Alison E. Harvey

Calories and carcinogenesis: lessons learned from 30 years of calorie restriction research

Calorie restriction (CR) is arguably the most potent, broadly acting dietary regimen for suppressing the carcinogenesis process, and many of the key studies in this field have been published in Carcinogenesis. Translation of the knowledge gained from CR research in animal models to cancer prevention strategies in humans is urgently needed given the worldwide obesity epidemic and the established link between obesity and increased risk of many cancers. This review synthesizes the evidence on key biological mechanisms underlying many of the beneficial effects of CR, with particular emphasis on the impact of CR on growth factor signaling pathways and inflammatory processes and on the emerging development of pharmacological mimetics of CR. These approaches will facilitate the translation of CR research into effective strategies for cancer prevention in humans.

The growing challenge of obesity and cancer: an inflammatory issue

The prevalence of obesity, an established risk factor for many cancers, has risen steadily for the past several decades in the United States and in many parts of the world. This review synthesizes the evidence on key biological mechanisms underlying the obesity–cancer link, with particular emphasis on the impact of energy balance modulation, such as diet‐induced obesity and calorie restriction, on growth factor signaling pathways and inflammatory processes. Particular attention is placed on the proinflammatory environment associated with the obese state, specifically highlighting the involvement of obesity‐associated hormones/growth factors in crosstalk between macrophages, adipocytes, and epithelial cells in many cancers. Understanding the contribution of obesity to growth factor signaling and chronic inflammation provides mechanistic targets for disrupting the obesity–cancer link.

Differential susceptibility to obesity between male, female and ovariectomized female mice

BackgroundThe prevalence of obesity has increased dramatically. A direct comparison in the predisposition to obesity between males, premenopausal females, and postmenopausal females with various caloric intakes has not been made. To determine the effects of sex and ovarian hormones on the susceptibility to obesity, we conducted laboratory studies with mice. To eliminate confounders that can alter body weight gain, such as age and food consumption; we used mice with the same age and controlled the amount of calories they consumed.MethodsWe determined sex-specific susceptibility to obesity between male, non-ovariectomized female, and ovariectomized female mice. To compare susceptibility to gaining body weight between males and females, animals from each sex were exposed to either a 30% calorie-restricted, low-fat (5% fat), or high-fat (35% fat) diet regimen. To establish the role of ovarian hormones in weight gain, the ovaries were surgically removed from additional female mice, and then were exposed to the diets described above. Percent body fat and percent lean mass in the mice were determined by dual energy x-ray absorptiometry (DEXA).ResultsIn all three diet categories, male mice had a greater propensity of gaining body weight than female mice. However, ovariectomy eliminated the protection of female mice to gaining weight; in fact, ovariectomized female mice mimicked male mice in their susceptibility to weight gain. In summary, results show that male mice are more likely to become obese than female mice and that the protection against obesity in female mice is eliminated by ovariectomy.ConclusionUnderstanding metabolic differences between males and females may allow the discovery of better preventive and treatment strategies for diseases associated with body weight such as cancer and cardiovascular disease.

Energy Balance Modulates Colon Tumor Growth: Interactive Roles of Insulin and Estrogen

Obesity increases colorectal cancer (CRC) risk and progression. However, the impact of obesity on CRC in women is dependent on ovarian hormone status. The purpose of this study was to determine the interactive roles of obesity and ovarian hormones on serum markers of inflammation, cell signaling, and transplanted colon tumor growth. Female C57BL/6 mice (6 wk) were either ovariectomized (OVX) or ovaries left intact (nonovariectomized, NOVX) and randomized to receive a (1) control, (2) 30% calorie‐restricted (CR), or (3) diet‐induced obese (DIO) diet regimen for 20 wk to induce differing levels of adiposity. Serum was collected and inflammatory and metabolic markers were measured using an antibody array (62 proteins) and ELISAs. Mice were subcutaneously injected with syngeneic MC38 colon cancer cells after 20 wk and sacrificed 4 wk later. CR mice had the smallest tumors irrespective of hormone status, whereas the largest tumors were observed in DIO‐OVX mice. Glucose tolerance was impaired in OVX mice, being most severe in the DIO‐OVX group. Cytokine arrays suggested that in CR animals, inhibition of tumor growth paralleled insulin sensitivity and associated changes in leptin, adiponectin, and IGF‐BPs. Conversely, in DIO‐OVX animals, tumor growth was associated with insulin and leptin resistance as well as higher levels of pro‐inflammatory proteins. In vitro, leptin and adiponectin had no effect, whereas insulin induced MC38 cell proliferation and MAPK activation. Co‐treatment with estrogen blocked the stimulatory effects of insulin. Thus, our in vitro and in vivo data indicate female reproductive hormones have a modulating effect on obesity‐induced insulin resistance and inflammation, which may directly or indirectly influence CRC progression. ©2010 Wiley‐Liss, Inc.

Calorie restriction decreases murine and human pancreatic tumor cell growth, nuclear factor-κB activation, and inflammation-related gene expression in an insulin-like growth factor-1 - Dependent manner

Calorie restriction (CR) prevents obesity and has potent anticancer effects that may be mediated through its ability to reduce serum growth and inflammatory factors, particularly insulin-like growth factor (IGF)-1 and protumorigenic cytokines. IGF-1 is a nutrient-responsive growth factor that activates the inflammatory regulator nuclear factor (NF)-κB, which is linked to many types of cancers, including pancreatic cancer. We hypothesized that CR would inhibit pancreatic tumor growth through modulation of IGF-1-stimulated NF-κB activation and protumorigenic gene expression. To test this, 30 male C57BL/6 mice were randomized to either a control diet consumed ad libitum or a 30% CR diet administered in daily aliquots for 21 weeks, then were subcutaneously injected with syngeneic mouse pancreatic cancer cells (Panc02) and tumor growth was monitored for 5 weeks. Relative to controls, CR mice weighed less and had decreased serum IGF-1 levels and smaller tumors. Also, CR tumors demonstrated a 70% decrease in the expression of genes encoding the pro-inflammatory factors S100a9 and F4/80, and a 56% decrease in the macrophage chemoattractant, Ccl2. Similar CR effects on tumor growth and NF-κB-related gene expression were observed in a separate study of transplanted MiaPaCa-2 human pancreatic tumor cell growth in nude mice. In vitro analyses in Panc02 cells showed that IGF-1 treatment promoted NF-κB nuclear localization, increased DNA-binding of p65 and transcriptional activation, and increased expression of NF-κB downstream genes. Finally, the IGF-1-induced increase in expression of genes downstream of NF-κB (Ccdn1, Vegf, Birc5, and Ptgs2) was decreased significantly in the context of silenced p65. These findings suggest that the inhibitory effects of CR on Panc02 pancreatic tumor growth are associated with reduced IGF-1-dependent NF-κB activation.

Decreased systemic IGF‐1 in response to calorie restriction modulates murine tumor cell growth, nuclear factor‐κB activation, and inflammation‐related gene expression

Calorie restriction (CR) prevents obesity and has potent anticancer effects associated with altered hormones and cytokines. We tested the hypothesis that CR inhibits MC38 mouse colon tumor cell growth through modulation of hormone‐stimulated nuclear factor (NF)‐κB activation and protumorigenic gene expression. Female C57BL/6 mice were randomized (n = 30/group) to receive control diet or 30% CR diet. At 20 wk, 15 mice/group were killed for body composition analysis. At 21 wk, serum was obtained for hormone analysis. At 22 wk, mice were injected with MC38 cells; tumor growth was monitored for 24 d. Gene expression in excised tumors and MC38 cells was analyzed using real‐time RT‐PCR. In vitro MC38 NF‐κB activation (by p65 ELISA and immunofluorescence) were measured in response to varying IGF‐1 concentrations (1–400 ng/mL). Relative to controls, CR mice had decreased tumor volume, body weight, body fat, serum IGF‐1, serum leptin, and serum insulin, and increased serum adiponectin (P < 0.05, each). Tumors from CR mice, versus controls, had downregulated inflammation‐ and/or cancer‐related gene expression, including interleukin (IL)‐6, IL‐1β, tumor necrosis factor‐α, cyclooxygenase‐2, chemokine (C–C motif) ligand‐2, S100A9, and F4/80, and upregulated 15‐hydroxyprostaglandin dehydrogenase expression. In MC38 cells in vitro, IGF‐1 increased NF‐κB activation and NF‐κB downstream gene expression (P < 0.05, each). We conclude that CR, in association with reduced systemic IGF‐1, modulates MC38 tumor growth, NF‐κB activation, and inflammation‐related gene expression. Thus, IGF‐1 and/or NF‐κB inhibition may pharmacologically mimic the anticancer effects of CR to break the obesity–colon cancer link.

Ethanol Consumption Does Not Promote Weight Gain in Female Mice

Background: The prevalence of obese adult women has increased dramatically in the United States. Individuals consuming alcoholic beverages may obtain as much as 6–10% of their calories from ethanol; consequently, ethanol may contribute to a positive energy balance and weight gain in women consuming ethanol. The objective of these studies is to determine if ethanol consumption affects weight gain or body fat levels in female mice. Methods: In order to determine the effects of ethanol consumption on weight gain, female mice were given either water or 20% w/v ethanol in the drinking water; mice were then placed on 1 of 3 diets for 20 weeks: (1) 30% calorie-restricted diet, (2) low-fat diet or (3) high-fat diet. Mice were scanned using a GE Lunar Piximus Densitometer to determine body fat, lean body mass and bone mineral density. Results: Mice consuming the high-fat diet had the highest body weight. Moreover, ovariectomy exacerbated the effects of the high-fat diet. That is, ovariectomized female mice consuming the high-fat diet gained a higher amount of body weight and adipose tissue than nonovariectomized mice consuming the high-fat diet. Ethanol-consuming mice did not have a higher susceptibility to gaining body weight or body fat, even though they tended to have higher caloric intake than water-consuming mice. Conclusions: In female mice that consumed a high-fat diet, chronic ethanol consumption did not increase susceptibility to gaining weight or becoming obese.

Calories and Cancer: The Role of Insulin-Like Growth Factor-1

The poem “On the Nature of Things,” written around 55 bc by Titus Lucretius Carus, is considered to be the first published statement about the potential impact of the overconsumption of food on risk of chronic diseases such as cancer (Lucretius 2008). This apparent connection between excess calorie intake and cancer began to develop into a working hypothesis in the mid-to-late 1800s, following the writings of John Hughes Bennett (1849) and William Lambe (1850). The first tests of the hypothesis that a calorie restriction (CR) dietary regimen can suppress tumors in animal models were reported in 1909 by Moreschi (1909) and extended by Sweet et al. in (1913) and Peyton Rous in (1914). These investigators showed that a low-calorie diet, relative to ad libitum (AL)-fed controls, inhibited the growth of transplanted tumors in mice. Intense interest in the comparison of CR versus AL-fed animals developed in the 1930s, when McCay and Crowell showed that reduced energy intake also increased lifespan in rodents (1934). CR research was further catalyzed by Tannenbaum et al., who consistently showed that the incidence of tumors in mice decreased when food intake was reduced (1944).

Abstract 4100: The anticancer effects of calorie restriction on Panc 02 pancreatic tumors are associated with decreased activation of NF-kB

Calorie restriction (CR), an anti-obesity dietary regimen with potent anti-cancer effects, reduces serum levels of metabolic hormones and protumorigenic cytokines. Independent of diet, metabolism-related hormones such as insulin-like growth factor (IGF)-1 and leptin have been shown to activate the inflammatory signal transduction pathway regulated by nuclear factor (NF)-κB. NF-κB is a transcription factor that is upregulated in over 70% of human pancreatic cancer cell lines and primary tumors. In this study we tested the hypothesis that CR inhibits pancreatic tumor cell growth in part through modulation of hormone-stimulated NF-κB activation and downstream protumorigenic gene expression. Six-week-old male C57BL/6 mice (n=12 per diet) were singly housed and randomized to either a control diet consumed ad libitum (which results in an overweight phenotype) or a 30% CR diet regimen for 22 weeks, at which time mice were fasted and blood was collected for serum metabolic hormone analysis. At week 23, mice were injected subcutaneously (left flank) with 1 × 10^6 syngeneic mouse pancreatic cancer cells (Panc 02) and maintained on their diet regimens while tumor growth was monitored for an additional 4 weeks. Excised tumors underwent real-time RT-PCR analysis of protumorigenic cytokine gene expression. CR mice had lower body weight (19.0 g +/− 0.4 vs. 33.0 +/− 0.6 (mean +/− SEM) p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4100.

Abstract B54: The anticancer effects of calorie restriction on MC38 colon tumors are associated with decreased macrophage infiltration

Obesity is a risk factor for a number of chronic diseases including many types of cancer. The chronic, low‐grade inflammation that develops as a result of obesity may be a primary mechanistic target underlying obesity‐associated carcinogenesis. While the link between obesity and inflammation is not well understood, increases in adipose tissue macrophages may perpetuate and exacerbate a chronic inflammatory environment through production of inflammatory cytokines such as interleukin (IL)‐6. In this study we tested the hypothesis that macrophage infiltration underlies our previously observed anticancer effects of calorie restriction (CR) in a mouse model of colon cancer. Six‐week‐old female C57BL/6 mice (n=30 per diet) were singly housed and randomized to either a control diet consumed ad libitum (which results in an overweight phenotype) or a 30% calorie‐restricted diet regimen (CR) for 22 weeks, at which time 15 mice per group were euthanized and adiposity was measured using dual‐energy X‐ray absorptiometry (DXA). The remaining 15 mice/diet were then injected subcutaneously (flank) with 50,000 syngeneic mouse colon (MC)‐38‐adenocarcinoma cells and continued on their diet regimens while tumor growth was monitored for an additional 5 weeks. Excised tumors underwent histological analysis of macrophage infiltration (hematoxylin and eosin staining) and real‐time RT‐PCR analyses of macrophage (F4/80 and S100A9) and IL‐6 gene expression. CR mice had lower body weight (20.3g +/− 0.2 vs. 26.0g +/− 0.5 (mean +/− SEM), p Citation Information: Cancer Prev Res 2010;3(1 Suppl):B54.