In our study, we present experimental evidence suggesting that curcumin exerts multiple different suppressive effects on human breast carcinoma cells in vitro. Our experiments demonstrate that curcumin's antiproliferative effects are estrogen dependent in ER (estrogen receptor)-positive MCF-7 cells, being more pronounced in estrogen-containing media and in the presence of exogenous 17-␤ estradiol. Curcumin inhibits the expression of ER downstream genes including pS2 and TGF-␤ (transforming growth factor) in ER-positive MCF-7 cells, and this inhibition is also dependent on the presence of estrogen. Curcumin also decreases ERE (estrogen responsive element)-CAT activities induced by 17-␤ estradiol. In addition, we demonstrate that curcumin exerts strong antiinvasive effects in vitro that are not estrogen dependent in the ER-negative MDA-MB-231 breast cancer cells. These antiinvasive effects appear to be mediated through the downregulation of MMP-2 (matrix metalloproteinase) and the upregulation of TIMP-1 (tissue inhibitor of metalloproteinase), 2 common effector molecules that have been implicated in regulating tumor cell invasion. Our study also demonstrates that curcumin inhibits the transcript levels of 2 major angiogenesis factors VEGF (vascular endothelial growth factor) and b-FGF (basic fibroblast growth factor) mainly in ER-negative MDA-MB-231 cells. © 2002 Wiley-Liss, Inc. Key words: curcumin; chemoprevention; breast cancer; angiogenesis; estrogenBreast cancer chemoprevention is the subject of substantial research efforts to improve the health of women in the United States. Epidemiologic surveys suggest that diet has an impact on cancer incidence. Frequent consumption of vegetables and fruits decreases the risk for human cancer. 1,2 Although risk reduction by nutritional intervention alone may not be sufficient to protect high-risk individuals against cancer development, it would be very useful to identify agents with chemopreventive potency and to evaluate them in combination with nutritional intervention. 3,4 Recently, attention has been focused on identifying dietary phytochemicals that have the ability to inhibit the processes of carcinogenesis. Extracts of plants or their fractionated ingredients are found to possess inhibitory effects against chemically induced carcinogenesis. 5 Curcumin is a major component of turmeric, the dried rhizome of Curcuma longa L., which is commonly used as a yellow coloring and flavoring agent in food items in Asian countries. Commercial-grade curcumin has shown anticarcinogenic activity in animals as indicated by the ability to block colon tumor initiation induced by azoxymethans 6 and skin tumor promotion induced by phorbol ester. 7 Curcumin also has been reported to possess antiinflammatory activity and is a potent inhibitor of reactive oxygen-generating enzymes such as lipooxygenase/cyclooxygenase, 8,9 xanthine dehydroxygenase/oxidase 10 and nitric oxide synthase (NOS). 11,12 Recently, it has been shown that the administration of synthetic curcumin in the diet during t...

Our group has shown in a phase II clinical trial that pomegranate juice (PJ) increases prostate specific antigen (PSA) doubling time in prostate cancer (CaP) patients with a rising PSA. Ellagitannins (ETs) are the most abundant polyphenols present in PJ and contribute greatly towards its reported biological properties. On consumption, ETs hydrolyze to release ellagic acid (EA), which is then converted by gut microflora to 3,8-dihydroxy-6H-dibenzo [b,d]pyran-6-one (urolithin A, UA) derivatives. Despite the accumulating knowledge of ET metabolism in animals and humans, there is no available data on the pharmacokinetics and tissue disposition of urolithins. Using a standardized ET-enriched pomegranate extract (PE), we sought to further define the metabolism and tissue distribution of ET metabolites. PE and UA (synthesized in our laboratory) were administered to C57BL/6 wild-type male mice, and metabolite levels in plasma and tissues were determined over 24 h. ET metabolites were concentrated at higher levels in mouse prostate, colon, and intestinal tissues as compared to other tissues after administration of PE or UA. We also evaluated the effects of PE on CaP growth in severe combined immunodeficient (SCID) mice injected subcutaneously with human CaP cells (LAPC-4). PE significantly inhibited LAPC-4 xenograft growth in SCID mice as compared to vehicle control. Finally, EA and several synthesized urolithins were shown to inhibit the growth of human CaP cells in vitro. The chemopreventive potential of pomegranate ETs and localization of their bioactive metabolites in mouse prostate tissue suggest that pomegranate may play a role in CaP treatment and chemoprevention. This warrants future human tissue bioavailability studies and further clinical studies in men with CaP.

Investigators have shown that green tea and its main catechin epigallocatechin-3 gallate (EGCG) may decrease the risk of cancer. Our previous study showed that green tea extract (GTE) as well as its individual catechin components inhibited MDA-MB231 breast cancer cell and human umbilical vein endothelial cell (HUVEC) proliferation. Further, GTE suppressed breast cancer xenograft size and decreased the tumor vessel density in vivo. In the current study, we investigated the effect of GTE on the major angiogenic factor vascular endothelial growth factor (VEGF) in an in vitro experiment. GTE or EGCG (40 mg/L) significantly decreased the levels of the VEGF peptide secreted into conditioned media. This occurred in both HUVEC and human breast cancer cells and the effect was dose dependent. Furthermore, GTE and EGCG decreased the RNA levels of VEGF in MDA-MB231 cells. This inhibition occurred at the transcriptional regulation level and was accompanied by a significant decrease in VEGF promoter activity. We also showed that GTE decreased c-fos and c-jun RNA transcripts, suggesting that activator protein (AP)-1-responsive regions present in the human VEGF promoter may be involved in the inhibitory effect of GTE. Furthermore, GTE suppressed the expression of protein kinase C, another VEGF transcription modulator, in breast cancer cells. Inhibition of VEGF transcription appeared to be one of the molecular mechanism(s) involved in the antiangiogenic effects of green tea, which may contribute to its potential use for breast cancer treatment and/or prevention.

Nitroxyl (HNO) can inhibit the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Because of the importance of glycolysis in many malignant cells, we thus propose that HNO can adversely affect tumor growth. This hypothesis was tested using in vitro and in vivo models of breast cancer. We report here for the first time that HNO suppresses the proliferation of both estrogen receptor (ER)-positive and ER-negative human breast cancer cell lines, in a dose dependent manner. Mice treated with HNO either injected into the tumor itself or via the intraperitoneal approach had smaller xenograft tumor size. In addition to significantly decreased blood vessel density in the HNO-treated tumors, we observed lower levels of circulating serum vascular endothelial growth factor (VEGF). Accordingly, there was a decrease in total HIF-1a (hypoxia-inducible factor) protein in HNO-treated tumor cells. Further studies showed inhibition of GAPDH activity in HNO-treated human breast cancer cell lines and in HNO-treated tumor tissue derived from xenografts. One explanation for the multiplicity of actions observed after HNO treatment could be the effect from the initial inhibition of GAPDH, providing a potential therapeutic avenue based upon blocking glycolysis resulting in decreased HIF-1a, thus leading to angiogenesis inhibition. Therefore, HNO appears to act via mechanism(s) different from those of existing breast cancer drugs, making it a potential candidate to overcome known and emerging drug resistance pathways. ' 2007 Wiley-Liss, Inc.

Abstract. Angiogenesis is critical to tumor growth and is stimulated by tissue hypoxia due to poor oxygen delivery. In turn, cellular hypoxia leads to angiogenesis via the induction of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) at a cellular level. Pomegranate juice and extracts, which are rich sources of ellagitannins, have been shown to have chemopreventive potential against prostate cancer, but there have been no studies on the effects of an ellagitannin-rich pomegranate extract on angiogenesis. Human prostate cancer cells (LNCaP) and human umbilical vein endothelial cells (HUVEC) were incubated with a pomegranate extract standardized to ellagitannin content (POMx), under normoxic and hypoxic conditions in vitro. Human prostate cancer cells (LAPC4) were injected subcutaneously into severe combined immunodeficient (SCID) mice and the effects of oral administration of POMx on tumor growth, microvessel density, and HIF-1α and VEGF expression were determined after 4 weeks of treatment. POMx inhibited the proliferation of LNCaP and HUVEC cells significantly under both normoxic and hypoxic conditions. HIF-1α and VEGF protein levels were also reduced by POMx under hypoxic conditions. POMx decreased prostate cancer xenograft size, tumor vessel density, VEGF peptide levels and HIF-1α expression after 4 weeks of treatment in SCID mice. These results demonstrate that an ellagitannin-rich pomegranate extract can inhibit tumor-associated angiogenesis as one of several potential mechanisms for slowing the growth of prostate cancer in chemopreventive applications. Further studies in humans are needed to confirm that angiogenesis can be inhibited by an ellagitannin-rich pomegranate extract administered orally as a dietary supplement.

Investigators have shown that green tea may decrease the risk of cancer. It is widely accepted that the main active component of green tea is epigallocatechin-3-gallate (EGCG). In this study, we examined the effect of green tea on breast cancer growth and endothelial cells in in vitro assays and in animal models. Furthermore, we compared the potency of the different catechin components of green tea extract (GTE), including EGCG. Our data showed that mixed GTE and its individual catechin components were effective in inhibiting breast cancer and endothelial cell proliferation. In mouse experiments, GTE suppressed xenograft size and decreased the tumor vessel density. Our results demonstrated the value of all catechins and argued for the use of a mixed GTE as a botanical dietary supplement, rather than purified EGCG, in future clinical trials.

Epidemiologic data have suggested that green tea may prevent breast cancer. Studies in our laboratory have provided evidence that green tea extract inhibits breast cancer growth by a direct anti-proliferative effect on the tumor cells, as well as by indirect suppressive effects on the tumor-associated endothelial cells. In this study, we asked whether concurrent administration of green tea may add to the anti-tumor effects of standard breast cancer therapy. We observed that green tea increased the inhibitory effect of tamoxifen on the proliferation of the ER (estrogen receptor)-positive MCF-7, ZR75, T47D human breast cancer cells in vitro. This combination regimen was also more potent than either agent alone at increasing cell apoptosis. In animal experiments, mice treated with both green tea and tamoxifen had the smallest MCF-7 xenograft tumor size, and the highest levels of apoptosis in tumor tissue, as compared with either agent administered alone. Moreover, the suppression of angiogenesis in vivo correlated with larger areas of necrosis and lower tumor blood vessel density in treated xenografts. Green tea decreased levels of ER-alpha in tumors both in vitro and in vivo. We also observed that green tea blocked ER-dependent transcription, as well as estradiol-induced phosphorylation and nuclear localization of mitogen-activated protein kinase. To our knowledge, this study is the first to show the interaction of green tea with the ER pathway, as well as provide mechanistic evidence that the combination of green tea and tamoxifen is more potent than either agent alone in suppressing breast cancer growth. These results may lead to future improvements in breast cancer treatment and prevention.

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