Testable Alternatives Research Articles

Abstract PO1-08-09: Drynaria fortunie, a nutritional herb for prevention in triple negative breast cancer

Abstract Study Rationale: The triple negative breast cancer (TNBC) lacks the expression of hormone and growth factor receptors and responds only to anthracycline/taxol-based conventional chemotherapy. Major therapeutic limitations include systemic toxicity and acquired resistance to chemo-therapeutics. Relatively non-toxic nutritional herbs from traditional Chinese medicine (TCM) may represent testable alternatives against TNBC. These herbs effectively target multiple signaling pathways (Yang et al: J. Ethno-pharmacology 264: 113249, 2021). Growth inhibitory efficacy of nutritional herbs in a cellular model for TNBC is associated with downregulation of RB, RAS, PI3K and AKT signaling pathways (Telang et al: Pharmaceuticals 14: 1318, 2021). Drynaria fortunie (DF) is a nutritional herb that is used in many TCM herbal formulations. The present study is designed to examine the effects of DF in a cellular model for TNBC and to identify mechanistic leads for its efficacy. Experimental Model, herbal extract and biomarkers: Estrogen receptor negative, progesterone receptor negative and human epidermal growth factor receptor-2 negative MDA-MB-231 human breast cancer cell line represented a cellular model for TNBC. Non-fractionated aqueous extract from the bark of DF represented the test agent. Cell cycle progression, RB signaling and caspase 3/7 activity represented quantitative end point biomarkers. Study Outcome: Treatment with DF at cytostatic concentrations induced S phase arrest and inhibited RB signaling via downregulated expression of cyclin E, CDK2, pRB and E2F1. DF treatment also induced caspase 3/7 activity, a marker for cellular apoptosis. Collectively, these data provide potential mechanistic leads for anti-proliferative and pro-apoptotic effects of DF in the present model. Conclusion: Present data validate a mechanism-driven experimental approach to prioritize efficacious nutritional herbs as testable alternatives for prevention/therapy of TNBC. Citation Format: Nitin TELANG, Hareesh Nair, George Wong. Drynaria fortunie, a nutritional herb for prevention in triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-08-09.

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.

Drug-resistant Stem Cell Models for the Hormone-responsive Luminal A Breast Cancer

Background: Progression of early stage breast cancer to advanced stage metastatic disease represents a major cause of death in women. The Luminal A breast cancer subtype exhibits acceptable response to Chemo-endocrine and targeted therapy. However, these treatment options are associated with intrinsic/acquired therapy resistance and emergence chemo-resistant cancer initiating stem cell population, and resultant progression to advanced stage metastatic disease. These limitations emphasize an unmet need for the development of reliable models for cancer stem cells that facilitate identification of efficacious therapeutic alternatives. Documented human consumption, low systemic toxicity, preclinical cancer growth inhibitory efficacy and stem cell targeting efficacy of natural products, such as dietary phytochemicals and nutritional herbs, provide mechanistic leads for these agents as testable therapeutic alternatives. Objectives: The objectives of the present review are to i.) Provide a systematic discussion of published evidence relevant conceptual background of conventional/targeted therapy and nutritional herbs as testable alternatives, ii.) Growth inhibitory efficacy of nutritional herbs in a cellular model for the Luminal A breast cancer, iii.) Breast cancer stem cell biology and stem cell models for therapy-resistant breast cancer, and iv.) Future research directions. Conclusions: Collectively, all the elements discussed in the present review validate mechanism-based experimental approaches to identify and prioritize potential therapeutic alternatives. Future Research: This review provides a rationale for investigations on patient-derived tumor samples that may minimize extrapolation of the preclinical data for their clinical relevance and translatability.

Stem Cell Models for Cancer Therapy.

Metastatic progression of female breast and colon cancer represents a major cause of mortality in women. Spontaneous/acquired resistance to conventional and targeted chemo-endocrine therapy is associated with the emergence of drug-resistant tumor-initiating cancer stem cell populations. The cancer-initiating premalignant stem cells exhibit activation of select cancer cell signaling pathways and undergo epithelial–mesenchymal transition, leading to the evolution of a metastatic phenotype. The development of reliable cancer stem cell models provides valuable experimental approaches to identify novel testable therapeutic alternatives for therapy-resistant cancer. Drug-resistant stem cell models for molecular subtypes of clinical breast cancer and for genetically predisposed colon cancer are developed by selecting epithelial cells that survive in the presence of cytostatic concentrations of relevant therapeutic agents. These putative stem cells are characterized by the expression status of select cellular and molecular stem cell markers. The stem cell models are utilized as experimental approaches to examine the stem-cell-targeted growth inhibitory efficacy of naturally occurring dietary phytochemicals. The present review provides a systematic discussion on (i) conceptual and experimental aspects relevant to the chemo-endocrine therapy of breast and colon cancer, (ii) molecular/cellular aspects of cancer stem cells and (iii) potential stem-cell-targeting lead compounds as testable alternatives against the progression of therapy-resistant breast and colon cancer.

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 17β-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.

Drug-Resistant Stem Cells: Novel Approach for Colon Cancer Therapy.

Background: Next to breast cancer, advanced stage metastatic colon cancer represents a major cause for mortality in women. Germline or somatic mutations in tumor suppressor genes or in DNA mismatch repair genes represent risk factors for genetic predisposition of colon cancer that are also detectable in sporadic colon cancer. Conventional chemotherapy for colon cancer includes combination of 5-fluoro-uracil with oxaliplatin and irinotecan or targeted therapy with non-steroid anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors. Major limitations of these therapeutic interventions are associated with systemic toxicity, acquired tumor resistance and the emergence of drug resistant stem cells that favor initiation, progression and metastasis of therapy-resistant disease. These limitations emphasize an unmet need to identify tumor stem cell selective testable alternatives. Drug-resistant stem cell models facilitate the identification of new testable alternatives from natural phytochemicals and herbal formulations. The goal of this review is to provide an overview relevant to the current status of conventional/targeted therapy, the role of cancer stem cells and the status of testable alternatives for therapy-resistant colon cancer. Experimental models: Hyper-proliferative and tumorigenic cell lines from genetically predisposed colonic tissues of female mice represent experimental models. Chemotherapeutic agents select drug-resistant phenotypes that exhibit upregulated expressions of cellular and molecular stem cell markers. Mechanistically distinct natural phytochemicals effectively inhibit stem cell growth and downregulate the expressions of stem cell markers. Conclusions: The present review discusses the status of colon cancer therapy and inherent limitations, cancer stem cell biology, potential lead compounds and their advantages over chemotherapy. The present experimental approaches will facilitate the identification of pharmacological and naturally-occurring agents as lead compounds for stem cell targeted therapy of colon cancer.

Anti-Proliferative and Pro-Apoptotic Effects Of Dipsacus Asperoides in a Cellular Model for Triple-Negative Breast Cancer

Background: Triple negative breast cancer (TNBC) lacks expressions of estrogen receptor-α (ER-α), progesterone receptor (PR) and amplified human epidermal growth factor receptor-2 (HER-2). Current treatment for TNBC includes anthracyclin, taxol and cisplatin-based conventional chemotherapy and survival pathway PARP, PI3K, AKT and mTOR selective targeted therapy. These treatments exhibit dose-limiting systemic toxicity and presence of drug resistant cancer stem cells, which highlight the need for identification of efficacious testable alternatives that are not toxic to non-tumorigenic cells. Dipsacus asperoides (DA) is a Chinese nutritional herb and its root represents a common ingredient in Chinese herbal formulations used in women for estrogen related health issues, osteoporosis and breast diseases. This study aims to investigate the growth inhibitory effects of DA, and to detect mechanisms for its efficacy. Methods: Human mammary carcinoma derived triple negative MDA-MB-231 cell line represented the TNBC model. Non-fractionated aqueous extract from DA represented the test agent. Anchorage dependent growth, anchorage independent (AI) colony formation and cell cycle progression quantified growth inhibition. Western blot-based analysis for inhibition of RAS, PI3K and AKT and RB signaling identified mechanistic leads. Results: Treatment with DA induced a dose dependent cytostatic growth arrest (IC50:15 µg/ml; IC90: 30 µg/ml), reduced AI growth and inhibited cell cycle progression via G2/M arrest. DA affected the RAS, PI3K, AKT and RB signaling pathways, and functioned as a natural inhibitor of cyclin dependent kinase 4/6. Cellular apoptosis paralleled increase in pro-apoptotic Caspase 3/7 activity. Conclusion: These results demonstrate that DA inhibited growth, affected cell cycle progression, induced apoptosis and inhibited cancer cell survival pathways. This study validates a mechanism-based approach to identifying testable substitutes for secondary prevention/therapy of TNBC.

Cancer Stem Cells: Innovative Approach for Testable Alternatives against Therapy Resistant Cancer

Chemo-resistant cancer stem cells represent a sub-population of cancer initiating phenotype in primary cancer. These cells evolve in to a metastatic phenotype via activation of multiple cell signaling pathways for cancer cell survival and epithelial-mesenchymal transition. Reliable cancer stem cell models represent a valuable experimental approach for drug discovery platform to identify efficacious testable alternatives against therapy-resistant cancer. Present commentary provides a systematic discussion of relevant conceptual and technical aspects of cancer stem cell biology and its significance for therapeutic alternatives.

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 Tabebuia Avellanedae in a Model for Triple Negative Breast Cancer

Background: Tabebuia avellanedae (TA) is a tree that is indigenous to the Amazon rainforest. The experiments in the present study were designed to examine the inhibitory effects of TA, and to identify mechanistic targets for its efficacy in the estrogen-α receptor (ER-α), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) negative MDA-MB-231 model for triple negative breast cancer (TNBC). Methods: Non-fractionated aqueous extract from the inner bark of TA was used in the experiments. Anchorage dependent growth, anchorage independent (AI) colony formation, cell cycle progression, and expressions of relevant regulatory proteins represented quantitative end points. Results: Long-term treatment for 21 days with the maximum cytostatic concentration of 2.5% TA resulted in a 90% inhibition (P=0.014) in AI colony number. Short-term treatment for 2 days with 1.0% TA (IC50) resulted in about a 1.3 fold increase (P=0.014) in G1: S+G2/M ratio, about a 1.48 fold increase (P=0.010) in the sub G0 (apoptotic) cells and about a 3.2 fold increase (P=0.014) in the pro-apoptotic caspase 3/7 activity. Mechanistically, the short-term treatment with 2.5% TA decreased Cyclin D1 expression by about 83.3%, and pRB expression by about 73.3%. Conclusion: TNBC represents an aggressive cancer notable for its resistance to conventional and targeted therapy. Non-toxic natural substances may represent testable alternatives. This study identifies potential mechanistic leads for TA as a novel naturally occurring testable alternative for secondary prevention/therapy of TNBC, and validates a novel mechanistic approach to evaluate efficacious non-toxic phytochemicals and herbs as testable alternatives against therapy resistant breast cancer.

Growth inhibitory efficacy of the nutritional herb Psoralea corylifolia in a model of triple‑negative breast cancer

Triple‑negative breast cancer (TNBC) lacks the expression of estrogen receptor‑α (ER‑α), progesterone receptor (PR) and amplified human epidermal growth factor receptor‑2 (HER‑2). Current treatments involve chemotherapy and molecular targeted therapy. These options lead to dose‑limiting systemic toxicity and acquired tumor resistance. Moreover, no drug is yet available for the secondary prevention of TNBC. Such limitations underscore a need to investigate non‑toxic testable alternatives for the secondary prevention/treatment of TNBC. The Chinese nutritional herb, <em>Psoralea corylifolia</em> (PC), has been shown to be effective against the MCF‑7 cell line, a cellular model for ER‑α‑positive breast cancer. The present study aimed to investigate the growth inhibitory effects of PC against TNBC using the MDA‑MB‑231 cell line. Anchorage‑independent colony formation, cell cycle progression, cellular apoptosis, RB signaling and caspase‑3/7 activity assays were performed to determine the quantitative parameters for the efficacy of PC. Treatment with PC induced a 3.5‑fold increase in the G₁: S + G₂/M ratio, an 11‑fold increase in the apoptotic cell number and a 149‑fold increase in caspase‑3/7 activity. PC treatment also decreased cyclin D1, CDK4/6 and p‑RB expression levels. On the whole, the data of the present study identify mechanistic leads for PC as a naturally occurring testable alternative, and validate a mechanistic approach to prioritize efficacious nontoxic herbal extracts as nutritional supplements for the secondary prevention/therapy of TNBC.

Stem cell models for genetically predisposed colon cancer.

Negative growth regulatory tumor suppressor genes and positive growth regulatory oncogenes serve important roles in initiation/progression of colon cancer. Germline mutation in the adenomatous polyposis coli (APC) tumor suppressor gene represents a primary genetic defect for familial adenomatous polyposis (FAP) syndrome, a predisposing factor for clinical colon cancer. Somatic mutations in the APC gene are common in sporadic colon cancer. Preclinical and clinical efficacy is documented for targeted therapy with non-steroidal anti-inflammatory drugs, selective cyclo-oxygenase-2 inhibitors for prostaglandin biosynthesis and selective inhibitor of ornithine decarboxylase for polyamine biosynthesis. However, these therapeutic options lead to systemic toxicity, acquired tumor resistance and emergence of therapy resistant cancer stem cells. By contrast, non-toxic natural products are unlikely to exhibit drug resistance and may represent testable alternatives for therapy resistant colon cancer. Tumorigenic Apc [-/-] colonic epithelial cell lines derived from preclinical FAP models provide novel cellular models for drug resistant cancer stem cells. Apc [-/-] Sulindac resistant (SUL-R) cells exhibit upregulated expression levels of cancer stem cell markers. Natural products, such as naturally occurring vitamin A derivative all-trans retinoic acid (ATRA) and the anti-cancer agent from Turmeric root curcumin (CUR), represent testable alternatives. Relative to the non-tumorigenic Apc [+/+] C57 COL colonic epithelial cells, the tumorigenic Apc [-/-] 1638N COL and Apc [-/-] 850 MIN COL cells exhibit aneuploid cell hyper-proliferation and upregulated expression of Apc target genes β-catenin, cyclin D1, c-myc and COX-2. The SUL-R phenotypes exhibit enhanced tumor spheroid formation and upregulated expression levels of stem cell markers CD44, CD133 and c-Myc. Treatment of the SUL-R stem cells with ATRA and CUR inhibits tumor spheroid formation and reduces the expression of stem cell markers. Stem cell models developed for FAP syndrome provide a novel experimental approach to identify mechanistic leads for efficacious natural products as testable alternatives for therapy-resistant, genetically predisposed colon cancer.

Growth inhibitory efficacy and anti-aromatase activity of Tabebuia avellanedae in a model for post-menopausal Luminal A breast cancer.

Aromatase inhibitors (AIs) represent a treatment option for post-menopausal estrogen receptor-positive (ER+) breast cancer as monotherapy, or in combination with cyclin-dependent kinase 4/6 or mTOR inhibitors. Long-term treatment with these agents leads to dose-limiting toxicity and drug resistance. Natural substances provide testable alternatives to current therapy. Tabebuia avellanedae (TA) tree is indigenous to the Amazon rainforest. The inner bark of TA represents a medicinal dietary supplement known as Taheebo. Non-fractionated aqueous extract from TA is an effective growth inhibitor in the Luminal A and triple negative breast cancer models. The quinone derivative naphthofurandione (NFD) is a major bioactive agent in TA. The present study examined the efficacy of finely ground powder from the inner bark of TA, available under the name of Taheebo-NFD-Marugoto (TNM). The ER+ MCF-7 cells stably transfected with the aromatase gene MCF-7AROM represented a model for aromatase-expressing post-menopausal breast cancer. Anchorage-independent colony formation, cell cycle progression, pro-apoptotic caspase 3/7 activity, apoptosis-specific gene expression, aromatase activity and select estradiol (E2) target gene expression represented the mechanistic end points. Treatment of MCF-7AROM cells with TNM induced a dose-dependent reduction in E2-promoted anchorage-independent colony number. Mechanistic assays on TNM-treated MCF-7AROM cells demonstrated that TNM at a concentration of 10 µg (NFD content: 2 ng), induced S-phase arrest, increased pro-apoptotic caspase 3/7 activity, increased pro-apoptotic BAX and decreased anti-apoptotic BCL-2 gene expression, and inhibited aromatase activity. Additionally, TNM treatment downregulated ESR-1 (gene for ER-α), aromatase and progesterone gene expression and reduced mRNA levels of E2 target genes pS2, GRB2 and cyclin D1. Inhibition of aromatase activity, based on the NFD content of TNM was superior to the clinical AIs Letrozole and Exemestane. These data demonstrated the potential efficacy of TNM as a nutritional alternative for current therapy of aromatase positive, post-menopausal breast cancer.

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.

Challenges to the social motivation theory of autism: The dangers of counteracting an imprecise theory with even more imprecision

Abstract The arguments offered by Jaswal &amp; Akhtar to counter the social motivation theory (SMT) do not appear to be directly related to the SMT tenets and predictions, seem to not be empirically testable, and are inconsistent with empirical evidence. To evaluate the merits and shortcomings of the SMT and identify scientifically testable alternatives, advances are needed on the conceptualization and operationalization of social motivation across diagnostic boundaries.

Stem cell targeted therapeutic approaches for molecular subtypes of clinical breast cancer (Review)

The global profiling of differentially expressed genes in subtypes of clinical breast cancer identifies predictive and prognostic biomarkers for disease progression and rationalizes breast cancer subtype‑based treatment options. The expression status of hormone and growth factor receptors dictates the options for chemo‑endocrine and/or pathway selective small molecule inhibitor‑based treatments. Overall, these treatment options are associated with long‑term systemic toxicity and acquired tumor resistance, predominantly due to the emergence of drug‑resistant cancer stem cell population and due to therapy‑resistant disease progression. These limitations emphasize the identification of non‑toxic testable therapeutic alternatives for the efficacious targeting of breast cancer stem cells. The present review summarizes published evidence focused on i) developing cellular models for molecular subtypes of breast cancer; ii) isolating and characterizing drug‑resistant cancer stem cells from the developed models; and iii) identifying mechanistic leads for potential stem cell‑targeting lead compounds. Cellular models for Luminal A, human epidermal growth factor receptor‑2 (HER‑2) enriched and triple‑negative breast cancer subtypes represented the experimental models. Prototypic chemo‑endocrine therapeutic agents were used to select the drug‑resistant stem cell phenotype. The vitamin A derivative, all‑trans retinoic acid, and the rosemary terpenoid, carnosol, respectively representing a mechanistically distinct natural product and a potential bio‑active constituent of a nutritional herb provided stem cell‑selective lead compounds. The cellular models for Luminal A, HER‑2‑enriched and triple‑negative breast cancer subtypes exhibited growth inhibitory effects in response to treatment with prototypic chemo‑endocrine therapeutics, natural products and nutritional herbs. Drug‑resistant phenotypes exhibited an upregulated expression of stem cell‑specific cellular and molecular markers. Lead compounds induced the downregulated expression of the stem cell markers in drug‑resistant phenotypes. These data validate an experimental approach with which to identify potential non‑toxic natural products and nutritional herbs that may represent testable alternatives for the stem cell targeted therapy of breast cancer.

Stem cell targeted therapeutic approaches for molecular subtypes of clinical breast cancer (Review)

The global profiling of differentially expressed genes in subtypes of clinical breast cancer identifies predic‑ tive and prognostic biomarkers for disease progression and rationalizes breast cancer subtype‑based treatment options. The expression status of hormone and growth factor recep‑ tors dictates the options for chemo‑endocrine and/or pathway selective small molecule inhibitor‑based treatments. Overall, these treatment options are associated with long‑term systemic toxicity and acquired tumor resistance, predominantly due to the emergence of drug‑resistant cancer stem cell population and due to therapy‑resistant disease progression. These limitations emphasize the identification of non‑toxic testable therapeutic alternatives for the efficacious targeting of breast cancer stem cells. The present review summarizes published evidence focused on i) developing cellular models for molecular subtypes of breast cancer; ii) isolating and characterizing drug‑resistant cancer stem cells from the developed models; and iii) identi‑ fying mechanistic leads for potential stem cell‑targeting lead compounds. Cellular models for Luminal A, human epidermal growth factor receptor‑2 (HER‑2) enriched and triple‑negative breast cancer subtypes represented the experimental models. Prototypic chemo‑endocrine therapeutic agents were used to select the drug‑resistant stem cell phenotype. The vitamin A derivative, all‑trans retinoic acid, and the rosemary terpenoid, carnosol, respectively representing a mechanistically distinct natural product and a potential bio‑active constituent of a nutritional herb provided stem cell‑selective lead compounds. The cellular models for Luminal A, HER‑2‑enriched and triple‑negative breast cancer subtypes exhibited growth inhibitory effects in response to treatment with prototypic chemo‑endocrine therapeutics, natural products and nutritional herbs. Drug‑resistant phenotypes exhibited an upregulated expression of stem cell‑specific cellular and molecular markers. Lead compounds induced the downregulated expression of the stem cell markers in drug‑resistant phenotypes. These data validate an experimental approach with which to identify potential non‑toxic natural products and nutritional herbs that may represent testable alternatives for the stem cell targeted therapy of breast cancer.

Anti-proliferative and pro-apoptotic effects of rosemary and constituent terpenoids in a model for the HER-2-enriched molecular subtype of clinical breast cancer.

Neoadjuvant treatment options for human epidermal growth factor receptor-2 (HER-2)-enriched and luminal B molecular subtypes of clinical breast cancer include HER-2-targeted therapy with chemotherapy or anti-hormonal therapy. These treatment options result in systemic toxicity and acquired tumor resistance. Minimally toxic naturally occurring phytochemicals may represent testable alternatives to conventional therapy. HER-2-overexpressing tumorigenic human mammary epithelial 184-B5/HER cells represent a model for the HER-2-enriched breast cancer subtype. Non-fractionated rosemary extract (RME) and constituent phenolic terpenoids ursolic acid (UA), carnosol (CSOL) and carnosic acid (CA) represented the test agents. Anchorage-independent (AI) proliferation, cell cycle progression, cellular apoptosis and expression of cell cycle-regulatory and apoptosis-specific proteins represented the mechanistic end point biomarkers. Relative to the parental non-tumorigenic 184-B5 cells, tumorigenic 184-B5/HER cells exhibited decreased population doubling, increased saturation density, accelerated cell cycle progression and downregulated cellular apoptosis, confirming the loss of homeostatic control of proliferation. Treatment with the test agents resulted in a dose-dependent decrease in AI colony number, indicating a decrease in cancer risk. Mechanistically, RME and UA inhibited G1-S phase transition resulting in an increased G1:S+G2/M ratio and decreased cyclin D1 expression. The pro-apoptotic effect of RME and UA was indicated by increased sub-G0 (apoptotic) cell population, and relevant reciprocal modulation, as demonstrated by decreased anti-apoptotic B-cell lymphoma-2 (Bcl-2) and increased pro-apoptotic Bcl-2-associated X protein expression. In contrast, treatment with CA and CSOL resulted in cytostatic G2/M arrest and an increase in cyclin B1 expression; thus, naturally-occurring rosemary and its constitutive terpenoids re-establish homeostatic control of proliferation and decrease cancer risk via distinct mechanisms. These data validate an experimental approach to prioritize efficacious natural compounds as testable alternatives for conventional chemo-endocrine and HER-2-targeted therapies in HER-2-enriched breast cancer.

Growth inhibitory efficacy of natural products in a model for triple negative molecular subtype of clinical breast cancer.

Global gene expression profiling identifies predictive and prognostic biomarkers and rationalizes breast cancer subtype-targeted treatment. The Anthracyclin/Taxol and survival pathway specific small molecular inhibitors, constitute current treatment options. These options are associated with acquired tumor resistance and emergence of drug-resistant cancer stem cells. Dietary supplements and constitutive bioactive phytochemicals with relatively low systemic toxicity may provide testable alternatives for current therapy. Human breast epithelial cell lines 184-B5 (non-tumorigenic triple negative cell type) and MDA-MB-231 (breast carcinoma derived triple negative cell type) were used as the experimental models. Putative cancer chemo-preventive natural products and their constitutive bioactive agents represented the test agents. Anchorage independent growth, cell cycle progression and cell apoptosis quantified the treatment efficacy. Compared to the 184-B5 cells, the MDA-MB-231 cells exhibited anchorage-independent growth indicative of persistent cancer risk. Additionally, the MDA-MB-231 cells exhibited hyper-proliferation, accelerated cell cycle progression and inhibited apoptosis indicative of loss of homeostatic growth control. The test agents inhibited anchorage-independent growth via cytostatic and pro-apoptotic effects. The triple negative carcinoma-derived Doxorubicin-resistant phenotype exhibited cancer stem cell markers, including tumor spheroid formation and expression of CD44, NANOG and c-Myc. These data identify clinically relevant mechanistic leads for the efficacy of natural products in the aggressive therapy-resistant breast cancer subtype and suggests a testable approach for cancer stem cell-targeted therapy.

Beyond Reasonable Doubt: Evolution from DNA Sequences

We demonstrate quantitatively that, as predicted by evolutionary theory, sequences of homologous proteins from different species converge as we go further and further back in time. The converse, a non-evolutionary model can be expressed as probabilities, and the test works for chloroplast, nuclear and mitochondrial sequences, as well as for sequences that diverged at different time depths. Even on our conservative test, the probability that chance could produce the observed levels of ancestral convergence for just one of the eight datasets of 51 proteins is ≈1×10−19 and combined over 8 datasets is ≈1×10−132. By comparison, there are about 1080 protons in the universe, hence the probability that the sequences could have been produced by a process involving unrelated ancestral sequences is about 1050 lower than picking, among all protons, the same proton at random twice in a row. A non-evolutionary control model shows no convergence, and only a small number of parameters are required to account for the observations. It is time that that researchers insisted that doubters put up testable alternatives to evolution.