Omar Rahal

Repression of Mammosphere Formation of Human Breast Cancer Cells by Soy Isoflavone Genistein and Blueberry Polyphenolic Acids Suggests Diet-Mediated Targeting of Cancer Stem-Like/Progenitor Cells

Mammary stem cells are undifferentiated epithelial cells, which initiate mammary tumors and render them resistant to anticancer therapies, when deregulated. Diets rich in fruits and vegetables are implicated in breast cancer risk reduction, yet underlying mechanisms are poorly understood. Here, we addressed whether dietary factors selectively target mammary epithelial cells that display stem-like/progenitor subpopulations with previously recognized tumor-initiating potential. Using estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231 human breast cancer cell lines and freshly isolated epithelial cells from MMTV-Wnt-1 transgenic mouse mammary tumors, we demonstrate that sera of adult mice consuming soy isoflavone genistein (GEN) or blueberry (BB) polyphenol-containing diets alter the population of stem-like/progenitor cells, as measured by their functional ability to self-renew and form anchorage-independent spheroid cultures in vitro at low frequency (1-2%). Serum effects on mammosphere formation were dose-dependently replicated by GEN (40 nM >2 μM) and targeted the basal stem-like CD44+/CD24-/ESA+ and the luminal progenitor CD24+ subpopulations in MDA-MB-231 and MCF-7 cells. GEN inhibition of mammosphere formation was mimicked by the Akt inhibitor perifosine and was associated with enhanced tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) expression. In contrast, a selected mixture of BB phenolic acids was only active in MDA-MD-231 cells and its CD44+/CD24-/ESA+ subpopulation, and this activity was independent of induction of PTEN expression. These findings delineate a novel and selective function of distinct dietary factors in targeting stem/progenitor cell populations in estrogen receptor-dependent and -independent breast cancers.

The emerging role of Krüppel-like factors in endocrine-responsive cancers of female reproductive tissues

Krüppel-like factors (KLFs), of which there are currently 17 known protein members, belong to the specificity protein (Sp) family of transcription factors and are characterized by the presence of Cys(2)/His(2) zinc finger motifs in their carboxy-terminal domains that confer preferential binding to GC/GT-rich sequences in gene promoter and enhancer regions. While previously regarded to simply function as silencers of Sp1 transactivity, many KLFs are now shown to be relevant to human cancers by their newly identified abilities to mediate crosstalk with signaling pathways involved in the control of cell proliferation, apoptosis, migration, and differentiation. Several KLFs act as tumor suppressors and/or oncogenes under distinct cellular contexts, underscoring their prognostic potential for cancer survival and outcome. Recent studies suggest that a number of KLFs can influence steroid hormone signaling through transcriptional networks involving steroid hormone receptors and members of the nuclear receptor family of transcription factors. Since inappropriate sensitivity or resistance to steroid hormone actions underlies endocrine-related malignancies, we consider the intriguing possibility that dysregulation of expression and/or activity of KLF members is linked to the pathogenesis of endometrial and breast cancers. In this review, we focus on recently described mechanisms of actions of several KLFs (KLF4, KLF5, KLF6, and KLF9) in cancers of the mammary gland and uterus. We suggest that understanding the mode of actions of KLFs and their functional networks may lead to the development of novel therapeutics to improve current prospects for cancer prevention and cure.

PTEN and p53 cross-regulation induced by soy isoflavone genistein promotes mammary epithelial cell cycle arrest and lobuloalveolar differentiation

The tumor suppressors phosphatase and tensin homologue deleted on chromosome ten (PTEN) and p53 are closely related to the pathogenesis of breast cancer, yet pathway-specific mechanisms underlying their participation in mediating the protective actions of dietary bioactive components on breast cancer risk are poorly understood. We recently showed that dietary exposure to the soy isoflavone genistein (GEN) induced PTEN expression in mammary epithelial cells in vivo and in vitro, consistent with the breast cancer preventive effects of soy food consumption. Here, we evaluated PTEN and p53 functional interactions in the nuclear compartment of mammary epithelial cells as a mechanism for mammary tumor protection by GEN. Using the non-tumorigenic human mammary epithelial cells MCF10-A, we demonstrate that GEN increased PTEN expression and nuclear localization. We show that increased nuclear PTEN levels initiated an autoregulatory loop involving PTEN-dependent increases in p53 nuclear localization, PTEN-p53 physical association, PTEN-p53 co-recruitment to the PTEN promoter region and p53 transactivation of PTEN promoter activity. The PTEN-p53 cross talk induced by GEN resulted in increased cell cycle arrest; decreased pro-proliferative cyclin D1 and pleiotrophin gene expression and the early formation of mammary acini, indicative of GEN promotion of lobuloalveolar differentiation. Our findings provide support to GEN-induced PTEN as both a target and regulator of p53 action and offer a mechanistic basis for PTEN pathway activation to underlie the antitumor properties of dietary factors, with important implications for reducing breast cancer risk.

Copper chelation selectively kills colon cancer cells through redox cycling and generation of reactive oxygen species

BackgroundMetals including iron, copper and zinc are essential for physiological processes yet can be toxic at high concentrations. However the role of these metals in the progression of cancer is not well defined. Here we study the anti-tumor activity of the metal chelator, TPEN, and define its mechanism of action.MethodsMultiple approaches were employed, including cell viability, cell cycle analysis, multiple measurements of apoptosis, and mitochondrial function. In addition we measured cellular metal contents and employed EPR to record redox cycling of TPEN–metal complexes. Mouse xenografts were also performed to test the efficacy of TPEN in vivo.ResultsWe show that metal chelation using TPEN (5μM) selectively induces cell death in HCT116 colon cancer cells without affecting the viability of non-cancerous colon or intestinal cells. Cell death was associated with increased levels of reactive oxygen species (ROS) and was inhibited by antioxidants and by prior chelation of copper. Interestingly, HCT116 cells accumulate copper to 7-folds higher levels than normal colon cells, and the TPEN-copper complex engages in redox cycling to generate hydroxyl radicals. Consistently, TPEN exhibits robust anti-tumor activity in vivo in colon cancer mouse xenografts.ConclusionOur data show that TPEN induces cell death by chelating copper to produce TPEN-copper complexes that engage in redox cycling to selectively eliminate colon cancer cells.

The soybean peptide lunasin promotes apoptosis of mammary epithelial cells via induction of tumor suppressor PTEN: similarities and distinct actions from soy isoflavone genistein.

Breast cancer is the leading cause of cancer deaths in women. Diet and lifestyle are major contributing factors to increased breast cancer risk. While mechanisms underlying dietary protection of mammary tumor formation are increasingly elucidated, there remains a dearth of knowledge on the nature and precise actions of specific bioactive components present in foods with purported health effects. The 43-amino acid peptide lunasin (LUN) is found in soybeans, is bioavailable similar to the isoflavone genistein (GEN), and thus may mediate the beneficial effects of soy food consumption. Here, we evaluated whether LUN displays common and distinct actions from those of GEN in non-malignant (mouse HC11) and malignant (human MCF-7) mammary epithelial cells. In MCF-7 cells, LUN up-regulated tumor suppressor phosphatase and tensin homolog deleted in chromosome ten (PTEN) promoter activity, increased PTEN transcript and protein levels and enhanced nuclear PTEN localization, similar to that shown for GEN in mammary epithelial cells. LUN-induced cellular apoptosis, akin to GEN, was mediated by PTEN, but unlike that for GEN, was p53-independent. LUN promoted E-cadherin and β-catenin non-nuclear localization similar to GEN, but unlike GEN, did not influence the proliferative effects of oncogene Wnt1 on HC11 cells. Further, LUN did not recapitulate GEN inhibitory effects on expansion of the cancer stem-like/progenitor population in MCF-7 cells. Results suggest the concerted actions of GEN and LUN on cellular apoptosis for potential mammary tumor preventive effects and highlight whole food consumption rather than intake of specific dietary supplements with limited biological effects for greater health benefits.

Repression of mammary adipogenesis by genistein limits mammosphere formation of human MCF-7 cells.

Mammary adipose tissue may contribute to breast cancer development and progression by altering neighboring epithelial cell behavior and phenotype through paracrine signaling. Dietary exposure to soy foods is associated with lower mammary tumor risk and reduced body weight and adiposity in humans and in rodent breast cancer models. Despite the suggested linkage between obesity and breast cancer, the local influence of bioactive dietary components on mammary adiposity for antitumor effects remains unknown. Herein, we report that post-weaning dietary exposure to soy protein isolate and its bioactive isoflavone genistein (GEN) lowered mammary adiposity and increased mammary tumor suppressor PTEN and E-cadherin expression in female mice, relative to control casein diet. To ascertain GENs role in mammary adipose deposition that may affect underlying epithelial cell phenotype, we evaluated GENs effects on SV40-immortalized mouse mammary stromal fibroblast-like (MSF) cells during differentiation into adipocytes. MSF cells cultured in a differentiation medium with 40 nM GEN showed reductions in mature adipocyte numbers, triglyceride accumulation, and Pparγ (Pparg) and fatty acid synthase transcript levels. GEN inhibition of adipose differentiation was accompanied by increased estrogen receptor β (Erβ (Esr2)) gene expression and was modestly recapitulated by ERβ-selective agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN). Reduction of Erβ expression by siRNA targeting increased Pparγ transcript levels and stromal fibroblast differentiation into mature adipocytes; the latter was reversed by GEN but not by DPN. Conditioned medium from GEN-treated adipocytes diminished anchorage-independent mammosphere formation of human MCF-7 breast cancer cells. Our results suggest a mechanistic pathway to support direct regulation of mammary adiposity by GEN for breast cancer prevention.

In utero and lactational exposure to blueberry via maternal diet promotes mammary epithelial differentiation in prepubescent female rats

Early developmental events influence the fine tuning of later susceptibility to adult diseases. Diet is a determinant of breast cancer risk, and our previous studies showed that diet-mediated changes in transcriptional programs promote early mammary gland differentiation. Although consumption of fruits is considered to elicit multiple health benefits, little is known on whether associated bioactive components modify the early differentiation program in developing mammary glands. Here, we evaluated the hypothesis that early exposure (in utero and lactational) to blueberry through maternal diet enhances mammary epithelial differentiation in female offspring. Pregnant Sprague-Dawley rats beginning at gestation day 4 were fed American Institute of Nutrition-based diets containing casein and whole blueberry powders added to casein at 2.5%, 5.0%, and 10% weight/weight. Female pups at weaning were evaluated for growth and mammary tissue parameters. Blueberry at 5% dose increased body and adipose fat weights, relative to the other diets. Mammary branch density and terminal end bud size were highest for the 5% blueberry group, whereas terminal end bud numbers were not affected by all diets. Mammary ductal epithelial cells of the 5% blueberry group had lower nuclear phosphorylated histone 3 and higher nuclear tumor suppressor phosphatase and tensin homolog deleted in chromosome 10 (PTEN) levels than the casein group. Although sera of both diet groups had similar antioxidant capacity, 5% blueberry sera elicited higher nuclear PTEN accumulation in human MCF-10A mammary epithelial cells. Our studies identify developing mammary glands as early targets of blueberry-associated bioactive components, possibly through systemic effects on epithelial PTEN signaling.

Emerging Cytotoxic Alkaloids in the Battle against Cancer: Overview of Molecular Mechanisms

Considered as the second deadliest disease globally, cancer has captured the attention of researchers who have been trying with perseverance to decode its hidden aspects, to find new prognosis methods, and to develop better and more effective treatments. Plants have continuously offered an excess of unique secondary metabolites with remarkable biological applications. Alkaloids, one of the most abundant metabolites, constitute a large conglomerate of basic heterocyclic nitrogen-containing natural compounds which are normally produced by plants as toxic substances. Out of the 27,000 different alkaloids, more than 17,000 have displayed diversified pharmacological properties including anticancer activities. These metabolites have been classified either according to their chemical structures or their taxonomic origin. None of the researched alkaloids have been classified according to their molecular mechanism of action against cancer. In fact, only a fraction of the tremendous number of anticancer alkaloids has been copiously mentioned in journals. Here, we aim to provide a summary of the literature on some of the promising anticancer alkaloids that have not been well discussed previously and to classify them according to their molecular mechanisms of action. This review will provide a better understanding of the anticancer mechanisms of these promising natural products that are a rich reservoir for drug discovery.

Suppression of Wnt1-induced mammary tumor growth and lower serum insulin in offspring exposed to maternal blueberry diet suggest early dietary influence on developmental programming

Despite the well-accepted notion that early maternal influences persist beyond fetal life and may underlie many adult diseases, the risks imposed by the maternal environment on breast cancer development and underlying biological mechanisms remain poorly understood. In this study, we investigated whether early exposure to blueberry (BB) via maternal diet alters oncogene Wnt1-induced mammary tumorigenesis in offspring. Wnt1-transgenic female mice were exposed to maternal Casein (CAS, control) or blueberry-supplemented (CAS + 3%BB) diets throughout pregnancy and lactation. Offspring were weaned to CAS and mammary tumor development was followed until age 8 months. Tumor incidence and latency were similar for both groups; however, tumor weight at killing and tumor volume within 2 weeks of initial detection were lower (by 50 and 60%, respectively) in offspring of BB- versus control-fed dams. Dietary BB exposure beginning at weaning did not alter mammary tumor parameters. Tumors from maternal BB-exposed offspring showed higher tumor suppressor (Pten and Cdh1) and lower proproliferative (Ccnd1), anti-apoptotic (Bcl2) and proangiogenic (Figf, Flt1 and Ephb4) transcript levels, and displayed attenuated microvessel density. Expression of Pten and Cdh1 genes was also higher in mammary tissues of maternal BB-exposed offspring. Mammary tissues and tumors of maternal BB-exposed offspring showed increased chromatin-modifying enzyme Dnmt1 and Ezh2 transcript levels. Body weight, serum insulin and serum leptin/adiponectin ratio were lower for maternal BB-exposed than control tumor-bearing offspring. Tumor weights and serum insulin were positively correlated. Results suggest that dietary influences on the maternal environment contribute to key developmental programs in the mammary gland to modify breast cancer outcome in adult progeny.

Dietary suppression of the mammary CD29hiCD24+ epithelial subpopulation and its cytokine/chemokine transcriptional signatures modifies mammary tumor risk in MMTV-Wnt1 transgenic mice

Diet is highly linked to breast cancer risk, yet little is known about its influence on mammary epithelial populations with distinct regenerative and hence, tumorigenic potential. To investigate this, we evaluated the relative frequency of lineage-negative CD29(hi)CD24(+), CD29(lo)CD24(+) and CD29(hi)Thy1(+)CD24(+) epithelial subpopulations in pre-neoplastic mammary tissue of adult virgin MMTV-Wnt1-transgenic mice fed either control (Casein) or soy-based diets. We found that mammary epithelial cells exposed to soy diet exhibited a lower percentage of CD29(hi)CD24(+)Lin(-) population, decreased ability to form mammospheres in culture, lower mammary outgrowth potential when transplanted into cleared fat pads, and reduced appearance of tumor-initiating CD29(hi)Thy1(+)CD24(+) cells, than in those of control diet-fed mice. Diet had no comparable influence on the percentage of the CD29(lo)CD24(+)Lin(-) population. Global gene expression profiling of the CD29(hi)CD24(+)subpopulation revealed markedly altered expression of genes important to inflammation, cytokine and chemokine signaling, and proliferation. Soy-fed relative to casein-fed mice showed lower mammary tumor incidence, shorter tumor latency, and reduced systemic levels of estradiol 17-β, progesterone and interleukin-6. Our results provide evidence for the functional impact of diet on specific epithelial subpopulations that may relate to breast cancer risk and suggest that diet-regulated cues can be further explored for breast cancer risk assessment and prevention.