Natural products as drug candidates for breast cancer (Review).

Progression of early-stage breast cancer to advanced-stage metastatic disease represents a major cause of death in women. Long-term conventional and targeted therapy for breast cancer includes multi-drug combinations of cytotoxic chemotherapeutics and pathway-selective small molecule inhibitors. These treatment options are frequently associated with systemic toxicity, intrinsic/acquired therapy resistance and emergence of a drug-resistant cancer stem cell population. This stem cell population has a chemo-resistant, cancer-initiating, premalignant phenotype that is accompanied by cellular plasticity and metastatic potential. These limitations emphasize an unmet need to identify testable alternatives against therapy-resistant metastatic breast cancer. Natural products such as dietary phytochemicals, nutritional herbs and their constitutive bioactive agents have documented human consumption, and lack detectable systemic toxicity and resultant off-target unfavorable side effects. Because of these advantages, natural products may represent testable alternatives for therapy-resistant breast cancer. The present review discusses published evidence for growth inhibitory efficacy of natural products on cellular models for molecular subtypes of clinical breast cancer and development of drug-resistant stem cell models. Collectively, this evidence validates mechanism-based experimental approaches to screen and prioritize efficacious bioactive agents from natural products as novel drug candidates that may function as therapeutic alternatives for breast cancer.

Cancer Cell Models for the Development of Anti-Cancer Drugs.

In the multi-factorial etiology of organ-site cancers by suspect human chemical carcinogens, oncogenic virus, activation of RAS, Myc and HER-2 oncogenes, inactivation of TP53, RB and APC tumor suppressor genes represent early-occurring genetic events [...].

Stem Cell Models for Breast and Colon Cancer: Experimental Approach for Drug Discovery.

The progression of the early stages of female breast and colon cancer to metastatic disease represents a major cause of mortality in women. Multi-drug chemotherapy and/or pathway selective targeted therapy are notable for their off-target effects and are associated with spontaneous and/or acquired chemotherapy resistance and the emergence of premalignant chemo-resistant cancer-initiating stem cells. The stem cell populations are responsible for the evolution of therapy-resistant metastatic disease. These limitations emphasize an unmet need to develop reliable drug-resistant cancer stem cell models as novel experimental approaches for therapeutic alternatives in drug discovery platforms. Drug-resistant stem cell models for breast and colon cancer subtypes exhibit progressive growth in the presence of cytotoxic chemo-endocrine therapeutics. The resistant cells exhibit upregulated expressions of stem cell-selective cellular and molecular markers. Dietary phytochemicals, nutritional herbs and their constituent bioactive compounds have documented growth inhibitory efficacy for cancer stem cells. The mechanistic leads for the stem cell-targeted efficacy of naturally occurring agents validates the present experimental approaches for new drug discovery as therapeutic alternatives for therapy-resistant breast and colon cancer. The present review provides a systematic discussion of published evidence on (i) conventional/targeted therapy for breast and colon cancer, (ii) cellular and molecular characterization of stem cell models and (iii) validation of the stem cell models as an experimental approach for novel drug discovery of therapeutic alternatives for therapy-resistant cancers.

The Divergent Effects of Ovarian Steroid Hormones in the MCF-7 Model for Luminal A Breast Cancer: Mechanistic Leads for Therapy.

The growth modulating effects of the ovarian steroid hormones 17ß-estradiol (E2) and progesterone (PRG) on endocrine-responsive target tissues are well established. In hormone-receptor-positive breast cancer, E2 functions as a potent growth promoter, while the function of PRG is less defined. In the hormone-receptor-positive Luminal A and Luminal B molecular subtypes of clinical breast cancer, conventional endocrine therapy predominantly targets estrogen receptor function and estrogen biosynthesis and/or growth factor receptors. These therapeutic options are associated with systemic toxicity, acquired tumor resistance, and the emergence of drug-resistant cancer stem cells, facilitating the progression of therapy-resistant disease. The limitations of targeted endocrine therapy emphasize the identification of nontoxic testable alternatives. In the human breast, carcinoma-derived hormone-receptor-positive MCF-7 model treatment with E2 within the physiological concentration range of 1 nM to 20 nM induces progressive growth, upregulated cell cycle progression, and downregulated cellular apoptosis. In contrast, treatment with PRG at the equimolar concentration range exhibits dose-dependent growth inhibition, downregulated cell-cycle progression, and upregulated cellular apoptosis. Nontoxic nutritional herbs at their respective maximum cytostatic concentrations (IC90) effectively increase the E2 metabolite ratio in favor of the anti-proliferative metabolite. The long-term exposure to the selective estrogen-receptor modulator tamoxifen selects a drug-resistant phenotype, exhibiting increased expressions of stem cell markers. The present review discusses the published evidence relevant to hormone metabolism, growth modulation by hormone metabolites, drug-resistant stem cells, and growth-inhibitory efficacy of nutritional herbs. Collectively, this evidence provides proof of the concept for future research directions that are focused on novel therapeutic options for endocrine therapy-resistant breast cancer that may operate via E2- and/or PRG-mediated growth regulation.

Growth Inhibitory Efficacy of Chinese Herbs in a Cellular Model for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor-α progesterone receptor and human epidermal growth factor receptor-2. Treatment for this breast cancer subtype is restricted to multidrug chemotherapy and survival pathway-based molecularly targeted therapy. The long-term treatment options are associated with systemic toxicity, spontaneous and/or acquired tumor resistance and the emergence a of drug-resistant stem cell population. These limitations lead to advanced stage metastatic cancer. Current emphasis is on research directions that identify efficacious, naturally occurring agents representing an unmet need for testable therapeutic alternatives for therapy resistant breast cancer. Chinese herbs are widely used in traditional Chinese medicine in women for estrogen related health issues and also for integrative support for cancer treatment. This review discusses published evidence on a TNBC model for growth inhibitory effects of several mechanistically distinct nontoxic Chinese herbs, most of them nutritional in nature, and identifies susceptible pathways and potential molecular targets for their efficacy. Documented anti-proliferative and pro-apoptotic effects of these herbs are associated with downregulation of RB, RAS, PI3K, and AKT signaling, modulation of Bcl-2/BAX protein expressions and increased caspase activity. This review provides a proof of concept for Chinese herbs as testable alternatives for prevention/therapy of TNBC.

Growth inhibitory efficacy of Cornus officinalis in a cell culture model for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) lacks the expressions of estrogen receptor-α, progesterone receptor and human epidermal growth factor receptor-2. The treatment options for TNBC include anthracyclin/taxol based conventional chemotherapy and small molecular inhibitor based targeted therapy. However, the therapeutic efficacy is limited by systemic toxicity and acquired tumor resistance; identification of less toxic testable alternatives is urgently required. Non-toxic nutritional herbs are commonly used in traditional Chinese herbal medicine for general health management and may additionally represent a testable therapeutic alternative for TNBC. The present study examined the growth inhibitory efficacy of the nutritional herb Cornus officinalis (CO) in MDA-MB-231 cells, which represent a cell culture model for TNBC, and identified potential mechanistic leads. In MDA-MB-231 cells, CO induced dose-dependent cytostatic growth arrest [inhibitory concentration (IC)50, 0.1% and IC90, 0.5%], and inhibited anchorage independent colony formation. Mechanistically, CO inhibited G1 to S phase transition leading to G1 arrest and decreased the expression of cyclin D1 and phosphorylated-retinoblastoma proteins. CO additionally altered apoptosis specific BCL-2 associated X protein/B-cell lymphoma-2 expression and upregulated pro-apoptotic caspase-3/7 activity. Collectively, these data provided mechanistic evidence for the efficacy of CO, and validated a mechanism-based approach to prioritize efficacious nutritional herbs as testable alternatives for secondary prevention/treatment of TNBC.

The nutritional herb Epimedium grandiflorum inhibits the growth in a model for the Luminal A molecular subtype of breast cancer.

The Luminal A subtype of breast cancer expresses the estrogen receptor (ER)-α and progesterone receptor (PR), but not the human epidermal growth factor receptor (HER)-2 oncogene. This subtype of breast cancer responds to endocrine therapy involving the use of selective estrogen receptor modulators and/or inhibitors of estrogen biosynthesis. However, these therapeutic agents are frequently associated with long-term systemic toxicity and acquired tumor resistance, emphasizing the need to identify non-toxic alternative treatments for chemo-endocrine therapy responsive breast cancer. The present study utilized the human mammary carcinoma-derived, ER+/PR+/HER-2- MCF-7 cell line as a model of the Luminal A subtype of breast cancer to examine the growth inhibitory effect of the Chinese nutritional herb Epimedium grandiflorum (EG) and determine the mechanisms underlying this effect. MCF-7 cells maintained in a serum-depleted culture medium retained their ability to grow in response to 17ß-estradiol (E2). Treatment of the MCF-7 cells with EG resulted in dose-dependent inhibition of E2-promoted growth. Mechanistically, EG inhibited E2-promoted cell cycle progression through G1 stage arrest and modulated the cellular metabolism of E2, increasing the formation of the anti-proliferative metabolites 2-hydroxyestrone and estriol. Long-term treatment of MCF-7 cells with EG inhibited E2-promoted anchorage independent growth, a surrogate in vitro biomarker of tumorigenesis. In conclusion, the results of the present study demonstrate the growth inhibitory effects of EG on MCF-7 cells and identified clinically relevant mechanistic leads for its anti-tumorigenic efficacy.

Lycium barbarum inhibits growth of estrogen receptor positive human breast cancer cells by favorably altering estradiol metabolism.

Selective estrogen receptor modulators represent accepted therapy for estrogen receptor positive (ER+) breast cancer, exhibit adverse side effects, and reduce patient compliance. The use of phytoestrogen containing herbal medicines is limited because of efficacy and safety concerns. The ER+ MCF-7 model examined growth inhibitory effects of the medicinal herb Lycium barbarum (LB) and identified mechanistic leads for its efficacy. The MCF-7 cells maintained in 0.7% serum (17beta-estradiol, E2 < 1 nM) exhibited 11%-87% increased growth after treatment with 1nM to 20 nM E2. Growth promotion with 20 nM E2 exhibited 5.2-fold increased estrone (E1), 35.7% increased 2-hydroxyestrone (2-OHE1), 15.4% increased 16alpha-hydroxyestrone (16alpha-OHE1), and eightfold increased estriol (E3) formation. Treatment of E2 stimulated cells with LB exhibited a dose-dependent growth inhibition of 9.5%-42.8% at Day 3 and 33.9%-83.9% at Day 7. The 3-day inhibitory response to 1% LB (maximum cytostatic concentration) exhibited 84.8% increased E1, 3.6-fold increased 2-OHE1, 33.3% decreased 16alpha-OHE1, and 9.2-fold increased E3 formation. Thus, MCF-7 cells retain their mitogenic and metabolic response to E2 and LB downregulates E2-stimulated growth via the formation of antiproliferative 2-OHE1 and accelerated conversion of mitogenic 16alpha-OHE1 to antimitogenic E3.

Prevention of early-onset familial/hereditary colon cancer: new models and mechanistic biomarkers (review).

Human colon cancer is a multi-factorial, multi-step disease wherein genetic and dietary factors represent important regulators of initiation, promotion and progression. While the etiology of sporadic colon cancer remains largely unidentified, familial adenomatous polyposis (FAP) and hereditary non-polyposis colon cancer (HNPCC) represent predisposing genetic syndromes for early-onset familial/hereditary colon cancer. These syndromes are characterized by germ-line mutations in the adenomatous polyposis coli (APC) and/or DNA mismatch repair genes, respectively. Currently available preclinical animal models for human FAP and HNPCC syndromes, expressing clinically relevant germ-line mutations, exhibit adenomas in the small intestine rather than in the colorectum. These models are, therefore, subject to extrapolation for direct clinical translatability of the data for colon carcinogenesis and chemoprevention. Experimental models expressing clinically relevant genetic defects (APC and/or DNA mismatch repair gene mutations) in an appropriate target site (colon) may represent novel approaches that reduce extrapolation of the data for their clinical relevance. This report provides an overview on carcinogenesis and chemoprevention in preclinical models of FAP and HNPCC syndromes, and summarizes recent data on i) development of new cell culture models for FAP and HNPCC syndromes; and ii) validation of developed models for rapid, mechanism-based screening of new pharmacological or naturally occurring chemopreventive agents.

Novel cell culture models for prevention of human breast cancer (Review).

Human breast cancer is a multifactorial, multistep disease wherein genetic, endocrine and dietary factors represent crucial regulators of initiation, promotion and progression. Preclinical investigations utilizing human breast carcinoma derived cell lines either in culture, or upon xenotransplantation, have provided valuable leads for molecular pathogenesis of cancer progression and also for novel therapeutic modalities. The mechanistic significance of genetic factors on early events of initiation/promotion, however, is dependent on extrapolation, and is therefore, equivocal. Human tissue derived explant culture/cell culture models utilizing non-involved target tissue at risk for carcinogenic transformation provide a novel approach that minimizes extrapolation for clinical relevance and thereby maximizes the translational impact. This report provides an overview of laboratory investigations focused on: i) development of the model, ii) optimization of mechanistic biomarker assays for carcinogenic transformation, and iii) validation of the model as a high throughput mechanistic screen for preclinical efficacy of natural phytochemicals.

Soy isoflavone genistein modulates cell cycle progression and induces apoptosis in HER-2/neu oncogene expressing human breast epithelial cells.

In the multistep progressive pathogenesis of human breast cancer, comedo ductal carcinoma in situ (DCIS) represents a preinvasive precursor lesion for therapy resistant invasive cancer. Human tissue derived cell culture models exhibiting molecular similarities to clinical DCIS facilitate an important preclinical mechanistic approach for evaluation of preventive efficacy of natural and synthetic chemopreventive compounds. Natural phytochemicals present in fresh fruits, vegetables and grain products are likely to offer protection against cancer. The clinical efficacy of these natural phytochemicals, however, depends on extrapolation, and is therefore equivocal. The present study determined whether the natural soy isoflavone genistein (GEN) inhibited aberrant proliferation in 184-B5/HER cells (a model for human comedo DCIS) and identified possible mechanisms responsible for its efficacy. Human reduction mammoplasty derived HER-2/neu oncogene expressing preneoplastic 184-B5/HER cells represented the experimental system. Flow cytometry and cellular epifluorescence based assays were utilized to quantitate the alterations in cell cycle progression, cellular apoptosis, and in the status of cell cycle regulatory and apoptosis-associated gene product expression. The 184-B5/HER cells exhibited specific immunofluorescence to p185HER, p53, EGFR, but not to ERalpha, thus resembling comedo DCIS. Treatment of 184-B5/HER cells with GEN resulted in a dose-dependent decrease in the viable cell population, increase in the G0/G1:S + G2/M ratio and enhancement of sub G0/G1 (apoptotic population). Exposure to the maximum cytostatic 10 microM dose of GEN down-regulated HER-2/neu mediated signal transduction as evidenced by a 73.9% decrease (p=0.001) in p185HER specific, and a 89.8% decrease (p=0.001) in phosphotyrosine specific immunofluorescence. The increase in G0/G1:S + G2/M ratio in response to the treatment with 10 microM GEN was associated with a 85.5% decrease (p=0.001) in immunoreactivity to PCNA and a 128.6% increase (p=0.004) in immunoreactivity to the cyclin dependent kinase inhibitor p16INK4. The induction of apoptosis by GEN was associated with a 52.8% decrease (p=0.001) in the immunoreactivity to antiapoptotic Bcl-2 and with a 195.9% (p=0.001) increase in the immunoreactivity to proapoptotic Bax. Thus, preventive efficacy of GEN in HER-2/neu+/ER- 184-B5/HER cells may be due to its ability to down-regulate HER-2/neu mediated signal transduction, increase the expression of the cyclin dependent kinase inhibitor p16INK4, and induce Bcl-2 dependent apoptosis. These data provide evidence that GEN may be a potential chemopreventive lead compound for human comedo DCIS. The 184-B5/HER cells, may therefore, provide a high throughput mechanistic bioassay to identify new chemopreventive agents for human breast cancer.

Preventive efficacy of receptor class selective retinoids on HER-2/neu oncogene expressing preneoplastic human mammary epithelial cells.

Aberrant proliferation is an early-occurring event in vitro prior to tumorigenesis in vivo in the multistep process of carcinogenesis. Inhibition of aberrant proliferation therefore may represent a useful biomarker to evaluate the efficacy of chemopreventive agents. Retinoids have exhibited preventive efficacy in vitro and in vivo predominantly through the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Clinically relevant biochemical and cellular mechanistic endpoints for chemopreventive effects of retinoids should provide novel biomarkers. The present study was designed to examine the preventive efficacy of natural retinoids, all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9cisRA), and to identify the possible mechanisms for their effects using the HER-2/neu oncogene expressing preneoplastic human mammary epithelial 184-B5/HER cells. Seven-day treatment with ATRA and 9cisRA exhibited a dose-dependent growth inhibition. Long-term (21 days) treatment with IC20 doses of 50 nM ATRA and 100 nM 9cisRA inhibited anchorage-dependent colony forming efficiency by about 75.4% (p<0.01) and 84.9% (p<0.01), respectively. Cell cycle analysis revealed that a 24-h treatment with IC90 doses of 2 microM ATRA and 3 microM 9cisRA accumulates cells in the G0/G1 phase and inhibit S and/or G2/M phase of the cell cycle. ATRA and 9cisRA induced an 11-fold (p=0.03) and a 9-fold (p=0.04) increase in subG0/G1 (apoptotic) population relative to the solvent control, respectively. ATRA and 9cisRA induced 77% (p=0.01) and 51% (p=0.02) decrease in tyrosine kinase immunoreactivity, respectively. Similarly, the two retinoids caused almost a 50% (p=0.01) down-regulation of Bcl-2 immunoreactivity. Western blot analysis revealed that ATRA induced an increase in RARbeta expression and a decrease in RARgamma expression, while 9cisRA down-regulated RXRalpha expression. These data demonstrate that ATRA and 9cisRA may inhibit HER-2/neu induced aberrant proliferation in part by retarding cell cycle progression, down-regulating HER-2/neu-mediated signal transduction and inducing Bcl-2-dependent apoptosis through a retinoid receptor-mediated mechanism.