Weiqin Jiang

Dietary energy restriction modulates the activity of AMP-activated protein kinase, Akt, and mammalian target of rapamycin in mammary carcinomas, mammary gland, and liver.

Dietary energy restriction (DER) inhibits mammary carcinogenesis, yet mechanisms accounting for its protective activity have not been fully elucidated. In this study, we tested the hypothesis that DER exerts effects on intracellular energy sensing pathways, resulting in alterations of phosphorylated proteins that play a key role in the regulation of cancer. Experiments were conducted using the 1-methyl-1-nitrosourea-induced mammary cancer model in which rats were 0%, 20%, or 40% energy restricted during the postinitiation stage of carcinogenesis. Parallel experiments were done in non-carcinogen-treated rats in which effects of DER at 0%, 5%, 10%, 20%, or 40% in liver were investigated. In a DER dose-dependent manner, levels of Thr(172) phosphorylated AMP-activated protein kinase (AMPK) increased in mammary carcinomas with a concomitant increase in phosphorylated acetyl-CoA-carboxylase, a direct target of AMPK, the phosphorylation of which is regarded as an indicator of AMPK activity. Levels of phosphorylated mammalian target of rapamycin (mTOR) decreased with increasing DER, and down-regulation of mTOR activity was verified by a decrease in the phosphorylation state of two mTOR targets, 70-kDa ribosomal protein S6 kinase (p70S6K) and eukaryote initiation factor 4E binding protein 1 (4E-BP1). Coincident with changes in mTOR phosphorylation, levels of activated protein kinase B (Akt) were also reduced. Similar patterns were observed in mammary glands and livers of non-carcinogen-treated rats. This work identifies components of intracellular energy sensing pathways, specifically mTOR, its principal upstream regulators, AMPK and Akt, and its downstream targets, p70S6K and 4E-BP1, as candidate molecules on which to center mechanistic studies of DER.

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.

Dietary Energy Restriction in Breast Cancer Prevention

Dietary energy restriction (DER) inhibits the development of spontaneous, chemically, genetically, and virally induced mammary cancer in rats and/or mice. DER inhibits the initiation and postinitiation stages of mammary carcinogenesis and the development of both ovarian-hormone-dependent and -independent mammary carcinomas. The predominant effect of DER appears to be suppression of the clonal expansion of transformed cells, and this effect is most likely mediated via the coordinated regulation of cell proliferation, apoptosis, and angiogenesis. The effects of DER on cell cycle regulation and apoptosis are consistent with the limitation of one or more cell survival factors. Evidence is presented that the chemical mediators of this effect, glucocorticoids, insulin, and/or insulin-like growth factors, are elicited in response to the limitation in glucose availability imposed by DER. Investigation of DER is highly relevant to the misregulation of body weight which has been identified as a human health problem of global proportion. Mechanistic studies hold the promise of leading to the identification of DER mimetic approaches that can be used in the prevention and control of breast cancer.

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.

Effects of dietary energy repletion and IGF-1 infusion on the inhibition of mammary carcinogenesis by dietary energy restriction

Dietary energy restriction (DER) is a potent inhibitor of mammary carcinogenesis, but the responsible mechanisms are not fully understood. In a number of model systems, DER is associated with a decrease in circulating levels of IGF‐1. Moreover, we have recently reported that protection against cancer is lost, and plasma IGF‐1 levels are restored to control values when animals are re‐fed, i.e., energy repleted (DER‐REP). Accordingly, an experiment was designed to determine if infusion of IGF‐1 could mimic the effect of DER‐REP on the carcinogenic response in animals that were DER. Following 1‐methyl‐1‐nitrosourea injection (50 mg/kg), rats were fed either ad libitum (AL) or 40% DER. After 6 wk, the DER group was divided into three groups: (1) continued DER, (2) DER‐REP, or (3) continued DER and infused with 120 μg rh‐IGF‐1/d (INF) for a duration of 8 d. DER reduced mammary cancer incidence and multiplicity (P < 0.01) versus AL rats. In rats that were DER‐REP, cancer incidence increased 1.4‐fold and multiplicity increased by 3.6‐fold versus DER rats. Plasma IGF‐1 were reduced by DER (P < 0.01), an effect that was reversed by DER‐REP (P < 0.05). INF increased plasma IGF‐1 versus DER rats (P < 0.01) but did not reverse the carcinogenic response. Plasma IGFBP‐3 levels were reduced by DER (P < 0.01), but elevated by either REP or INF. Thus, an 8‐d period of refeeding following chronic DER (DER‐REP) reversed the anticancer effects of DER, and 8 d of IGF‐1 infusion without refeeding (INF) did not mimic the effects of the DER‐REP on the carcinogenic response.

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. (Cancer Epidemiol Biomarkers Prev 2008;17(8):1920–9)

Mechanisms by which energy restriction inhibits carcinogenesis.

Cancer that occurs at numerous organ sites, including the colon and breast, is inhibited by energy restriction, and the inhibition is proportional to the degree of restriction imposed. In an effort to identify the mechanism(s) by which energy restriction exerts this effect, a short term model system of experimentally induced mammary carcinogenesis was used. Given that carcinogenesis is known to involve a dysregulation to tissue size homeostasis in which cell proliferation and cell death are in dysequilibrium, we hypothesized that energy restriction exerts its effect by altering one or more aspects of cell cycle regulation. It was observed that energy restriction inhibited cell proliferation and increased cell death due to apoptosis. Thus attention was next focused on aspects of cell cycle regulation that might be affected by energy restriction. It was observed that the amount of p27 protein, one member of the Cip/Kip family of genes that are involved in cell cycle arrest, was increased dose dependently by energy restriction. Based on this and related observations, the hypothesis is advanced that energy restriction inhibits carcinogenesis, at least in part, by delaying cell cycle progression via shifting cell populations into a G(0)/G(1)state. Ongoing work indicates that corticosteroids, which are produced in increased amounts in response to energy restriction, may be involved in mediating this effect.

Identification of a Molecular Signature Underlying Inhibition of Mammary Carcinoma Growth by Dietary N-3 Fatty Acids

An increased ratio of dietary n-3 relative to n-6 fatty acids has been shown to inhibit the development of mammary cancer in animal models. However, the molecular mechanisms by which n-3 fatty acids affect tumor growth remain unknown. Here, we investigated the effects of varying dietary ratios of n-3:n-6 fatty acids on cell signaling in a rat model of chemically induced mammary carcinoma. Cell proliferation was reduced by 60% in carcinomas from the high n-3:n-6 treatment group compared with the low n-3:n-6 treatment group. These changes were associated with decreased cyclin-D1 and phospho-retinoblastoma protein expression and increased levels of cyclin-dependent kinase inhibitors, CIP1 (p21) and KIP1 (p27). In addition, the apoptotic index was increased in carcinomas from the high n-3:n-6 group and was associated with elevated apoptotic protease-activating factor 1 and a higher ratio of Bax/Bcl-2. Interestingly, changes in protein expression were consistent with reduced inflammation and suppressed mTOR activity, and the molecular signature associated with high n-3:n-6 treatment revealed changes in PPARγ activation and suppression of lipid synthesis. Together, our findings indicate that the molecular effects of high dietary n-3 to n-6 ratios are heterogeneous in nature but point to consistent changes in lipid metabolism pathways, which may serve as potential therapeutic targets for cancer prevention and control. This study identifies the pathways modulated by dietary fatty acid ratios in a rat model of breast cancer, with implications for cancer prevention.

Effect of energy restriction on the expression of cyclin D1 and p27 during premalignant and malignant stages of chemically induced mammary carcinogenesis.

The restriction of energy intake has a profound inhibitory effect on carcinogenesis, yet the mechanism or mechanisms that account for this effect are unknown. In this experiment, the hypothesis tested was that energy restriction upregulates the expression of p27/kip1, a gene product associated with cell‐cycle growth arrest, while downregulating cyclin D1, a protein that combines with cyclin‐dependent kinases to promote phosphorylation of retinoblastoma protein and the progression of cells through the cell cycle. We studied levels of these proteins in uninvolved mammary epithelial cells and in mammary intraductal proliferations, ductal carcinomas in situ, and adenocarcinomas induced in response to administration of 1‐methyl‐1‐nitrosourea in animals fed either ad libitum or 90%, 80%, or 60% of ad libitum intake. Protein levels were evaluated immunohistochemically by using computer‐assisted image analysis to quantify differences in protein expression among treatment groups. The expression of p27 increased and the expression of cyclin D1 decreased dose‐dependently in response to energy restriction. The effect was greater on p27 than on cyclin D1. The hypothesis proposed is that energy restriction inhibits carcinogenesis by arresting cell‐cycle progression by regulating p27/kip1. Mol. Carcinog. 24:241–245, 1999.

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 ∼2-fold higher in carcinomas from ER rats (P ≤ 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.