppression may underlie the chemopreventive activity of GTPs against photocarcinogenesis.

Epigenetic targets of bioactive dietary components for cancer prevention and therapy.

The emergent interest in cancer epigenetics stems from the fact that epigenetic modifications are implicated in virtually every step of tumorigenesis. More interestingly, epigenetic changes are reversible heritable changes that are not due to the alteration in DNA sequence but have potential to alter gene expression. Dietary agents consist of many bioactive ingredients which actively regulate various molecular targets involved in tumorigenesis. We present evidence that numerous bioactive dietary components can interfere with various epigenetic targets in cancer prevention and therapy. These agents include curcumin (turmeric), genistein (soybean), tea polyphenols (green tea), resveratrol (grapes), and sulforaphane (cruciferous vegetables). These bioactive components alter the DNA methylation and histone modifications required for gene activation or silencing in cancer prevention and therapy. Bioactive components mediate epigenetic modifications associated with the induction of tumor suppressor genes such as p21(WAF1/CIP1) and inhibition of tumor promoting genes such as the human telomerase reverse transcriptase during tumorigenesis processes. Here, we present considerable evidence that bioactive components and their epigenetic targets are associated with cancer prevention and therapy which should facilitate novel drug discovery and development. In addition, remarkable advances in our understanding of basic epigenetic mechanisms as well as the rapid progress that is being made in developing powerful new technologies, such as those for sensitive and quantitative detection of epigenetic and epigenomic changes in cancer biology, hold great promise for novel epigenetic approaches to cancer prevention and therapy.

Grape seed proanthocyanidins inhibit the growth of human non-small cell lung cancer xenografts by targeting insulin-like growth factor binding protein-3, tumor cell proliferation, and angiogenic factors.

PURPOSE: Lung cancer is a leading cause of cancer-related deaths worldwide. Here, we assessed the chemotherapeutic effect of grape seed proanthocyanidins (GSPs) on human non-small cell lung cancer (NSCLC) cells in vitro and in vivo using a tumor xenograft model. EXPERIMENTAL DESIGN: The effects of GSPs on human NSCLC cell lines in terms of cellular proliferation were determined. The chemotherapeutic effects of a GSP- supplemented AIN76A control diet fed to nude mice bearing tumor xenografts (A549 and H1299) were evaluated in terms of biomarkers of cell proliferation and angiogenesis and on insulin-like growth factor binding protein-3 using immunohistochemical detection, ELISA, and Western blotting. RESULTS: In vitro treatment of NSCLC cells with GSPs resulted in inhibition of cellular proliferation. Administration of GSPs (0.1%, 0.2%, and 0.5%, w/w) as a supplement of an AIN76A control diet resulted in a dose-dependent inhibition of the growth of NSCLC (A549 and H1299) tumor xenografts in athymic nude mice (25-76%; P < 0.05-0.001). The growth-inhibitory effect of GSPs on the NSCLC xenograft tumors was associated with the enhancement of the levels of insulin-like growth factor binding protein-3 in the tumor microenvironment and plasma and antiproliferative, antiangiogenic, and proapoptotic effects. CONCLUSIONS: This preclinical study reveals for the first time that dietary GSPs have the ability to inhibit the growth of human NSCLC tumor xenografts grown in vivo in athymic nude mice. More studies are needed to develop GSPs as a pharmacologically safe agent for the prevention of lung cancer in humans.

Dietary grape seed proanthocyanidins inhibit 12-O-tetradecanoyl phorbol-13-acetate-caused skin tumor promotion in 7,12-dimethylbenz[a]anthracene-initiated mouse skin, which is associated with the inhibition of inflammatory responses.

Grape seed proanthocyanidins (GSPs) possess anticarcinogenic activities. Here, we assessed the effects of dietary GSPs on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumor promotion in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. Administration of dietary GSPs (0.2 and 0.5%, wt/wt) supplemented with control AIN76A diet resulted in significant inhibition of TPA-induced skin tumor promotion in C3H/HeN mice. The mice treated with GSPs developed a significantly lower tumor burden in terms of the percentage of mice with tumors (P < 0.05), total number of tumors per group (P < 0.01, n = 20) and total tumor volume per tumor-bearing mouse (P < 0.01-0.001) as compared with the mice that received the control diet. GSPs also delayed the malignant progression of papillomas into carcinomas. As TPA-induced inflammatory responses are used routinely as markers of skin tumor promotion, we assessed the effect of GSPs on biomarkers of TPA-induced inflammation. Immunohistochemical analysis and western blotting revealed that GSPs significantly inhibited expression of cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)) and markers of proliferation (proliferating cell nuclear antigen and cyclin D1) in both the DMBA-initiated/TPA-promoted mouse skin and skin tumors. In short-term experiments in which the mouse skin was treated with acute or multiple TPA applications, we found that dietary GSPs inhibited TPA-induced edema, hyperplasia, leukocytes infiltration, myeloperoxidase, COX-2 expression and PGE(2) production in the mouse skin. The inhibitory effect of GSPs was also observed against other structurally different skin tumor promoter-induced inflammation in the skin. Together, our results show that dietary GSPs inhibit chemical carcinogenesis in mouse skin and that the inhibition of skin tumorigenesis by GSPs is associated with the inhibition of inflammatory responses caused by tumor promoters.

Inhibition of UVB-induced skin tumor development by drinking green tea polyphenols is mediated through DNA repair and subsequent inhibition of inflammation.

Consumption of green tea polyphenols (GTPs) in drinking water prevents photocarcinogenesis in mice; however, the molecular mechanisms underlying this effect have not been fully elucidated. Using IL-12p40 knockout (KO) mice and their wild-type counterparts and an established photocarcinogenesis protocol, we found that although administration of GTPs (0.2%, w/v) in drinking water significantly reduced UVB-induced tumor development in wild-type mice, this treatment had a nonsignificant effect in IL-12-KO mice. GTPs resulted in reduction in the levels of markers of inflammation (cyclooxygenase-2, prostaglandin E(2), proliferating cell nuclear antigen, and cyclin D1) and proinflammatory cytokines (tumor necrosis factor-alpha, IL-6, and IL-1beta) in chronically UVB-exposed skin and skin tumors of wild-type mice but less effective in IL-12p40-KO mice. UVB-induced DNA damage (cyclobutane pyrimidine dimers) was resolved rapidly in GTPs-treated wild-type mice than untreated wild-type mice and this resolution followed the same time course as the GTPs-induced reduction in the levels of inflammatory responses. This effect of GTPs was less pronounced in IL-12-KO mice. The above results were confirmed by treatment of IL-12-KO mice with murine recombinant IL-12 and treatment of wild-type mice with neutralizing anti-IL-12 antibody. To our knowledge, it is previously unreported that prevention of photocarcinogenesis by GTPs is mediated through IL-12-dependent DNA repair and a subsequent reduction in skin inflammation.

Dietary grape seed proanthocyanidins inhibit UVB-induced oxidative stress and activation of mitogen-activated protein kinases and nuclear factor-kappaB 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/cm(2)) or chronic irradiation of UVB inhibited depletion of glutathione peroxidase, catalase, and glutathione, and inhibited UVB-induced H(2)O(2), 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-kappaB (NF-kappaB) 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-NH(2)-kinase, and p38 proteins of MAPK family, which seems to be mediated through reactivation of MAPK phosphatases. GSPs inhibited UVB-induced activation of NF-kappaB/p65 through inhibition of degradation of IkappaBalpha and activation of IkappaB kinase alpha (IKKalpha). As NF-kappaB-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-kappaB signaling pathways and provide a molecular basis for the photoprotective effects of GSPs in an in vivo animal model.

Silymarin inhibits UV radiation-induced immunosuppression through augmentation of interleukin-12 in mice.

We have shown previously that silymarin, a plant flavonoid, inhibits UVB-induced photocarcinogenesis in mice. As UVB-induced immunosuppression has been implicated in the development of skin cancer, we investigated whether silymarin can modulate the effects of UVB radiation on the immune system. Treatment of C3H/HeN mice with topically applied silymarin (0.5 or 1.0 mg/cm(2)) or silibinin, a major component of silymarin, markedly inhibited UVB (180 mJ/cm(2))-induced suppression of contact hypersensitivity response in a local model of immunosuppression and had a moderate inhibitory effect in a systemic model of contact hypersensitivity. Silymarin reduced the UVB-induced enhancement of the levels of the immunosuppressive cytokine, interleukin (IL)-10, in the skin and draining lymph nodes and enhanced the levels of the immunostimulatory cytokine, IL-12. Intraperitoneal injection of mice treated with silymarin with an endotoxin-free neutralizing anti-IL-12 antibody abrogated the protective effects of the silymarin against UVB-induced suppression of the contact hypersensitivity response. Furthermore, the treatment of silymarin did not prevent UVB-induced suppression of the contact hypersensitivity response in IL-12 knockout mice but prevented it in their wild-type mice. Moreover, i.p. injection of IL-12 to silymarin-treated or non-silymarin-treated IL-12 knockout mice resulted in an enhanced response to contact hypersensitivity compared with the response in mice that were exposed to either UVB alone or silymarin plus UVB. These data indicate for the first time that silymarin has the ability to protect mice from UVB-induced immunosuppression and that this protective effect is mediated, at least in part, through IL-12.

Orally administered green tea polyphenols prevent ultraviolet radiation-induced skin cancer in mice through activation of cytotoxic T cells and inhibition of angiogenesis in tumors.

Green tea polyphenols (GTPs) show promise as anticarcinogenic agents and may prevent the development of solar UV radiation-induced skin cancer. Here we investigated the mechanisms by which GTPs prevent UVB-induced skin cancer in mice. Two groups of 6- to 7-wk-old female SKH-1 hairless mice were UVB irradiated (180 mJ/cm(2)) 3 times each week for 24 wk. One group consumed water and the other, water containing 2 g/L GTPs. A control group drank water and was not exposed to UVB radiation. UVB-induced tumors and skin biopsies from the control group were analyzed using immunostaining, Western blotting, and gelatinolytic zymography. Oral administration of GTPs reduced UVB-induced tumor incidence (35%), tumor multiplicity (63%), and tumor growth (55%). The GTPs+UVB group had reduced expression of the matrix metalloproteinases (MMP)-2 and MMP-9, which have crucial roles in tumor growth and metastasis, and enhanced expression of tissue inhibitor of MMP in the tumors compared with mice that were treated with UVB alone. The GTPs+UVB group also had reduced expressions of CD31 and vascular endothelial growth factor, which are essential for angiogenesis, and inhibited expression of proliferating cell nuclear antigen in the tumors compared with the UVB group. Additionally, there were more cytotoxic CD8(+) T cells in the tumors of the GTPs+UVB group than in the UVB group and their tumor cells exhibited greater activation of caspase-3, indicating the apoptotic death of the tumor cells. Taken together, these data suggest that in mice, administration of GTPs affects several biomarkers that are involved in UV-carcinogenesis, including inhibition of angiogenic factors and recruitment of cytotoxic T cells in the tumor microenvironment.