Overweight and obese individuals frequently restrict caloric intake to lose weight. The resultant weight loss, however, typically is followed by an equal or greater weight gain, a phenomenon called weight cycling. Most attention to weight cycling has focused on identifying its detrimental effects, but preclinical experiments indicating that intermittent caloric restriction or fasting can reduce cancer risk have raised interest in potential benefits of weight cycling. Although hypothesized adverse effects of weight cycling on energy metabolism remain largely unsubstantiated, there also is a lack of epidemiological evidence that intentional weight loss followed by regain of weight affects chronic-disease risk. In the limited studies of weight cycling and cancer, no independent effect on post-menopausal breast cancer but a modest enhancement of risk for renal cell carcinoma, endometrial cancer, and non-Hodgkin’s lymphoma have been reported. An effect of either intermittent caloric restriction or fasting in protecting against cancer is not supported by the majority of rodent carcinogenesis experiments. Collectively, the data argue against weight cycling and indicate that the objective of energy balance–based approaches to reduce cancer risk should be to strive to prevent adult weight gain and maintain body weight within the normal range defined by body mass index.
cancer risk; energetics; intermittent caloric restriction; weight cycling
Obese and sedentary persons have increased risk for cancer; inflammation is a hypothesized mechanism. We examined the effects of a caloric restriction weight loss diet and exercise on inflammatory biomarkers in 439 women. Overweight and obese postmenopausal women were randomized to 1-year: caloric restriction diet (goal of 10% weight loss, N=118), aerobic exercise (225 minutes/week of moderate-to-vigorous activity, N=117), combined diet+exercise (N=117) or control (N=87). Baseline and 1-year high-sensitivity C-reactive protein (hs-CRP), serum amyloid A (SAA), interleukin-6 (IL-6), leukocyte and neutrophil levels were measured by investigators blind to group. Inflammatory biomarker changes were compared using generalized estimating equations. Models were adjusted for baseline body mass index (BMI), race/ethnicity and age. 438 (N=1 in diet+exercise group was excluded) were analyzed. Relative to controls, hs-CRP decreased by geometric mean (95% confidence interval, p-value) 0.92mg/L (0.53–1.31, P<0.001) in the diet and 0.87mg/L (0.51–1.23, P<0.0001) in the diet+exercise groups. IL-6 decreased by 0.34pg/ml (0.13–0.55, P=0.001) in the diet and 0.32pg/ml (0.15–0.49, P<0.001) in the diet+exercise groups. Neutrophil counts decreased by 0.31×109/L (0.09–0.54, P=0.006) in the diet and 0.30×109/L (0.09–0.50, P=0.005) in the diet+exercise groups. Diet and diet+exercise participants with ≥5% weight loss reduced inflammatory biomarkers (hs-CRP, SAA, and IL-6) compared to controls. The diet and diet+exercise groups reduced hs-CRP in all subgroups of baseline BMI, waist circumference, CRP level, and fasting glucose. Our findings indicate that a caloric restriction weight loss diet with or without exercise reduces biomarkers of inflammation in postmenopausal women, with potential clinical significance for cancer risk reduction.
inflammation; postmenopausal women; obesity; exercise; dietary weight loss
Limiting energy availability via diet or physical activity has health benefits; however, it is not known if these interventions have similar effects on the development of cancer. Two questions were addressed: 1) does limiting energy availability by increasing physical activity have the same effect on mammary carcinogenesis as limiting caloric intake, and 2) are host systemic factors, implicated as risk biomarkers for breast cancer, similarly affected by these interventions? Female Sprague Dawley rats were injected with 50 mg 1-methyl-1-nitrosourea/kg body weight at 21 days of age and randomized to one of five groups (30 rats/gp): 1) sham running wheel control; 2) restricted fed to 85% of the sham-control, 3 and 4) voluntary running in a motorized activity wheel (37 m/min) to a maximum of 3500 m/day or 1750 m/day, and 5) sedentary ad libitum fed control with no access to a running wheel. The three energetics interventions inhibited the carcinogenic response, reducing cancer incidence (P=0.01), cancer multiplicity (P<0.001), and cancer burden (P<0.001), while prolonging cancer latency (P=0.004) although differences among energetics interventions were not significant. Of the plasma biomarkers associated with the development of cancer, the energetics interventions reduced bioavailable insulin-like growth factor-1(IGF-1), insulin, interleukin-6, serum amyloid protein, tumor necrosis factor-α, and leptin and increased IGF binding protein 3 (IGFBP3) and adiponection. Plasma fasting glucose, C-reactive protein, estradiol, and progesterone were unaffected. The plasma biomarkers of greatest value in predicting the carcinogenic response were: adiponectin > IGF-1/IGFBP-3 > IGFBP-3 > leptin > IGF-1.
physical activity; dietary energy restriction; mammary carcinogenesis; adipokines; insulin like growth factors; chronic inflammation
The purpose of this investigation was to identify pathways by which physical activity, implemented as running in an activity wheel, inhibits carcinogenesis. The focus of this analysis was on 20 plasma biomarkers for glucose homeostasis, inflammation, cytokine function and endocrine activity, known to be affected by a physically active lifestyle. Plasma for analysis was obtained from previously reported carcinogenesis experiments in which the effects on mammary carcinogenesis, induced by i.p. injection of 1-methyl-1-nitrosurea, of running on a motorized activity wheel or a non-motorized free wheel were compared to sedentary controls. Wheel running reduced cancer incidence (P=0.0004) and the number of cancers per animal (P=0.005). Principal components analysis (PCA) was used to reduce the 20 plasma biomarkers to a concise index that was significantly different by treatment group assignment (P<0.0001). Statistical analyses provided evidence that supported the hypothesis of a mediational role of these molecules in accounting for the protective effect of wheel running on the carcinogenic process. In addition, the plasma biomarker index derived from PCA was a good discriminator of treatment group assignment (only 4.5 % of animals were misclassified). These findings suggest that the plasma biomarkers evaluated have utility in assessing the breast cancer response to a physical activity intervention. Identification of such biomarkers is critical for clinical studies where evaluating the effects of physical activity on cancer outcomes (diagnosis, recurrence or mortality) is not possible.
Physical activity; plasma biomarkers; mammary carcinogenesis; insulin like growth factor-1; wheel running
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.
Healthy body weight is an important factor for prevention of breast cancer
recurrence. Yet, weight loss and weight gain are not currently included in
clinical-practice guidelines for posttreatment of breast cancer. The work reported
addresses one of the questions that must be considered in recommending weight loss
to patients: does it matter what diet plan is used, a question of particular
importance because breast cancer treatment can increase risk for cardiovascular
Women who completed treatment for breast cancer were enrolled in a nonrandomized,
controlled study investigating effects of weight loss achieved by using two
dietary patterns at the extremes of macronutrient composition, although both diet
arms were equivalent in protein: high fat, low carbohydrate versus low fat, high
carbohydrate. A nonintervention group served as the control arm; women were
assigned to intervention arms based on dietary preferences. During the 6-month
weight-loss program, which was menu and recipe defined, participants had monthly
clinical visits at which anthropometric data were collected and fasting blood was
obtained for safety monitoring for plasma lipid profiles and fasting glucose.
Results from 142 participants are reported.
Adverse effects on fasting blood lipids or glucose were not observed in either
dietary arm. A decrease in fasting glucose was observed with progressive weight
loss and was greater in participants who lost more weight, but the effect was not
statistically significant, even though it was observed across both diet groups
(P = 0.21). Beneficial effects of weight loss on cholesterol (4.7%;
P = 0.001), triglycerides (21.8%; P = 0.01), and low-density
lipoprotein (LDL) cholesterol (5.8%; P = 0.06) were observed in both
groups. For cholesterol (P = 0.07) and LDL cholesterol (P =
0.13), greater reduction trends were seen on the low-fat diet pattern; whereas,
for triglycerides (P = 0.01) and high-density lipoprotein (HDL)
cholesterol (P = 0.08), a decrease or increase, respectively, was greater
on the low-carbohydrate diet pattern.
Because an individual's dietary preferences can affect dietary adherence and
weight-loss success, the lack of evidence of a negative effect of dietary pattern
on biomarkers associated with cardiovascular risk is an important consideration in
the development of breast cancer practice guidelines for physicians who recommend
that their patients lose weight. Whether dietary pattern affects biomarkers that
predict long-term survival is a primary question in this ongoing clinical
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).
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.
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.
AMP-activated protein kinase; insulin; mammalian target of rapamycin; mammary carcinogenesis; metformin
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 versus72%, 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.
Rodent models have been used extensively to study mammary gland development and for studies of toxicology and carcinogenesis. Mammary gland gross morphology can visualized via the excision of intact mammary gland chains following fixation and staining with carmine using a tissue preparation referred to as a whole mount. Methods are described for the automated collection of digital images from an entire mammary gland whole mount and for the interrogation of digital data using a "masking" technique available with Image-Pro® plus image analysis software (Mediacybernetics. Silver Spring, MD).
Parallel to mammographic analysis in humans, measurements of rodent mammary gland density were derived from area-based or volume-based algorithms and included: total circumscribed mammary fat pad mass, mammary epithelial mass, and epithelium-free fat pad mass. These values permitted estimation of absolute mass of mammary epithelium as well as breast density. The biological plausibility of these measurements was evaluated in mammary whole mounts from rats and mice. During mammary gland development, absolute epithelial mass increased linearly without significant changes in mammographic density. Treatment of rodents with tamoxifen, 9-cis-retinoic acid, or ovariectomy, and occurrence of diet induced obesity decreased both absolute epithelial mass and mammographic density. The area and volumetric methods gave similar results.
Digital image analysis can be used for screening agents for potential impact on reproductive toxicity or carcinogenesis as well as for mechanistic studies, particularly for cumulative effects on mammary epithelial mass as well as translational studies of mechanisms that explain the relationship between epithelial mass and cancer risk.
Dry bean consumption has been reported to be associated with reduced risk for a number of chronic diseases including cancer. The extent to which these benefits are associated with primary versus secondary plant metabolites is not known. The work reported herein focuses on low molecular weight secondary metabolites and uses longevity extension of wild-type C. elegans nematodes as a surrogate marker for human health benefits. A modified Bligh and Dyer technique was used to extract freeze-dried bean and the resulting fractions were evaluated for longevity extension and metabolite fingerprinting using ultra performance liquid chromatography-mass spectrometry (UPLC-MS). Dry bean extracts extended adult C. elegans lifespan by as much as 16%. Hydrophilic fractions increased lifespan whereas the hydrophobic fraction induced longevity reduction. Metabolite fingerprinting revealed distinguishing spectral differences among the four chemical fractions evaluated and demonstrated that within each fraction chemical composition differed significantly based on dry bean genetic heritage.
Dry bean; Phaseolus vulgaris; C. elegans longevity; metabolomics
Genetic diversity among wild accessions and cultivars of common bean (Phaseolus vulgaris L.) has been characterized using plant morphology, seed protein allozymes, random amplified polymorphic DNA, restriction fragment length polymorphisms, DNA sequence analysis, chloroplast DNA, and microsatellite markers. Yet, little is known about whether these traits, which distinguish among genetically distinct types of common bean, can be evaluated using omics technologies.
Three 'omics' approaches: transcriptomics, proteomics, and metabolomics were used to qualitatively evaluate the diversity of common bean from two Centers of Domestication (COD). All three approaches were able to classify common bean according to their COD using unsupervised analyses; these findings are consistent with the hypothesis that differences exist in gene transcription, protein expression, and synthesis and metabolism of small molecules among common bean cultivars representative of different COD. Metabolomic analyses of multiple cultivars within two common bean gene pools revealed cultivar differences in small molecules that were of sufficient magnitude to allow identification of unique cultivar fingerprints.
Given the high-throughput and low cost of each of these 'omics' platforms, significant opportunities exist for their use in the rapid identification of traits of agronomic and nutritional importance as well as to characterize genetic diversity.
The use of architectural and morphological characteristics of cells for establishing prognostic indicators by which individual pathologies are assigned grade and stage is a well-accepted practice. Advances in automated micro- and macroscopic image acquisition and digital image analysis have created new opportunities in the field of prognostic assessment; but, one area in experimental pathology, animal models for colon cancer, has not taken advantage of these opportunities. This situation is primarily due to the methods available to evaluate the colon of the rodent for the presence of premalignant and malignant pathologies. We report a new method for the excision and processing of the entire colon of the rat and illustrate how this procedure permitted the quantitative assessment of aberrant crypt foci (ACF), a premalignant colon pathology, for characteristics consistent with progression to malignancy. ACF were detected by methylene blue staining and subjected to quantitative morphometric analysis. Colons were then restained with high iron diamine–alcian blue for assessment of mucin depletion using an image overlay to associate morphometric data with mucin depletion. The subsequent evaluation of ACF for beta-catenin staining is also demonstrated. The methods described are particularly relevant to the screening of compounds for cancer chemopreventive activity.
colon carcinogenesis; aberrant crypt foci; mucin depletion; beta-catenin; image analysis; morphometry
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.
Laser capture microdissection; Mammary epithelial cells; RNA quality
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 post initiation 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 mTOR decreased with increasing DER and down regulation of mTOR activity was verified by a decrease in the phosphorylation state of two mTOR’s targets, 70-kDa ribosomal protein S6 kinase (p70S6K) and 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 gland and in liver of non carcinogen treated rats. This work identifies components of intracellular energy sensing pathways, specifically mTOR, its principal up-stream regulators, AMPK and Akt, and its downstream targets, p70S6K and 4E-BP1 as candidate molecules on which to center mechanistic studies of DER.
dietary energy restriction; AMP-activated protein kinase; mammalian target of rapamycin; protein kinase B (Akt); mammary carcinogenesis
Genetic differences among major types of wheat are well characterized; however, little is known about how these distinctions affect the small molecule profile of the wheat seed. Ethanol/water (65% v/v) extracts of seed from 45 wheat lines representing 3 genetically distinct classes, tetraploid durum (Triticum turgidum subspecies durum) (DW) and hexaploid hard and soft bread wheat (T. aestivum subspecies aestivum) (BW) were subjected to ultraperformance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-TOF-MS). Discriminant analyses distinguished DW from BW with 100% accuracy due to differences in expression of nonpolar and polar ions, with differences attributed to sterol lipids/fatty acids and phospholipids/glycerolipids, respectively. Hard versus soft BW was distinguished with 100% accuracy by polar ions, with differences attributed to heterocyclic amines and polyketides versus phospholipid ions, respectively. This work provides a foundation for identification of metabolite profiles associated with desirable agronomic and human health traits and for assessing how environmental factors impact these characteristics.
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.
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.
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
biomarkers; dietary patterns; low fat; low carbohydrate; weight loss; breast cancer; long term survival