Anti-Inflammatory Mechanisms of Dietary Flavones: Tapping into Nature to Control Chronic Inflammation in Obesity and Cancer.

Flavones are natural phytochemicals broadly distributed in our diet. Their anti-inflammatory properties provide unique opportunities to control the innate immune system and inflammation. Here, we review the role of flavones in chronic inflammation with an emphasis on their impact on the molecular mechanisms underlying inflammatory diseases including obesity and cancer. Flavones can influence the innate immune cell repertoire restoring the immune landscape. Flavones impinge on NF-κB, STAT, COX-2, or NLRP3 inflammasome pathways reestablishing immune homeostasis. Devoid of adverse side effects, flavones could present alternative opportunities for the treatment and prevention of chronic inflammation that contributes to obesity and cancer.

Genomic and metabolomic analysis of step-wise malignant transformation in human skin fibroblasts.

Metabolic changes accompanying a step-wise malignant transformation was investigated using a syngeneic lineage of human fibroblasts. Cell immortalization was associated with minor alterations in metabolism. Consecutive loss of cell cycle inhibition in immortalized cells resulted in increased levels of oxidative phosphorylation (OXPHOS). Overexpression of the H-Ras oncoprotein produced cells forming sarcomas in athymic mice. These transformed cells exhibited increased glucose consumption, glycolysis and a further increase in OXPHOS. Because of the markedly increased OXPHOS in transformed cells, the impact of a transaminase inhibitor, aminooxyacetic acid (AOA), which decreases glutamine influx to the tricarboxylic acid (TCA) cycle, was tested. Indeed, AOA significantly decreased proliferation of malignantly transformed fibroblasts and fibrosarcoma-derived cells in vitro and in vivo. AOA also decreased proliferation of cells susceptible to malignant transformation. Metabolomic studies in normal and transformed cells indicated that, in addition to the anticipated effect on the TCA cycle, AOA decreased production of nucleotides adenosine triphosphate (ATP) and uridine monophosphate. Exogenous nucleotides partially rescued decreased proliferation of the malignant cells treated with AOA. Our data indicate that AOA blocks several metabolic pathways essential for growth of malignant cells. Therefore, OXPHOS may provide important therapeutic targets for treatment of sarcoma.

Benzophenone-3 alters expression of genes encoding vascularization and epithelial-mesenchymal transition functions during Trp53-null mammary tumorigenesis.

Benzophenone-3 (also referred to as oxybenzone) is a putative endocrine disrupting chemical and common ingredient in sunscreens and other personal care products. We previously showed that benzophenone-3 was promotional for epithelial tumorigenesis in mice fed adult high-fat diet, while protective against the incidence of more aggressive spindle cell tumors in the same treatment group. In this study, we show that benzophenone-3 reduces epithelial to mesenchymal transition in the epithelial tumors of these mice. This reduction in epithelial to mesenchymal transition is associated with altered expression of several genes involved in regulation of angiogenesis and epithelial to mesenchymal transition. Among the genes altered in expression, Timp1 is of particular interest because benzophenone-3 suppressed both migration and Timp1 expression in a mammary tumor cell line that displays epithelial to mesenchymal transition characteristics. These alterations in gene expression plausibly stabilize the vasculature of epithelial carcinomas and contribute to benzophenone-3 promotion of epithelial tumors, while at the same time suppress epithelial to mesenchymal transition and suppress incidence of spindle cell tumors.

Benzophenone-3 promotion of mammary tumorigenesis is diet-dependent.

Benzophenone-3 is a putative endocrine disrupting chemical and common ingredient in sunscreens. The potential of endocrine disrupting chemicals to act as agonists or antagonists in critical hormonally regulated processes, such as mammary gland development and mammary tumorigenesis, demands evaluation of its potential in promoting breast cancer. This study identifies the effects of BP-3 on mammary tumorigenesis with high-fat diet during puberty versus adulthood in Trp53-null transplant BALB/c mice. Benzophenone-3 exposure yielded levels in urine similar to humans subjected to heavy topical sunscreen exposure. Benzophenone-3 was protective for epithelial tumorigenesis in mice fed lifelong low-fat diet, while promotional for epithelial tumorigenesis in mice fed adult high-fat diet. Benzophenone-3 increased tumor cell proliferation, decreased tumor cell apoptosis, and increased tumor vascularity dependent on specific dietary regimen and tumor histopathology. Even in instances of an ostensibly protective effect, other parameters suggest greater risk. Although benzophenone-3 seemed protective on low-fat diet, spindle cell tumors arising in these mice showed increased proliferation and decreased apoptosis. This points to a need for further studies of benzophenone-3 in both animal models and humans as a potential breast cancer risk factor, as well as a more general need to evaluate endocrine disrupting chemicals in varying dietary contexts.

Progesterone receptor isoform functions in normal breast development and breast cancer.

Progesterone acting through two isoforms of the progesterone receptor (PR), PRA and PRB, regulates proliferation and differentiation in the normal mammary gland in mouse, rat, and human. Progesterone and PR have also been implicated in the etiology and pathogenesis of human breast cancer. The focus of this review is recent advances in understanding the role of the PR isoform-specific functions in the normal breast and in breast cancer. Also discussed is information obtained from rodent studies and their relevance to our understanding of the role of progestins in breast cancer etiology.

The Proliferative Response to p27 Down-Regulation in Estrogen Plus Progestin Hormonal Therapy is Lost in Breast Tumors.

Increased proliferation and breast cancer risk has been observed in postmenopausal women receiving estrogen (E) + progestin hormone replacement therapy (HRT). Progestin action is mediated through two progesterone receptor (PR) isoforms, PRA and PRB, with unique transcriptional activity and function. The current study examines hormonal regulation of PR isoforms in the normal postmenopausal human breast and the mechanism by which progestins increase proliferation and breast cancer risk. Archival benign breast biopsies from postmenopausal and premenopausal women, and luminal breast tumor biopsies from postmenopausal women, were analyzed for regulation of PRA and PRB expression by E and E+medroxyprogesterone acetate (MPA). In the postmenopausal breast without HRT, PRA and PRB expression was decreased compared to the premenopausal breast. Both E (n = 12) and E+MPA (n = 13) HRT in the postmenopausal breast were associated with increased PRA and PRB expression, increased nuclear cyclin E expression, and decreased nuclear p27 expression compared to no HRT (n = 16). With E+MPA HRT, there was a further decrease in nuclear p27 and increased Receptor Activator of NF-kappa B Ligand (RANKL) expression compared to E-alone HRT. In luminal breast cancers, E+MPA HRT (n = 6) was also associated with decreased nuclear expression of the cell cycle inhibitor p27 compared to E HRT (n = 6), but was not associated with increased proliferation. These results suggest that p27 mediates progestin-induced proliferation in the normal human breast and that regulation of this proliferative response by E+MPA is lost in breast tumors.

Progesterone receptor isoforms and proliferation in the rat mammary gland during development.

Progesterone (P), acting through progesterone receptor (PR) isoforms A and B, plays an important role in normal mammary gland development and is implicated in the etiology of breast cancer. Because of significant similarities between human and rat mammary gland development and hormonal responsiveness of mammary cancers, we investigated P action in the rat mammary gland. By immunohistochemical methods we determined PRA and PRB expression at puberty, sexual maturity, pregnancy, and lactation and after postlactational involution and their functional roles in the regulation of proliferation. PRA expression was restricted to luminal epithelial cells, whereas PRB was expressed in both luminal and myoepithelial cells, indicating a novel role of PRB in myoepithelial cell regulation. The majority of PRA-positive (PRA+) cells coexpressed PRB. In the pubertal and adult virgin mammary gland, PRA+PRB+ cells also expressed nuclear cyclin D1 but did not contain the proliferation marker bromodeoxyuridine. Based on a lack of phosphorylated retinoblastoma protein expression and the expression patterns of the cyclin-dependent kinase inhibitors p21 and p27 in these cells, we conclude that PRA+PRB+ cells appear to be cell cycle arrested and do not proliferate. PRA+ cells were decreased in the adult gland and during and after pregnancy. The percentage of PRB+ cells was relatively constant throughout development, and in a significant proportion of cells, only PRB was detected. During development, and especially during pregnancy, a high percentage of PRB+ cells were positive for bromodeoxyuridine. From this observation, we conclude that these cells proliferate and that P acting through PRB may directly stimulate proliferation.

Developmental exposure to corticosterone: behavioral changes and differential effects on leukemia inhibitory factor (LIF) and corticotropin-releasing hormone (CRH) gene expression in the mouse.

RATIONALE: Cytokines are found in both the peripheral and central nervous system. There has been increasing interest in their potential role in some of the behavioral features of depressive disorders. Leukemia inhibitory factor (LIF), a proinflammatory cytokine, produces stimulation of adrenocorticotropic hormone (ACTH) secretion in response to emotional and inflammatory stress and recently has been linked to depressive-type behavior. Both the hypothalamic-pituitary-adrenal axis and the immune system, including cytokine-mediated responses, appear to be susceptible to long-term programming during fetal and neonatal development. OBJECTIVE: The present study was designed to characterize the effects of perinatal exposure to corticostereone on behavior, hypothalamic LIF and corticotropin-releasing hormone (CRH) mRNA expression, and basal plasma corticosterone levels in adult female mice. METHODS: Corticosterone was added to the drinking water beginning the last week of gestation and continued until weaning. Behavior in the open field and forced swim tests, baseline plasma corticosterone levels, and hypothalamic LIF and CRH gene expression were evaluated in the adult offspring. RESULTS: Mice exposed to perinatal corticosterone showed increased immobility in the forced swim test and increased locomotor activity in the open field test. Although there were no differences between treatment groups in terms of basal plasma levels of corticosterone or hypothalamic CRH mRNA, LIF mRNA expression was increased in the hypothalamus. CONCLUSIONS: These results show that perinatal exposure to glucocorticoids can produce long-term behavioral changes and upregulation of central LIF mRNA expression.

Progesterone receptor isoforms A and B: temporal and spatial differences in expression during murine mammary gland development.

Progesterone is a potent mitogen in the mammary gland. Based on studies using cells and animals engineered to express progesterone receptor (PR) isoforms A or B, PRA and PRB are believed to have different functions. Using an immunohistochemical approach with antibodies specific for PRA only or PRB only, we show that PRA and PRB expression in mammary epithelial cells is temporally and spatially separated during normal mammary gland development in the BALB/c mouse. In the virgin mammary gland when ductal development is active, the only PR protein isoform expressed was PRA. PRA levels were significantly lower during pregnancy, suggesting a minor role at this stage of development. PRB was abundantly expressed only during pregnancy, during alveologenesis. PRA and PRB colocalization occurred in only a small percentage of cells. During pregnancy there was extensive colocalization of PRB with 5-bromo-2'-deoxyuridine (BrdU) and cyclin D1; 95% of BrdU-positive cells and 83% of cyclin D1-positive cells expressed PRB. No colocalization of PRA with either BrdU or cyclin D1 was observed at pregnancy. In the virgin gland, PRA colocalization with BrdU or cyclin D1 was low; only 27% of BrdU-positive cells and 4% of cyclin D1-positive cells expressed PRA. The implication of these findings is that different actions of progesterone are mediated in PRB positive vs. PRA-positive cells in vivo. The spatial and temporal separation of PR isoform expression in mouse mammary gland provides a unique opportunity to determine the specific functions of PRA vs. PRB in vivo.

Leukemia inhibitory factor signaling is implicated in embrionic development of the HPA axis.

Leukemia inhibitory factor (LIF) is expressed in the hypothalamic-pituitary adrenal (HPA) axis and stimulates pituitary POMC transcription. The role of LIF receptor signaling in HPA axis development was examined. Lifr -/- and Lifr +/+ fetuses were obtained by Cesarean section on E18.5. Despite a 3-fold induction of hypothalamic CRH mRNA, pituitary POMC mRNA and RU486-induced ACTH levels were decreased in Lifr -/- mice. High CRH may be caused by increased central TNFalpha and IL-6 expression. Lifr -/- mice demonstrate elevated pituitary glucocorticoid (GR) and mineralocorticoid (MR) receptor mRNA and protein levels, indicating the importance of LIF signaling for HPA axis development.

Progestins and breast cancer.

Progesterone (P) regulates proliferation and differentiation in the normal mammary gland in mouse, rat and human. Progesterone has also been implicated in the etiology and pathogenesis of human breast cancer. The focus of this review is on recent advances in understanding the role of the progesterone receptor (PR) and functional significance of PR isoforms, PRA and PRB, in the normal mammary gland and in mammary cancer in mouse, rat and human.

Hypothalamic-pituitary cytokine network.

Cytokines expressed in the brain and involved in regulating the hypothalamus-pituitary-adrenal (HPA) axis contribute to the neuroendocrine interface. Leukemia inhibitory factor (LIF) and LIF receptors are expressed in human pituitary cells and murine hypothalamus and pituitary. LIF potently induces pituitary proopiomelanocortin (POMC) gene transcription and ACTH secretion and potentiates CRH induction of POMC. In vivo, LIF, along with CRH, enhances POMC expression and ACTH secretion in response to emotional and inflammatory stress. To further elucidate specific roles for both CRH and LIF in activating the inflammatory HPA response, double-knockout mice (CRH/LIFKO) were generated by breeding the null mutants for each respective single gene. Inflammation produced by ip injection of lipopolysaccharide (1 microg/mouse) to double CRH and LIF-deficient mice elicited pituitary POMC induction similar to wild type and markedly higher than in single null animals (P<0.0.01). Double-knockout mice also demonstrated robust corticosterone response to inflammation. High pituitary POMC mRNA levels may reflect abundant TNFalpha, IL-1beta, and IL-6 activation observed in the hypothalamus and pituitary of these animals. Our results suggest that increased central proinflammatory cytokine expression can compensate for the impaired HPA axis function and activates inflammatory ACTH and corticosterone responses in mice-deficient in both CRH and LIF.

Reduced immobility in the forced swim test in mice with a targeted deletion of the leukemia inhibitory factor (LIF) gene.

Cytokines are a large and diverse group of polypeptides that are rapidly released in response to tissue injury, infection, and inflammation. Besides their effects in the periphery, cytokines also affect the central nervous system (CNS). There has been increasing interest in the potential role of cytokines in the behavioral features of depressive disorders. One cytokine that might be a candidate for a role in the etiology of depression is leukemia inhibitory factor (LIF). LIF mRNA has been detected in the hypothalamus, hippocampus, amygdala, cerebellum, cerebral cortex, and basal forebrain nuclei. The role of LIF in the CNS has not been fully elucidated. Based upon the hypothesis that cytokines might have a role in depression, the present study characterized the behavior of mice with a targeted disruption of the LIF gene (LIF knockouts) in the forced swim test, an animal model used to measure depressive-like behavior and the response to antidepressants. It was found that LIF knockout mice show reduced immobility in the forced swim test, suggesting that LIF might have a potential role in the etiology of some forms of depression.

Progesterone stimulates proliferation and promotes cytoplasmic localization of the cell cycle inhibitor p27 in steroid receptor positive breast cancers.

Progestins are reported to increase the risk of more aggressive estrogen receptor positive, progesterone receptor positive (ER+ PR+) breast cancers in postmenopausal women. Using an in vivo rat model of ER+ PR + mammary cancer, we show that tumors arising in the presence of estrogen and progesterone exhibit increased proliferation and decreased nuclear expression of the cell cycle inhibitor p27 compared with tumors growing in the presence of estrogen alone. In human T47D breast cancer cells, progestin increased proliferation and decreased nuclear p27 expression. The decrease of nuclear p27 protein was dependent on activation of Src and PI3K by progesterone receptor isoforms PRA or PRB. Importantly, increased proliferation and decreased nuclear p27 expression were observed in invasive breast carcinoma compared with carcinoma in situ. These results suggest that progesterone specifically regulates intracellular localization of p27 protein and proliferation. Therefore, progesterone-activated pathways can provide useful therapeutic targets for treatment of more aggressive ER+ PR+ breast cancers.

Progesterone decreases levels of the adhesion protein E-cadherin and promotes invasiveness of steroid receptor positive breast cancers.

Progestins are reported to increase the risk of invasive breast cancers in postmenopausal women receiving hormone therapy with estrogen plus progestin. We report here that estrogen and progesterone receptor positive (ER+PR+) rat mammary tumors arising in the presence of estrogen and progesterone exhibit increased invasiveness and decreased expression of E-cadherin protein compared with tumors growing in the presence of estrogen alone. A similar decrease of E-cadherin expression was observed in human ER+PR+ invasive ductal carcinoma compared with ductal carcinoma in situ. In agreement with findings in the rat, estrogen plus progestin R5020 treatment decreased E-cadherin expression in vitro in T47D human breast cancer cells. Decrease of E-cadherin protein was mediated by progesterone receptor B (PRB) and dependent on the activation of the Wnt pathway. These results suggest that progesterone signaling via PRB contributes to tumor invasiveness and can provide an important therapeutic target for treatment of invasive ER+PR+ breast cancers.

Regulation of estrogen receptor α N-terminus conformation and function by peptidyl prolyl isomerase Pin1.

Estrogen receptor alpha (ERα), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ERα AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifen-inducible activity of ERα and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ERα-positive rat mammary tumors. These results establish phosphorylation-coupled proline isomerization as a mechanism modulating AF1 functional activity and provide insight into the role of a conformational switch in the functional regulation of the intrinsically disordered transactivation domain of ERα.

Amphiregulin mediates estrogen, progesterone, and EGFR signaling in the normal rat mammary gland and in hormone-dependent rat mammary cancers.

Both estrogen (E) and progesterone (P) are implicated in the etiology of human breast cancer. Defining their mechanisms of action, particularly in vivo, is relevant to the prevention and therapy of breast cancer. We investigated the molecular and cellular mechanisms of E and/or P-induced in vivo proliferation, in the normal rat mammary gland and in hormone-dependent rat mammary cancers which share many characteristics with the normal human breast and hormone-dependent breast cancers. We show that E+P treatment induced significantly greater proliferation in both the normal gland and mammary cancers compared to E alone. In both the normal gland and tumors, E+P-induced proliferation was mediated through the increased production of amphiregulin (Areg), an epidermal growth factor receptor (EGFR) ligand, and the activation of intracellular signaling pathways (Erk, Akt, JNK) downstream of EGFR that regulate proliferation. In vitro experiments using rat primary mammary organoids or T47D breast cancer cells confirmed that Areg and the synthetic progestin, R5020, synergize to promote cell proliferation through EGFR signaling. Iressa, an EGFR inhibitor, effectively blocked this proliferation. These results indicate that mediators of cross talk between E, P, and EGFR pathways may be considered as relevant molecular targets for the therapy of hormone-dependent breast cancers, especially in premenopausal women.

Leukemia inhibitory factor regulates glucocorticoid receptor expression in the hypothalamic-pituitary-adrenal axis.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine belonging to the gp130 family. LIF is induced peripherally and within the brain during inflammatory or chronic autoimmune diseases and is a potent stimulator of the hypothalamic-pituitary-adrenal (HPA) axis. Here we investigated the role of LIF in mediating glucocorticoid receptor (GR) expression in the HPA axis. LIF treatment (3 microg/mouse, i.p.) markedly decreased GR mRNA levels in murine hypothalamus (5-fold, P < 0.01) and pituitary (1.7-fold, P < 0.01) and downregulated GR protein levels. LIF decreased GR expression in murine corticotroph cell line AtT20 within 2 h, and this effect was sustained for 8 h after treatment. LIF-induced GR mRNA reduction was abrogated in AtT20 cells overexpressing dominant-negative mutants of STAT3, indicating that intact JAK-STAT signaling is required to mediate LIF effects on GR expression. Conversely, mice with LIF deficiency exhibited increased GR mRNA levels in the hypothalamus and pituitary (3.5- and 3.5-fold, respectively; P < 0.01 for both) and increased GR protein expression when compared with wild-type littermates. The suppressive effects of dexamethasone on GR were more pronounced in LIF-null animals. These data suggest that LIF maintains the HPA axis activation by decreasing GR expression and raise the possibility that LIF might contribute to the development of central glucocorticoid resistance during inflammation.

Opposing effects of pituitary leukemia inhibitory factor and SOCS-3 on the ACTH axis response to inflammation.

We have shown that leukemia inhibitory factor (LIF) and suppressor of cytokine signaling (SOCS)-3 are expressed in the hypothalamus and pituitary and that LIF induces proopiomelanocortin (POMC) and ACTH, whereas SOCS-3 abrogates corticotroph POMC gene transcription and ACTH secretion. Here, we determined the role of pituitary LIF and SOCS-3 in regulating hypothalamo-pituitary-adrenal (HPA) axis inflammatory responses. Murine pituitary LIF expression was induced up to eightfold after intraperitoneal injection of lipopolysaccharide or tumor necrosis factor-alpha, concordant with elevated plasma levels of ACTH and corticosterone. In LIF knockout (LIFKO) mice, induction of both ACTH and corticosterone were attenuated. LIF deletion was associated with elevated (P < 0.05) levels of pituitary TNF-alpha, interleukin (IL)-1beta, and IL-6 mRNA and cytokine-inducible pituitary SOCS-3 expression. Abrogation of the HPA axis stress response and higher pituitary levels of proinflammatory cytokines observed in LIFKO mice resulted in a stronger inflammatory process, as evidenced by elevated erythrocyte sedimentation rate and increased serum amyloid A levels (P < 0.05). The results indicate that, although LIF induces ACTH, SOCS-3 acts to counterregulate the HPA axis response to inflammation.