Effects of limiting energy availability via diet and physical activity on mammalian target of rapamycin-related signaling in rat mammary carcinomas.

This study evaluated how different approaches to limiting energy availability (LEA) by 15% affected mammalian target of rapamycin (mTOR)-related signaling in mammary carcinomas. Female Sprague Dawley rats, injected with 50mg 1-methyl-1-nitrosourea per kilogram body weight, were randomized to a control or three LEA interventions: (i) sedentary and restricted rats fed to 85% of energy available to the control or motorized wheel running (37 m/min) for an average of (ii) 1621 ± 55 (WRL) or (iii) 3094 ± 126 (WRH) meters/day with food intake adjusted to provide the same net amount of available energy across LEA interventions. Under these conditions, LEA reduced overall cancer burden by 28% (P = 0.04) and down-regulated mTOR-related signaling (Hotelling multivariate, P = 0.002). Among the regulatory nodes assessed, reduced levels of activated protein kinase B (pAkt) and induction of sirtuin 1 (SIRT1) were the most influential factors in distinguishing between sham control and LEA carcinomas. P-Akt was predictive of observed changes in levels of proteins involved in cell cycle control (r = 0.698, P < 0.0001) and induction of apoptosis (r = -0.429, P = 0.014). Plasma insulin and leptin were strongly associated with carcinoma pAkt levels. Consistent with downregulation of mTOR-related signaling by LEA, evidence of decreased lipid synthesis in carcinomas was observed (Hotelling multivariate, P < 0.001) and was negatively correlated with SIRT1 induction. Despite large differences between control and LEA, effects on mTOR regulation were insufficient to distinguish among LEA intervention groups. Given the modest effects observed on the LKB1/AMP-activated protein kinase regulatory node, NADH and NADPH rather than ATP may be more limiting for tumor growth when LEA is 15%.

Cell signaling pathways associated with a reduction in mammary cancer burden by dietary common bean (Phaseolus vulgaris L.).

Emerging evidence indicates that common bean (Phaseolus vulgaris L.) is associated with reduced cancer risk in human populations and rodent carcinogenesis models. This study sought to identify cancer-associated molecular targets that mediate the effects of bean on cancer burden in a chemically induced rat model for breast cancer. Initial experiments were conducted using a high dietary concentration of bean (60% wt/wt) where carcinoma burden in bean-fed rats was reduced 62.2% (P < 0.001) and histological and western blot analyses revealed that the dominant cellular process associated with reduced burden was induction of apoptosis. Further analysis of mammary carcinomas revealed changes in the phosphorylation states of mammalian target of rapamycin (mTOR) substrates (4E-binding protein 1 and p70S6 kinase) and mTOR regulators adenosine monophosphate-activated protein kinase and protein kinase B (Akt) (P < 0.001). Effects on mTOR signaling in carcinomas were also found at lower dietary concentrations of bean (7.5-30% wt/wt). Liquid chromatography-time of flight-mass spectrometry analysis of plasma provided evidence of altered lipid metabolism consistent with reduced mTOR network activity in the liver (P < 0.001). Plasma concentrations of insulin and insulin-like growth factor-1 were reduced by 36.3 and 38.9%, respectively, (P < 0.001), identifying a link to Akt regulation. Plasma C-reactive protein, a prognostic marker for long-term survival in breast cancer patients, was reduced by 23% (P < 0.001) in bean-fed rats. Identification of a role for the mTOR signaling network in the reduction of cancer burden by dietary bean is highly relevant given that this pathway is deregulated in the majority of human breast cancers.

Edible dry bean consumption (Phaseolus vulgaris L.) modulates cardiovascular risk factors and diet-induced obesity in rats and mice.

Pulses are grain legumes that have sustained the civilisations of the world throughout their development; yet this staple food crop has fallen into disuse, particularly in Westernised societies, and decreased consumption parallels increased prevalence of CVD. The objective of the present study was to identify mechanisms that account for the cardioprotective activity of dry bean (Phaseolus vulgaris L.), one of the four primary pulse crops, which is widely produced and consumed globally. Laboratory assays that can be used for in vivo screening of dry beans and other pulses to identify those with the greatest potential to benefit human health are also reported. Sprague-Dawley rats and a diet-induced obesity model in C57Bl/6 mice were used to assess the effect of cooked dry bean incorporated into a purified diet formulation on plasma lipids and hepatic proteins involved in the regulation of lipid biosynthesis. In both animal species, short-term feeding of a bean-containing diet reduced plasma total cholesterol and LDL-cholesterol without affecting HDL-cholesterol or total TAG. Mechanisms associated with cholesterol catabolism and excretion are the likely targets of the bean effect. Unexpectedly, bean-fed obese mice experienced weight loss as well as an improved plasma lipid profile within a 12 d time frame. These findings support the use of short-term (7-14 d) assays to investigate mechanisms that account for the cardioprotective and weight regulatory effects of dry bean and to screen dry bean germplasm resources for types of bean with high protective activity. These same assays can be used to identify the bioactive components of bean that account for the observed effects.

Hypertriglyceridemic-waist phenotype predicts diabetes: a cohort study in Chinese urban adults.

BACKGROUND: Hypertriglycedemic-waist (HTGW) phenotype is a simple and inexpensive screening parameter to identify people at increased risk for cardiovascular disease. We evaluated whether the HTGW phenotype predicts prediabetes and diabetes in Chinese urban adults. METHODS: Two thousand nine hundred and eight (2908) subjects including 1957 men and 951 women, aged 20 years and older, free of prediabetes and diabetes at baseline were enrolled in 2008 and followed for 3 years. Meanwhile, new cases of prediabetes and diabetes were identified via annual physical examination. Cox proportional hazards models were used to assess the association of HTGW phenotype with the incidence of prediabetes and diabetes. RESULTS: One thousand five hundred and thirty-three (1533) new prediabetes and 90 new diabetes cases were diagnosed during the follow-up period. The accumulated incidence of prediabetes and diabetes was 52.7% and 3.1%, respectively. Compared with the normal waist normal triglyceride (NWNT) group, those in the HTGW group had higher incidence of prediabetes and diabetes for both men and women. The hazard ratio (HR) for developing prediabetes in the presence of HTGW phenotype at baseline was 1.51 (95% confidence interval [CI] =1.04-2.19) in women, not in men (HR=1.01; 95% CI = 0.82-1.24), after adjusting for age, body mass index, systolic blood pressure, total cholesterol and low density lipoprotein-cholesterol. The HR for developing diabetes were 4.46 (95% CI = 1.88-10.60) in men and 4.64 (95% CI = 1.20-17.97) in women for people who were HTGW phenotype at baseline, after adjusting for age, body mass index, systolic blood pressure, total cholesterol and low density lipoprotein-cholesterol. CONCLUSIONS: The HTGW phenotype can be used as a simple screening approach to predict diabetes. By using this approach, it is possible to identify individuals at high-risk for diabetes, which is of great significance in reducing the incidence of diabetes among Chinese urban adults.

Effect of a low fat versus a low carbohydrate weight loss dietary intervention on biomarkers of long term survival in breast cancer patients ('CHOICE'): study protocol.

BACKGROUND: Weight loss in overweight or obese breast cancer patients is associated with an improved prognosis for long term survival. However, it is not clear whether the macronutrient composition of the chosen weight loss dietary plan imparts further prognostic benefit. A study protocol is presented for a dietary intervention to investigate the effects of weight loss dietary patterns that vary markedly in fat and carbohydrate contents on biomarkers of exposure to metabolic processes that may promote tumorigenesis and that are predictive of long term survival. The study will also determine how much weight must be lost for biomarkers to change in a favorable direction. METHODS/DESIGN: Approximately 370 overweight or obese postmenopausal breast cancer survivors (body mass index: 25.0 to 34.9 kg/m²) will be accrued and assigned to one of two weight loss intervention programs or a non-intervention control group. The dietary intervention is implemented in a free living population to test the two extremes of popular weight loss dietary patterns: a high carbohydrate, low fat diet versus a low carbohydrate, high fat diet. The effects of these dietary patterns on biomarkers for glucose homeostasis, chronic inflammation, cellular oxidation, and steroid sex hormone metabolism will be measured. Participants will attend 3 screening and dietary education visits, and 7 monthly one-on-one dietary counseling and clinical data measurement visits in addition to 5 group visits in the intervention arms. Participants in the control arm will attend two clinical data measurement visits at baseline and 6 months. The primary outcome is high sensitivity C-reactive protein. Secondary outcomes include interleukin-6, tumor necrosis factor-α, insulin-like growth factor-1 (IGF), IGF binding protein-3, 8-isoprostane-F2-alpha, estrone, estradiol, progesterone, sex hormone binding globulin, adiponectin, and leptin. DISCUSSION: While clinical data indicate that excess weight for height is associated with poor prognosis for long term survival, little attention is paid to weight control in the clinical management of breast cancer. This study will provide information that can be used to answer important patient questions about the effects of dietary pattern and magnitude of weight loss on long term survival following breast cancer treatment. CLINICAL TRIAL REGISTRATION: CA125243.

Metformin as an energy restriction mimetic agent for breast cancer prevention.

BACKGROUND: This study examined whether metformin administration inhibited chemically induced mammary carcinogenesis in rats. In cancer prevention, metformin may act (1) indirectly through reducing systemic risk factors; or (2) directly through AMPK-mediated signaling. To begin to delineate clinically relevant mechanisms for breast cancer prevention, metformin was also studied along with dietary energy restriction. MATERIALS AND METHODS: Mammary cancer was induced in female Sprague--Dawley rats (50 mg/kg MNU, i.p.). Metformin was fed alone (AIN93G + 0.05 to 1.0% w/w metformin) or combined with 40% dietary energy restriction. Plasma analytes (e.g., insulin, glucose, IGF-1) and protein expression (e.g., AMPK, mTOR, Akt) in mammary carcinomas and liver were evaluated. Additional studies included (1) aldehyde dehydrogenase flow cytometry, to gauge potential for cancer-initiated cells in mammary carcinomas to respond to metformin; (2) cell culture, to understand dose response (0.02--20 mM) of different cancer cell line molecular subtypes to metformin; and (3) analysis of a rat mammary epithelial cell microarray database, to examine expression of genes related to metformin pharmacokinetics (e.g., organic cation transporters) and pharmacodynamics (e.g., complex I of electron transport). RESULTS: While a dosing regimen of 1.0%/0.25% metformin-reduced palpable mammary carcinoma incidence, multiplicity, and tumor burden and prolonged latency, lower doses of metformin failed to inhibit carcinogenesis despite effects on plasma insulin. Human breast cancer cell growth inhibition in response to metformin was only observed at high concentrations. Poor in vivo and in vitro response to metformin may be the result of pharmacokinetic (OCT-1 expression was low in rat mammary cells; OCT-3 was downregulated in mammary carcinoma) and pharmacodynamic (complex I transcripts were higher in mammary epithelial cells from carcinomas versus uninvolved gland) effects. In combination with dietary energy restriction, metformin offered protection against new tumor occurrence following release from combined treatment. Flow cytometry indicated the presence of cancer-initiated cells in mammary carcinomas. CONCLUSIONS: As a single agent, metformin possessed limited cancer inhibitory activity. However, metformin may be an effective component of multiagent interventions that target cancer-initiated cells. There is a clear need to identify the conditions under which metformin is likely to benefit prevention and control of breast cancer.

Wheel running, skeletal muscle aerobic capacity and 1-methyl-1-nitrosourea induced mammary carcinogenesis in the rat.

Emerging evidence indicates that intrinsic differences and induced changes in aerobic capacity are probably to play a critical role in the development of chronic diseases like cancer. This study was initiated: (i) to determine how citrate synthase activity, which is routinely used as a marker of aerobic capacity and mitochondrial density in skeletal muscle, was affected by voluntary running on either a motorized activity wheel or a non-motorized free wheel and (ii) to investigate the association between aerobic capacity and the carcinogenic response induced in the mammary gland by intraperitoneal injection of 1-methyl-1-nitrosurea. Overall, wheel running reduced cancer incidence (96 versus 72%, P = 0.0006) and the number of cancers per animal (2.84 versus 1.78, P < 0.0001) and induced citrate synthase activity (276 versus 353 U/mg, P < 0.0001, sedentary control versus wheel running,respectively). Both motorized and free wheel running increased citrate synthase activity (373 +/- 24, 329 +/- 11 and 276 +/- 9 U/mg protein, P < 0.0001) and reduced the average number of cancers per rat (2.84, 1.96 and 1.63, P < 0.01), sedentary control, free wheel and motorized wheel, respectively. However, regression analyses failed to provide evidence of a significant association between citrate synthase activity and either cancer incidence or cancer multiplicity. Citrate synthase activity is a single measure in a complex pathway that determines aerobic capacity. The multifaceted nature of intrinsic and inducible aerobic capacity limits the usefulness of citrate synthase activity alone in elucidating the relationship between aerobic capacity and the carcinogenic response.

Collection of epithelial cells from rodent mammary gland via laser capture microdissection yielding high-quality RNA suitable for microarray analysis.

Laser capture microdissection (LCM) enables collection of cell populations highly enriched for specific cell types that have the potential of yielding critical information about physiological and pathophysiological processes. One use of cells collected by LCM is for gene expression profiling. Samples intended for transcript analyses should be of the highest quality possible. RNA degradation is an ever-present concern in molecular biological assays, and LCM is no exception. This paper identifies issues related to preparation, collection, and processing in a lipid-rich tissue, rodent mammary gland, in which the epithelial to stromal cell ratio is low and the stromal component is primarily adipocytes, a situation that presents numerous technical challenges for high-quality RNA isolation. Our goal was to improve the procedure so that a greater probe set present call rate would be obtained when isolated RNA was evaluated using Affymetrix microarrays. The results showed that the quality of RNA isolated from epithelial cells of both mammary gland and mammary adenocarcinomas was high with a probe set present call rate of 65% and a high signal-to-noise ratio.

Discovery of Dihydropyrrolo[1,2-a]pyrazin-3(4H)-one-Based Second-Generation GluN2C- and GluN2D-Selective Positive Allosteric Modulators (PAMs) of the N-Methyl-d-Aspartate (NMDA) Receptor.

The N-methyl-d-aspartate receptor (NMDAR) is an ion channel that mediates the slow, Ca2+-permeable component of glutamatergic synaptic transmission in the central nervous system (CNS). NMDARs are known to play a significant role in basic neurological functions, and their dysfunction has been implicated in several CNS disorders. Herein, we report the discovery of second-generation GluN2C/D-selective NMDAR-positive allosteric modulators (PAMs) with a dihydropyrrolo[1,2-a]pyrazin-3(4H)-one core. The prototype, R-(+)-EU-1180-453, exhibits log unit improvements in the concentration needed to double receptor response, lipophilic efficiency, and aqueous solubility, and lowers cLogP by one log unit compared to the first-generation prototype CIQ. Additionally, R-(+)-EU-1180-453 was found to increase glutamate potency 2-fold, increase the response to maximally effective concentration of agonist 4-fold, and the racemate is brain-penetrant. These compounds are useful second-generation in vitro tools and a promising step toward in vivo tools for the study of positive modulation of GluN2C- and GluN2D-containing NMDA receptors.

Negative allosteric modulation of GluN1/GluN3 NMDA receptors.

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission. Most native NMDA receptors are tetrameric assemblies of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Co-assembly of the glycine-binding GluN1 with glycine-binding GluN3 subunits (GluN3A-B) creates glycine activated receptors that possess strikingly different functional and pharmacological properties compared to GluN1/GluN2 NMDA receptors. The role of GluN1/GluN3 receptors in neuronal function remains unknown, in part due to lack of pharmacological tools with which to explore their physiological roles. We have identified the negative allosteric modulator EU1180-438, which is selective for GluN1/GluN3 receptors over GluN1/GluN2 NMDA receptors, AMPA, and kainate receptors. EU1180-438 is also inactive at GABA, glycine, and P2X receptors, but displays inhibition of some nicotinic acetylcholine receptors. Furthermore, we demonstrate that EU1180-438 produces robust inhibition of glycine-activated current responses mediated by native GluN1/GluN3A receptors in hippocampal CA1 pyramidal neurons. EU1180-438 is a non-competitive antagonist with activity that is independent of membrane potential (i.e. voltage-independent), glycine concentration, and extracellular pH. Non-stationary fluctuation analysis of neuronal current responses provided an estimated weighted mean unitary conductance of 6.1 pS for GluN1/GluN3A channels, and showed that EU1180-438 has no effect on conductance. Site-directed mutagenesis suggests that structural determinants of EU1180-438 activity reside near a short pre-M1 helix that lies parallel to the plane of the membrane below the agonist binding domain. These findings demonstrate that structural differences between GluN3 and other glutamate receptor subunits can be exploited to generate subunit-selective ligands with utility in exploring the roles GluN3 in neuronal function.

Candidate mechanisms accounting for effects of physical activity on breast carcinogenesis.

Evidence is strong that a reduction in risk for breast cancer is associated with moderate to vigorous physical activity (PA); however, there is limited understanding of the role of type, intensity, duration, and frequency of PA and their mechanisms in accounting for this health benefit. The objective of this review is to stimulate investigations of candidate mechanisms that may account for the effects of the intensity and duration of aerobic PA on breast cancer risk and tumor burden. Three hypotheses are considered: 1) the mTOR network hypothesis: PA inhibits carcinogenesis by suppressing the activation of the mTOR signaling network in mammary carcinomas; 2) the hormesis hypothesis: the carcinogenic response to PA is nonlinear and accounted for by a physiological cellular stress response; and 3) the metabolic reprogramming hypothesis: PA limits the amount of glucose and glutamine available to mammary carcinomas thereby inducing apoptosis because tumor-associated metabolic programming is reversed. To link these hypotheses to systemic effects of PA, it is recommended that consideration be given to determining: 1) what contracting muscle releases into circulation or removes from circulation that would directly modulate the carcinogenic process in epithelial cells; 2) whether the effects of muscle contraction on epithelial cell carcinogenesis are exerted in an endocrine, paracrine, autocrine, or intracrine manner; and 3) if the effects of muscle contraction on malignant cells differ from effects on normal or premalignant cells that do not manifest the hallmarks of malignancy.

Energetics and mammary carcinogenesis: effects of moderate-intensity running and energy intake on cellular processes and molecular mechanisms in rats.

The objective of this experiment was to determine the effects on mammary carcinogenesis of similar limitations in energy availability either by energy expenditure due to moderate-intensity running (physical activity, PA) or by regulating dietary energy (RE) intake relative to a sedentary control (SC) group that ate ad libitum. A total of 90 female Sprague-Dawley rats were injected with 1-methyl-1-nitrosourea (50 mg/kg) and 7 days thereafter were randomized to either SC, a PA group given free access to a motorized running wheel, or a RE group whose food intake limited growth to the rate observed in PA. Compared with SC, mammary carcinogenesis was inhibited by RE or PA. Cancer incidence, 92.6%, 77.8%, and 66.7% (P = 0.06), and cancer multiplicity, 3.44, 2.11, and 1.62 cancers/rat (P = 0.006), in SC, RE, and PA, respectively, were reduced to a similar extent by RE and PA. Histological and Western blot analyses of mammary carcinomas provided evidence that RE and PA induced apoptosis via the mitochondrial pathway, that cell cycle progression was suppressed at the G(1)/S transition, and that intratumoral blood vessel density was reduced, although it remains to be determined whether PA and RE exert these effects via the same mechanisms.

Di-aryl Sulfonamide Motif Adds π-Stacking Bulk in Negative Allosteric Modulators of the NMDA Receptor.

The N-methyl-d-aspartate receptor plays a critical role in central nervous system processes. Its diverse properties, as well as hypothesized role in neurological disease, render NMDA receptors a target of interest for the development of therapeutically relevant modulators. A number of subunit-selective modulators have been reported in the literature, one of which is TCN-201, a GluN2A-selective negative allosteric modulator. Recently, it was determined from a cocrystallization study of TCN-201 with the NMDA receptor that a unique active pose exists in which the sulfonamide group of TCN-201 incorporates a π-π stacking interaction between the two adjacent aryl rings that allows it to make important contacts with the protein. This finding led us to investigate whether this unique structural feature of the diaryl sulfonamide could be incorporated into other modulators that act on distinct pockets. To test whether this idea might have more general utility, we added an aryl ring plus the sulfonamide linker modification to a previously published series of GluN2C- and GluN2D-selective negative allosteric modulators that bind to an entirely different pocket. Herein, we report data suggesting that this structural modification of the NAB-14 series of modulators was tolerated and, in some instances, enhanced potency. These results suggest that this motif may be a reliable means for introducing a π-π stacking element to molecular scaffolds that could improve activity if it allowed access to ligand-protein interactions not accessible from one planar aromatic group.

Structural elements of a pH-sensitive inhibitor binding site in NMDA receptors.

Context-dependent inhibition of N-methyl-D-aspartate (NMDA) receptors has important therapeutic implications for the treatment of neurological diseases that are associated with altered neuronal firing and signaling. This is especially true in stroke, where the proton concentration in the afflicted area can increase by an order of magnitude. A class of allosteric inhibitors, the 93-series, shows greater potency against GluN1-GluN2B NMDA receptors in such low pH environments, allowing targeted therapy only within the ischemic region. Here we map the 93-series compound binding site in the GluN1-GluN2B NMDA receptor amino terminal domain and show that the interaction of the N-alkyl group with a hydrophobic cage of the binding site is critical for pH-dependent inhibition. Mutation of residues in the hydrophobic cage alters pH-dependent potency, and remarkably, can convert inhibitors into potentiators. Our study provides a foundation for the development of highly specific neuroprotective compounds for the treatment of neurological diseases.

Effect of nonmotorized wheel running on mammary carcinogenesis: circulating biomarkers, cellular processes, and molecular mechanisms in rats.

The objective of this experiment was to identify circulating growth factors, hormones, and cellular and molecular mechanisms that account for the effects of physical activity on mammary carcinogenesis. A total of 120 female Sprague-Dawley rats were injected with 1-methyl-1-nitrosourea (50 mg/kg) and 7 days thereafter were randomized to either a physically active or a sedentary control group. Individually housed rats were given free access to a nonmotorized, computer-controlled activity wheel and running behavior was reinforced by food reward. Rats self-determined their daily intensity and duration of running. Sedentary control rats received the same amount of food as the physically active rats to which they were paired. Physical activity reduced mammary cancer incidence (P = 0.015) and cancer multiplicity (P = 0.01). Physical activity induced changes in plasma insulin, insulin-like growth factor-I, and corticosterone, suggesting that mechanisms regulating glucose homeostasis were affected. Western blot analyses of mammary carcinomas revealed that proteins involved in cell proliferation were reduced (P < 0.001) and those involved in apoptosis via the mitochondrial pathway were elevated (P < 0.001) by physical activity. The hypothesis that these effects were mediated by activation of AMP-activated protein kinase, and down-regulation of protein kinase B, which collectively down-regulate the activity of the mammalian target of rapamycin, was evaluated. Evidence in support of this hypothesis was found in the Western blot analyses of mammary carcinomas, mammary gland, liver, and skeletal muscle. Collectively, these findings provide a rationale for additional studies of energy-sensing pathways in the elucidation of mechanisms that account for the inhibition of carcinogenesis by physical activity.

Modulation of the activities of AMP-activated protein kinase, protein kinase B, and mammalian target of rapamycin by limiting energy availability with 2-deoxyglucose.

2-Deoxyglucose (2-DG), which has been shown to inhibit mammary carcinogenesis, was used as a metabolic probe to investigate effects of limiting energy availability (reduced cellular ATP) on patterns of proteins' phosphorylation that play a role in the development of cancer. Experiments were conducted using a human breast cancer cell line, MDA-MB-468, and 1-methyl-1-nitrosourea-induced rat model for mammary carcinogenesis. Under in vitro conditions in which cellular ATP concentration decreased rapidly with increasing 2-DG in a dose and time dependent manner, levels of phosphorylated mammalian target of rapamycin (P-mTOR) decreased in parallel to decreases in ATP concentration. Concomitantly, phosphorylation of two upstream regulators of mTOR, AMP-activated protein kinase (AMPK) and Akt/protein kinase B were increased and decreased, respectively, with increased levels of phosphorylated acetyl-CoA carboxylase as an indicator of AMPK activation. Levels of insulin like growth factor 1-receptor and phosphoinositide-3 kinase p110 alpha were also reduced. Similar effects were observed in mammary carcinomas in vivo at concentration of 0.03% (w/w) dietary 2-DG that inhibited carcinogenesis. In vitro, downregulation of mTOR was accompanied by decreases in phosphorylation of two of mTOR's targets, 70-kDa ribosomal protein S6 kinase and eukaryote initiation factor 4E binding protein 1. Glucose treatment reversed 2-DG effects. When cells were transfected with dominant-negative AMPK alpha 2, effects of 2-DG on mTOR and its downstream effectors were diminished, providing evidence of a link between AMPK and mTOR when energy availability was limited. This work indicates that AMPK, Akt, and mTOR are candidate targets for efforts to inhibit the carcinogenic process by limiting energy availability.

Mechanisms associated with dose-dependent inhibition of rat mammary carcinogenesis by dry bean (Phaseolus vulgaris, L.).

The purpose of this study was to determine whether a dry bean (Phaseolus vulgaris, L.) containing diet exerts an inhibitory effect on mammary carcinogenesis in a well-characterized rodent model for breast cancer. Twenty-one-d-old female Sprague Dawley rats were given an intraperitoneal injection of 1-methyl-1-nitrosourea and 7 d after carcinogen injection were randomized to 1 of 5 groups fed a modification of the AIN-93G diet formulation containing 0, 7.5, 15, 30, or 60% (wt:wt) small red dry bean incorporated as cooked, freeze-dried, and milled powder. All experimental diets had the same macronutrient content based on proximate analysis. Compared with the control group, dry bean consumption resulted in dose-dependent reductions in mammary cancer incidence (P = 0.046), cancer multiplicity (P = 0.001), and tumor burden (P = 0.01). Dry bean consumption was associated with dose-dependent reductions in plasma concentrations of glucose, insulin, insulin-like growth factor-1, C-reactive protein, and interleukin-6 in food-deprived rats. Analysis of mammary adenocarcinomas indicated that a dominant mechanism accounting for reduced tumor burden was the induction of apoptosis. B cell lymphoma 2 and X-linked inhibitor of apoptosis protein levels decreased and BCL-2-associated X protein increased with increasing dry bean consumption, findings consistent with the induction of apoptosis via the mitochondrial pathway. These data demonstrate that a legume without noteworthy content of isoflavones inhibits the development of mammary carcinogenesis and are consistent with a recent report from the Nurses Health Study that bean or lentil intake is associated with a lower risk for breast cancer.

2-Deoxyglucose as an energy restriction mimetic agent: effects on mammary carcinogenesis and on mammary tumor cell growth in vitro.

Dietary energy restriction (DER) is a potent inhibitor of carcinogenesis, but chronic DER in human populations is difficult to sustain. Consequently, interest exists in identifying energy restriction mimetic agents (ERMAs), agents that provide the health benefits of DER without reducing caloric intake. The selection of a candidate ERMAs for this study was based on evidence that DER inhibits carcinogenesis by limiting glucose availability. The study objective was to determine if 2-deoxyglucose (2-DG), a glucose analogue that blocks its metabolism, would inhibit mammary carcinogenesis. Pilot studies were done to establish a dietary concentration of 2-DG that would not affect growth. For the carcinogenesis study, ninety 21-day-old female Sprague-Dawley rats were injected i.p. with 50 mg of 1-methyl-1-nitrosourea per kilogram of body weight. Following injection, animals were ad libitum fed AIN-93G diet containing 0.00%, 0.02%, or 0.03% (w/w) 2-DG for 5 weeks. 2-DG decreased the incidence and multiplicity of mammary carcinomas and prolonged cancer latency (P < 0.05). The 0.02% dose of 2-DG had no effect on circulating levels of glucose, insulin, insulin-like growth factor-I, IGF binding protein-3, leptin, or body weight gain. Using MCF-7 human breast cancer cells to investigate the signaling pathways perturbed by disruption of glucose metabolism, 2-DG reduced cell growth and intracellular ATP in a dose- and time-dependent manner (P < 0.01). Treatment with 2-DG increased levels of phosphorylated AMP-activated protein kinase and Sirt-1 and reduced phosphorylated Akt (P < 0.05). These studies support the hypothesis that DER inhibits carcinogenesis, in part, by limiting glucose availability and that energy metabolism is a target for the development of ERMA for chemoprevention.

Identification of the apoptosis activation cascade induced in mammary carcinomas by energy restriction.

Energy restriction (ER) inhibits mammary carcinogenesis and results in a marked reduction in tumor size, effects likely to be explained by ER-mediated induction of apoptosis. The goal of this study was to investigate the molecular mechanism(s) accounting for apoptosis induction. To do this, chemically induced mammary carcinomas were evaluated from rats that were ad libitum fed (control), 40% ER, or 40% ER but energy repleted for 7 days before study termination (ER-REP); the ER-REP group permitted the determination of the reversibility of ER-mediated effects. Cleaved products of poly(ADP-ribose) polymerase 1 were elevated by ER (P < 0.025) providing biochemical evidence of apoptosis induction. cDNA microarray analysis identified the Bcl-2, CARD, and IAP functional gene groupings as being involved in apoptosis induction. Consistent with the microarray data, the activities of caspases 9 and 3 were observed to be approximately 2-fold higher in carcinomas from ER rats (P < or =0.01), whereas caspase 8 activity was similar in carcinomas from all three of the groups. This evidence that ER-induced apoptosis mediated by the mitochondrial pathway was additionally supported by the finding that levels of Bcl-2, Bcl-xl, and XIAP protein were significantly lower (P < 0.01), and levels of Bax and Apaf-1 were elevated (P < 0.02) in ER carcinomas versus those carcinomas from control or ER-REP rats. Additional studies revealed that Akt phosphorylation (activation) was reduced in mammary carcinomas from ER rats. Thus, it appears that ER induces apoptosis in mammary carcinomas via a cell survival factor-dependent pathway.

Effect of energy restriction on cell cycle machinery in 1-methyl-1-nitrosourea-induced mammary carcinomas in rats.

Energy restriction (ER) results in a profound inhibition of chemically induced mammary carcinogenesis. The cancer inhibitory activity of ER has been shown to be associated with lower rates of cell proliferation during both premalignant and malignant stages of this disease process. Moreover, inhibition of carcinogenesis and suppression of cell proliferation occur in animals in which plasma concentrations of insulin-like growth factor (IGF)-I are reduced, and plasma corticosterone levels are increased concomitantly. Given the role of both hormones in signal transduction pathways that can modulate cell cycle progression, albeit via different regulatory mechanisms, we report experiments conducted to determine whether hypothesized effects of changes in plasma levels of IGF-I and corticosterone on cell cycle regulation could be detected in mammary carcinomas occurring in 40% ER rats in comparison to ad libitum fed control rats or 40% ER rats that were energy repleted for 7 days (ER-REP). As determined by appropriate combinations of immunoprecipitations, Western blots, and kinase activity assays, it was found that levels of phosphorylated retinoblastoma and E2F-1 were significantly reduced by ER (approximately 40 and 75%, respectively; P < 0.01), an effect that was partially reversed by ER-REP. Reductions in cyclin-dependent kinase (CDK)2 (82%) and CDK4 (77%) kinase activity in ER carcinomas were likely to account for the observed effects on retinoblastoma and E2F-1. Both Cip1/p21 and Kip1/p27 and levels of these proteins complexed with CDK2 were significantly elevated in ER carcinomas (P < 0.01), and levels of cyclin E were reduced. On the other hand, regulation of CDK4 kinase activity by ER was likely attributable to effects on cyclin D1 as well as increased binding of P16 and P19 to CDK4. The majority of changes induced by ER were reversed by ER-REP. These observations are consistent with the hypothesis that ER exerts its profound cancer inhibitory activity, in part, by multifaceted regulation of cell cycle machinery, possibly via concomitant changes in corticosterone and IGF-1 metabolism, although the role of other hormones and growth factors should not be dismissed.