Ketone Bodies Induce Unique Inhibition of Tumor Cell Proliferation and Enhance the Efficacy of Anti-Cancer Agents.

The ketone bodies, sodium and lithium salts of acetoacetate (AcAc) and sodium 3-hydroxybutyrate (3-HB; commonly called beta-hydroxybutyrate) have been found to inhibit the proliferation of cancer cells. Previous studies have suggested that lithium itself may be an inhibiting agent but may be additive or synergistic with the effect of AcAc. We previously found that sodium acetoacetate (NaAcAc) inhibits the growth of human colon cancer cell line SW480. We report here similar results for several other cancer cell lines including ovarian, cervical and breast cancers. We found that NaAcAc does not kill cancer cells but rather blocks their proliferation. Similar inhibition of growth was seen in the effect of lithium ion alone (as LiCl). The effect of LiAcAc appears to be due to the combined effects of acetoacetate and the lithium ion. The ketone bodies, when given together with chemotherapeutic agents, rapamycin, methotrexate and the new peptide anti-cancer agent, PNC-27, substantially lowers their IC50 values for cancer cell, killing suggesting that ketone bodies and ketogenic diets may be powerful adjunct agents in treating human cancers.

The effect of a ketogenic diet and synergy with rapamycin in a mouse model of breast cancer.

BACKGROUND: The effects of diet in cancer, in general, and breast cancer in particular, are not well understood. Insulin inhibition in ketogenic, high fat diets, modulate downstream signaling molecules and are postulated to have therapeutic benefits. Obesity and diabetes have been associated with higher incidence of breast cancer. Addition of anti-cancer drugs together with diet is also not well studied. METHODS: Two diets, one ketogenic, the other standard mouse chow, were tested in a spontaneous breast cancer model in 34 mice. Subgroups of 3-9 mice were assigned, in which the diet were implemented either with or without added rapamycin, an mTOR inhibitor and potential anti-cancer drug. RESULTS: Blood glucose and insulin concentrations in mice ingesting the ketogenic diet (KD) were significantly lower, whereas beta hydroxybutyrate (BHB) levels were significantly higher, respectively, than in mice on the standard diet (SD). Growth of primary breast tumors and lung metastases were inhibited, and lifespans were longer in the KD mice compared to mice on the SD (p<0.005). Rapamycin improved survival in both mouse diet groups, but when combined with the KD was more effective than when combined with the SD. CONCLUSIONS: The study provides proof of principle that a ketogenic diet a) results in serum insulin reduction and ketosis in a spontaneous breast cancer mouse model; b) can serve as a therapeutic anti-cancer agent; and c) can enhance the effects of rapamycin, an anti-cancer drug, permitting dose reduction for comparable effect. Further, the ketogenic diet in this model produces superior cancer control than standard mouse chow whether with or without added rapamycin.

Synthesis and in vivo evaluation of a radiofluorinated ketone body derivative.

The ketone bodies d-beta-hydroxybutyric acid and acetoacetic acid represent the principal oxidative energy sources of most tissues when dietary glucose is scarce. An 18F-labeled ketone body could be a useful tool for studying ketone body metabolism using positron emission tomography (PET). Here, we report the first radiofluorinated ketone body derivative (3S)-4-[18F]fluoro-3-hydroxybutyric acid ([18F]FBHB) as well as its enantiomer and l-beta-hydroxybutyric acid derivative, (3R)-4-[18F]fluoro-3-hydroxybutyric acid ((R)-[18F]F3HB). PET imaging in mice showed biodistribution profiles of the radiotracers that were consistent with the biodistribution of the respective endogenous compounds. Moreover, both enantiomers visualized breast cancer xenografts in vivo. Fasting over 24 h showed significantly enhanced brain and heart uptake of [18F]FBHB and tumor uptake of (R)-[18F]F3HB. Disorders exhibiting altered energy substrate utilization, such as Alzheimer's disease, epilepsy, diabetes, and cancer may be of interest for PET imaging studies using [18F]FBHB.

A Nutritional Perspective of Ketogenic Diet in Cancer: A Narrative Review.

The predominant use of glucose anaerobically by cancer cells (Warburg effect) may be the most important characteristic the majority of these cells have in common and, therefore, a potential metabolic pathway to be targeted during cancer treatment. Because this effect relates to fuel oxidation, dietary manipulation has been hypothesized as an important strategy during cancer treatment. As such, the concept of a ketogenic diet (KD) in cancer emerged as a metabolic therapy (ie, targeting cancer cell metabolism) rather than a dietary approach. The therapeutic mechanisms of action of this high-fat, moderate-to-low protein, and very-low-carbohydrate diet may potentially influence cancer treatment and prognosis. Considering the lack of a dietetics-focused narrative review on this topic, we compiled the evidence related to the use of this diet in humans with diverse cancer types and stages, also focusing on the nutrition and health perspective. The use of KD in cancer shows potentially promising, but inconsistent, results. The limited number of studies and differences in study design and characteristics contribute to overall poor quality evidence, limiting the ability to draw evidence-based conclusions. However, the potential positive influences a KD may have on cancer treatment justify the need for well-designed clinical trials to better elucidate the mechanisms by which this dietary approach affects nutritional status, cancer prognosis, and overall health. The role of registered dietitian nutritionists is demonstrated to be crucial in planning and implementing KD protocols in oncology research settings, while also ensuring patients' adherence and optimal nutritional status.

Insulin, carbohydrate restriction, metabolic syndrome and cancer.

We propose that dietary carbohydrate restriction, particularly ketogenic diets, may provide benefit as a therapeutic or preventive strategy in cancer, alone or as an adjunct to pharmacology. The argument derives from several points of evidence: There is a close association between cancer and both diabetes and obesity. Extensive evidence shows that low carbohydrate diets are the most effective dietary treatment of Type 2 diabetes and dietary adjunct in Type 1. Such diets also target all the markers of metabolic syndrome. Finally, de facto reduction in carbohydrate likely contributes to total dietary restriction, which is effective in the prevention and treatment of cancer. The idea is consistent with recent interest in treating cancer with drugs that target diabetes. To move forward, we must understand obesity and diabetes as response to a hyperglycemic state rather than simply a cause of downstream effects.

Dietary carbohydrate restriction as the first approach in diabetes management: critical review and evidence base.

The inability of current recommendations to control the epidemic of diabetes, the specific failure of the prevailing low-fat diets to improve obesity, cardiovascular risk, or general health and the persistent reports of some serious side effects of commonly prescribed diabetic medications, in combination with the continued success of low-carbohydrate diets in the treatment of diabetes and metabolic syndrome without significant side effects, point to the need for a reappraisal of dietary guidelines. The benefits of carbohydrate restriction in diabetes are immediate and well documented. Concerns about the efficacy and safety are long term and conjectural rather than data driven. Dietary carbohydrate restriction reliably reduces high blood glucose, does not require weight loss (although is still best for weight loss), and leads to the reduction or elimination of medication. It has never shown side effects comparable with those seen in many drugs. Here we present 12 points of evidence supporting the use of low-carbohydrate diets as the first approach to treating type 2 diabetes and as the most effective adjunct to pharmacology in type 1. They represent the best-documented, least controversial results. The insistence on long-term randomized controlled trials as the only kind of data that will be accepted is without precedent in science. The seriousness of diabetes requires that we evaluate all of the evidence that is available. The 12 points are sufficiently compelling that we feel that the burden of proof rests with those who are opposed.

Small-animal research imaging devices.

The scientific study of living animals may be dated to Aristotle's original dissections, but modern animal studies are perhaps a century in the making, and advanced animal imaging has emerged only during the past few decades. In vivo imaging now occupies a growing role in the scientific research paradigm. Imaging of small animals has been particularly useful to help understand human molecular biology and pathophysiology using rodents, especially using genetically engineered mice (GEM) with spontaneous diseases that closely mimic human diseases. Specific examples of GEM models of veterinary diseases exist, but in general, GEM for veterinary research has lagged behind human research applications. However, the development of spontaneous disease models from GEM may also hold potential for veterinary research. The imaging techniques most widely used in small-animal research are CT, PET, single-photon emission CT, MRI, and optical fluorescent and luminescent imaging.

Bisected, complex N-glycans and galectins in mouse mammary tumor progression and human breast cancer.

Bisected, complex N-glycans on glycoproteins are generated by the glycosyltransferase MGAT3 and cause reduced cell surface binding of galectins. Previously, we showed that MGAT3 reduces growth factor signaling and retards mammary tumor progression driven by the Polyoma middle T antigen (PyMT) expressed in mammary epithelium under the mouse mammary tumor virus (MMTV) promoter. However, the penetrance of the tumor phenotype became variable in mixed FVB/N and C57BL/6 female mice and we therefore investigated a congenic C57BL/6 Mgat3(-/-)/MMTV-PyMT model. In the absence of MGAT3, C57BL/6 Mgat3(-/-)/MMTV-PyMT females exhibited accelerated tumor appearance and increased tumor burden, glucose uptake in tumors and lung metastasis. Nevertheless, activation of extracellular signal-regulated kinase (ERK)1/2 or protein kinase B (AKT) was reduced in ∼20-week C57BL/6 MMTV-PyMT tumors lacking MGAT3. Activation of focal adhesion kinase (FAK), protein tyrosine kinase Src, and p38 mitogen-activated protein kinase were similar to that of controls. All the eight mouse galectin genes were expressed in mammary tumors and tumor epithelial cells (TECs), but galectin-2 and -12 were not detected by western analysis in tumors, and galectin-7 was not detected in 60% of the TEC lines. From microarray data reported for human breast cancers, at least 10 galectin and 7 N-glycan N-acetylglucosaminyl (GlcNAc)-transferase (MGAT) genes are expressed in tumor tissue, and expression often varies significantly between different breast cancer subtypes. Thus, in summary, while MGAT3 and bisected complex N-glycans retard mouse mammary tumor progression, genetic background may modify this effect; identification of key galectins that promote mammary tumor progression in mice is not straightforward because all the eight galectin genes are expressed; and high levels of MGAT3, galectin-4, -8, -10, -13 and -14 transcripts correlate with better relapse-free survival in human breast cancer.

Fructose in perspective.

Whether dietary fructose (as sucrose or high fructose corn syrup) has unique effects separate from its role as carbohydrate, or, in fact, whether it can be considered inherently harmful, even a toxin, has assumed prominence in nutrition. Much of the popular and scientific media have already decided against fructose and calls for regulation and taxation come from many quarters. There are conflicting data, however. Outcomes attributed to fructose - obesity, high triglycerides and other features of metabolic syndrome - are not found in every experimental test and may be more reliably caused by increased total carbohydrate. In this review, we try to put fructose in perspective by looking at the basic metabolic reactions. We conclude that fructose is best understood as part of carbohydrate metabolism. The pathways of fructose and glucose metabolism converge at the level of the triose-phosphates and, therefore, any downstream effects also occur with glucose. In addition, a substantial part of ingested fructose is turned to glucose. Regulation of fructose metabolism per se, is at the level of substrate control - the lower Km of fructokinase compared to glucokinase will affect the population of triose-phosphates. Generally deleterious effects of administering fructose alone suggest that fructose metabolism is normally controlled in part by glucose. Because the mechanisms of fructose effects are largely those of a carbohydrate, one has to ask what the proper control should be for experiments that compare fructose to glucose. In fact, there is a large literature showing benefits in replacing total carbohydrate with other nutrients, usually fat, and such experiments sensibly constitute the proper control for comparisons of the two sugars. In terms of public health, a rush to judgement analogous to the fat-cholesterol-heart story, is likely to have unpredictable outcome and unintended consequences. Popular opinion cannot be ignored in this problem and comparing fructose to ethanol, for example, is without biochemical correlates. Also, nothing in the biochemistry suggests that sugar is a toxin. Dietary carbohydrate restriction remains the best strategy for obesity, diabetes and metabolic syndrome. The specific contribution of the removal of fructose or sucrose to this effect remains unknown.

MicroPET/SPECT/CT imaging of small animal models of disease.

For the greater part of the last century, basic science research has been limited to in vitro studies of cellular processes and ex vivo tissue examination from suitable animal models of disease. In the last three decades, however, new techniques have been developed that permit the imaging of live animals using X-rays, radiotracer emissions, magnetic resonance signals, sound waves and optical fluorescence, and bioluminescence. The objective of this review is to provide a broad overview of common animal imaging modalities, with a focus on positron emission tomography (PET), single photon emission computed tomography (SPECT), and computed tomography (CT). Important examples, benefits, and limits of microPET/SPECT/CT technologies in current use, and their central role in improving our understanding of biological behavior and in facilitating the development of treatments from bench to bedside are included.

Targeting insulin inhibition as a metabolic therapy in advanced cancer: a pilot safety and feasibility dietary trial in 10 patients.

OBJECTIVE: Most aggressive cancers demonstrate a positive positron emission tomographic (PET) result using ¹8F-2-fluoro-2-deoxyglucose (FDG), reflecting a glycolytic phenotype. Inhibiting insulin secretion provides a method, consistent with published mechanisms, for limiting cancer growth. METHODS: Eligible patients with advanced incurable cancers had a positive PET result, an Eastern Cooperative Oncology Group performance status of 0 to 2, normal organ function without diabetes or recent weight loss, and a body mass index of at least 20 kg/m². Insulin inhibition, effected by a supervised carbohydrate dietary restriction (5% of total kilocalories), was monitored for macronutrient intake, body weight, serum electrolytes, ß-hydroxybutyrate, insulin, and insulin-like growth factors-1 and -2. An FDG-PET scan was obtained at study entry and exit. RESULTS: Ten subjects completed 26 to 28 d of the study diet without associated unsafe adverse effects. Mean caloric intake decreased 35 ± 6% versus baseline, and weight decreased by a median of 4% (range 0.0-6.1%). In nine patients with prior rapid disease progression, five with stable disease or partial remission on PET scan after the diet exhibited a three-fold higher dietary ketosis than those with continued progressive disease (n = 4, P = 0.018). Caloric intake (P = 0.65) and weight loss (P = 0.45) did not differ in those with stable disease or partial remission versus progressive disease. Ketosis was associated inversely with serum insulin levels (P = 0.03). CONCLUSION: Preliminary data demonstrate that an insulin-inhibiting diet is safe and feasible in selected patients with advanced cancer. The extent of ketosis, but not calorie deficit or weight loss, correlated with stable disease or partial remission. Further study is needed to assess insulin inhibition as complementary to standard cytotoxic and endocrine therapies.

Aspirin treatment of mice infected with Trypanosoma cruzi and implications for the pathogenesis of Chagas disease.

Chagas disease, caused by infection with Trypanosoma cruzi, is an important cause of cardiovascular disease. It is increasingly clear that parasite-derived prostaglandins potently modulate host response and disease progression. Here, we report that treatment of experimental T. cruzi infection (Brazil strain) beginning 5 days post infection (dpi) with aspirin (ASA) increased mortality (2-fold) and parasitemia (12-fold). However, there were no differences regarding histopathology or cardiac structure or function. Delayed treatment with ASA (20 mg/kg) beginning 60 dpi did not increase parasitemia or mortality but improved ejection fraction. ASA treatment diminished the profile of parasite- and host-derived circulating prostaglandins in infected mice. To distinguish the effects of ASA on the parasite and host bio-synthetic pathways we infected cyclooxygenase-1 (COX-1) null mice with the Brazil-strain of T. cruzi. Infected COX-1 null mice displayed a reduction in circulating levels of thromboxane (TX)A(2) and prostaglandin (PG)F(2α). Parasitemia was increased in COX-1 null mice compared with parasitemia and mortality in ASA-treated infected mice indicating the effects of ASA on mortality potentially had little to do with inhibition of prostaglandin metabolism. Expression of SOCS-2 was enhanced, and TRAF6 and TNFα reduced, in the spleens of infected ASA-treated mice. Ablation of the initial innate response to infection may cause the increased mortality in ASA-treated mice as the host likely succumbs more quickly without the initiation of the "cytokine storm" during acute infection. We conclude that ASA, through both COX inhibition and other "off-target" effects, modulates the progression of acute and chronic Chagas disease. Thus, eicosanoids present during acute infection may act as immunomodulators aiding the transition to and maintenance of the chronic phase of the disease. A deeper understanding of the mechanism of ASA action may provide clues to the differences between host response in the acute and chronic T. cruzi infection.

Micro-positron emission tomography in the evaluation of Trypanosoma cruzi-induced heart disease: Comparison with other modalities.

Noninvasive assessment of cardiac structure and function is essential to understand the natural course of murine infection with Trypanosoma cruzi. Magnetic resonance imaging (MRI) and echocardiography have been used to monitor anatomy and function; positron emission tomography (PET) is ideal for monitoring metabolic events in the myocardium. Mice infected with T. cruzi (Brazil strain) were imaged 15-100 days post infection (dpi). Quantitative (18)F-FDG microPET imaging, MRI and echocardiography were performed and compared. Tracer ((18)F-FDG) uptake was significantly higher in infected mice at all days of infection, from 15 to 100 dpi. Dilatation of the right ventricular chamber was observed by MRI from 30 to 100 dpi in infected mice. Echocardiography revealed significantly reduced ejection fraction by 60 dpi. Combination of these three complementary imaging modalities makes it possible to noninvasively quantify cardiovascular function, morphology, and metabolism from the earliest days of infection through the chronic phase.

Chronic effects of dietary carbohydrate variation on [18F]-2-fluoro-2-deoxyglucose uptake in rodent heart.

PURPOSE: Measured cardiac [F]-2-fluoro-2-deoxyglucose (FDG) activity in human PET scans is variable despite efforts to standardize patient preparation. Heart uptake can obscure chest disease, and is of physiologic interest. Short-term carbohydrate (CHO) restriction can reduce FDG uptake, although unreliably, whereas long-term restriction of CHO has not been systematically studied. It would be valuable to understand FDG hearts' chronic dietary dependence. METHODS: Fifteen Wistar rats (age 4 weeks) were randomized to three diet groups (n = 5) of low (0.1% of total energy), intermediate (52%), and high (78%) CHO content (LC, IC, and HC, respectively). After 4 weeks, blood for ketone bodies (KB), glucose, insulin, and glucagon was obtained, followed in 2 days by whole-body PET with 37 MBq FDG. Diet groups were switched every 4 weeks to control for the effects of dietary order. Heart maximal standardized uptake value was compared among animals. RESULTS: Heart mean maximal standardized uptake value was dramatically reduced for LC (3.4+/-0.4; P<0.001) compared with either IC (10.9+/-0.7) or HC (11.0+/-0.7) (P=NS, IC vs. HC). KB (mumol/l) differed widely (P<0.001) in LC (718.6+/-40.0) versus IC (120.3+/-34.0) and HC (99.2+/-32.1) (P=NS, IC vs. HC), whereas glucose, insulin, and glucagon did not differ among the groups. CONCLUSION: Sustained CHO-restriction results in marked, reproducibly reduced cardiac FDG uptake. Six-fold to seven-fold increased KB concentrations provide alternative substrate to glucose.

Acetoacetate reduces growth and ATP concentration in cancer cell lines which over-express uncoupling protein 2.

BACKGROUND: Recent evidence suggests that several human cancers are capable of uncoupling of mitochondrial ATP generation in the presence of intact tricarboxylic acid (TCA) enzymes. The goal of the current study was to test the hypothesis that ketone bodies can inhibit cell growth in aggressive cancers and that expression of uncoupling protein 2 is a contributing factor. The proposed mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration. METHODS: Seven aggressive human cancer cell lines, and three control fibroblast lines were grown in vitro in either 10 mM glucose medium (GM), or in glucose plus 10 mM acetoacetate [G+AcA]. The cells were assayed for cell growth, ATP production and expression of UCP2. RESULTS: There was a high correlation of cell growth with ATP concentration (r = 0.948) in a continuum across all cell lines. Controls demonstrated normal cell growth and ATP with the lowest density of mitochondrial UCP2 staining while all cancer lines demonstrated proportionally inhibited growth and ATP, and over-expression of UCP2 (p < 0.05). CONCLUSION: Seven human cancer cell lines grown in glucose plus acetoacetate medium showed tightly coupled reduction of growth and ATP concentration. The findings were not observed in control fibroblasts. The observed over-expression of UCP2 in cancer lines, but not in controls, provides a plausible molecular mechanism by which acetoacetate spares normal cells but suppresses growth in cancer lines. The results bear on the hypothesized potential for ketogenic diets as therapeutic strategies.

Proangiogenic contribution of adiponectin toward mammary tumor growth in vivo.

PURPOSE: Adipocytes represent one of the most abundant constituents of the mammary gland. They are essential for mammary tumor growth and survival. Metabolically, one of the more important fat-derived factors ("adipokines") is adiponectin (APN). Serum concentrations of APN negatively correlate with body mass index and insulin resistance. To explore the association of APN with breast cancer and tumor angiogenesis, we took an in vivo approach aiming to study its role in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) mammary tumor model. EXPERIMENTAL DESIGN: We compared the rates of tumor growth in MMTV-PyMT mice in wild-type and APN-null backgrounds. RESULTS: Histology and micro-positron emission tomography imaging show that the rate of tumor growth is significantly reduced in the absence of APN at early stages. PyMT/APN knockout mice exhibit a reduction in their angiogenic profile resulting in nutrient deprivation of the tumors and tumor-associated cell death. Surprisingly, in more advanced malignant stages of the disease, tumor growth develops more aggressively in mice lacking APN, giving rise to a larger tumor burden, an increase in the mobilization of circulating endothelial progenitor cells, and a gene expression fingerprint indicative of more aggressive tumor cells. CONCLUSIONS: These observations highlight a novel important contribution of APN in mammary tumor development and angiogenesis, indicating that APN has potent angio-mimetic properties in tumor vascularization. However, in tumors deprived of APN, this antiangiogenic stress results in an adaptive response that fuels tumor growth through mobilization of circulating endothelial progenitor cells and the development of mechanisms enabling massive cell proliferation despite a chronically hypoxic microenvironment.

Analysis of dietary interventions. A simple payoff matrix for display of comparative dietary trials.

OBJECTIVE: To provide a simple method for presentation of data in comparative dietary trials. METHODS: Individual data from each diet are ranked and all possible paired comparisons are made and displayed in a pay-off matrix which can be color-coded according to the magnitude of the differences between the two diets. Probability of outcome can be calculated from the fraction of matrix elements corresponding to specified conditions. The method has the advantage of emphasizing differences and providing the maximum amount of information. RESULTS: The method was tested with values from the literature and allows intuitive sense of the comparative effectiveness of the two diets. In a test case in which a cross-over study had been performed the matrix derived from theoretical paired comparisons (treating the data as two parallel studies) was consistent with the results from the actual pairing in the cross-over. CONCLUSION: The matrix method is a simple way of providing access to the differences between dietary trials. It exaggerates differences but can be used in combination with group statistics that, conversely, provide reliability at the expense of detailed information.