Anti-Cancer Peptide PNC-27 Kills Cancer Cells by Unique Interactions with Plasma Membrane-Bound hdm-2 and with Mitochondrial Membranes Causing Mitochondrial Disruption.

OBJECTIVE: We have previously shown that the anti-cancer peptide PNC-27 kills cancer cells by co-localizing with membrane-expressed HDM-2, resulting in transmembrane pore formation causing extrusion of intracellular contents. We have also observed cancer cell mitochondrial disruption in PNC-27-treated cancer cells. Our objectives are to determine: 1. if PNC-27 binds to the p53 binding site of HDM-2 (residues 1-109) in the cancer cell membrane and 2. if this peptide causes selective disruption of cancer cell mitochondria. METHODS: For aim 1, we incubated MIA-PaCa-2 human pancreatic carcinoma cells with PNC-27 in the presence of a monoclonal antibody against the amino terminal p53 binding site of HDM-2 to determine if it, but not negative control immune serum, blocks PNC-27-induced tumor cell necrosis. For the second aim, we incubated these cells with PNC-27 in the presence of two specific dyes that highlight normal organelle function: mitotracker for mitochondria and lysotracker for lysosomes. We also performed immuno-electron microscopy (IEM) with gold-labeled anti-PNC-27 antibody on the mitochondria of these cells treated with PNC-27. RESULTS: Monoclonal antibody to the p53 binding site of HDM-2 blocks PNC-27-induced cancer cell necrosis, whereas negative control immune serum does not. The mitochondria of PNC-27-treated cancer cells fail to retain mitotracker dye while their lysosomes retain lysotracker dye. IEM of the mitochondria cancer cells reveals gold particles present on the mitochondrial membranes. CONCLUSIONS: PNC-27 binds to the p53 binding site of HDM-2 (residues 1-109) inducing transmembrane pore formation and cancer cell necrosis. Furthermore, this peptide enters cancer cells and binds to the membranes of mitochondria, resulting in their disruption.

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.

The biochemistry of low-carbohydrate and ketogenic diets.

PURPOSE OF REVIEW: To summarize the underlying biochemical basis for low-carbohydrate and ketogenic diets (LC/KD) and provide mechanisms to account for demonstrated effectiveness. RECENT FINDINGS: LC/KD continue to have success, to outperform other diets as well as most drugs for weight loss and diabetes treatment. In many cases, LC/KD can effect remission (absence of drugs) or reversal (only metformin or nondiabetes drugs) of type 2 diabetes and can provide a significant adjunct to pharmacology in type 1. Medication is reduced or eliminated in most cases. The results are consistent with the biochemical rationale which stresses the global effects of the glucose-insulin axis. SUMMARY: Evidence for the superior effectiveness of LC/KD for metabolic disease is now overwhelming. At the same time, the approach has received only limited support, and in many cases, persistence of the traditional opposition. Clinical practice or research must confront this crisis in order to bring practice in line with current science and to avoid continued harm to medicine and ultimately, the patient.

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.

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.

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.

Fad diets in the treatment of diabetes.

Use of the term "fad diet" reflects the contentious nature of the debate in the treatment of diabetes and generally targets diets based on carbohydrate restriction, the major challenge to traditional dietary therapy. Although standard low-fat diets more accurately conform to the idea of a practice supported by social pressure rather than scientific data, it is suggested that we might want to give up altogether unscientific terms like "fad" and "healthy." Far from faddish, diets based on carbohydrate restriction have been the historical treatment for diabetes and are still supported by basic biochemistry, and it is argued that they should be considered the "default" diet, the one to try first, in diseases of carbohydrate intolerance or insulin resistance. The barrier to acceptance of low-carbohydrate diets in the past has been concern about saturated fat, which might be substituted for the carbohydrate that is removed. However, recent re-analysis of much old data shows that replacing carbohydrate with saturated fat is, if anything, beneficial. The dialectic of impact of continued hemoglobin A(1c) versus effect of dietary saturated fat in the risk of cardiovascular disease is resolved in direction of glycemic control. Putting biased language behind us and facing the impact of recent results that point to the value of low-carbohydrate diets would offer patients the maximum number of options.

Limited effect of dietary saturated fat on plasma saturated fat in the context of a low carbohydrate diet.

We recently showed that a hypocaloric carbohydrate restricted diet (CRD) had two striking effects: (1) a reduction in plasma saturated fatty acids (SFA) despite higher intake than a low fat diet, and (2) a decrease in inflammation despite a significant increase in arachidonic acid (ARA). Here we extend these findings in 8 weight stable men who were fed two 6-week CRD (12%en carbohydrate) varying in quality of fat. One CRD emphasized SFA (CRD-SFA, 86 g/d SFA) and the other, unsaturated fat (CRD-UFA, 47 g SFA/d). All foods were provided to subjects. Both CRD decreased serum triacylglycerol (TAG) and insulin, and increased LDL-C particle size. The CRD-UFA significantly decreased plasma TAG SFA (27.48 ± 2.89 mol%) compared to baseline (31.06 ± 4.26 mol%). Plasma TAG SFA, however, remained unchanged in the CRD-SFA (33.14 ± 3.49 mol%) despite a doubling in SFA intake. Both CRD significantly reduced plasma palmitoleic acid (16:1n-7) indicating decreased de novo lipogenesis. CRD-SFA significantly increased plasma phospholipid ARA content, while CRD-UFA significantly increased EPA and DHA. Urine 8-iso PGF(2α), a free radical-catalyzed product of ARA, was significantly lower than baseline following CRD-UFA (-32%). There was a significant inverse correlation between changes in urine 8-iso PGF(2α) and PL ARA on both CRD (r = -0.82 CRD-SFA; r = -0.62 CRD-UFA). These findings are consistent with the concept that dietary saturated fat is efficiently metabolized in the presence of low carbohydrate, and that a CRD results in better preservation of plasma ARA.

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.

Intention-to-treat. What is the question?

It has become commonplace for Randomized Controlled Trials (RCTs) to be analyzed according to Intention-to-Treat (ITT) principles in which data from all subjects are used regardless of the subjects' adherence to protocol. While ITT analyses can provide useful information in some cases, they do not answer the question that motivates many RCTs, namely, whether the treatments differ in efficacy. ITT tends to reduce information by combining two questions, whether the intervention is effective and whether, as implemented, it has good compliance. Because these questions may be separate there is a risk of misuse. Two examples are presented that demonstrate this potential for abuse: a study on the effectiveness of vitamin E in reducing cardiovascular risk and comparisons of low fat and low carbohydrate diets. In the first case, a treatment that is demonstrably effective is described as without merit. In the second, ITT describes as the same, two diets that actually have different outcomes. These misuses of ITT are not atypical and are not technical problems in statistics but have real consequences for scientific principles and health recommendations. ITT analyses may answer the question of what happens when treatments are recommended but are inappropriate where separate information on adherence and performance is available. It is proposed that results of RCTs, or any experimental study, be reported, not in terms of the analyses that were performed, but rather in terms of the questions that the analyses can answer properly.

Carbohydrate restriction has a more favorable impact on the metabolic syndrome than a low fat diet.

We recently proposed that the biological markers improved by carbohydrate restriction were precisely those that define the metabolic syndrome (MetS), and that the common thread was regulation of insulin as a control element. We specifically tested the idea with a 12-week study comparing two hypocaloric diets (approximately 1,500 kcal): a carbohydrate-restricted diet (CRD) (%carbohydrate:fat:protein = 12:59:28) and a low-fat diet (LFD) (56:24:20) in 40 subjects with atherogenic dyslipidemia. Both interventions led to improvements in several metabolic markers, but subjects following the CRD had consistently reduced glucose (-12%) and insulin (-50%) concentrations, insulin sensitivity (-55%), weight loss (-10%), decreased adiposity (-14%), and more favorable triacylglycerol (TAG) (-51%), HDL-C (13%) and total cholesterol/HDL-C ratio (-14%) responses. In addition to these markers for MetS, the CRD subjects showed more favorable responses to alternative indicators of cardiovascular risk: postprandial lipemia (-47%), the Apo B/Apo A-1 ratio (-16%), and LDL particle distribution. Despite a threefold higher intake of dietary saturated fat during the CRD, saturated fatty acids in TAG and cholesteryl ester were significantly decreased, as was palmitoleic acid (16:1n-7), an endogenous marker of lipogenesis, compared to subjects consuming the LFD. Serum retinol binding protein 4 has been linked to insulin-resistant states, and only the CRD decreased this marker (-20%). The findings provide support for unifying the disparate markers of MetS and for the proposed intimate connection with dietary carbohydrate. The results support the use of dietary carbohydrate restriction as an effective approach to improve features of MetS and cardiovascular risk.

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.

Carbohydrate restriction as the default treatment for type 2 diabetes and metabolic syndrome.

Dietary carbohydrate restriction in the treatment of diabetes and metabolic syndrome is based on an underlying principle of control of insulin secretion and the theory that insulin resistance is a response to chronic hyperglycemia and hyperinsulinemia. As such, the theory is intuitive and has substantial experimental support. It has generally been opposed by health agencies because of concern that carbohydrate will be replaced by fat, particularly saturated fat, thereby increasing the risk of cardiovascular disease as dictated by the so-called diet-heart hypothesis. Here we summarize recent data showing that, in fact, substitution of fat for carbohydrate generally improves cardiovascular risk factors. Removing the barrier of concern about dietary fat makes carbohydrate restriction a reasonable, if not the preferred method for treating type 2 diabetes and metabolic syndrome. We emphasize the ability of low carbohydrate diets to improve glycemic control, hemoglobin A1C and to reduce medication. We review evidence that such diets are effective even in the absence of weight loss.

Dietary carbohydrate restriction induces a unique metabolic state positively affecting atherogenic dyslipidemia, fatty acid partitioning, and metabolic syndrome.

Abnormal fatty acid metabolism and dyslipidemia play an intimate role in the pathogenesis of metabolic syndrome and cardiovascular diseases. The availability of glucose and insulin predominate as upstream regulatory elements that operate through a collection of transcription factors to partition lipids toward anabolic pathways. The unraveling of the details of these cellular events has proceeded rapidly, but their physiologic relevance to lifestyle modification has been largely ignored. Here we highlight the role of dietary input, specifically carbohydrate intake, in the mechanism of metabolic regulation germane to metabolic syndrome. The key principle is that carbohydrate, directly or indirectly through the effect of insulin, controls the disposition of excess dietary nutrients. Dietary carbohydrate modulates lipolysis, lipoprotein assembly and processing and affects the relation between dietary intake of saturated fat intake and circulating levels. Several of these processes are the subject of intense investigation at the cellular level. We see the need to integrate these cellular mechanisms with results from low-carbohydrate diet trials that have shown reduced cardiovascular risk through improvement in hepatic, intravascular, and peripheral processing of lipoproteins, alterations in fatty acid composition, and reductions in other cardiovascular risk factors, notably inflammation. From the current state of the literature, however, low-carbohydrate diets are grounded in basic metabolic principles and the data suggest that some form of carbohydrate restriction is a candidate to be the preferred dietary strategy for cardiovascular health beyond weight regulation.

Dietary carbohydrate restriction in type 2 diabetes mellitus and metabolic syndrome: time for a critical appraisal.

Current nutritional approaches to metabolic syndrome and type 2 diabetes generally rely on reductions in dietary fat. The success of such approaches has been limited and therapy more generally relies on pharmacology. The argument is made that a re-evaluation of the role of carbohydrate restriction, the historical and intuitive approach to the problem, may provide an alternative and possibly superior dietary strategy. The rationale is that carbohydrate restriction improves glycemic control and reduces insulin fluctuations which are primary targets. Experiments are summarized showing that carbohydrate-restricted diets are at least as effective for weight loss as low-fat diets and that substitution of fat for carbohydrate is generally beneficial for risk of cardiovascular disease. These beneficial effects of carbohydrate restriction do not require weight loss. Finally, the point is reiterated that carbohydrate restriction improves all of the features of metabolic syndrome.