Association of lifestyle and demographic factors with estrogenic and glucocorticogenic activity in Mexican American women.

Breast cancer risk is higher in US-born than in foreign-born Hispanics/Latinas and also increases with greater length of US residency. It is only partially known what factors contribute to these patterns of risk. To gain new insights, we tested the association between lifestyle and demographic variables and breast cancer status, with measures of estrogenic (E) and glucocorticogenic (G) activity in Mexican American women. We used Chemical-Activated LUciferase gene eXpression assays to measure E and G activity in total plasma from 90 Mexican American women, without a history of breast cancer at the time of recruitment, from the San Francisco Bay Area Breast Cancer Study. We tested associations of nativity, lifestyle and sociodemographic factors with E and G activity using linear regression models. We did not find a statistically significant difference in E or G activity by nativity. However, in multivariable models, E activity was associated with Indigenous American ancestry (19% decrease in E activity per 10% increase in ancestry, P = 0.014) and with length of US residency (28% increase in E activity for every 10 years, P = 0.035). G activity was associated with breast cancer status (women who have developed breast cancer since recruitment into the study had 21% lower G activity than those who have not, P = 0.054) and alcohol intake (drinkers had 25% higher G activity than non-drinkers, P = 0.015). These associations suggest that previously reported breast cancer risk factors such as genetic ancestry and alcohol intake might in part be associated with breast cancer risk through mechanisms linked to the endocrine system.

Follicular lymphoma-protective HLA class II variants correlate with increased HLA-DQB1 protein expression.

Multiple follicular lymphoma (FL) susceptibility single-nucleotide polymorphisms in the human leukocyte antigen (HLA) class I and II regions have been identified, including rs6457327, rs3117222, rs2647012, rs10484561, rs9268853 and rs2621416. Here we validated previous expression quantitative trait loci results with real-time reverse transcription quantitative PCR and investigated protein expression in B-lymphoblastoid cell lines and primary dendritic cells using flow cytometry, cell-based enzyme-linked immunosorbent assay and western blotting. We confirmed that FL-protective rs2647012-linked variants, in high linkage disequilibrium with the extended haplotype DRB1*15:01-DQA1*01:02-DQB1*06:02, correlate with increased HLA-DQB1 expression. This association remained significant at the protein level and was reproducible across different cell types. We also found that differences in HLA-DQB1 expression were not related to changes in activation markers or class II, major histocompatibility complex, transactivator expression, suggesting the role of an alternative regulatory mechanism. However, functional analysis using RegulomeDB did not reveal any relevant regulatory candidates. Future studies should focus on the clinical relevance of increased HLA-DQB1 protein expression facilitating tumor cell removal through increased immune surveillance.

Ligand-independent activation of estrogen receptor function by 3, 3'-diindolylmethane in human breast cancer cells.

3,3'-Diindolylmethane (DIM), a major in vivo product of acid-catalyzed oligomerization of indole-3-carbinol (I3C), is a promising anticancer agent present in vegetables of the Brassica genus. We investigated the effects of DIM on estrogen-regulated events in human breast cancer cells and found that DIM was a promoter-specific activator of estrogen receptor (ER) function in the absence of 17beta-estradiol (E(2)). DIM weakly inhibited the E(2)-induced proliferation of ER-containing MCF-7 cells and induced proliferation of these cells in the absence of steroid, by approximately 60% of the E(2) response. DIM had little effect on proliferation of ER-deficient MDA-MB-231 cells, suggesting that it is not generally toxic at these concentrations. Although DIM did not bind to the ER in this concentration range, as shown by a competitive ER binding assay, it activated the ER to a DNA-binding species. DIM increased the level of transcripts for the endogenous pS2 gene and activated the estrogen-responsive pERE-vit-CAT and pS2-tk-CAT reporter plasmids in transiently transfected MCF-7 cells. In contrast, DIM failed to activate transcription of the simple E(2)- and diethylstilbesterol-responsive reporter construct pATC2. The estrogen antagonist ICI 182780 (7alpha-[9-[(4,4,5,5, 5-pentafluoropentyl)sulfonyl]nonyl]-estra-1,3,5(10)-triene-3, 17beta-diol) was effective against DIM-induced transcriptional activity of the pERE-vit-CAT reporter, which further supports the hypothesis that DIM is acting through the ER. We demonstrated that ligand-independent activation of the ER in MCF-7 cells could be produced following treatment with the D1 dopamine receptor agonist SKF-82958 [(+/-)6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepinehydrobromide]. We also demonstrated that the agonist effects of SKF-82958 and DIM, but not of E(2), could be blocked by co-treatment with the protein kinase A (PKA) inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide). These results have uncovered a promoter-specific, ligand-independent activation of ER signaling for DIM that may require activation by PKA, and suggest that this major I3C product may be a selective activator of ER function.

The major cyclic trimeric product of indole-3-carbinol is a strong agonist of the estrogen receptor signaling pathway.

Indole-3-carbinol (I3C), a component of Brassica vegetables, is under study as a preventive agent of cancers of the breast and other organs. Following ingestion, I3C is converted to a series of oligomeric products that presumably are responsible for the in vivo effects of I3C. We report the effects of the major trimeric product, 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b':7,8-b' ']triindole (CTr), on the estrogen receptor (ER) signaling pathways. Tumor-promoting effects of high doses of I3C may be due to activation of aryl hydrocarbon receptor (AhR)-mediated pathways; therefore, we also examined the effects of CTr on AhR activated processes. We observed that CTr is a strong agonist of ER function. CTr stimulated the proliferation of estrogen-responsive MCF-7 cells to a level similar to that produced by estradiol (E(2)) but did not affect the growth of the estrogen-independent cell line, MDA-MD-231. CTr displaced E(2) in competitive-binding studies and activated ER-binding to an estrogen responsive DNA element in gel mobility shift assays with EC(50)s of about 0.1 microM. CTr activated transcription of an E(2)-responsive endogenous gene and exogenous reporter genes in transfected MCF-7 cells, also with high potency. CTr failed to activate AhR-mediated pathways, consistent with the low-binding affinity of CTr for the AhR reported previously. Comparisons of the conformational characteristics of CTr with other ER ligands indicated a remarkable similarity with tamoxifen, a selective ER antagonist used as a breast cancer therapeutic agent and suggest an excellent fit of CTr into the ligand-binding site of the ER.

Cytostatic and antiestrogenic effects of 2-(indol-3-ylmethyl)-3,3'-diindolylmethane, a major in vivo product of dietary indole-3-carbinol.

Under acidic conditions, indole-3-carbinol (13C) is converted to a series of oligomeric products thought to be responsible for the biological effects of dietary 13C. Chromatographic separation of the crude acid mixture of 13C, guided by cell proliferation assay in human MCF-7 cells, resulted in the isolation of 2-(indol-3-ylmethyl)-3,3'-diindolylmethane (LTr-1) as a major antiproliferative component. LTr-1 inhibited the growth of both estrogen-dependent (MCF-7) and -independent (MDA-MB-231) breast cancer cells by approximately 60% at a non-lethal concentration of 25 microM. LTr-1 had no apparent effect on the proliferation of MCF-7 cells in the absence of estrogen. LTr-1 was a weak ligand for the estrogen receptor (ER) (IC50 70 microM) and efficiently inhibited the estradiol (E2)-induced binding of the ER to its cognate DNA responsive element. The antagonist effects of LTr-1 also were exhibited in assays of endogenous pS2 gene expression and in cells transiently transfected with an estrogen-responsive reporter construct (pERE-vit-CAT). LTr-1 activated both binding of the aryl hydrocarbon (Ah) receptor to its cognate DNA responsive element and expression of the Ah receptor-responsive gene CYP1A1. LTr-1 was a competitive inhibitor of CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity. In summary, these results demonstrated that LTr-1, a major in vivo product of I3C, could inhibit the proliferation of both estrogen-dependent and -independent breast tumor cells and that LTr-1 is an antagonist of estrogen receptor function and a weak agonist of Ah receptor function.

Lipoxin A4: a new class of ligand for the Ah receptor.

The Ah receptor is a ligand-activated transcription factor that mediates many of the biological actions of a large class of environmental compounds. Support for a role of the Ah receptor in normal physiology also has been reported, but an endogenous regulating ligand has not been identified. We have examined candidate endogenous lipophilic substances and report here the ability of the arachidonic acid metabolite, lipoxin A4, to bind to and activate the Ah receptor in Hepa-1 cells. Lipoxin A4 produced a concentration-dependent response in a DRE-driven CAT reporter construct, with a greater than 10-fold increase in CAT activity at 0. 3 microM. Lipoxin A4 transformed the Ah receptor to an active DRE-binding form in a concentration-dependent manner as indicated by gel mobility shift analysis. Results of Ah receptor competitive binding experiments indicated that at a concentration of 100 nM, lipoxin A4 produced a half-maximum displacement (EC50) of [3H]TCDD binding. Results of Northern blot analyses indicated a transient increase in mRNA levels of the Ah receptor-responsive gene CYP1A1, which peaked at 4 h, consistent with the kinetics observed for lipoxin A4-induced CYP1A1 enzyme activity. Further, lipoxin A4 was found to be a competitive inhibitor for the CYP1A1 enzyme, with a calculated Ki = 1.1 microM. These results establish lipoxin A4 as a new class of Ah receptor ligand, one that differs dramatically from classical Ah receptor ligands.

Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells.

The current options for treating breast cancer are limited to excision surgery, general chemotherapy, radiation therapy, and, in a minority of breast cancers that rely on estrogen for their growth, antiestrogen therapy. The naturally occurring chemical indole-3-carbinol (I3C), found in vegetables of the Brassica genus, is a promising anticancer agent that we have shown previously to induce a G1 cell cycle arrest of human breast cancer cell lines, independent of estrogen receptor signaling. Combinations of I3C and the antiestrogen tamoxifen cooperate to inhibit the growth of the estrogen-dependent human MCF-7 breast cancer cell line more effectively than either agent alone. This more stringent growth arrest was demonstrated by a decrease in adherent and anchorage-independent growth, reduced DNA synthesis, and a shift into the G1 phase of the cell cycle. A combination of I3C and tamoxifen also caused a more pronounced decrease in cyclin-dependent kinase (CDK) 2-specific enzymatic activity than either compound alone but had no effect on CDK2 protein expression. Importantly, treatment with I3C and tamoxifen ablated expression of the phosphorylated retinoblastoma protein (Rb), an endogenous substrate for the G1 CDKs, whereas either agent alone only partially inhibited endogenous Rb phosphorylation. Several lines of evidence suggest that I3C works through a mechanism distinct from tamoxifen. I3C failed to compete with estrogen for estrogen receptor binding, and it specifically down-regulated the expression of CDK6. These results demonstrate that I3C and tamoxifen work through different signal transduction pathways to suppress the growth of human breast cancer cells and may, therefore, represent a potential combinatorial therapy for estrogen-responsive breast cancer.

Regulation of CYP1A1 by indolo[3,2-b]carbazole in murine hepatoma cells.

To determine the basis for unexpected differences in CYP1A1 inducing potencies and efficacies for the diet-derived indole derivative, indolo[3,2-b]carbazole (ICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we conducted a systematic analysis of events involved in the induced expression of CYP1A1 in murine hepatoma-derived cell lines (Hepa-1). In contrast to the effects of TCDD, induction kinetics and CYP1A1 mRNA half-life were dependent on ICZ concentration, and the response from low doses of inducer was transient due to rapid clearance of ICZ. TCDD and ICZ produced the same maximum response (i.e. equal efficacies) from a TCDD-responsive CAT reporter construct in Hepa-1 cells. When measured by the immediate responses associated with CYP1A1 expression, including cellular uptake of inducer, receptor transformation and binding to DRE (gel mobility shift assay), initiation of transcription (nuclear run-on assay), and short-term accumulation of mRNA (Northern blot assay), ICZ also exhibited an efficacy equal to that of TCDD and a potency that corresponds to its receptor affinity. ICZ is a potent and selective noncompetitive inhibitor of ethoxyresorufin O-deethylase activity (Ki = 1.5 nM). Taken together these results indicate that ICZ is a bifunctional modulator of CYP1A1 expression with intrinsic efficacy equal to that of TCDD.

Carbohydrate malabsorption.

Carbohydrate malabsorption is a very important clinical entity, particularly in pediatrics, where, if untreated, it can lead to malnutrition and failure to thrive. Malabsorption that can be treated readily with elimination of the offending carbohydrate. Knowledge by the physician of the specific mechanisms involved in the physiology of carbohydrate absorption and digestion will help in the handling of the clinical situation of malabsorption.

Absorption of fructose by isolated small intestine of rats is via a specific saturable carrier in the absence of glucose and by the disaccharidase-related transport system in the presence of glucose.

Previous studies have shown that the absorption of fructose is aided by simultaneous ingestion of glucose. The aim of the present study was to reproduce this finding in vitro to better understand the mechanism of the effect of glucose on absorption of fructose. The phenomena could not be reproduced with everted sleeves of rat intestine or brush border vesicles. In a perfused segment of isolated intestine, it was possible to demonstrate that the transport of fructose was accelerated when glucose was present in the perfusion medium. The enhanced transport was inhibited by sucrose and also by acarbazone, an inhibitor of intestinal alpha-disaccharidases. Phlorizin had no effect on the transport of fructose. The results of these studies indicate that there is a specific carrier for fructose saturated with a low concentration of the sugar, and that in the presence of glucose there is joint absorption of the two sugars by the disaccharidase-related transport system.

Fructose absorption.

Fructose found in modern diets as a constituent of the disaccharide sucrose is absorbed by a well-characterized absorptive system integrating enzymatic hydrolysis of the disaccharide and transfer of the resulting two monosaccharides through the apical membrane of the epithelial cell. The increasing use of high-fructose syrups and crystalline fructose prompted new studies aimed at the determination of the absorptive capacity for free fructose in the human gut. Results indicate that the capacity for fructose absorption is small compared with that for sucrose and glucose and is much less than previously estimated. The unexpected finding that the simultaneous ingestion of glucose can prevent fructose malabsorption suggests that the pair of monosaccharides might be absorbed by the disaccharidase-related transport system as if they were the product of the enzymatic hydrolysis of sucrose. This absorptive mechanism might not be able to transport fructose when ingested without glucose.

The effect of exercise on fructose absorption.

The effect of exercise on intestinal absorption of fructose was evaluated in 10 subjects after they consumed four beverages, each containing a total of 50 g carbohydrate: 100% fructose (100F), 95% fructose and 5% glucose (95F), 70% fructose and 30% glucose (70F), and 100% glucose (100G), as well as a water placebo. With 100F and 95F, breath hydrogen, which is an index of incomplete absorption, increased significantly in all subjects. In contrast, hydrogen excretion did not increase in any subject after consumption of 100G or water, or in five of seven subjects who consumed 70F. The rapid increase in hydrogen excretion observed when consumption of 100F was followed by exercise was not noted during a comparable nonexercise trial. These data suggest that intestinal capacity for absorption of fructose is readily saturated after ingestion of amounts as small as 50 g and that exercise, which reduces intestinal transit time, can cause incomplete absorption of fructose.

Intestinal absorption of fructose in the rat.

The objective of this study was to investigate the mechanisms involved in intestinal absorption of fructose. The results indicate that adult rats readily absorbed 0.4 g of fructose, an amount equivalent to 1.4-1.6 g fructose/kg body wt. Acute malabsorption of fructose occurred with doses greater than 0.6 g (2.1-2.4 g/kg body wt). Continued exposure to dietary fructose resulted in a decrease in the evidence of colonic fermentation. Glucose or galactose administered with fructose enhanced the absorption of fructose. The greatest absorption was observed when equal amounts of fructose and glucose were given simultaneously. If glucose was ingested as a polymer (starch or dextrin), the stimulatory effect was dependent on the digestibility of the polymer. Sucrose given with the fructose and glucose diminished the absorption of fructose. Acarbazone, a specific inhibitor of alpha-glucosidases, including sucrase, also inhibited the facilitating effect of glucose and galactose in absorption of fructose. These results give evidence for joint absorption of the two monosaccharides, fructose and glucose.

Development of ornithine metabolism in the mouse intestine.

Circulating arginine available for synthesis of protein is produced in the kidney of the adult mammal by the action of the last two enzymes of the urea cycle, argininosuccinate synthase and argininosuccinate lyase. In a previous publication, we reported the presence of a complete biosynthetic pathway for arginine in the intestine of the neonatal mouse at a time when no other endogenous sources of arginine were available. Our present study was aimed at the determination of the source of ornithine used by the intestine of the neonatal mouse for the synthesis of arginine. We established the developmental profile of the two intestinal mitochondrial enzymes, pyrroline 5-carboxylate synthase and ornithine aminotransferase, responsible for the conversion of glutamate to ornithine. Both enzymatic activities were found to be significantly elevated throughout the suckling period with a peak of activity during the 2nd wk of life. Glutamate dehydrogenase activity in the intestine did not appear to be developmentally regulated during the suckling and weaning periods; therefore, this enzyme was used as a convenient marker to quantify mitochondrial preparations. Ornithine decarboxylase activity was undetectable in the intestine of the mouse during the suckling period and was detected briefly at weaning, indicating that ornithine synthesized in the intestinal mitochondria is probably not diverted actively into the polyamine pathway and is available for synthesis of arginine by the enzymes of the urea cycle.

Renal trehalase: function and development.

1. Following injection of trehalose into the bloodstream, no trehalose was found in urine of rabbits until the concentration of trehalose in blood exceeded 0.6 mg/ml. 2. Absence of trehalose in urine of the rabbit when the concentration of the sugar in blood is elevated supports the hypothesis that renal trehalase functions as a digestive enzyme in kidney. 3. The rat does not possess renal trehalase, and excretion of trehalose was in direct relation to the concentration of trehalose in blood. 4. There are differences in expression of the disaccharidases in kidney and intestine although they share many structural and enzymatic characteristics.

Participation of pancreatic enzymes in the degradation of intestinal sucrase-isomaltase.

The pancreatic ducts of the rats were bypassed with a catheter placed within the common bile duct to prevent the entry of pancreatic enzymes into the duodenum without interrupting bile flow. For 8 days, the animals were fed a diet (peptones, sucrose, coconut oil, vitamins, and minerals) that could be digested without pancreatic enzymes. Control animals were sham operated and pair-fed with the same diet. Relative rates of synthesis and degradation were estimated by pulse labeling and double labeling, respectively, for sucrase and for total protein, in intestinal mucosa and along the gradient of cells collected from the tip of the villus to the bottom of the crypt. The rate of degradation of sucrase was 1.7 times greater than that of total protein in controls, whereas in animals with the pancreatic bypass it was equal to that of total protein. This decrease in rate of degradation produced a proportional increase of activity of sucrase in experimental animals. The hydrolytic effect of pancreatic enzymes on sucrase was apparent along the entire length of the villus but not in the crypt. These data support the hypothesis that pancreatic proteases release sucrase-isomaltase from the brush border membrane, resulting in the observed increase of the rate of degradation. Electrophoretic separation of immunoprecipitated sucrase-isomaltase showed that the intact pro-sucrase-isomaltase observed in operated animals is split into two subunits (sucrase and isomaltase) by action of pancreatic proteases in control animals.

Rat intestinal brush border membrane trehalase: some properties of the purified enzyme.

Rat intestinal brush border trehalase (EC 3.2.1.28) solubilized by Triton X-100 or Emulphogen BC 720 has been purified almost to homogeneity in a five steps procedure including DEAE cellulose, Sephadex G-200, preparative flat bed electrofocusing and hydroxylapatite. The apparent molecular weight was estimated to be about 65,500 daltons by mannitol density gradient ultracentrifugation. The optimum pH of the enzyme was between 5.5 and 5.7 in phosphate, maleate or citrate buffers. The apparent Km for trehalose was found to be 10 mM in maleate buffer pH 6.0. The isoelectric point was 4.9. Tris, P-aminophenylglucoside, sucrose and maltose are fully competitive inhibitors with Kis of 2.2, 1.8, 7.7 and 170 mM, respectively. The inhibition by Phloridzin appeared to be of the mixed type with a Ki of 1.7 mM. Trehalase is heat stable up to 50 degrees C and the activation energy is 10.96 kcal/mol. Schiff's staining on polyacrylamide gel and interaction with Con-A-Sepharose indicate that rat trehalase is a glycoprotein.

Effect of dietary sucrose on synthesis and degradation of intestinal sucrase.

Rates of synthesis and degradation of sucrase-isomaltase were measured along the crypt-villus unit of intestinal mucosa of rats fed either a high-sucrose or a carbohydrate-free diet. The objective of the study was to investigate i) the biochemical basis for the accumulation of sucrase during migration and differentiation of the enterocyte, leading to changes in distribution of activity of sucrase along the villus, and ii) the mechanism for the adaptation of sucrase activity to the amount of dietary carbohydrate. The results indicate that synthesis of sucrase is more rapid than degradation at the crypt-villus junction and in the lower part of the villus, producing a progressive accumulation of enzyme. The decreased activity at the tip of the villus is the consequence of a decided diminution of synthesis while the rate of degradation remains elevated. In rats fed a diet high in sucrose, the increased activity (3.25 times) is associated with much more rapid synthesis (2.6 times), while degradation is only slightly slower (0.8 times) than in those animals deprived of carbohydrate.