Effects of high-fat diet and intestinal aryl hydrocarbon receptor deletion on colon carcinogenesis.

Consumption of a high-fat diet has been associated with an increased risk of developing colorectal cancer (CRC). However, the effects of the interaction between dietary fat content and the aryl hydrocarbon receptor (AhR) on colorectal carcinogenesis remain unclear. Mainly known for its role in xenobiotic metabolism, AhR has been identified as an important regulator for maintaining intestinal epithelial homeostasis. Although previous research using whole body AhR knockout mice has revealed an increased incidence of colon and cecal tumors, the unique role of AhR activity in intestinal epithelial cells (IECs) and modifying effects of fat content in the diet at different stages of sporadic CRC development are yet to be elucidated. In the present study, we have examined the effects of a high-fat diet on IEC-specific AhR knockout mice in a model of sporadic CRC. Although loss of AhR activity in IECs significantly induced the development of premalignant lesions, in a separate experiment, no significant changes in colon mass incidence were observed. Moreover, consumption of a high-fat diet promoted cell proliferation in crypts at the premalignant colon cancer lesion stage and colon mass multiplicity as well as ß-catenin expression and nuclear localization in actively proliferating cells in colon masses. Our data demonstrate the modifying effects of high-fat diet and AhR deletion in IECs on tumor initiation and progression.NEW & NOTEWORTHY Through the use of an intestinal-specific aryl hydrocarbon receptor (AhR) knockout mouse model, this study demonstrates that the expression of AhR in intestinal epithelial cells is required to reduce the formation of premalignant colon cancer lesions. Furthermore, consumption of a high-fat diet and the loss of AhR in intestinal epithelial cells influences the development of colorectal cancer at various stages.

Estradiol Has Differential Effects on Acute Colonic Inflammation in the Presence and Absence of Estrogen Receptor ß Expression.

BACKGROUND: Inflammatory bowel disease (IBD) increases the risk of developing colon cancer. This risk is higher in men compared to women, implicating a role for female hormones in the protection against this disease. Studies from our laboratory demonstrated that estradiol (E2) protects against inflammation-associated colon tumor formation when administered following chemical carcinogen and induction of chronic colitis. AIM: This study seeks to better understand the effect of E2 on acute colitis in the presence and absence of estrogen receptor ß (ERß). METHODS: Inflammation was induced by 2,4,6-trinitrobenzenesulfonic acid in wild-type (WT) and ERß knockout (ERßKO) mice implanted with a control or E2-containing pellet and killed 5 days later. Inflammation and injury were scored by a pathologist. Apoptosis and proliferation were assessed by immunohistochemistry. Cytokines were measured by multiplex analysis. RESULTS: E2 treatment reduced inflammation in the middle colon in WT mice and the distal colon in ERßKO mice compared to control mice. WT mice had reduced IL-6, IL-12, IL-17, GM-CSF, IFN-γ, MCP-1, MIP-1α, and TNF-α, and ERßKO had reduced IL-6 and IFN-γ expression in response to E2. Injury scores were lower in E2-treated ERßKO mice compared to control ERßKO mice. ERßKO mice had increased proliferation in the basal third of crypts in the distal colon and decreased apoptosis in the proximal colon. CONCLUSIONS: These data suggest that E2 has differential protective effects against acute colitis in the presence or absence of ERß and provide insight into how E2 may protect against IBD.

Sorghum phenolics demonstrate estrogenic action and induce apoptosis in nonmalignant colonocytes.

Evidence indicates sorghum may be protective against colon cancer; however, the mechanisms are unknown. Estrogen is believed to protect against colon cancer development by inducing apoptosis in damaged nonmalignant colonocytes. Three sorghum extracts (white, red, and black) were screened for estrogenic activity using cell models expressing estrogen receptor α (ER-α; MCF-7 breast cancer cells) and ß [ER-ß; nonmalignant young adult mouse colonocytes (YAMC)]. Black and white sorghum extracts had significant estrogenic activity mediated through both estrogen receptors at 1-5 and 5-10 µg/mL, respectively; but red sorghum did not. Activation of ER-ß in YAMC reduced cell growth via induction of apoptosis. Only the black and red sorghums contained 3-deoxyanthocyanins; however, these compounds were non-estrogenic. Flavones with estrogenic properties, luteolin (0.41-2.12 mg/g) and apigenin (1.1-1.4 mg/g), and their O-methyl derivatives (0.70-0.95 mg/g) were detected in white and black sorghums, but not in the red sorghum. On the other hand, naringenin, a flavanone known to interfere with transcriptional activities of estrogen, was only detected in the red sorghum extract (as its 7-O-glycoside) at relatively high concentration (11.8 mg/g). Sorghum flavonoid composition has important implications on possible modes of chemoprotection by sorghum against colon carcinogenesis.

Estrogen Protects Cardiac Function and Energy Metabolism in Dilated Cardiomyopathy Induced by Loss of Cardiac IRS1 and IRS2.

BACKGROUND: Type 2 diabetes (T2D) is a high-risk factor for incident of cardiovascular diseases. Women at young ages show a reduced incidence of both T2D and cardiovascular diseases compared with men, but these disparities disappear in postmenopausal women versus age-matched men. Thus, ovaries and ovarian hormones, such as estrogen, are expected to protect from T2D and cardiovascular diseases. In this study, we aimed to investigate the role of ovaries and ovarian hormone estrogen in cardiac function and energy metabolism using the cardiac IRS (insulin receptor substrate) 1 and IRS2 double genes knockout mice that mimic cardiac insulin resistance. METHODS: Control and heart-specific IRS1/2 double genes knockout mice were treated with placebo or 17ß-estradiol (E2) pellets, respectively, through subcutaneous implantation. Female mice were subjected to a bilateral ovariectomy surgery to remove endogenous E2. The cardiac function and energy metabolism were determined using echocardiography and indirect calorimeter, respectively. RESULTS: All male heart-specific IRS1/2 double genes knockout mice died of heart failure at 6 to 8 weeks as we previously described (Qi et al), but all female heart-specific IRS1/2 double genes knockout mice survived >1 year. Removal of ovaries in heart-specific IRS1/2 double genes knockout female mice resulted in cardiac dysfunction, and ultimately animal death. However, E2 supplementation prevented the dilated cardiomyopathy, improved cardiac function and energy metabolism, and enhanced lifespan in both male and ovariectomy female mice deficient for cardiac IRS1 and IRS2 genes, largely owing to the activation of Akt (protein kinase B)-Foxo1 (O1 class of forkhead/winged helix transcription factor) signaling cascades. CONCLUSIONS: These results show that estrogen protects mice from cardiac insulin resistance-induced diabetic cardiomyopathy. This may provide a fundamental mechanism for the gender difference for the incidence of both T2D and cardiovascular diseases. This study highlights that estrogen signaling could be a potential target for improving cardiac function and energy metabolism in humans with T2D.

Dietary fish oil reduces DNA adduct formation while estradiol upregulates apoptosis in response to DNA damage in the rat colon.

BACKGROUND: Dietary fish oil is associated with a decrease in colon cancer incidence: in part through a reduction in DNA adduct formation and an induction of colonocyte apoptosis. Estradiol (E(2)) has also been demonstrated to be protective against colon cancer incidence. Studies evaluating fish oil diets and DNA adduct formation in the colon have been conducted in male models without regard to possible interactions with E(2). AIMS: The aim of this study was to evaluate the effects of E(2) and fish oil both together and separately in female rats at the point of DNA damage. METHODS: Ovariectomized female Sprague-Dawley rats were fed either a corn oil or fish oil diet in the presence or absence of E(2) for two weeks prior to being sacrificed at four time points following injection with azoxymethane. O(6)-methyldeoxyguanosine (O(6)-MedG) DNA adducts and apoptosis were examined using immunohistochemistry. RESULTS: Dietary fish oil reduced DNA adduct formation independent of the presence of E(2) at both 9 and 12 h post carcinogen. E(2) itself did not suppress adduct formation. E(2) significantly induced apoptosis 12 h after carcinogen independent of diet, primarily in the luminal third of the crypts. Fish oil was not associated with increased colonocyte apoptosis. CONCLUSIONS: These data demonstrate that fish oil is protective against DNA damage in the colon regardless of gender through reduction of O(6)-MedG adduct formation. Additionally, E(2) is capable of inducing apoptosis directly at the point of DNA damage.

Trigonelline is a novel phytoestrogen in coffee beans.

Drinking coffee has been associated with the development of several endocrine-related cancers. The interpretation of these data has often been limited to the role that caffeine plays. Trigonelline (Trig), a niacin-related compound, is a natural constituent of coffee accounting for approximately 1% dry matter in roasted beans. Studies exploring the effects of this bioactive compound on mammalian cells are limited. The initial purpose of our studies was to determine whether Trig alters the actions of estradiol (E(2)), using proliferation of estrogen-dependent human breast cancer (MCF-7) cells as a model system. When cells were cotreated with suboptimal doses of E(2) (10 pmol/L) and Trig (100 pmol/L), an additive enhancement of MCF-7 growth was observed. In the absence of E(2), Trig stimulated MCF-7 cell proliferation in a dose-responsive manner and significantly enhanced cell growth at concentrations as low as 100 pmol/L. Cotreatment of MCF-7 cells with Trig and ICI 182,780, an estrogen receptor (ER) antagonist, inhibited Trig-induced cell proliferation. Trig treatment also induced activation of estrogen response element reporter assays in MCF-7 cells and increased expression of ER target genes (pS2, progesterone receptor, and cyclin D1) similar to E(2). While our data demonstrate that Trig activates the ER, competitive binding assays showed that Trig does not compete E(2) off of the ER at any concentration. This suggests that Trig is activating the ER through a separate mechanism. Collectively, these data demonstrate that Trig even at low concentrations stimulates MCF-7 cell growth and that this effect is mediated through ER, clearly identifying Trig as a novel phytoestrogen.

Estrogen Improves Insulin Sensitivity and Suppresses Gluconeogenesis via the Transcription Factor Foxo1.

Premenopausal women exhibit enhanced insulin sensitivity and reduced incidence of type 2 diabetes (T2D) compared with age-matched men, but this advantage disappears after menopause with disrupted glucose homeostasis, in part owing to a reduction in circulating 17ß-estradiol (E2). Fasting hyperglycemia is a hallmark of T2D derived largely from dysregulation of hepatic glucose production (HGP), in which Foxo1 plays a central role in the regulation of gluconeogenesis. Here, we investigated the action of E2 on glucose homeostasis in male and ovariectomized (OVX) female control and liver-specific Foxo1 knockout (L-F1KO) mice and sought to understand the mechanism by which E2 regulates gluconeogenesis via an interaction with hepatic Foxo1. In both male and OVX female control mice, subcutaneous E2 implant improved insulin sensitivity and suppressed gluconeogenesis; however, these effects of E2 were abolished in L-F1KO mice of both sexes. In our use of mouse primary hepatocytes, E2 suppressed HGP and gluconeogenesis in hepatocytes from control mice but failed in hepatocytes from L-F1KO mice, suggesting that Foxo1 is required for E2 action on the suppression of gluconeogenesis. We further demonstrated that E2 suppresses hepatic gluconeogenesis through activation of estrogen receptor (ER)α-phosphoinositide 3-kinase-Akt-Foxo1 signaling, which can be independent of insulin receptor substrates 1 and 2 (Irs1 and Irs2), revealing an important mechanism for E2 in the regulation of glucose homeostasis. These results may help explain why premenopausal women have lower incidence of T2D than age-matched men and suggest that targeting ERα can be a potential approach to modulate glucose metabolism and prevent diabetes.

Estrogen prevents cholesteryl ester accumulation in macrophages induced by the HIV protease inhibitor ritonavir.

Individuals with HIV can now live long lives with drug therapy that often includes protease inhibitors such as ritonavir. Many patients, however, develop negative long-term side effects such as premature atherosclerosis. We have previously demonstrated that ritonavir treatment increases atherosclerotic lesion formation in male mice to a greater extent than in female mice. Furthermore, peripheral blood monocytes isolated from ritonavir-treated females had less cholesteryl ester accumulation. In the present study, we have investigated the molecular mechanisms by which female hormones influence cholesterol metabolism in macrophages in response to the HIV protease inhibitor ritonavir. We have utilized the human monocyte cell line, THP-1 as a model to address this question. Briefly, cells were differentiated for 72 h with 100 nM PMA to obtain a macrophage-like phenotype in the presence or absence of 1 nM 17beta-estradiol (E2), 100 nM progesterone or vehicle (0.01% ethanol). Cells were then treated with 30 ng/ml ritonavir or vehicle in the presence of aggregated LDL for 24 h. Cell extracts were harvested, and lipid or total RNA was isolated. E2 decreased the accumulation of cholesteryl esters in macrophages following ritonavir treatment. Ritonavir increased the expression of the scavenger receptor, CD36 mRNA, responsible for the uptake of LDL. Additionally, ritonavir treatment selectively increased the relative levels of PPARgamma mRNA, a transcription factor responsible for the regulation of CD36 mRNA expression. Treatment with E2, however, failed to prevent these increases at the mRNA level. E2 did, however, significantly suppress CD36 protein levels as measured by fluorescent immunocytochemistry. This data suggests that E2 modifies the expression of CD36 at the level of protein expression in monocyte-derived macrophages resulting in reduced cholesteryl ester accumulation following ritonavir treatment.

Bisphenol-A alters microbiota metabolites derived from aromatic amino acids and worsens disease activity during colitis.

Inflammatory bowel disease is a complex collection of disorders. Microbial dysbiosis as well as exposure to toxins including xenoestrogens are thought to be risk factors for inflammatory bowel disease development and relapse. Bisphenol-A has been shown to exert estrogenic activity in the colon and alter intestinal function, but the role that xenoestrogens, such as bisphenol-A , play in colonic inflammation has been previously described but with conflicting results. We investigated the ability of bisphenol-A to exacerbate colonic inflammation and alter microbiota metabolites derived from aromatic amino acids in an acute dextran sulfate sodium-induced colitis model. Female C57BL/6 mice were ovariectomized and exposed to bisphenol-A daily for 15 days. Disease activity measures include body weight, fecal consistency, and rectal bleeding. Colons were scored for inflammation, injury, and nodularity. Alterations in the levels of microbiota metabolites derived from aromatic amino acids known to reflect phenotypic changes in the gut microbiome were analyzed. Bisphenol-A exposure increased mortality and worsened disease activity as well as inflammation and nodularity scores in the middle colon region following dextran sulfate sodium exposure. Unique patterns of metabolites were associated with bisphenol-A consumption. Regardless of dextran sulfate sodium treatment, bisphenol-A reduced levels of tryptophan and several metabolites associated with decreased inflammation in the colon. This is the first study to show that bisphenol-A treatment alone can reduce microbiota metabolites derived from aromatic amino acids in the colon which may be associated with increased colonic inflammation and inflammatory bowel disease. Impact statement As rates of inflammatory bowel disease rise, discovery of the mechanisms related to the development of these conditions is important. Environmental exposure is hypothesized to play a role in etiology of the disease, as are alterations in the gut microbiome and the metabolites they produce. This study is the first to show that bisphenol-A alone alters tryptophan and microbiota metabolites derived from aromatic amino acids in a manner consistent with autoimmune diseases, specifically inflammatory bowel diseases, regardless of dextran sulfate sodium treatment. These findings indicate a potential mechanism by which bisphenol-A negatively affects gut physiology to exacerbate inflammation.

Gender-specific effects of HIV protease inhibitors on body mass in mice.

Protease inhibitors, as part of highly active anti-retroviral therapy (HAART), have significantly increased the lifespan of human immunodeficiency virus (HIV) infected patients. Several deleterious side effects including dyslipidemia and lipodystrophy, however, have been observed with HAART. Women are at a higher risk of developing adipose tissue alterations and these alterations have different characteristics as compared to men. We have previously demonstrated that in mice the HIV protease inhibitor, ritonavir, caused a reduction in weight gain in females, but had no effect on male mice. In the present study, we examined the potential causes of this difference in weight gain. Low-density lipoprotein receptor (LDL-R) null mice or wild-type C57BL/6 mice, were administered 15 mug/ml ritonavir or vehicle (0.01% ethanol) in the drinking water for 6 weeks. The percent of total body weight gained during the treatment period was measured and confirmed that female LDL-R gained significantly less weight with ritonavir treatment than males. In wild type mice, however, there was no effect of ritonavir treatment in either sex. Despite the weight loss in LDL-R null mice, ritonavir increased food intake, but no difference was observed in gonadal fat weight. Serum leptin levels were significantly lower in females. Ritonavir further suppressed leptin levels in (p < 0.05). Ritonavir did not alter serum adiponectin levels in either gender. To determine the source of these differences, female mice were ovariectomized remove the gonadal sex hormones. Ovariectomy prevented the weight loss induced by ritonavir (p < 0.05). Furthermore, leptin levels were no longer suppressed by ritonavir (p < 0.05). This study demonstrates that gonadal factors in females influence the hormonal control of weight gain changes induced by HIV protease inhibitors in an environment of elevated cholesterol.

Estradiol negatively regulates HIV-LTR promoter activity in glial cells.

HIV-associated dementia results from neuronal loss and an alteration of neuronal function due to a loss of synapses. While HIV infection in astrocytes is limited, astrocytes exhibit a chronic nonproductive infection that can lead to the release of neurotoxic proteins. Additionally, infection can disrupt the normal neurotrophic role of astrocytes that results in neuronal death. Gonadal steroid hormones are known to act as trophic and protective factors in the brain under a variety of normal and pathological conditions. In the present study, to determine if estrogen plays a role in the ability of Tat to function as a transcriptional activator within astrocytes, we examined the effect of estrogen on regulation of viral transcription. We utilized an immortalized human astrocyte cell line (SVGA) stably transfected with a reporter plasmid containing the HIV-1IIIB LTR driving the chloramphenicol acetyltransferase (CAT) gene. The amount of transcriptional activity was measured by quantifying the amount of CAT produced. We determined that 17beta-estradiol treatment (1 nM) had no effect on basal LTR activity. Following transfection with a Tat-expressing plasmid, there was a 100-fold increase in CAT production. This induction was reduced by 40% in cells pretreated with 17beta-estradiol. 17beta- Estradiol only suppressed transcription stimulated by Tat. Furthermore, we determined that this effect was specific to 17beta-estradiol and estrogen receptor agonists. This activity was limited to astrocytes as no effect was observed in a monocytic cell line. Finally, the mechanism of action did not involve an alteration in levels of Cdk9 or Cyclin T1 proteins necessary for Tat activation of the HIV-1 LTR. This study demonstrates a novel activity of 17beta-estradiol in glial cells that could play a role in the maintenance of neuronal health during HIV infection of the central nervous system.

Dietary genistein negates the inhibitory effect of tamoxifen on growth of estrogen-dependent human breast cancer (MCF-7) cells implanted in athymic mice.

The use of dietary isoflavone supplements by postmenopausal women with breast cancer is increasing. We investigated interactions between the soy isoflavone, genistein, and an antiestrogen, tamoxifen (TAM), on the growth of estrogen (E)-dependent breast cancer (MCF-7) cells implanted in ovariectomized athymic mice. We hypothesized that weakly estrogenic genistein negate/overwhelm the inhibitory effect of TAM on the growth of E-dependent breast tumors. Six treatment groups were used: control (C); 0.25 mg estradiol (E2) implant (E); E2 implant + 2.5 mg TAM implant (2.5 TE); E2 implant + 2.5 mg TAM implant + 1000 ppm genistein (2.5 TEG); E2 implant + 5 mg TAM implant (5 TE), and E2 implant +5 mg TAM implant +1000 ppm genistein (5 TEG). Treatment with TAM (2.5 TE and 5 TE) suppressed E2-stimulated MCF-7 tumor growth in ovariectomized athymic mice. Dietary genistein negated/overwhelmed the inhibitory effect of TAM on MCF-7 tumor growth, lowered E2 level in plasma, and increased expression of E-responsive genes (e.g., pS2, PR, and cyclin D1). Therefore, caution is warranted for postmenopausal women consuming dietary genistein while on TAM therapy for E-responsive breast cancer.

Soy processing affects metabolism and disposition of dietary isoflavones in ovariectomized BALB/c mice.

Soy foods and nutritional supplements are widely consumed for potential health benefits. It was previously shown that isoflavone-supplemented diets, which contained equal genistein equivalents, differentially stimulated mammary tumor growth in athymic mice based on the degree of processing. This paper reports plasma pharmacokinetic analysis and metabolite identification using the parental mouse strain fed the same diets, which contained genistin, mixed isoflavones, Novasoy, soy molasses, or soy flour plus mixed isoflavones. Whereas the degree of soy processing did affect several parameters reflecting isoflavone bioavailability and gut microflora metabolism of daidzein to equol, stimulation of tumor growth correlated significantly with only the plasma concentration of aglycon genistein produced by the diets. This conclusion is consistent with the known estrogen agonist activity of genistein aglycon on mammary tumor growth. Conversely, plasma equol concentration was inversely correlated with the degree of soy processing. Although antagonism of genistein-stimulated tumor growth by equol could explain this result, the very low concentration of aglycon equol in plasma (12-fold lower relative to genistein) is inconsistent with any effect. These findings underscore the importance of food processing, which can remove non-nutritive components from soy, on the pharmacokinetics and pharmacodynamics of isoflavones. Such changes in diet composition affect circulating, and presumably target tissue, concentrations of genistein aglycon, which initiates estrogen receptor-mediated processes required for the stimulation of tumor growth in a mouse model for postmenopausal breast cancer.

Enhanced action of apigenin and naringenin combination on estrogen receptor activation in non-malignant colonocytes: implications on sorghum-derived phytoestrogens.

Activation of estrogen receptor-ß (ERß) is an important mechanism for colon cancer prevention. Specific sorghum varieties that contain flavones were shown to activate ER in non-malignant colonocytes at low concentrations. This study aimed to determine positive interactions among estrogenic flavonoids most relevant in sorghum. Apigenin and naringenin were tested separately and in combination for their ability to influence ER-mediated cell growth in non-malignant young adult mouse colonocytes (YAMC). Sorghum extracts high in specific flavanones and flavones were also tested. Apigenin reduced ER-mediated YAMC cell growth comparable to physiological levels of estradiol (E2, 1 nM) at 1 µM; naringenin had similar effect at 10 µM. However, when combined, 0.1 µM apigenin plus 0.05 µM naringenin produced similar effect as 1 nM E2; these concentrations represented 1/10th and 1/200th, respectively, of the active concentrations of apigenin and naringenin, demonstrating a strong enhanced action. A sorghum extract higher in flavones (apigenin and luteolin) (4.8 mg g(-1)) was more effective (5 µg mL(-1)) at activating ER in YAMC than a higher flavanone (naringenin and eriodictyol) (28.1 mg g(-1)) sorghum extract (10 µg mL(-1)). Enhanced actions observed for apigenin and naringenin were adequate to explain the level of effects produced by the high flavone and flavanone sorghum extracts. Strong positive interactions among sorghum flavonoids may enhance their ability to contribute to colon cancer prevention beyond what can be modeled using target compounds in isolation.

A novel shift in estrogen receptor expression occurs as estradiol suppresses inflammation-associated colon tumor formation.

Postmenopausal women on estrogen replacement therapy (ERT) have a reduced risk of developing colon cancer compared with postmenopausal women not on ERT, suggesting a role for estradiol (E2) in protection against this disease. To determine whether E2 protects against inflammation-associated colon cancer when administered following the initiation of colonic DNA damage, in this study, we implanted E2-containing pellets into mice after co-treatment with azoxymethane and two rounds of dextran sulfate sodium (DSS). Wild-type (WT) E2-treated mice had reduced numbers and average area of adenocarcinomas compared with the control mice. These effects were lost in estrogen receptor-ß (Erß (Esr2)) knockout mice. Surprisingly, apoptosis was reduced and cell proliferation was increased in sections from tumors of the WT E2 mice compared with the WT control mice. These findings are probably due, in part, to a reduction in ERß expression in colonic epithelial cells as the cells progressed from a non-malignant to a cancerous state as enhanced apoptosis was observed in normal colonocytes expressing higher levels of ERß. Furthermore, epithelial cells within the tumors had dramatically increased ERα mRNA and protein expression compared with the non-diseased mice. We conclude that while E2 treatment resulted in an overall suppression of colonic adenocarcinoma formation, reduced ERß expression accompanied by enhanced ERα expression caused an altered colonocyte response to E2 treatment compared with the earlier stages of colon cancer development. These data are the first examples of decreased ERß expression concurrent with increased ERα expression as a disease develops and highlight the importance of understanding the timing of E2 exposure with regard to the prevention of inflammation-associated colon cancer.

P53 mediates estradiol induced activation of apoptosis and DNA repair in non-malignant colonocytes.

Clinical and animal studies have shown a strong link between estrogen status in women and decreased risk of colon cancer. However, little research has been done into the mechanism of protection that estrogen provides. Our laboratory has demonstrated that estradiol (E2) inhibits the development of pre-neoplastic lesions through an estrogen receptor ß (ERß) mediated mechanism in mice. Our data also suggest that the primary protective role of E2 treatment is increased apoptosis in non-malignant colonocytes that are damaged and at risk of becoming cancerous. The p53 protein plays a crucial role in the cellular response to stress by inducing cell cycle arrest, DNA repair mechanisms, and/or apoptosis. Due to the observed induction of apoptosis in response to E2, we are investigating the role of p53 in this chemo-protective mechanism. E2 suppressed growth of young adult mouse colonocytes (YAMCs) by inducing apoptosis and these physiological responses were completely lost in YAMCs lacking a functional p53 protein. Western blot analysis demonstrated increases in p53 protein levels in YAMCs after treatment with E2 likely due to protein stabilization. E2 was shown to enhance the transcriptional activity of p53, resulting in up-regulation of pro-apoptotic p53 target genes (Bax, Noxa, and PUMA). Finally, repair of DNA double stranded breaks was shown to be increased by E2 treatment. Collectively, these data are the first to demonstrate that p53 is a primary mediator of the protective actions of E2 in the colon.

Estradiol alters cell growth in nonmalignant colonocytes and reduces the formation of preneoplastic lesions in the colon.

Numerous clinical and animal studies show that hormone replacement therapy reduces the risk of colon tumor formation. However, the majority of experiments have shown that estradiol (E(2)) does not inhibit the growth of malignantly transformed colon epithelia. As such, the presented studies focused on evaluating the effects of E(2) in noncancerous colonocytes. E(2) treatments (0-10 nmol/L) reduced cell growth and increased apoptotic activity in young adult mouse colonocytes (YAMC), a nonmalignant cell line, in a dose-responsive manner. These effects were lost in the YAMC-Ras cells, an isogenic cell line with a single malignant transformation. Cotreatment with an estrogen receptor (ER) antagonist inhibited the physiologic effects of E(2) in YAMC cells, suggesting that the response is ER mediated. To further study the effect of E(2) on colonic epithelia, we evaluated the development of preneoplastic lesions in ovariectomized wild-type (WT) and ERbeta knockout (ERbetaKO) mice treated with either vehicle or E(2). WT E(2)-treated animals exhibited significantly fewer aberrant crypt foci and increased apoptotic activity in colonic epithelia when compared with WT control mice or ERbetaKO animals receiving either treatment. For the first time, we showed that E(2) alters the growth of nontransformed colonocytes in vitro and that, through an ERbeta-mediated mechanism, E(2) influences the physiology of noncancerous colonocytes, resulting in fewer preneoplastic lesions. Collectively, these data show that the protective actions of E(2) occur primarily during the initiation/promotion stages of disease development and identify the hormone as an important chemoprotective agent.

Intercepting the synthesis of triazine dendrimers with nucleophilic pharmacophores: a general strategy toward drug delivery vehicles.

The camptothecin ester of isonipecotic acid is installed on a triazine dendrimer intermediate obtained through an iterative, scalable route to ultimately yield cationic and PEGylated targets with activities in cell culture comparable to free drug.

Estrogen receptor-alpha mediates gender differences in atherosclerosis induced by HIV protease inhibitors.

As part of highly active antiretroviral therapy, protease inhibitor treatment has significantly increased the lifespan of human immunodeficiency virus (HIV)-infected individuals. Many patients, however, develop negative side effects, including premature atherosclerosis. We have previously demonstrated that in male low density lipoprotein receptor (LDL-R) null mice, HIV protease inhibitors induce atherosclerotic lesions and cholesterol accumulation in macrophages in the absence of changes in plasma lipid levels. We determined that these increases were due to an up-regulation of the scavenger receptor, CD36. In the present study, we examined the effects of HIV protease inhibitors in female LDL-R null mice. Female mice given ritonavir and amprenavir (23 and 10 microg/mouse/day, respectively) developed fewer atherosclerotic lesions than males. Furthermore, peritoneal macrophages isolated from ritonavir-treated females had reduced levels of cholesterol accumulation as compared with males, and CD36 protein levels were increased to a significantly lesser degree in females than in males. To investigate the molecular mechanisms of this gender difference, we examined the effect of genetically removing estrogen receptor-alpha (ERalpha). In female mice lacking both LDL-R and ERalpha, the protective effect of gender was lost. Additionally, the reduced levels of cholesterol accumulation in macrophages observed in females was reversed. Furthermore, the absence of ERalpha resulted in increased expression of CD36 protein in a macrophage-specific manner in mice treated with ritonavir. These data demonstrate that ERalpha is directly involved in the regulation of cholesterol metabolism in macrophages and plays an important role in the gender differences observed in HIV protease inhibitor-induced atherosclerosis.

Genistein stimulates growth of human breast cancer cells in a novel, postmenopausal animal model, with low plasma estradiol concentrations.

We have demonstrated that genistein (GEN) stimulates growth of estrogen-dependent breast tumors in vivo. In this study, we evaluated whether dietary GEN can act in an additive manner with low circulating levels of 17beta-estradiol (E2). We developed an E2 delivery system using silastic implants that yield low circulating plasma E2 levels similar to those observed in postmenopausal women. We inserted various concentrations of E2 silastic implants (1:127, 1:63, 1:31, 1:15 and 1:7 = E2:cholesterol) and injected estrogen-dependent human breast cancer (MCF-7) cells into ovariectomized athymic mice. The E2 implants tested (1:127-1:7) generated 30.1-101.6 pM E2 in plasma, which is comparable to the E2 levels observed in postmenopausal women. The E2 implants stimulated MCF-7 tumor growth in a dose-dependent manner. We selected the 1:31 ratio of E2 implant to evaluate if dietary GEN acts in an additive manner with low E2 levels to influence the growth of MCF-7 tumors. Ovariectomized mice were divided into four groups: MCF-7 control, 500 ppm GEN, 1:31 E2, and 1:31 E2 + 500 ppm GEN. At week 17, the average tumor sizes were 7.6, 32.1, 67.4 and 106.8 mm2 for these groups, respectively (P < 0.05), demonstrating that 500 ppm GEN additively stimulated MCF-7 tumor growth in the presence of low levels of E2. In summary, we established a preclinical mouse model that results in E2 blood concentrations similar to those found in postmenopausal women. Further, we observed that these concentrations regulate the growth rate of MCF-7 breast tumors. Using this model, we demonstrated that dietary GEN in the presence of low levels of circulating E2 act in an additive manner to stimulate estrogen-dependent tumor growth in vivo. Results from this study suggest that consumption of products containing GEN may not be safe for postmenopausal women with estrogen-dependent breast cancer.