The estrogen receptor beta agonist liquiritigenin enhances the inhibitory effects of the cholesterol biosynthesis inhibitor RO 48-8071 on hormone-dependent breast-cancer growth.

PURPOSE: Most hormone-dependent human breast cancers develop resistance to anti-hormone therapy over time. Our goal was to identify novel treatment strategies to avoid this drug resistance and thereby control hormone-dependent breast cancer. METHODS: Sulforhodamine B assays were used to measure viability of cultured human breast-cancer cells. BT-474 cell tumor xenografts in nude mice were used to evaluate tumor growth. Immunohistochemistry was used to assess estrogen-receptor and angiogenesis-marker expression, as well as apoptosis, in tumor-xenograft tissues. RESULTS: MCF-7 and BT-474 breast-cancer cells treated with either RO 48-8071 <[4'-[6-(Allylmethylamino)hexyloxy]-4-bromo-2'-fluorobenzophenone fumarate] [RO]; a small-molecule inhibitor of oxidosqualene cyclase, a key enzyme in cholesterol biosynthesis> or liquiritigenin [LQ; an estrogen receptor (ER) ß agonist] exhibited significantly reduced viability in vitro. RO + LQ treatment further significantly reduced cell viability. Administration of RO, LQ, or RO + LQ significantly inhibited growth of BT-474 tumor xenografts in vivo. RO, LQ, or RO + LQ reduced ERα but induced ER ß expression in tumor xenografts. Both compounds significantly reduced angiogenesis-marker expression and increased apoptosis in tumor xenografts; use of RO + LQ significantly enhanced the effects observed with a single agent. CONCLUSION: The ERß ligand LQ significantly enhanced the inhibition of breast-cancer cell viability and tumor-xenograft growth by RO. The anti-tumor properties of RO may in part be due to an off-target effect that reduces ERα and increases ERß, the latter of which can then interact with LQ to promote anti-proliferative effects. The RO + LQ combination may have value when considering novel treatment strategies for hormone-dependent breast cancer.

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.

Cholesterol Biosynthesis Inhibitor RO 48-8071 Suppresses Growth of Epithelial Ovarian Cancer Cells in Vitro and In Vivo.

Introduction: Epithelial Ovarian Cancer (EOC) cells express enzymes in the cholesterol biosynthetic pathway, making this pathway an attractive therapeutic target for controlling ovarian cancer. Potent small molecule inhibitors of one biosynthetic enzyme, Oxidosqualene Cyclase (OSC), have been identified, and RO 48-8071 (4'-[6-(allylmethylamino)hexyloxy]-4-bromo-2'-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for breast and prostate cancer. Methods: Cell viability assays were performed to determine effects of RO 48-8071 on growth of EOC cells. Aldehyde Dehydrogenase (ALDH) assay was conducted to determine the effects of drug on reducing stem cell like properties of EOC cells. Finally, xenograft studies were performed to assess the ability of RO 48-8071 to inhibit the growth of EOC cells in vivo. Results: We found that short-term (24-48 h) administration of pharmacological doses of RO effectively reduced the viability of drug-resistant EOC cells (SK-OV-3 and OVCAR-3), as determined with sulforhodamine B colorimetric assays. In 7-day assays, nanomolar concentrations of RO effectively inhibited the growth of EOC cells. RO also suppressed ALDH activity, a marker of stem cells. Importantly, RO significantly suppressed growth of xenografts derived from EOC cells when given to mice intraperitoneally (20-40 mg kg-1 day-1) for 27 days once tumors reached 100 mm3 (controls: 336 + 60 mm3; treated: 171 + 20 mm3) with no toxicity to the experimental animals. Mechanistically, RO induced apoptosis in tumor cells in vivo as shown with immunohistochemistry. Conclusion: Cholesterol biosynthesis inhibitor RO 48-8071 is thus a novel and potent inhibitor of human EOC, including EOC stem cells.

Targeting mutant p53 protein and the tumor vasculature: an effective combination therapy for advanced breast tumors.

Breast cancer progression depends upon the elaboration of a vasculature sufficient for the nourishment of the developing tumor. Breast tumor cells frequently contain a mutant form of p53 (mtp53), a protein which promotes their survival. The aim of this study was to determine whether combination therapy targeting mtp53 and anionic phospholipids (AP) on tumor blood vessels might be an effective therapeutic strategy for suppressing advanced breast cancer. We examined the therapeutic effects, singly, or in combination, of p53 reactivation and induction of massive apoptosis (PRIMA-1), which reactivates mtp53 and induces tumor cell apoptosis, and 2aG4, a monoclonal antibody that disrupts tumor vasculature by targeting AP on the surface of tumor endothelial cells and causes antibody-dependent destruction of tumor blood vessels, leading to ischemia and tumor cell death. Xenografts from two tumor cell lines containing mtp53, BT-474 and HCC-1428, were grown in nude mice to provide models of advanced breast tumors. After treatment with PRIMA-1 and/or 2aG4, regressing tumors were analyzed for vascular endothelial growth factor (VEGF) expression, blood vessel loss, and apoptotic markers. Individual drug treatment led to partial suppression of breast cancer progression. In contrast, combined treatment with PRIMA-1 and 2aG4 was extremely effective in suppressing tumor growth in both models and completely eradicated approximately 30% of tumors in the BT-474 model. Importantly, no toxic effects were observed in any treatment group. Mechanistic studies determined that PRIMA-1 reactivated mtp53 and also exposed AP on the surface of tumor cells as determined by enhanced 2aG4 binding. Combination treatment led to significant induction of tumor cell apoptosis, loss of VEGF expression, as well as destruction of tumor blood vessels. Furthermore, combination treatment severely disrupted tumor blood vessel perfusion in both tumor models. The observed in vitro PRIMA-1-induced exposure of tumor epithelial cell AP might provide a target for 2aG4 and contribute to the increased effectiveness of such combination therapy in vivo. We conclude that the combined targeting of mtp53 and the tumor vasculature is a novel effective strategy for combating advanced breast tumors.

Estrogen regulation of thrombospondin-1 in human breast cancer cells.

Expression of thrombospondin-1 (TSP-1), a large extracellular matrix protein, has been associated with modulation of angiogenesis and tumor growth. Both pro and antiangiogenic properties of TSP-1 have been described, and the role of TSP-1 expression in the growth and progression of human breast cancer is not clear. Because estrogens cause progression of many breast cancers, and estradiol (E2) downregulates a TSP-1 receptor, we examined whether TSP-1 is regulated by estrogen and involved in tumor progression. E2 induced TSP-1 expression in T47-D and MCF-7 breast cancer cells in vitro within 3 to 6 hr; the induction was blocked by the anti-estrogen ICI 182,780, indicating that estrogen receptors (ER) are necessary for this effect. Furthermore, E2 caused the production of TSP-1 protein from tumor cells in an ER-alpha-dependent manner. The E2-mediated TSP-1 RNA induction was dose-dependent and blocked by actinomycin D, indicating that the response to E2 was at least partly transcriptional. Transfection studies with deletion constructs of the TSP-1 promoter identified an estrogen-responsive region in the human TSP-1 promoter, located between -2,200 and -1,792 bp upstream of the transcription start site. An antibody against TSP-1 restricted the proliferation of E2-dependent MCF-7 cells in vitro and in vivo. A panel of breast cancer cells proliferated in the presence of low concentrations of exogenous TSP-1, whereas higher concentrations inhibited proliferation. A real-time PCR analysis showed that E2 also induced TSP-1 mRNA in the normal mammary glands of immature ovariectomized mice in an ER-dependent manner. In summary, we report the novel observation that TSP-1 production is directly controlled by estrogens in ER-positive breast cancer cells, and the released protein has pro-growth regulatory functions. Consequently, we propose that TSP-1 could be a therapeutic target for anti-tumor therapy in early-stage tumors. (c) 2009 UICC.

Progestin-dependent progression of human breast tumor xenografts: a novel model for evaluating antitumor therapeutics.

Recent clinical trials indicate that synthetic progestins may stimulate progression of breast cancer in postmenopausal women, a result that is consistent with studies in chemically-induced breast cancer models in rodents. However, progestin-dependent progression of breast cancer tumor xenografts has not been shown. This study shows that xenografts obtained from BT-474 and T47-D human breast cancer cells without Matrigel in estrogen-supplemented nude mice begin to regress within days after tumor cell inoculation. However, their growth is resumed if animals are supplemented with progesterone. The antiprogestin RU-486 blocks progestin stimulation of growth, indicating involvement of progesterone receptors. Exposure of xenografts to medroxyprogesterone acetate, a synthetic progestin used in postmenopausal hormone replacement therapy and oral contraception, also stimulates growth of regressing xenograft tumors. Tumor progression is dependent on expression of vascular endothelial growth factor (VEGF); growth of progestin-dependent tumors is blocked by inhibiting synthesis of VEGF or VEGF activity using a monoclonal anti-VEGF antibody (2C3) or by treatment with PRIMA-1, a small-molecule compound that reactivates mutant p53 into a functional protein and blocks VEGF production. These results suggest a possible model system for screening potential therapeutic agents for their ability to prevent or inhibit progestin-dependent human breast tumors. Such a model could potentially be used to screen for safer antiprogestins, antiangiogenic agents, or for compounds that reactivate mutant p53 and prevent progestin-dependent progression of breast disease.

APR-246 alone and in combination with a phosphatidylserine-targeting antibody inhibits lung metastasis of human triple-negative breast cancer cells in nude mice.

BACKGROUND: Approximately 15-20% of all human breast cancers are classified as triple-negative because they lack estrogen and progesterone receptors and Her-2-neu, which are commonly targeted by chemotherapeutic drugs. New treatment strategies are therefore urgently needed to combat triple-negative breast cancers (TNBCs). Almost 80% of the triple-negative tumors express mutant p53 (mtp5), a functionally defective tumor suppressor protein. Whereas wild-type p53 (wtp53) promotes cell-cycle arrest and apoptosis and inhibits vascular endothelial growth factor-dependent angiogenesis, mtp53 fails to regulate these functions, resulting in tumor vascularization, growth, resistance to chemotherapy, and metastasis. Restoration of p53 function is therefore a promising drug-targeted strategy for suppressing TNBC metastasis. METHODS: APR-246 is a small-molecule drug that reactivates mtp53, thereby restoring p53 function. In this study, we sought to determine whether administration of APR-246, either alone or in combination with 2aG4, an antibody that targets phosphatidylserine residues on tumor blood vessels and disrupts tumor vasculature, effectively inhibits stem cell-like characteristics of tumor cells and migration in vitro, and metastasis of human mtp53-expressing TNBC cells to the lungs in mouse models. RESULTS: APR-246 reduced both the stem cell-like properties and migration of TNBC cells in vitro. In mouse models, administration of either APR-246 or 2aG4 reduced metastasis of TNBC cells to the lungs; a combination of the two diminished lung metastasis to the same extent as either agent alone. Combination treatment significantly reduced the incidence of lung metastasis compared either single agent alone. CONCLUSION: Metastasis of human mtp53-expressing TNBC cells to the lungs of nude mice is inhibited by the treatment that combines activation of mtp53 with targeting of phosphatidylserine residues on tumor blood vessels. We contend therefore that our findings strongly support the use of combination treatment involving mtp53 activation and immunotherapy in patients with TNBC.

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.

Re-activation of the p53 pathway inhibits in vivo and in vitro growth of hormone-dependent human breast cancer cells.

Mutations in wild-type p53 (wtp53) protein lead to loss of its tumor suppressor function in breast cancer cells, facilitating uncontrolled tumor growth. Consequently, procedures to repair defective p53 functions in tumor cells are being actively pursued. We sought to determine whether expression of wtp53 protein, or conversion of endogenous mutant p53 (mtp53) into a functional p53 protein with small molecule PRIMA-1, can override the tumor-promoting effects of naturally occurring mtp53 protein in hormone-responsive T47-D human breast cancer cells. We show that transfection of wtp53 gene into T47-D cells suppresses their proliferation in regular media, and inhibits estrogen-dependent cell proliferation in media containing dextran-coated charcoal treated serum. Growth inhibition was not due to the absence of estrogen receptor-alpha or estrogen receptor-beta though receptor levels for estrogen receptor-alpha were drastically reduced in wtp53 expressing cells. Focused microarray analysis of wtp53 expressing cells revealed suppression of PCNA cell-cycle regulatory mRNA and protein. Wild-type p53 transfected T47-D cells also failed to grow in vivo in estrogen supplemented nude mice. Furthermore, xenografts obtained with parental T47-D cells expressing mtp53 grew poorly in nude mice treated with PRIMA-1. PRIMA-1 treated tumors exhibited a low proliferation index, even though mice were estrogen-supplemented. PRIMA-1 treatment of tumor cells suppressed VEGF and induced expression of estrogen receptor-beta though expression of estrogen receptor-alpha and progesterone receptors was unaffected. These data indicate that alteration of the p53 signal transduction pathway by re-expression of wtp53 protein in T47-D cells, or treatment of parental cells with PRIMA-1, can prevent in vivo and in vitro proliferation of T47-D breast cancer cells.

Cholesterol biosynthesis inhibitor RO 48-8071 reduces progesterone receptor expression and inhibits progestin-dependent stem cell-like cell growth in hormone-dependent human breast cancer cells.

Clinical trials and studies have shown that postmenopausal women undergoing combination hormone replacement therapy containing estrogen and progestin have an increased risk of breast cancer compared with women taking estrogen or placebo alone. Using animal models, we have previously shown that synthetic progestins, including medroxyprogesterone acetate (MPA), which is widely used clinically, accelerate breast cancer tumor growth and promote metastasis. Furthermore, we have found that MPA elevates CD44 protein expression and aldehyde dehydrogenase (ALDH) activity, two markers of cancer stem cells (CSCs), and increases mammosphere formation, another hallmark of stem cells, in hormone-dependent T47-D human breast cancer cells. Herein, we show that RO 48-8071 (RO), an inhibitor of cholesterol synthesis, reduced MPA-induced CD44 protein expression in two hormone-dependent human breast cancer cell lines, T47-D and BT-474. Because we have previously shown that MPA induction of CD44 is progesterone receptor (PR) dependent, we examined RO's effects on PR protein and mRNA expressions in T47-D cells. PR mRNA levels remained unchanged after RO treatment; however, RO significantly reduced the protein expression of both PR receptor isoforms, PR-A and PR-B. Using the proteasome inhibitor MG-132, we demonstrated that RO decreases PR protein expression in T47-D cells via the proteasomal degradation pathway. Importantly, treatment of T47-D cells with RO abolished MPA-induced mammosphere formation. Based on our observations, we contend that RO may represent a novel means of preventing MPA-induced CSC expansion. RO could be used clinically to both treat and prevent hormone-dependent breast cancers, which represent the majority of human breast cancers. RO may also have clinical utility in reducing resistance to antihormone therapy.

Luteolin inhibits lung metastasis, cell migration, and viability of triple-negative breast cancer cells.

Most breast cancer-related deaths from triple-negative breast cancer (TNBC) occur following metastasis of cancer cells and development of tumors at secondary sites. Because TNBCs lack the three receptors targeted by current chemotherapeutic regimens, they are typically treated with extremely aggressive and highly toxic non-targeted treatment strategies. Women with TNBC frequently develop metastatic lesions originating from drug-resistant residual cells and have poor prognosis. For this reason, novel therapeutic strategies that are safer and more effective are sought. Luteolin (LU) is a naturally occurring, non-toxic plant compound that has proven effective against several types of cancer. With this in mind, we conducted in vivo and in vitro studies to determine whether LU might suppress metastasis of TNBC. In an in vivo mouse metastasis model, LU suppressed metastasis of human MDA-MB-435 and MDA-MB-231 (4175) LM2 TNBC cells to the lungs. In in vitro assays, LU inhibited cell migration and viability of MDA-MB-435 and MDA-MB-231 (4175) LM2 cells. Further, LU induced apoptosis in MDA-MB-231 (4175) LM2 cells. Relatively low levels (10 µM) of LU significantly inhibited vascular endothelial growth factor (VEGF) secretion in MDA-MB-231 (4175) LM2 cells, suggesting that it has the ability to suppress a potent angiogenic and cell survival factor. In addition, migration of MDA-MB-231 (4175) LM2 cells was inhibited upon exposure to an antibody against the VEGF receptor, KDR, but not by exposure to a VEGF165 antibody. Collectively, these data suggest that the anti-metastatic properties of LU may, in part, be due to its ability to block VEGF production and KDR-mediated activity, thereby inhibiting tumor cell migration. These studies suggest that LU deserves further investigation as a potential treatment option for women with TNBC.

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.

Vascular endothelial growth factor induces proliferation of breast cancer cells and inhibits the anti-proliferative activity of anti-hormones.

Increased levels of vascular endothelial growth factor (VEGF) are associated with a poor response of breast cancer to anti-hormone treatment. Although VEGF is regarded as an endothelial-specific growth factor, recent reports have shown that VEGF can promote proliferation of other cell types, including breast tumor cells. We have characterized the proliferative effects of VEGF in breast cancer cell lines that are commonly used for understanding the role of estrogens, progestins, and anti-hormones on tumor growth. Since steroid hormones can increase the level of VEGF in certain breast cancer cells, we evaluated the effects of exogenous VEGF on the growth-suppressive effects of anti-estrogen (ICI 182,780) and RU-486 (anti-progestin mifepristone) in human breast cancer cells. VEGF165 and VEGF121 increased the proliferation of tumor cell lines that expressed VEGFR-2 (VEGF receptor 2) (flk/kdr) via the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK) pathway. Furthermore, VEGF induced the expression of the anti-apoptotic protein Bcl-2 and blocked down-regulation of Bcl-2 by ICI 182,780 and induced Bcl-2 in BT-474 and T47-D cells even in the presence of RU-486. Increased Bcl-2 levels in response to VEGF were associated with increased proliferation and survival of tumor cells even in the presence of anti-hormones. These results suggest that VEGF stimulates proliferation of VEGFR2-positive tumor cells, promotes survival via the expression and activity of Bcl-2 and overrides the growth-suppressive effects of anti-hormones. This represents a potential explanation for anti-hormone resistance and tumor progression in clinical samples. Thus, it may be useful to use combined modality treatment involving anti-hormones and anti-angiogenic agents to treat breast cancers that express elevated levels of VEGF.

Mitochondria from TRAIL-resistant prostate cancer cells are capable of responding to apoptotic stimuli.

TNFalpha-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in prostate cancer cells. However, some prostate cancer cells, such as LNCaP are resistant to TRAIL. In addition to the involvement of several pathways in the TRAIL-resistance of LNCaP, it has been shown that mitochondrial response to TRIAL is low in these cells. Therefore, in this study, using in vitro cell free and reconstitution models, we have demonstrated that mitochondria from these cells are capable of responding to apoptotic stimuli. Furthermore, experiments to determine the influence of cytochrome c on apoptotic response noted that incubation of cytosol with exogenous cytochrome c induced truncation of Bid. We have demonstrated that truncation of Bid by exogenous cytochrome c is mediated through the activation of caspases-9 and -3. Incubation of cytosol with recombinant caspases-9 and -3 in the absence or presence of inhibitors showed that activation of caspase-9, leading to the activation of caspase-3 was necessary for the truncation of Bid. Published results indicate that in apoptotic cells cytochrome c is released from the mitochondria in two installments, an early small amount and a late larger amount. Our results suggest that the initial release of cytochrome generates tBid that is capable of translocation into the mitochondria causing further release of cytochrome c. Thus, in addition to providing functional explanation for the biphasic release of cytochrome c from mitochondria, we demonstrate the presence of a feedback amplification of mitochondrial apoptotic signal.

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.

Proliferation of endothelial and tumor epithelial cells by progestin-induced vascular endothelial growth factor from human breast cancer cells: paracrine and autocrine effects.

Angiogenesis, the formation of new blood vessels, is essential for tumor expansion, and vascular endothelial growth factor (VEGF) is one of the most potent angiogenic growth factors known. We have previously shown that natural and synthetic progestins, including those used in hormone replacement therapy and oral contraception, induce the synthesis and secretion of VEGF in a subset of human breast cancer cells in a progesterone receptor-dependent manner. We now report that conditioned medium from progestin-treated breast tumor cells can induce the proliferation of endothelial cells in a paracrine manner and induce the proliferation of tumor epithelial cells in a paracrine and an autocrine manner. The use of an anti-VEGF antibody and SU-1498, an inhibitor of VEGF receptor-2 (VEGFR-2 or flk/kdr) tyrosine kinase activity, demonstrated that these effects involve interactions between VEGF and VEGFR-2. Also, blockage of progestin-induced VEGF by the antiprogestin RU-486 (mifepristone) eliminated VEGF-induced proliferative effects. The ability of VEGF to increase the proliferation of endothelial cells and tumor cells, including those that do not release VEGF in response to progestins, suggests that these effects are mediated by amplification of the progestin signal, which culminates in angiogenesis and tumor growth. These novel findings suggest that targeting the release of VEGF from tumor epithelial cells as well as blocking interactions between VEGF and VEGFR-2 on both endothelial and tumor epithelial cells may facilitate the development of new antiangiogenic therapies for progestin-dependent breast tumors. Furthermore, these data indicate that it would be useful to develop selective progesterone receptor modulators that prevent the release of angiogenic growth factors from breast cancer cells.

Complex agonist-like properties of ICI 182,780 (Faslodex) in human breast cancer cells that predominantly express progesterone receptor-B: implications for treatment resistance.

ICI 182,780 (Faslodex), considered a pure anti-estrogen, is approved for treatment of post-menopausal breast cancer patients who fail to respond to tamoxifen therapy. We recently reported that, like mifepristone, ICI 182,780 exhibits anti-progestin activity, blocking the progestin-dependent increase in endogenous vascular endothelial growth factor (VEGF) mRNA and protein release. Some anti-progestins have partial agonist-like activity in breast cancer cells expressing high levels of progesterone receptor B (PRB). Our results show that ICI 182,780 can also induce reporter activity from a plasmid containing a simple progestin responsive element (PRE) in these cells. Using small interfering RNA, we determined that induction is dependent on the presence of PR, estrogen receptor and SRC-1. Regulation of more complex progestin-responsive promoters was context-dependent; induction was observed from the MMTV promoter but not from the VEGF promoter. In contrast, ICI 182,780 increased the release of angiogenically active VEGF from cells expressing elevated levels of PRB. This effect was dependent on the phosphatidylinositol-3 kinase and ERK/MAPK signaling pathways. We hypothesize that these agonist-like properties of ICI 182,780 (one genomic and one non-genomic) may contribute to the acquisition of drug resistance, suggesting that both anti-hormonal and anti-angiogenic treatment may be appropriate in these patients.

p53-dependent inhibition of progestin-induced VEGF expression in human breast cancer cells.

VEGF, a potent angiogenic growth factor, is up-regulated in many tumors including human breast tumors and stimulates growth of vascular networks that support tumor growth and metastasis. We previously reported that natural and synthetic progestins (P) increased VEGF mRNA and protein levels in progesterone receptor (PR) containing T47-D human breast cancer cells in a PR dependent manner, but not in PR positive ZR-75 and MCF-7, or in PR negative MDA-MB-231 cells. This indicated that factors beside PR are involved in progesterone-dependent VEGF regulation. We, therefore, tested additional tumor cell lines reported to contain PR for progestin-dependent VEGF induction. Out of nine PR-positive breast tumor cell lines, progestins induced VEGF in three cell lines that lack wild-type p53 (T47-D, BT-474, and HCC-1428) but not in cell lines that contained the wild-type p53 protein. The T47-D and BT-474 cells express mutant p53, while the p53 protein is absent HCC-1428 cells. The anti-progestin RU-486 blocked progestin-dependent induction of VEGF in T47-D and BT-474 cells but not in HCC-1428 cells. However, RU-486 partially blocked medroxyprogesterone acetate-dependent induction of VEGF in HCC-1428 cells. Estrogen receptor (ER) and PR agonists and antagonists also induce VEGF in HCC-1428 cells and this effect was partially blocked by anti-estrogen ICI-182, 780. Progestin-dependent VEGF induction was completely inhibited by PRIMA-1-activated p53 in all cell-types, but progestin-dependent transcription of a progesterone-regulated minimal promoter was only partially inhibited. PRIMA-1 induced activation of p53 in tumor cell lines was confirmed with a p53-responsive p21 reporter plasmid and by detecting increased levels of p21 proteins in cell lysates. PRIMA-1 induced p53 protein in the HCC-1428 cells while levels of mutant p53 protein in T47-D and BT-474 remained unaltered. Progestin-dependent induction of VEGF was also inhibited by stable transfection of wild-type p53 in T47-D cells. These results are consistent with the hypothesis that wild-type p53 blocks progestin-dependent induction of VEGF in breast cancer cells and this may be a novel anti-angiogenic mechanism for controlling the growth of progestin-dependent tumors.

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.

Estradiol down-regulates CD36 expression in human breast cancer cells.

CD36 is a trans-membrane receptor that regulates apoptosis and angiogenesis in response to its ligand thrombospondin-1 (TSP-1). This study measures expression of CD36 and TSP-1 in breast cancer cell lines. Expression of TSP-1 was approximately 50-fold higher in the aggressive cell line MDA-MB-231 than in less aggressive MCF-7, BT-474, ZR-75 and T47-D cells. In contrast, MDA-MB-231 express 30 to 100-fold less CD36 than less aggressive cells. Hormone-dependent T47-D and MCF-7 cells down-regulate CD36 in response to estradiol, and anti-hormone ICI 182,780 block this effect. These results suggest that the estrogen receptors play a role in regulating CD36 expression and ICI 182,780 prevents loss of CD36 as a novel mechanism for its anti-estrogen effect in breast cancer cells.