ß-Hydroxy-ß-Methylbutyrate Supplementation Promotes Antitumor Immunity in an Obesity Responsive Mouse Model of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States, and effective therapies for PDAC are currently lacking. Moreover, PDAC is promoted and exacerbated by obesity, while cachexia and sarcopenia are exceptionally common comorbidities that predict both poor survival and treatment response. Managing PDAC with immunotherapies has thus far proven ineffective, partly due to the metabolically hostile tumor microenvironment. ß-hydroxy-ß-methylbutyrate (HMB), a metabolite of leucine commonly used as a dietary supplement to boost muscle growth and immune function, may be an attractive candidate to augment PDAC therapy. We therefore sought to test the hypothesis that HMB would enhance antitumor immunity while protecting mouse muscle mass. Control and diet-induced obese C57BL/6 male mice bearing subcutaneously injected Panc02 tumors were supplemented with 1% HMB and treated with or without 50 mg/kg gemcitabine (n = 15/group). HMB was associated with reduced muscle inflammation and increased muscle fiber size. HMB also reduced tumor growth and promoted antitumor immunity in obese, but not lean, mice, independent of the gemcitabine treatment. Separately, in lean tumor-bearing mice, HMB supplementation promoted an anti-PD1 immunotherapy response (n = 15/group). Digital cytometry implicated the decreased abundance of M2-like macrophages in PDAC tumors, an effect that was enhanced by anti-PD1 immunotherapy. We confirmed that HMB augments M1-like macrophage (antitumor) polarization. These preclinical findings suggest that HMB has muscle-sparing and antitumor activities against PDAC in the context of obesity, and that it may sensitize otherwise nonresponsive PDAC to immunotherapy.

Circadian rhythms and the gut microbiome synchronize the host's metabolic response to diet.

The molecular circadian clock and symbiotic host-microbe relationships both evolved as mechanisms that enhance metabolic responses to environmental challenges. The gut microbiome benefits the host by breaking down diet-derived nutrients indigestible by the host and generating microbiota-derived metabolites that support host metabolism. Similarly, cellular circadian clocks optimize organismal physiology to the environment by influencing the timing and coordination of metabolic processes. Host-microbe interactions are influenced by dietary quality and timing, as well as daily light/dark cycles that entrain circadian rhythms in the host. Together, the gut microbiome and the molecular circadian clock play a coordinated role in neural processing, metabolism, adipogenesis, inflammation, and disease initiation and progression. This review examines the bidirectional interactions between the circadian clock, gut microbiota, and host metabolic systems and their effects on obesity and energy homeostasis. Directions for future research and the development of therapies that leverage these systems to address metabolic disease are highlighted.

Starving cancer from the outside and inside: separate and combined effects of calorie restriction and autophagy inhibition on Ras-driven tumors.

BACKGROUND: Calorie restriction (CR) prevents obesity and exerts anticancer effects in many preclinical models. CR is also increasingly being used in cancer patients as a sensitizing strategy prior to chemotherapy regimens. While the beneficial effects of CR are widely accepted, the mechanisms through which CR affects tumor growth are incompletely understood. In many cell types, CR and other nutrient stressors can induce autophagy, which provides energy and metabolic substrates critical for cancer cell survival. We hypothesized that limiting extracellular and intracellular substrate availability by combining CR with autophagy inhibition would reduce tumor growth more effectively than either treatment alone. RESULTS: A 30 % CR diet, relative to control diet, in nude mice resulted in significant decreases in body fat, blood glucose, and serum insulin, insulin-like growth factor-1, and leptin levels concurrent with increased adiponectin levels. In a xenograft model in nude mice involving H-Ras(G12V)-transformed immortal baby mouse kidney epithelial cells with (Atg5 (+/+) ) and without (Atg5 (-/-)) autophagic capacity, the CR diet (relative to control diet) genetically induced autophagy inhibition and their combination, each reduced tumor development and growth. Final tumor volume was greatest for Atg5 (+/+) tumors in control-fed mice, intermediate for Atg5 (+/+) tumors in CR-fed mice and Atg5 (-/-) tumors in control-fed mice, and lowest for Atg5 (-/-) tumors in CR mice. In Atg5 (+/+) tumors, autophagic flux was increased in CR-fed relative to control-fed mice, suggesting that the prosurvival effects of autophagy induction may mitigate the tumor suppressive effects of CR. Metabolomic analyses of CR-fed, relative to control-fed, nude mice showed significant decreases in circulating glucose and amino acids and significant increases in ketones, indicating CR induced negative energy balance. Combining glucose deprivation with autophagy deficiency in Atg5 (-/-) cells resulted in significantly reduced in vitro colony formation relative to glucose deprivation or autophagy deficiency alone. CONCLUSIONS: Combined restriction of extracellular (via CR in vivo or glucose deprivation in vitro) and intracellular (via autophagy inhibition) sources of energy and nutrients suppresses Ras-driven tumor growth more effectively than either CR or autophagy deficiency alone. Interventions targeting both systemic energy balance and tumor-cell intrinsic autophagy may represent a novel and effective anticancer strategy.

Stage-Specific MicroRNAs and Their Role in the Anticancer Effects of Calorie Restriction in a Rat Model of ER-Positive Luminal Breast Cancer.

MicroRNAs have emerged as ubiquitous post-transcriptional regulators that coordinate many fundamental processes within cells, including those commonly linked to cancer when dysregulated. Profiling microRNAs across stages of cancer progression provides focus as to which microRNAs are key players in cancer development and are therefore important to manipulate with interventions to delay cancer onset and progression. Calorie restriction is one of the most effective preventive interventions across many types of cancer, although its effects on microRNAs have not been well characterized. We used the dimethylbenz[a]-anthracene-induced model of luminal mammary cancer in Sprague Dawley rats to elucidate which microRNAs are linked to progression in this type of cancer and, subsequently, to study how calorie restriction affects such microRNAs. We identified eight microRNAs (miR-10a, miR-10b, miR-21, miR-124, miR-125b, miR-126, miR-145 and miR-200a) to be associated with DMBA-induced mammary tumor progression. Calorie restriction, which greatly increased tumor-free survival and decreased the overall size of tumors that did develop, significantly decreased the expression of one microRNA, miR-200a, which was positively associated with tumor progression. We further showed that inhibition of miR-200a function, mimicking the effect of calorie restriction on this microRNA, inhibited proliferation in both rat (LA7) and human (MCF7) luminal mammary cancer cell lines. These findings present, for the first time, a stage-specific profile of microRNAs in a rodent model of luminal mammary cancer. Furthermore, we have identified the regulation of miR-200a, a microRNA that is positively associated with progression in this model, as a possible mechanism contributing to the anticancer effects of calorie restriction.

Synthesis, Characterization, and In Vitro and In Vivo Evaluations of 4-(N)-Docosahexaenoyl 2', 2'-Difluorodeoxycytidine with Potent and Broad-Spectrum Antitumor Activity.

In this study, a new compound, 4-(N)-docosahexaenoyl 2', 2'-difluorodeoxycytidine (DHA-dFdC), was synthesized and characterized. Its antitumor activity was evaluated in cell culture and in mouse models of pancreatic cancer. DHA-dFdC is a poorly soluble, pale yellow waxy solid, with a molecular mass of 573.3Da and a melting point of about 96°C. The activation energy for the degradation of DHA-dFdC in an aqueous Tween 80-based solution is 12.86kcal/mol, whereas its stability is significantly higher in the presence of vitamin E. NCI-60 DTP Human Tumor Cell Line Screening revealed that DHA-dFdC has potent and broad-spectrum antitumor activity, especially in leukemia, renal, and central nervous system cancer cell lines. In human and murine pancreatic cancer cell lines, the IC50 value of DHA-dFdC was up to 10(5)-fold lower than that of dFdC. The elimination of DHA-dFdC in mouse plasma appeared to follow a biexponential model, with a terminal phase t1/2 of about 58minutes. DHA-dFdC significantly extended the survival of genetically engineered mice that spontaneously develop pancreatic ductal adenocarcinoma. In nude mice with subcutaneously implanted human Panc-1 pancreatic tumors, the antitumor activity of DHA-dFdC was significantly stronger than the molar equivalent of dFdC alone, DHA alone, or the physical mixture of them (1:1, molar ratio). DHA-dFdC also significantly inhibited the growth of Panc-1 tumors orthotopically implanted in the pancreas of nude mice, whereas the molar equivalent dose of dFdC alone did not show any significant activity. DHA-dFdC is a promising compound for the potential treatment of cancers in organs such as the pancreas.

Metformin and Rapamycin Reduce Pancreatic Cancer Growth in Obese Prediabetic Mice by Distinct MicroRNA-Regulated Mechanisms.

Metformin treatment is associated with a decreased risk and better prognosis of pancreatic cancer (PC) in patients with type 2 diabetes, but the mechanism of metformin's PC growth inhibition in the context of a prediabetic state is unknown. We used a Panc02 pancreatic tumor cell transplant model in diet-induced obese (DIO) C57BL/6 mice to compare the effects of metformin and the direct mammalian target of rapamycin (mTOR) inhibitor rapamycin on PC growth, glucose regulation, mTOR pathway signaling, and candidate microRNA (miR) expression. In DIO/prediabetic mice, metformin and rapamycin significantly reduced pancreatic tumor growth and mTOR-related signaling. The rapamycin effects centered on decreased mTOR-regulated growth and survival signaling, including increased expression of let-7b and cell cycle-regulating miRs. Metformin (but not rapamycin) reduced glucose and insulin levels and expression of miR-34a and its direct targets Notch, Slug, and Snail. Metformin also reduced the number and size of Panc02 tumor spheres in vitro and inhibited the expression of Notch in spheroids. Our results suggest that metformin and rapamycin can both inhibit pancreatic tumor growth in obese, prediabetic mice through shared and distinct mechanisms. Metformin and direct mTOR inhibitors, alone or possibly in combination, represent promising intervention strategies for breaking the diabetes-PC link.

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.

Leucine supplementation differentially enhances pancreatic cancer growth in lean and overweight mice.

BACKGROUND: The risk of pancreatic cancer, the 4th deadliest cancer for both men and women in the United States, is increased by obesity. Calorie restriction (CR) is a well-known dietary regimen that prevents or reverses obesity and suppresses tumorigenesis in a variety of animal models, at least in part via inhibition of mammalian target of rapamycin (mTOR) signaling. Branched-chain amino acids (BCAA), especially leucine, activate mTOR and enhance growth and proliferation of myocytes and epithelial cells, which is why leucine is a popular supplement among athletes. Leucine is also increasingly being used as a treatment for pancreatic cancer cachexia, but the effects of leucine supplementation on pancreatic tumor growth have not been elucidated. RESULTS: Supplementation with leucine increased pancreatic tumor growth in both lean (104 ± 17 mm3 versus 46 ± 13 mm3; P <0.05) and overweight (367 ± 45 mm3 versus 230 ± 39 mm3; P <0.01) mice, but tumor enhancement was associated with different biological outcomes depending on the diet. In the lean mice, leucine increased phosphorylation of mTOR and downstream effector S6 ribosomal protein, but in the overweight mice, leucine reduced glucose clearance and thus increased the amount of circulating glucose available to the tumor. CONCLUSIONS: These findings show that leucine supplementation enhances tumor growth in both lean and overweight mice through diet-dependent effects in a murine model of pancreatic cancer, suggesting caution against the clinical use of leucine supplementation for the purposes of skeletal muscle enhancement in cachectic patients.

Interacting inflammatory and growth factor signals underlie the obesity-cancer link.

The prevalence of obesity, an established risk factor for many chronic diseases (including diabetes, cardiovascular disease, stroke, and several types of cancer), has risen steadily for the past several decades in the United States and many parts of the world. Today, ∼70% of U.S. adults and 30% of children are at an unhealthy weight. The evidence on key biologic mechanisms underlying the obesity-cancer link, with an emphasis on local and systemic inflammatory processes and their crosstalk with energy-sensing growth factor signaling pathways, will be discussed. Understanding the influence and underlying mechanisms of obesity on chronic inflammation and cancer will identify promising mechanistic targets and strategies for disrupting the obesity-cancer link and provide important lessons regarding the associations between obesity, inflammation, and other chronic diseases.

Calorie restriction and cancer prevention: a mechanistic perspective.

Calorie restriction (CR) is one of the most potent broadly acting dietary interventions for inducing weight loss and for inhibiting cancer in experimental models. Translation of the mechanistic lessons learned from research on CR to cancer prevention strategies in human beings is important given the high prevalence of excess energy intake, obesity, and metabolic syndrome in many parts of the world and the established links between obesity-associated metabolic perturbations and increased risk or progression of many types of cancer. This review synthesizes findings on the biological mechanisms underlying many of the anticancer effects of CR, with emphasis on the impact of CR on growth factor signaling pathways, inflammation, cellular and systemic energy homeostasis pathways, vascular perturbations, and the tumor microenvironment. These CR-responsive pathways and processes represent targets for translating CR research into effective cancer prevention strategies in human beings.

Deconvoluting the obesity and breast cancer link: secretome, soil and seed interactions.

Obesity is associated with increased risk of breast cancer in postmenopausal women and is linked with poor prognosis in pre- and postmenopausal breast cancer patients. The mechanisms underlying the obesity-breast cancer connection are becoming increasingly clear and provide multiple opportunities for primary to tertiary prevention. Several obesity-related host factors can influence breast tumor initiation, progression and/or response to therapy, and these have been implicated as key contributors to the complex effects of obesity on cancer incidence and outcomes. These host factors include components of the secretome, including insulin, insulin-like growth factor-1, leptin, adiponectin, steroid hormones, cytokines, vascular regulators, and inflammation-related molecules, as well as the cellular and structural components of the tumor microenvironment. These secreted and structural host factors are extrinsic to, and interact with, the intrinsic molecular characteristics of breast cancer cells (including breast cancer stem cells), and each will be considered in the context of energy balance and as potential targets for cancer prevention.

Mechanistic targets and phytochemical strategies for breaking the obesity-cancer link.

The prevalence of obesity, an established risk and progression factor for many cancers, has increased dramatically in many countries over the past three decades. Worldwide, an estimated 600 million adults are currently obese. Thus, a better understanding of the mechanistic links between obesity and cancer is urgently needed to identify intervention targets and strategies to offset the procancer effects of obesity. This review synthesizes the evidence on key biological mechanisms underlying the obesity-cancer association, with particular emphasis on obesity-associated enhancements in growth factor signaling, inflammation, and perturbations in the tumor microenvironment. These interrelated pathways and processes that are aberrantly regulated in obese individuals represent mechanism-based targets for disrupting the obesity-cancer link using phytochemicals.

Dietary energy balance modulation of Kras- and Ink4a/Arf+/--driven pancreatic cancer: the role of insulin-like growth factor-I.

New molecular targets and intervention strategies for breaking the obesity-pancreatic cancer link are urgently needed. Using relevant spontaneous and orthotopically transplanted murine models of pancreatic cancer, we tested the hypothesis that dietary energy balance modulation impacts pancreatic cancer development and progression through an insulin-like growth factor (IGF)-I-dependent mechanism. In LSL-Kras(G12D)/Pdx-1-Cre/Ink4a/Arf(lox/+) mice, calorie restriction versus overweight- or obesity-inducing diet regimens decreased serum IGF-I, tumoral Akt/mTOR signaling, pancreatic desmoplasia, and progression to pancreatic ductal adenocarcinoma (PDAC), and increased pancreatic tumor-free survival. Serum IGF-I, Akt/mTOR signaling, and orthotopically transplanted PDAC growth were decreased in liver-specific IGF-I-deficient mice (vs. wild-type mice), and rescued with IGF-I infusion. Thus, dietary energy balance modulation impacts spontaneous pancreatic tumorigenesis induced by mutant Kras and Ink4a deficiency, the most common genetic alterations in human pancreatic cancer. Furthermore, IGF-I and components of its downstream signaling pathway are promising mechanistic targets for breaking the obesity-pancreatic cancer link.

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-1ß, 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.

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.

Rapamycin partially mimics the anticancer effects of calorie restriction in a murine model of pancreatic cancer.

Etiologic factors for pancreatic cancer, the 4th deadliest malignant neoplasm in the United States, include obesity and abnormal glucose metabolism. Calorie restriction (CR) and rapamycin each affect energy metabolism and cell survival pathways via inhibition of mammalian target of rapamycin (mTOR) signaling. By using a Panc02 murine pancreatic cancer cell transplant model in 45 male C57BL/6 mice, we tested the hypothesis that rapamycin mimics the effects of CR on pancreatic tumor growth. A chronic regimen of CR, relative to an ad libitum-fed control diet, produced global metabolic effects such as reduced body weight (20.6 ± 1.6 g vs. 29.3 ± 2.3 g; P < 0.0001), improved glucose responsiveness, and decreased circulating levels of insulin-like growth factor (IGF)-1 (126 ± 8 ng/mL vs. 199 ± 11 ng/mL; P = 0.0006) and leptin (1.14 ± 0.2 ng/mL vs. 5.05 ± 1.2 ng/mL; P = 0.01). In contrast, rapamycin treatment (2.5 mg/kg intraperitoneal every other day, initiated in mice following 20 weeks of ad libitum control diet consumption), relative to control diet, produced no significant change in body weight, IGF-1 or leptin levels, but decreased glucose responsiveness. Pancreatic tumor volume was significantly reduced in the CR group (221 ± 107 mm(3); P < 0.001) and, to a lesser extent, the rapamycin group (374 ± 206 mm(3); P = 0.04) relative to controls (550 ± 147 mm(3)), and this differential inhibition correlated with expression of the proliferation marker Ki-67. Both CR and rapamycin decreased phosphorylation of mTOR, p70/S6K, and S6 ribosomal protein, but only CR decreased phosphorylation of Akt, GSK-3ß, extracellular signal regulated kinase/mitogen-activated protein kinase, and STAT3(TYR705). These findings suggest that rapamycin partially mimics the anticancer effects of CR on tumor growth in a murine model of pancreatic cancer.

Genetic reduction of insulin-like growth factor-1 mimics the anticancer effects of calorie restriction on cyclooxygenase-2-driven pancreatic neoplasia.

Risk of pancreatic cancer, the fourth deadliest cancer in the United States, is increased by obesity. Calorie restriction (CR) prevents obesity, suppresses carcinogenesis in many models, and reduces serum levels of IGF-1. In the present study, we examined the impact of CR on a model of inflammation-associated pancreatitis and pancreatic dysplasia, with a focus on the mechanistic contribution of systemic IGF-1. Administration of a 30% CR diet for 14 weeks decreased serum IGF-1 levels and hindered pancreatic ductal lesion formation and dysplastic severity, relative to a higher calorie control diet, in transgenic mice overexpressing COX-2 [bovine keratin-5 promoter (BK5.COX-2)]. These findings in CR mice correlated with reductions in Ki-67-positive cells, vascular luminal size, VEGF expression, and phosphorylation and total expression of downstream mediators of the IGF-1 pathway. Cell lines derived from BK5.COX-2 ductal lesions (JC101 cells) formed pancreatic tumors in wild-type FVB mice that were significantly reduced in size by a 14-week CR regimen, relative to the control diet. To further understand the impact of circulating levels of IGF-1 on tumor growth in this model, we orthotopically injected JC101 cells into liver-specific IGF-1-deficient (LID) mice. The approximate 65% reduction of serum IGF-1 levels in LID mice resulted in significantly decreased burden of JC101 tumors, despite modestly elevated levels of circulating insulin and leptin. These data show that CR prevents development of dysplasia and growth of pancreatic cancer through alterations in IGF-1, suggesting that modulation of this pathway with dietary and/or pharmacologic interventions is a promising pancreatic cancer prevention strategy.

Smac mimetic reverses resistance to TRAIL and chemotherapy in human urothelial cancer cells.

PURPOSE: inhibitors of apoptosis proteins (IAPs) have been shown to contribute to resistance of neoplastic cells to chemotherapy and to biologic antineoplastic agents. Consequently, new agents are being developed targeting this family of proteins. In a panel of bladder cancer cell lines, we evaluated a Smac mimetic that antagonizes several IAPs for its suitability for bladder cancer therapy. Experimental design: A panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-A alone, TRAIL alone, chemotherapy alone, compound-A plus TRAIL, and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally, siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting. RESULTS: single-agent compound-A had little effect, but compound-A augmented TRAIL- and chemotherapy-induced apoptosis. Immunoblotting showed that combination treatment with compound-A and TRAIL resulted in cleavage of procaspase-3 and procaspase-7, activation of which irreversibly commits cells to apoptosis. Immunoprecipitation of XIAP showed displacement of active caspase-3 fragments from XIAP, supporting the proposed mechanism of action. Furthermore, siRNA-mediated silencing of XIAP similarly sensitized these cells to apoptosis. EXPERIMENTAL DESIGN: a panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-Alone, TRAIL alone, Chemotherapy alone, compound-A plus TRAIL and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting. CONCLUSION: our results suggest that targeting of XIAP with the Smac mimetic compound-A has the potential to augment the effects of a variety of chemotherapeutic and biologic therapies in bladder cancer.

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.

Reducing the weight of cancer: mechanistic targets for breaking the obesity-carcinogenesis link.

The prevalence of obesity, an established epidemiologic risk factor for many cancers, has risen steadily for the past several decades in the US. The increasing rates of obesity among children are especially alarming and suggest continuing increases in the rates of obesity-related cancers for many years to come. Unfortunately, the mechanisms underlying the association between obesity and cancer are not well understood. In particular, the effects on the carcinogenesis process and mechanistic targets of interventions that modulate energy balance, such as reduced-calorie diets and physical activity, have not been well characterized. The purpose of this review is to provide a strong foundation for the translation of mechanism-based research in this area by describing key animal and human studies of energy balance modulations involving diet or physical activity and by focusing on the interrelated pathways affected by alterations in energy balance. Particular attention is placed on signaling through the insulin and insulin-like growth factor-1 receptors, including components of the Akt and mammalian target of rapamycin (mTOR) signaling pathways downstream of these growth factor receptors. These pathways have emerged as potential targets for disrupting the obesity-cancer link. The ultimate goal of this work is to provide the missing mechanistic information necessary to identify targets for the prevention and control of cancers related to or caused by excess body weight.