A combination of p53-activating APR-246 and phosphatidylserine-targeting antibody potently inhibits tumor development in hormone-dependent mutant p53-expressing breast cancer xenografts.

BACKGROUND: Between 30 and 40% of human breast cancers express a defective tumor suppressor p53 gene. Wild-type p53 tumor suppressor protein promotes cell-cycle arrest and apoptosis and inhibits vascular endothelial growth factor-dependent angiogenesis, whereas mutant p53 protein (mtp53) lacks these functions, resulting in tumor cell survival and metastasis. Restoration of p53 function is therefore a promising drug-targeted strategy for combating mtp53-expressing breast cancer. METHODS: In this study, we sought to determine whether administration of APR-246, a small-molecule drug that restores p53 function, in combination with 2aG4, an antibody that targets phosphatidylserine residues on tumor blood vessels and disrupts tumor vasculature, effectively inhibits advanced hormone-dependent breast cancer tumor growth. RESULTS: APR-246 reduced cell viability in mtp53-expressing BT-474 and T47-D human breast cancer cells in vitro, and significantly induced apoptosis in a dose-dependent manner. However, APR-246 did not reduce cell viability in MCF-7 breast cancer cells, which express wild-type p53. We next examined APR-246's anti-tumor effects in vivo using BT-474 and T47-D tumor xenografts established in female nude mice. Tumor-bearing mice were treated with APR-246 and/or 2aG4 and tumor volume followed over time. Tumor growth was more effectively suppressed by combination treatment than by either agent alone, and combination therapy completely eradicated some tumors. Immunohistochemistry analysis of tumor tissue sections demonstrated that combination therapy more effectively induced apoptosis and reduced cell proliferation in tumor xenografts than either agent alone. Importantly, combination therapy dramatically reduced the density of blood vessels, which serve as the major route for tumor metastasis, in tumor xenografts compared with either agent alone. CONCLUSION: Based on our findings, we contend that breast tumor growth might effectively be controlled by simultaneous targeting of mtp53 protein and tumor blood vessels in mtp53-expressing cancers.

Natural and synthetic progestins enrich cancer stem cell-like cells in hormone-responsive human breast cancer cell populations in vitro.

Clinical trials and studies have shown that combination estrogen/progestin hormone replacement therapy, but not estrogen therapy alone or placebo, increases breast cancer risk in postmenopausal women. Using animal models, we have previously shown that both natural and synthetic progestins (including medroxyprogesterone acetate [MPA], a synthetic progestin used widely in the clinical setting) accelerate the development of breast tumors in vivo and increase their metastasis to lymph nodes. Based on these observations, we have hypothesized that progestin-induced breast cancer tumor growth and metastasis may be mediated by an enrichment of the cancer stem cell (CSC) pool. In this study, we used T47-D and BT-474 hormone-responsive human breast cancer cells to examine the effects of progestin on phenotypic and functional markers of CSCs in vitro. Both natural and synthetic progestins (10 nM) significantly increased protein expression of CD44, an important CSC marker in tumor cells. MPA increased the levels of both CD44 variants v3 and v6 associated with stem cell functions. This induction of CD44 was blocked by the antiprogestin RU-486, suggesting that this process is progesterone receptor (PR) dependent. CD44 induction was chiefly progestin dependent. Because RU-486 can bind other steroid receptors, we treated PR-negative T47-DCO-Y cells with MPA and found that MPA failed to induce CD44 protein expression, confirming that PR is essential for progestin-mediated CD44 induction in T47-D cells. Further, MPA treatment of T47-D cells significantly increased the activity of aldehyde dehydrogenase (ALDH), another CSC marker. Finally, two synthetic progestins, MPA and norethindrone, significantly increased the ability of T47-D cells to form mammospheres, suggesting that enrichment of the CD44high, ALDHbright subpopulation of cancer cells induced by MPA exposure is of functional significance. Based on our observations, we contend that exposure of breast cancer cells to synthetic progestins leads to an enrichment of the CSC pool, supporting the development of progestin-accelerated tumors in vivo.

Cholesterol biosynthesis inhibitor RO 48-8071 suppresses growth of hormone-dependent and castration-resistant prostate cancer cells.

Standard treatment for primary prostate cancer includes systemic exposure to chemotherapeutic drugs that target androgen receptor or antihormone therapy (chemical castration); however, drug-resistant cancer cells generally emerge during treatment, limiting the continued use of systemic chemotherapy. Patients are then treated with more toxic standard therapies. Therefore, there is an urgent need for novel and more effective treatments for prostate cancer. The cholesterol biosynthetic pathway is an attractive therapeutic target for treating endocrine-dependent cancers because cholesterol is an essential structural and functional component of cell membranes as well as the metabolic precursor of endogenous steroid hormones. In this study, we have examined the effects of RO 48-8071 (4'-[6-(allylmethylamino)hexyloxy]-4-bromo-2'-fluorobenzophenone fumarate; Roche Pharmaceuticals internal reference: RO0488071) (RO), which is an inhibitor of 2, 3-oxidosqualene cyclase (a key enzyme in the cholesterol biosynthetic pathway), on prostate cancer cells. Exposure of both hormone-dependent and castration-resistant human prostate cancer cells to RO reduced prostate cancer cell viability and induced apoptosis in vitro. RO treatment reduced androgen receptor protein expression in hormone-dependent prostate cancer cells and increased estrogen receptor ß (ERß) protein expression in both hormone-dependent and castration-resistant prostate cancer cell lines. Combining RO with an ERß agonist increased its ability to reduce castration-resistant prostate cancer cell viability. In addition, RO effectively suppressed the growth of aggressive castration-resistant human prostate cancer cell xenografts in vivo without any signs of toxicity to experimental animals. Importantly, RO did not reduce the viability of normal prostate cells in vitro. Our study is the first to demonstrate that the cholesterol biosynthesis inhibitor RO effectively suppresses growth of human prostate cancer cells. Our findings suggest that cholesterol biosynthesis inhibitors such as RO, when used in combination with commonly used chemotherapeutic drugs or ERß specific ligands, could represent a novel therapeutic approach to prevent the growth of prostate cancer tumors.

Luteolin suppresses development of medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a)anthracene-induced mammary tumors in Sprague-Dawley rats.

Postmenopausal women undergoing hormone-replacement therapy containing both progestins and estrogens are at an increased risk of developing breast cancer compared with women taking estrogen alone. We recently demonstrated that medroxyprogesterone acetate, a progestin commonly used for hormone-replacement therapy, accelerates development of mammary carcinogenesis in 7,12-dimethylbenz(a)anthracene­treated Sprague-Dawley rats. Synthetic antiprogestins used to block the deleterious effects of progestins, are themselves associated with toxic side-effects. In order to circumvent this, we used the aforementioned model to identify less toxic natural compounds that may prevent the development of progestin-accelerated tumors. Luteolin, a naturally-occurring flavonoid commonly found in fruits and vegetables, has previously been shown to possess anticancer properties. In our studies, both low (1 mg/kg) and high (25 mg/kg) doses of luteolin significantly suppressed progestin-dependent increases in tumor incidence, while increasing tumor latency and reducing the occurrence of large (>300 mm3) mammary tumors. However, an intermediate dose of luteolin (10 mg/kg), while suppressing the development of large tumors, did not affect either tumor incidence or latency. Immunohistochemical analysis of tumor tissues revealed that all concentrations of luteolin (1, 10, and 25 mg/kg) significantly reduced levels of VEGF within tumors. The suppressive effects of luteolin on tumor incidence and volume, together with its ability to reduce VEGF and blood vessels, persisted even after treatment was terminated. This suggests that luteolin possesses anti­angiogenic properties which could mechanistically explain its capacity to control tumor progression. Thus luteolin may be a valuable, non-toxic, naturally-occurring anticancer compound which may potentially be used to combat progestin-accelerated mammary tumors.

TGF-ß Negatively Regulates CXCL1 Chemokine Expression in Mammary Fibroblasts through Enhancement of Smad2/3 and Suppression of HGF/c-Met Signaling Mechanisms.

Fibroblasts are major cellular components of the breast cancer stroma, and influence the growth, survival and invasion of epithelial cells. Compared to normal tissue fibroblasts, carcinoma associated fibroblasts (CAFs) show increased expression of numerous soluble factors including growth factors and cytokines. However, the mechanisms regulating expression of these factors remain poorly understood. Recent studies have shown that breast CAFs overexpress the chemokine CXCL1, a key regulator of tumor invasion and chemo-resistance. Increased expression of CXCL1 in CAFs correlated with poor patient prognosis, and was associated with decreased expression of TGF-ß signaling components. The goal of these studies was to understand the role of TGF-ß in regulating CXCL1 expression in CAFs, using cell culture and biochemical approaches. We found that TGF-ß treatment decreased CXCL1 expression in CAFs, through Smad2/3 dependent mechanisms. Chromatin immunoprecipitation and site-directed mutagenesis assays revealed two new binding sites in the CXCL1 promoter important for Smad2/3 modulation of CXCL1 expression. Smad2/3 proteins also negatively regulated expression of Hepatocyte Growth Factor (HGF), which was found to positively regulate CXCL1 expression in CAFs through c-Met receptor dependent mechanisms. HGF/c-Met signaling in CAFs was required for activity of NF-κB, a transcriptional activator of CXCL1 expression. These studies indicate that TGF-ß negatively regulates CXCL1 expression in CAFs through Smad2/3 binding to the promoter, and through suppression of HGF/c-Met autocrine signaling. These studies reveal novel insight into how TGF-ß and HGF, key tumor promoting factors modulate CXCL1 chemokine expression in CAFs.

Luteolin inhibits progestin-dependent angiogenesis, stem cell-like characteristics, and growth of human breast cancer xenografts.

PURPOSE: Clinical trials and epidemiological evidence have shown that combined estrogen/progestin hormone replacement therapy, but not estrogen therapy alone, increases breast cancer risk in post-menopausal women. Previously we have shown that natural and synthetic progestins, including the widely used synthetic progestin medroxyprogesterone acetate (MPA), increase production of a potent angiogenic factor, vascular endothelial growth factor (VEGF), in human breast cancer cells, potentially providing an explanation for progestin's mechanism of action. Here, we tested the effects of luteolin (LU), a flavonoid commonly found in fruits and vegetables, on inhibiting progestin-dependent VEGF induction and angiogenesis in human breast cancer cells, inhibiting stem cell-like characteristics, as well as breast cancer cell xenograft tumor growth in vivo and expression of angiogenesis markers. METHODS: Viability of both T47-D and BT-474 cells was measured using sulforhodamine B assays. Enzyme-linked immunosorbent assays were used to monitor VEGF secretion from breast cancer cells. Progestin-dependent xenograft tumor growth was used to determine LU effects in vivo. CD31 immunohistochemistry was used to determine blood-vessel density in xenograft tumors. CD44 expression, aldehyde dehydrogenase activity, and mammosphere-formation assays were used to monitor stem cell-like characteristics of breast cancer cells. RESULTS: Luteolin treatment reduced breast cancer cell viability, progestin-dependent VEGF secretion from breast cancer cells, and growth of MPA-dependent human breast cancer cell xenograft tumors in nude mice. LU treatment also decreased xenograft tumor VEGF expression and blood-vessel density. Furthermore, LU blocked MPA-induced acquisition of stem cell-like properties by breast cancer cells. CONCLUSIONS: Luteolin effectively blocks progestin-dependent human breast cancer tumor growth and the stem cell-like phenotype in human breast cancer cells.

Cholesterol synthesis inhibitor RO 48-8071 suppresses transcriptional activity of human estrogen and androgen receptor.

Breast cancer cells express enzymes that convert cholesterol, the synthetic precursor of steroid hormones, into estrogens and androgens, which then drive breast cancer cell proliferation. In the present study, we sought to determine whether oxidosqualene cyclase (OSC), an enzyme in the cholesterol biosynthetic pathway, may be targeted to suppress progression of breast cancer cells. In previous studies, we showed that the OSC inhibitor RO 48-8071 (RO) may be a ligand which could potentially be used to control the progression of estrogen receptor-α (ERα)-positive breast cancer cells. Herein, we showed, by real-time PCR analysis of mRNA from human breast cancer biopsies, no significant differences in OSC expression at various stages of disease, or between tumor and normal mammary cells. Since the growth of hormone-responsive tumors is ERα-dependent, we conducted experiments to determine whether RO affects ERα. Using mammalian cells engineered to express human ERα or ERß protein, together with an ER-responsive luciferase promoter, we found that RO dose-dependently inhibited 17ß-estradiol (E2)-induced ERα responsive luciferase activity (IC50 value, ~10 µM), under conditions that were non-toxic to the cells. RO was less effective against ERß-induced luciferase activity. Androgen receptor (AR) mediated transcriptional activity was also reduced by RO. Notably, while ERα activity was reduced by atorvastatin, the HMG-CoA reductase inhibitor did not influence AR activity, showing that RO possesses broader antitumor properties. Treatment of human BT-474 breast cancer cells with RO reduced levels of estrogen-induced PR protein, confirming that RO blocks ERα activity in tumor cells. Our findings demonstrate that an important means by which RO suppresses hormone-dependent growth of breast cancer cells is through its ability to arrest the biological activity of ERα. This warrants further investigation of RO as a potential therapeutic agent for use against hormone-dependent breast cancers.

Cholesterol biosynthesis inhibitors as potent novel anti-cancer agents: suppression of hormone-dependent breast cancer by the oxidosqualene cyclase inhibitor RO 48-8071.

In most human breast cancers, tumor cell proliferation is estrogen dependent. Although hormone-responsive tumors initially respond to anti-estrogen therapies, most of them eventually develop resistance. Our goal was to identify alternative targets that might be regulated to control breast cancer progression. Sulforhodamine B assay was used to measure the viability of cultured human breast cancer cell lines exposed to various inhibitors. Protein expression in whole-cell extracts was determined by Western blotting. BT-474 tumor xenografts in nude mice were used for in vivo studies of tumor progression. RO 48-8071 ([4'-[6-(Allylmethylamino)hexyloxy]-4-bromo-2'-fluorobenzophenone fumarate]; RO), a small-molecule inhibitor of oxidosqualene cyclase (OSC, a key enzyme in cholesterol biosynthesis), potently reduced breast cancer cell viability. In vitro exposure of estrogen receptor (ER)-positive human breast cancer cells to pharmacological levels of RO or a dose close to the IC50 for OSC (nM) reduced cell viability. Administration of RO to mice with BT-474 tumor xenografts prevented tumor growth, with no apparent toxicity. RO degraded ERα while concomitantly inducing the anti-proliferative protein ERß. Two other cholesterol-lowering drugs, Fluvastatin and Simvastatin, were less effective in reducing breast cancer cell viability and were found not to induce ERß. ERß inhibition or knockdown prevented RO-dependent loss of cell viability. Importantly, RO had no effect on the viability of normal human mammary cells. RO is a potent inhibitor of hormone-dependent human breast cancer cell proliferation. The anti-tumor properties of RO appear to be in part due to an off-target effect that increases the ratio of ERß/ERα in breast cancer cells.

Effects of dietary apigenin on tumor latency, incidence and multiplicity in a medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a)anthracene-induced breast cancer model.

Medroxyprogesterone acetate (MPA) is a synthetic progestin commonly administered to postmenopausal women for hormone replacement therapy and has been associated with increased risk of breast cancer. MPA has been shown to accelerate the development of mammary tumors in a 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer animal model. Previously, we have shown that intraperitoneally administered apigenin effectively treated and prevented the progression of MPA-accelerated breast cancer in DMBA-induced and xenograft mammary cancer models. Here we used the DMBA model to examine the chemopreventive effect of dietary apigenin against MPA-accelerated tumors with 3 different levels of apigenin (0.02%, 0.1%, and 0.5% w/w) incorporated into a phytoestrogen-free diet. Results showed that 0.1% dietary apigenin reduced MPA-dependent tumor incidence; however, the same dietary level increased tumor multiplicity in animals that developed tumors. Neither 0.02% nor 0.5% dietary apigenin reduced MPA-dependent tumor incidence or latency, and tumor multiplicity increased significantly in response to 0.5% apigenin. These results contrast with previous chemopreventive effects observed when apigenin was administered intraperitoneally, suggesting that route of administration may influence its action. Consequently, until further research clarifies the effect of dietary apigenin on progestin-accelerated mammary tumors, caution should be exercised when considering the flavonoid as a dietary supplement for preventing hormone-dependent breast cancer.

Dental caries and oral health practice among 12 year old school children from low socio-economic status background in Zimbabwe.

INTRODUCTION: Dental caries is one of the most prevalent chronic diseases affecting children in Sub-Saharan Africa. Previous studies show a higher prevalence of dental caries in children from low socio-economic status backgrounds. The purpose of this study was to determine the prevalence of dental caries among 12 year old children in urban and rural areas of Zimbabwe and establish preliminary baseline data. METHODS: A descriptive cross-sectional study was conducted among 12 year old children at primary schools in Harare and Bikita district. A Pre-tested questionnaire was administered to elicit information from the participants on tooth cleaning, dietary habits and dental experience. Dental caries status was assessed using the DMFT index following World Health Organization (WHO) guidelines. RESULTS: Our results showed a high prevalence of dental caries in both urban (59.5%) and rural (40.8%) children. The mean DMFT in urban and rural areas was 1.29 and 0.66, respectively. Furthermore, our data showed a general lack of knowledge on oral health issues by the participants. CONCLUSION: There is high prevalence of dental caries among 12 years old school children in both urban and rural areas of Zimbabwe. This calls for early preventive strategies and treatment services. We recommend incorporation of oral health education in the elementary school curricula.

Breast cancer knowledge and awareness among university students in Angola.

BACKGROUND: The high breast cancer mortality rate in Sub-Saharan Africa has been attributed to a lack of public awareness of the disease which often leads to late diagnosis of the disease. Little is known about the level of knowledge and awareness of breast cancer in Angola. Previous studies have shown that breast cancer awareness is higher among well-educated people. The goal of this study was to assess breast cancer knowledge and awareness among university students in Angola. METHODS: We conducted a cross-sectional survey of university students using a self-administered questionnaire to investigate participants' awareness and knowledge of breast cancer. A total of 595 university students in medical and non-medical programs successfully completed the survey. RESULTS: Our results showed insufficient knowledge of breast cancer among university students in Angola irrespective of whether they were in medical or non-medical programs. The majority of the participants were not aware of some of the early signs of breast cancer such as change in color or shape of the nipple, even though they appreciated the need for monthly breast self-examination. Overall most of the participants indicated the need for increased breast cancer awareness among university students. CONCLUSION: The study points to the insufficient knowledge of university students in Angola about breast cancer. We expect that our results may provide useful data that may be used by the department of health in Angola and other African countries to formulate health education programs aimed at increasing awareness and promote screening and early detection of breast cancer in the continent.

Apigenin induces apoptosis and blocks growth of medroxyprogesterone acetate-dependent BT-474 xenograft tumors.

Recent clinical and epidemiological evidence shows that hormone replacement therapy (HRT) containing both estrogen and progestin increases the risk of primary and metastatic breast cancer in post-menopausal women while HRT containing only estrogen does not. We and others previously showed that progestins promote the growth of human breast cancer cells in vitro and in vivo. In this study, we sought to determine whether apigenin, a low molecular weight anti-carcinogenic flavonoid, inhibits the growth of aggressive Her2/neu-positive BT-474 xenograft tumors in nude mice exposed to medroxyprogesterone acetate (MPA), the most commonly used progestin in the USA. Our data clearly show that apigenin (50 mg/kg) inhibits progression and development of these xenograft tumors by inducing apoptosis, inhibiting cell proliferation, and reducing expression of Her2/neu. Moreover, apigenin reduced levels of vascular endothelial growth factor (VEGF) without altering blood vessel density, indicating that continued expression of VEGF may be required to promote tumor cell survival and maintain blood flow. While previous studies showed that MPA induces receptor activator of nuclear factor kappa-B ligand (RANKL) expression in rodent mammary gland, MPA reduced levels of RANKL in human tumor xenografts. RANKL levels remained suppressed in the presence of apigenin. Exposure of BT-474 cells to MPA in vitro also resulted in lower levels of RANKL; an effect that was independent of progesterone receptors since it occurred both in the presence and absence of the antiprogestin RU-486. In contrast to our in vivo observations, apigenin protected against MPA-dependent RANKL loss in vitro, suggesting that MPA and apigenin modulate RANKL levels differently in breast cancer cells in vivo and in vitro. These preclinical findings suggest that apigenin has potential as an agent for the treatment of progestin-dependent breast disease.

Differential expression of FGF family members in a progestin-dependent BT-474 human breast cancer xenograft model.

Members of the fibroblast growth factor (FGF) family have been associated with tumor progression and angiogenesis, though the mechanism through which they affect the progression of breast cancer remains elusive. We recently showed that progestins increase the production of the potent angiogenic factor VEGF in an in vivo BT-474 human breast cancer cell-derived xenograft model. In this study we sought to determine the effect of progesterone (P) on regulation of specific FGF family members (FGF-2, FGF-4 and FGF-8) in the same model. Using immunohistochemistry we found that treatment with P significantly reduced FGF-2 and FGF-8 levels, while modestly increasing the levels of FGF-4 in tumors collected at the termination of the study or soon after P treatment began. The in vivo observations with FGF-2 were confirmed in cultured BT-474 cells, though the P-mediated reduction in FGF-2 was not blocked by the anti-progestin RU-486, suggesting that classical progesterone receptors (PR) are not involved in FGF-2 down-regulation. Also, P did not affect levels of FGF-2 mRNA in BT-474 cells, indicating that P exerts its effects on FGF-2 post-transcriptionally. Our observations suggest that the in vivo stimulation of BT-474 cell growth by P is associated with down-regulation of FGF-2 and FGF-8. Furthermore, since FGF-4 levels increased during P-treatment, FGF-4 may be required for tumor growth and maintenance and might therefore be a potential therapeutic target through which to suppress P-dependent tumor growth.

Breast Cancer Knowledge and Awareness among University Students in Angola

BACKGROUND:The high breast cancer mortality rate in Sub-Saharan Africa has been attributed to a lack of public awareness of the disease which often leads to late diagnosis of the disease. Little is known about the level of knowledge and awareness of breast cancer in Angola. Previous studies have shown that breast cancer awareness is higher among well-educated people. The goal of this study was to assess breast cancer knowledge and awareness among university students in Angola.METHODS: We conducted a cross-sectional survey of university students using a self-administered questionnaire to investigate participants' awareness and knowledge of breast cancer. A total of 595 university students in medical and non-medical programs successfully completed the survey. RESULTS: Our results showed insufficient knowledge of breast cancer among university students in Angola irrespective of whether they were in medical or non-medical programs. The majority of the participants were not aware of some of the early signs of breast cancer such as change in color or shape of the nipple; even though they appreciated the need for monthly breast self-examination. Overall most of the participants indicated the need for increased breast cancer awareness among university students.CONCLUSION: The study points to the insufficient knowledge of university students in Angola about breast cancer. We expect that our results may provide useful data that may be used by the department of health in Angola and other African countries to formulate health education programs aimed at increasing awareness and promote screening and early detection of breast cancer in the continent

Apigenin prevents development of medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a)anthracene-induced mammary tumors in Sprague-Dawley rats.

The use of progestins as a component of hormone replacement therapy has been linked to an increase in breast cancer risk in postmenopausal women. We have previously shown that medroxyprogesterone acetate (MPA), a commonly administered synthetic progestin, increases production of the potent angiogenic factor vascular endothelial growth factor (VEGF) by tumor cells, leading to the development of new blood vessels and tumor growth. We sought to identify nontoxic chemicals that would inhibit progestin-induced tumorigenesis. We used a recently developed progestin-dependent mammary cancer model in which tumors are induced in Sprague-Dawley rats by 7,12-dimethylbenz(a)anthracene (DMBA) treatment. The flavonoid apigenin, which we previously found to inhibit progestin-dependent VEGF synthesis in human breast cancer cells in vitro, significantly delayed the development of, and decreased the incidence and multiplicity of, MPA-accelerated DMBA-induced mammary tumors in this animal model. Whereas apigenin decreased the occurrence of such tumors, it did not block MPA-induced intraductal and lobular epithelial cell hyperplasia in the mammary tissue. Apigenin blocked MPA-dependent increases in VEGF, and suppressed VEGF receptor-2 (VEGFR-2) but not VEGFR-1 in regions of hyperplasia. No differences were observed in estrogen or progesterone receptor (ER/PR) levels, or the number of estrogen receptor-positive cells, within the mammary gland of MPA-treated animals administered apigenin, MPA-treated animals, and placebo treated animals. However, the number of progesterone receptor-positive cells was reduced in animals treated with MPA or MPA and apigenin compared with those treated with placebo. These findings suggest that apigenin has important chemopreventive properties for those breast cancers that develop in response to progestins.

Apigenin blocks induction of vascular endothelial growth factor mRNA and protein in progestin-treated human breast cancer cells.

OBJECTIVE: The results of recent clinical trials indicate that hormone therapy with estrogen and progestin is associated with higher risk of breast cancer in postmenopausal women than treatment with estrogen alone or placebo. This observation is consistent with studies showing that progestins stimulate the expression of vascular endothelial growth factor (VEGF), which in turn stimulates angiogenesis. The objective of this study was to examine whether apigenin, a natural flavone, inhibits the progestin-dependent induction of VEGF in human breast cancer cells. METHODS: T47-D human breast cancer cells were treated with medroxyprogesterone acetate (MPA; 10 nM) or other synthetic progestins in the presence or absence of antiprogestin RU-486 and variable doses of apigenin (1-100 microM). BT-474 cells were also treated with MPA +/- 100 microM apigenin. Secreted VEGF was quantified by enzyme-linked immunosorbent assay, and total cellular VEGF mRNA was quantified by reverse transcriptase polymerase chain reaction. The expression of VEGF receptor-1 (flt), VEGF receptor-2 (flk), progesterone receptor, and estrogen receptor-α was also quantified by Western blot analysis and/or reverse transcriptase polymerase chain reaction. RESULTS: Apigenin (50 or 100 microM) prevented progestin-dependent induction of both VEGF mRNA and protein and reduced progesterone receptor levels in T47-D cells. Apigenin also blocked MPA-dependent secretion of VEGF from BT-474 cells. mRNA levels of progesterone and estrogen receptor-α were unaffected by apigenin, which did exert somewhat suppressive, although complex, effects on VEGF receptor expression in MPA-treated T47-D cells. CONCLUSIONS: Apigenin blocks progestin-dependent induction of VEGF mRNA and protein and broadly inhibits the ability of progestins to alter the expression of other components of the angiogenesis pathway, including progesterone receptor and VEGF receptors, in human breast cancer cells. Further studies are warranted to explore the potential of apigenin as a chemopreventive agent in postmenopausal women exposed to oral progestins.