Clinton D. Allred

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.

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.

Diet Complexity and Estrogen Receptor β Status Affect the Composition of the Murine Intestinal Microbiota

ABSTRACT Intestinal microbial dysbiosis contributes to the dysmetabolism of luminal factors, including steroid hormones (sterones) that affect the development of chronic gastrointestinal inflammation and the incidence of sterone-responsive cancers of the breast, prostate, and colon. Little is known, however, about the role of specific host sterone nucleoreceptors, including estrogen receptor β (ERβ), in microbiota maintenance. Herein, we test the hypothesis that ERβ status affects microbiota composition and determine if such compositionally distinct microbiota respond differently to changes in diet complexity that favor Proteobacteria enrichment. To this end, conventionally raised female ERβ+/+ and ERβ−/− C57BL/6J mice (mean age of 27 weeks) were initially reared on 8604, a complex diet containing estrogenic isoflavones, and then fed AIN-76, an isoflavone-free semisynthetic diet, for 2 weeks. 16S rRNA gene surveys revealed that the fecal microbiota of 8604-fed mice and AIN-76-fed mice differed, as expected. The relative diversity of Proteobacteria, especially the Alphaproteobacteria and Gammaproteobacteria, increased significantly following the transition to AIN-76. Distinct patterns for beneficial Lactobacillales were exclusive to and highly abundant among 8604-fed mice, whereas several Proteobacteria were exclusive to AIN-76-fed mice. Interestingly, representative orders of the phyla Proteobacteria, Bacteroidetes, and Firmicutes, including the Lactobacillales, also differed as a function of murine ERβ status. Overall, these interactions suggest that sterone nucleoreceptor status and diet complexity may play important roles in microbiota maintenance. Furthermore, we envision that this model for gastrointestinal dysbiosis may be used to identify novel probiotics, prebiotics, nutritional strategies, and pharmaceuticals for the prevention and resolution of Proteobacteria-rich dysbiosis.

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 β [ER-β; 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-β 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.

Transactivation of ERα by Rosiglitazone induces proliferation in breast cancer cells

In the present study, we demonstrate that Rosiglitazone (Rosi), a thiazolidinedione and PPARγ agonist, induces ERE (Estrogen Receptor Response Element) reporter activity, pS2 (an endogenous ER gene target) expression, and proliferation of ER positive breast cancer (MCF-7) cells. By performing a dose–response assay, we determined that high concentrations of Rosi inhibit proliferation, while low concentrations of Rosi induce proliferation. Using the anti-estrogen ICI, ER negative breast cancer (MDA-MB-231) cells, and a prostate cancer cell line (22Rv1) deficient in both ERα and PPARγ, we determined that Rosiglitazone-induced ERE reporter activation and proliferation is through an ERα dependent mechanism. Rosiglitazone-induced ERE activation is also dependent on activation of the Extracellular Signal-Regulated Kinase–Mitogen Activated Protein Kinase (ERK–MAPK) pathway, since it is inhibited by co-treatment with U0126, a specific inhibitor of this pathway. We also demonstrate that when ERα and PPARγ are both present, they compete for Rosi, inhibiting each others transactivation. To begin to unravel the pharmacological mechanism of Rosi-induced ER activation, sub-maximally effective concentrations of E2 were used in combination with increasing concentrations of Rosi in luciferase reporter assays. From these assays it appears that E2 and Rosi both activate ERα via similar pharmacological mechanisms. Furthermore sub-maximally effective concentrations of E2 and Rosi additively increase both ERE reporter activity and MCF-7 cell proliferation. The results of this study may have clinical relevancy for Rosi’s use both as an anti-diabetic in post-menopausal women and as an anti-cancer drug in women with ER positive breast cancer

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.

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-β (Erβ (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 ERβ 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 ERβ. 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 ERβ 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 ERβ 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 β (ERβ) 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.

Rapidly cycling Lgr5 + stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk

The majority of colon tumors are driven by aberrant Wnt signaling in intestinal stem cells, which mediates an efficient route toward initiating intestinal cancer. Natural lipophilic polyphenols and long-chain polyunsaturated fatty acids (PUFAs) generally suppress Wnt- and NF-κB- (nuclear factor-κ light-chain enhancer of activated B-cell) related pathways. However, the effects of these extrinsic agents on colonic leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5+) stem cells, the cells of origin of colon cancer, have not been documented to date. Therefore, we examined the effect of n-3 PUFA and polyphenol (curcumin) combination on Lgr5+ stem cells during tumor initiation and progression in the colon compared with an n-6 PUFA-enriched control diet. Lgr5-EGFP-IRES-creERT2 knock-in mice were fed diets containing n-6 PUFA (control), n-3 PUFA, n-6 PUFA+curcumin or n-3 PUFA+curcumin for 3 weeks, followed by 6 azoxymethane (AOM) injections, and terminated 17 weeks after the last injection. To further elucidate the effects of the dietary bioactives at the tumor initiation stage, Lgr5+ stem cells were also assessed at 12 and 24 h post AOM injection. Only n-3 PUFA+curcumin feeding reduced nuclear β-catenin in aberrant crypt foci (by threefold) compared with control at the progression time point. n-3 PUFA+curcumin synergistically increased targeted apoptosis in DNA-damaged Lgr5+ stem cells by 4.5-fold compared with control at 12 h and maximally reduced damaged Lgr5+ stem cells at 24 h, down to the level observed in saline-treated mice. Finally, RNAseq analysis indicated that p53 signaling in Lgr5+ stem cells from mice exposed to AOM was uniquely upregulated only following n-3 PUFA+curcumin cotreatment. These novel findings demonstrate that Lgr5+ stem cells are uniquely responsive to external dietary cues following the induction of DNA damage, providing a therapeutic strategy for eliminating damaged Lgr5+ stem cells to reduce colon cancer initiation.

Targeted Deletion of p53 in Lgr5-Expressing Intestinal Stem Cells Promotes Colon Tumorigenesis in a Preclinical Model of Colitis-Associated Cancer.

p53 has been shown to mediate cancer stem-like cell function by suppressing pluripotency and cellular dedifferentiation. However, there have been no studies to date that have addressed the specific effects of p53 loss in colonic adult stem cells. In this study, we investigated the consequences of conditionally ablating p53 in the highly relevant Lgr5(+) stem cell population on tumor initiation and progression in the colon. In a mouse model of carcinogen (AOM)-induced colon cancer, tamoxifen-inducible Lgr5-driven deletion of p53 reduced apoptosis and increased proliferation of crypt stem cells, but had no effect on tumor incidence or size. Conversely, in a mouse model of colitis-associated cancer, in which mice are exposed to AOM and the potent inflammation inducer DSS, stem cell-specific p53 deletion greatly enhanced tumor size and incidence in the colon. These novel findings suggest that the loss of p53 function in stem cells enables colonic tumor formation only when combined with DNA damage and chronic inflammation. Furthermore, we propose that stem cell targeting approaches are valuable for interrogating prevention and therapeutic strategies that aim to specifically eradicate genetically compromised stem cells.