Mitochondrial metabolism and cancer

Most cancer therapies are designed to take advantage of the metabolic and physiological differences that exist between cancer cells and normal cells. Compared to normal cells, cancer cells exhibit increased glucose metabolism as well as alterations in mitochondrial oxidative metabolism that are believed to be the result of chronic metabolic oxidative stress [3,4,45] (Fig. 2). Mitochondria are involved in the regulation of cellular energy production through the process of oxidative phosphorylation where electron transport chain (ETC) activity is used in the generation of cellular ATP [46]. In the mitochondrial ETC, electrons are shuttled down complexes I–IV, resulting in the generation of transmembrane proton gradient that is coupled to ATP production through ATP synthase (Complex V). Studies have shown increased prevalence of mitochondrial DNA mutations as well as alterations in the expression of nuclear encoded mitochondrial proteins in many human cancers [47–49] including head and neck [50], prostate [51], ovary [52], and liver cancers [53]. Previous data suggests that the susceptibility of mitochondrial DNA to mutations is largely due to the increase in ROS levels in this organelle [6,49,54–57]. Furthermore, recent studies have shown that breast and colon cancer cells demonstrate significantly increased steady-state levels of ROS relative to normal colon and breast cells [3]. These differences were even more pronounced in the presence of mitochondrial ETC blockers, suggesting dysfunctional mitochondrial ETCs as the major source of elevated ROS production in cancer cells [3]. Overall, there is substantial literature indicating that there is a significant increase in intracellular O2•− and H2O2 in cancer cell mitochondria relative to normal cells and that this could represent a target for enhancing cancer therapy [5,7,9–14,16,17].

Fig. 2

Comparison of normal cell and tumor cell metabolism on an American diet and a ketogenic diet. Relative to normal cells, tumor cells have been hypothesized to have increased mitochondrial DNA mutations as well as alterations in the expression of nuclear encoded mitochondrial proteins, resulting in increased production of reactive oxygen species (ROS) during mitochondrial respiration. Increased tumor cell ROS increases tumor cell dependence upon glucose metabolism, resulting in generation of NADPH and pyruvate via the pentose phosphate shunt and pyruvate from glycolysis. NADPH and pyruvate reduce hydroperoxides. Ketogenic diets decrease the capability of tumor cells to produce NADPH because, in most tissues, fat metabolism is unable to undergo gluconeogenesis to form glucose-6-phosphate (G-6-P) necessary to enter the pentose phosphate shunt. Thus, ketogenic diets should further increase the oxidative stress in tumor cells relative to normal cells by limiting NADPH regeneration.

Glucose dependence of cancer cells

Glycolysis mediates the enzymatic breakdown of glucose to pyruvate, which in the presence of oxygen is converted to acetyl-CoA and enters the Citric Acid Cycle in the mitochondria. In the absence of oxygen, pyruvate is alternatively converted into lactate. Normal cells link pyruvate production to mitochondrial respiration to efficiently generate ATP via oxidative phosphorylation and usually demonstrate low levels of glycolysis as well as lactate production. In contrast to normal cells, cancer cells demonstrate increased glucose consumption, even in the presence of oxygen [1] which was suggested to occur because of defective mitochondrial respiration requiring increased glycolysis as a compensatory response.

Numerous animal studies over the past 60 years have not only confirmed the observation of increased glucose consumption in cancer cells but also demonstrate the importance of glucose for tumor survival and metastasis. The flux of energy-yielding substrates across colon carcinomas in patients has demonstrated that net glucose uptake and lactate release by malignant tumors exceeds the peripheral non-malignant exchange rates by 30 and 43 fold, respectively, while no significant differences existed between tumor and peripheral tissue in fatty acid or ketone balance. FDG PET conclusively demonstrates that most human carcinomas have an increased glucose demand when compared to the surrounding normal tissue [58].

In addition to abnormal aerobic glycolysis, cancer cells have increased pentose phosphate pathway activity [3,59]. The pentose phosphate pathway oxidizes glucose to produce two molecules of the reducing equivalent nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphate. NADPH acts as a cofactor for the glutathione/glutathione peroxidase system as well as the thioredoxin/thioredoxin peroxidase system [60]. These thiol systems are responsible for detoxifying H2O2 and organic peroxides, thereby maintaining the redox balance by preventing and repairing oxidative damage.

Glucose metabolism is known to play a major role in the detoxification of peroxides both through the formation of pyruvate (which scavenges peroxides directly through a deacetylation reaction) and the regeneration of the redox cofactor NADPH. Previous studies have shown glucose deprivation selectively causes oxidative stress and toxicity in human cancer cells relative to normal cells that is reversed upon addition of superoxide and peroxide scavengers [2,3]. Furthermore, many in vitro and in vivo studies have successfully investigated the use of glycolytic inhibitors to cause selective cancer cell toxicity via a mechanism involving metabolic oxidative stress [3,7,61–65].

Ketogenic diets increase cancer cell oxidative stress

Ketogenic diets may act as an adjuvant cancer therapy by two different mechanisms that both increase the oxidative stress inside cancer cells. Lipid metabolism limits the availability of glucose for glycolysis restricting the formation of pyruvate and glucose-6 phosphate which can enter the pentose phosphate pathway forming NADPH necessary for reducing hydroperoxides (Fig. 2). Additionally, lipid metabolism forces cells to derive their energy from mitochondrial metabolism. Because cancer cells are believed to have dysfunctional mitochondrial ETCs resulting in increased one electron reductions of O2 leading to ROS production, cancer cells will be predicted to selectively experience oxidative stress, relative to normal cells, when glucose metabolism is restricted in the case of feeding ketogenic diets (Fig. 2). Similar to fat metabolism, protein derived energy production, such as in glutaminolysis, forces cells to derive their energy from mitochondrial metabolism and would be expected to increase cancer cell oxidative stress. However, many amino acids enter the Citric Acid Cycle through alpha-keto-glutarate which may undergo gluconeogenesis allowing for the production of NADPH. Thus, protein metabolism may not lead to the same levels of increased tumor cell oxidative stress as fat metabolism.

Evidence of ketogenic diets increasing cancer cell oxidative stress is present both clinically and in animal models. Hyperketotic diabetic humans have a higher level of lipid peroxidation in red blood cells and a significant decrease in cellular glutathione relative to normal ketonic diabetic controls [66]. Jain et al. also found elevated indices of lipid peroxidation in cultured human endothelial cells treated with acetoacetate [66]. Acetoacetate was also found to deplete cellular glutathione and increase intracellular peroxides in primary rat hepatocytes [67]. Chronic exposure to β-hydroxybutyrate was shown to increase ROS production in cardiomyocytes [68]. Combining a ketogenic diet with hyperbaric oxygen therapy decreased tumor growth rate, increased mean survival time, and increased β-hydroxybutyrate compared to controls in a metastatic mouse cancer model [69]. Thus combining a ketogenic diet with hyperbaric oxygen may further increase the oxidative stress inside of tumor cells. Furthermore, animals bearing lung cancer xenografts fed a ketogenic diet and treated with chemotherapy and radiation had increased 4-hydroxy-2-nonenal (4HNE)-modified protein relative to tumors treated with chemotherapy and radiation therapy alone [5]. 4HNE is a product of lipid peroxidation that damages proteins by forming adducts and is therefore both a marker of lipid and protein damage during oxidative stress.

Potential risks of ketogenic diets

Ketogenic diets haves been recognized to be effective at controlling seizures and inducing weight loss but have been suggested to cause some potential side effects. The acute side effects of high fat intake are typically lethargy, nausea, and vomiting due to intolerance of the diet, especially in children [70] (Fig. 3). Children can be prone to hypoglycemia due to low glucose intake and nausea [70]. In contrast, gastrointestinal discomfort is a common side effect in adults due to the high fat content of the diet [71]. A prospective pilot study on ketogenic diets reported a substantial and progressive increase in the cholesterol levels in patients after 1 year [72]. Past studies have also reported some deficiencies in trace minerals like selenium, copper, and zinc in the serum levels of patients on ketogenic diets, suggesting that appropriate supplementation of trace minerals is needed while on the diet [73].

Fig. 3

Possible acute and chronic side effects associated with the ketogenic diet.

Although no severe adverse changes have been reported with long term consumption of a ketogenic diet, renal damage due to excretion of nitrogenous waste products is also a possible side effect [74]. While no studies report absolute renal damage associated with ketogenic diet use, 6% of cases involving children with intractable epilepsy have reported the presence of kidney stones following eating the ketogenic diet for 1–5 years [75,76]. Most studies that examine the adverse effects of ketogenic diets have been done in children with epilepsy that had prolonged consumption of the diet over a period of 1–6 years. Most adverse effects reported in children only occur in patients who are on the ketogenic diet for greater than 1 year and include hypertriglyceridemia, decreased growth (decreased levels of insulin-like growth factor–1) and progressive bone mineral content loss. In addition, the most serious adverse effects of ketogenic diets can be prevented or corrected with appropriate measures such as vitamin supplements, assessment of bone function, and use of oral potassium citrate to decrease the risk of kidney stones [76,77].

In contrast, studies of ketogenic diets in adults show fewer and more minor adverse effects. In a 6-month study of adults on low-carbohydrate ketogenic diets, the only adverse effects noted were an increase in low-density lipoprotein (LDL) cholesterol levels, shakiness, and uneasiness [78]. In another trial, only 3 out of 72 adult patients on ketogenic diets for 1 year had adverse effects, with two showing elevated LDL cholesterol and one developing a kidney stone [79]. Another expected change associated with ketogenic diets is elevated blood ketones. This raises some concern in diabetic patients who are at an increased risk of developing ketoacidosis, a potentially life-threatening condition. However, the level of blood ketones as a result of ketogenic diet use in most adult patients is modest and is not accompanied with high blood glucose and therefore presents a low risk for ketoacidosis.

Clinical trials using the ketogenic diet for cancer control

There are currently 62 trials assessing low carbohydrate diets as a potential therapy for a variety of diseases of which 11 trials are assessing ketogenic diets as an adjuvant cancer therapy. In the University of Würzburg, Germany, patients having failed traditional cancer therapy and with no other salvage options have been enrolled in trials involving the ketogenic diet. Preliminary reports indicate that patients who were able to continue the ketogenic diet therapy for over 3 months showed improvement with a stable physical condition, tumor shrinkage, or slowed growth [44].

At the University Hospital in Tübingen, Germany a Phase 1 ERGO study designed to determine whether a mild ketogenic diet can influence quality of life and survival of patients with glutamino recurrent glioblastoma was conducted by Dr. Johannes Rieger and Dr. J. Steinbachand. No severe adverse events were reported (http://ClinicalTrials.gov/show/NCT00575146).

At the University of Iowa, three phase I trials assessing the tolerability of a ketogenic diet in combination with chemotherapy and radiation therapy are on-going in locally advanced pancreas, lung cancer as well as head and neck cancer (http://ClinicalTrials.gov). The typical schema is shown in Fig. 4a. While receiving standard of care radiation and chemotherapy, patients are consuming a ketogenic diet for 5 weeks; serum glucose and ketone levels are assessed daily in combination with weekly oxidative stress markers. A sample ketogenic diet is demonstrated in Fig. 4b.

Fig. 4

(A) Ketogenic diet phase I clinical trial schema and (B) sample ketogenic diet meal with a similar 4:1 ratio of fat to carbohydrate+protein as provided in the nutritionally complete KetoCal©. Ketosis is confirmed by laboratory measurement prior to beginning radiation therapy.

The authors would like to thank the manufacturers of KetoCal for providing their 4:1 diet formulation for ongoing pre-clinical and clinical trials. This work was supported in part by the Carver Research Program of Excellence in Redox Biology and Medicine (R01CA133114, R21CA161182, R01182804-01, R21CA139182, P30CA086862, and UL1TR000442), RSNA Research and Educational Foundation Grant RR1020, as well as a generous gift from Mrs. Nellie K. Spitz, Mrs. Marie Foster, and the IBM Corporation.