Non-Toxic Metabolic Management of Metastatic Cancer in VM Mice: Novel Combination of Ketogenic Diet, Ketone Supplementation, and Hyperbaric Oxygen Therapy
The Warburg effect and tumor hypoxia underlie a unique cancer metabolic phenotype characterized by glucose dependency and aerobic fermentation. We previously showed that two non-toxic metabolic therapies – the ketogenic diet with concurrent hyperbaric oxygen (KD+HBOT) and dietary ketone supplementation – could increase survival time in the VM-M3 mouse model of metastatic cancer.
We hypothesized that combining these therapies could provide an even greater therapeutic benefit in this model. Mice receiving the combination therapy demonstrated a marked reduction in tumor growth rate and metastatic spread, and lived twice as long as control animals.
To further understand the effects of these metabolic therapies, we characterized the effects of high glucose (control), low glucose (LG), ketone supplementation (βHB), hyperbaric oxygen (HBOT), or combination therapy (LG+βHB+HBOT) on VM-M3 cells. Individually and combined, these metabolic therapies significantly decreased VM-M3 cell proliferation and viability. HBOT, alone or in combination with LG and βHB, increased ROS production in VM-M3 cells.
This study strongly supports further investigation into this metabolic therapy as a potential non-toxic treatment for late-stage metastatic cancers.
Academic Editor: Dhyan Chandra, Roswell Park Cancer Institute, UNITED STATES
Received: June 4, 2014; Accepted: April 14, 2015; Published: June 10, 2015
Funding: Patent 12B152PRWO; University of South Florida; D. P. D’Agostino, A. Poff, P. Arnold, “Targeting cancer with metabolic therapy and hyperbaric oxygen.” P. Arnold (Savind Inc.) has received financial support (ONR N000140910244) from D. D’Agostino (USF) to synthesize ketone esters. The remaining authors have no conflicts of interest. Funding for this project was supported in part by a charitable contribution from Scivation, Inc.; however, the company did not influence study design, data collection, analysis, or interpretation of results. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: Patent 12B152PRWO; University of South Florida; D. P. D’Agostino, A. Poff, P. Arnold, “Targeting cancer with metabolic therapy and hyperbaric oxygen.” P. Arnold (Savind Inc.) has received financial support (ONR N000140910244) from D. D’Agostino (USF) to synthesize ketone esters. The remaining authors have no conflicts of interest. Author (P. Arnold) affiliations with Savind Inc. and funding support from Scivation, Inc. does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.
Cancers exhibit a dysregulated metabolic phenotype characterized by lactate fermentation in the presence of oxygen, a phenomenon known as the Warburg effect . Interest in cancer metabolism has increased over the past decade, with researchers suggesting numerous causes and consequences of the Warburg effect, and investigating novel therapeutic strategies to exploit it [1–5]. Glucose dependency and lactate production, two key features of the Warburg effect, correlate strongly with aggressive capacity and invasive potential [3, 4, 6, 7]. Metastasis, or the spread of tumor cells from a primary site to distal tissues, is responsible for over 90 percent of cancer-related deaths. There are no cancer therapies currently available that can effectively manage systemic metastasis. Since the Warburg effect is a prominent phenotype in metastatic cells, metabolic therapies which exploit this phenomenon may offer new therapeutic options for patients with aggressive or late-stage cancers.
The glycolytic-dependency associated with the Warburg effect has led researchers to investigate dietary therapies which decrease glucose availability to the tumor. The ketogenic diet (KD) is a high fat, adequate protein, very low carbohydrate diet which has been used in preclinical and clinical studies to slow cancer progression [8–21]. The KD forces a physiological shift to fat metabolism, lowing blood glucose, suppressing insulin, and elevating blood ketone levels by stimulating ketogenesis from dietary and stored fats. Although the anti-cancer effects of the KD are largely attributed to a reduction in the glycolytic substrates and insulin signaling which fuel cancer metabolism, emerging evidence suggests that ketones have a therapeutic potential of their own [17, 22, 23]. While healthy cells readily adapt to ketones as an efficient energy substrate, many cancers are not able to make this adaptation [24, 25].
The expression of ketone utilization enzymes is often reduced in malignant cancers compared to their normal tissue counterparts [26, 27]. Indeed, unlike in healthy neurons, ketones fail to rescue glioma cells from glucose deprivation-induced death . We recently described this phenomenon and investigated the potential use of dietary ketone supplementation in a mouse model of metastatic cancer . Our study showed that exogenous ketone supplementation exerts potent anti-cancer effects in vivo, even when administered with a high carbohydrate diet, with similar effects on cancer cells in the presence of high glucose media in vitro .
Cellular energy metabolism is intricately linked to the oxygenation status of the tissue [29, 30]. When oxygen is readily available, normal cells produce up to 90 percent of their ATP by mitochondrial respiration. When oxygen availability becomes limited, such as in the exercising muscle, cells convert to using anaerobic fermentation to preserve ATP production. This metabolic switch sustains cellular function and promotes survival in the face of transient hypoxia, and its mechanism is largely driven by the HIF-1 transcription factor [30, 31]. HIF-1 enhances the expression of over 60 genes, many involved in glycolysis and fermentation, angiogenesis, growth, and survival [30, 32]. The aberrant signaling that drives tumor angiogenesis creates immature and leaky blood vessels which are unable to adequately perfuse the entire tumor . This leads to the formation of hypoxic regions inside the tumor which enhance the Warburg effect and promote cancer progression, invasion, and metastasis [33–35]. Tumor hypoxia and HIF-1 signaling are both strongly correlate with aggressive capacity and poor prognosis . Tumor hypoxia is also known to mediate some chemo- and radio-resistance [37–40]. Because these therapies work in large part by stimulating the overproduction of reactive oxygen species (ROS) within the tumor, limited oxygen availability lessens their efficacy [41, 42].
Hyperbaric oxygen therapy (HBOT) is the administration of 100% oxygen at elevated pressure. In vivo, HBOT saturates blood plasma with oxygen, allowing it to diffuse further into the tissues and oxygenate hypoxic tumor regions [43–45]. Similarly, HBOT increases oxygen diffusion into cells in culture and thus its effects can be readily evaluated in the in vitro environment. HBOT has been shown to inhibit angiogenesis and tumor growth and increase survival time as a stand-alone or adjuvant therapy to standard care in a variety of cell, animal, and human studies [46–52]. We previously showed that the ketogenic diet with concurrent hyperbaric oxygen therapy was an effective combination therapy against metastatic cancer .
The ketogenic diet, ketone supplementation, and HBOT target overlapping metabolic pathways which are especially prominent in metastatic cells. We hypothesized that combining these three metabolic therapies could provide a safe, cost-effective adjuvant treatment for metastatic cancer. We tested this hypothesis in vivo and in vitro using the VM-M3 mouse model of metastatic cancer [9, 22, 53].
Metastasis remains the largest obstacle in identifying effective therapies for the long-term management of cancer. A lack of animal models which reflect the metastatic phenotype prevents major improvements in patient care. The major goal of this study was to investigate the anti-cancer efficacy of a novel combination of metabolic therapies—the ketogenic diet, ketone supplementation, and hyperbaric oxygen—in a mouse model of aggressive metastatic cancer that recapitulates much of the metastatic phenotype seen in invasive human cancers [53, 57–59]. Individually, these therapies inhibited proliferation and viability in vitro, slowed disease progression in vivo, and when combined, elicited potent anti-cancer effects by inhibiting metastatic spread and doubling the survival time of mice with systemic, metastatic disease.
We previously found that ketone supplementation and combination ketogenic diet with HBOT slowed cancer progression in the VM-M3 model of metastatic cancer [9, 22]. We hypothesized that combining these therapeutic regimens could provide an even more significant response in this model. The KD has been used clinically to treat pediatric refractory epilepsy for nearly a century, and is known to be a safe and feasible option for patients [60, 61]. HBOT is an approved therapy for several disease states, including decompression sickness, carbon monoxide poisoning, and radionecrosis . Its use and safety is well-characterized and understood [62, 63]. Exogenous ketone ester supplementation is a novel therapy that is also safe and feasible [64, 65]. When ketone supplementation is administered properly, blood ketone levels remain in normal physiological levels and do not approach the dangerous levels associated with diabetic ketoacidosis (15–20 mM). Ketone supplementation can be used, however, to elevate blood ketone levels to a therapeutic range of 1-5mM , which is reported to be beneficial in an array of disease states, including epilepsy, Alzheimer’s disease, and other neurological disorders [60, 66–72]. Importantly, these treatments represent non-toxic alternative or adjuvant therapies, which could be readily implemented clinically if their effects hold up in human trials, especially in cases where standard of care is not advised or has limited efficacy.
The VM-M3 model exhibits rapid, systemic metastasis following s.c. inoculation . As demonstrated by the in vivo bioluminescent imaging (Figs 1 and 2), primary tumor growth and pattern of metastatic spread can be highly variable, such as is seen in many human metastatic cancers. Combination KD, ketone supplementation, and HBOT treatment induced a trend of slowed tumor growth rate in vivo (Fig 1). Overall tumor burden of these combination therapy animals was significantly decreased compared to control animals at all applicable time points (weeks 1, 2, and 3), and was also significantly decreased compared to KD treated mice at weeks 3 and 5 and KD+KE treated mice at weeks 1, 2, 3, and 5 (Fig 1). Livers of KD+KE+HBOT treated mice exhibited only micrometastatic lesions with little notable vascularization compared to controls at day 21 (Fig 4). These results are consistent with the literature where others have reported an anti-angiogenic effect of HBOT in tumors. Stuhr et. al reported that HBOT decreased vascular density and tumor growth in DMBA-induced rat mammary tumors [50, 73]. Pro-angiogenic genes, including VEGF, FGF, PDGF, and TGF-α were down-regulated in the HBOT-treated tumors. Therefore, our data suggest that KD+KE+HBOT combination therapy may work in part by inhibiting tumor vascularization, which likely contributes to its potent ability to prolong survival time in VM-M3 mice (Fig 4).
Importantly, the data presented in this manuscript can be directly compared to our two previous works investigating the proposed therapies individually (S1 and S2 Files) [9, 22]. The animal studies from these three manuscripts were performed in back-to-back phases, in which a portion of the control animals were implanted during each phase. We had previously reported that combining the KD with HBOT increased survival time in the VM-M3 model by 77.9% (S1 File) and that KE treatment alone increased survival time by 69.2% (S2 File) [9, 22]. Not surprisingly considering the variability in animal models, while mean survival time of animals receiving the multi-combination therapy was increased by 103% compared to controls, it was not significantly different than those receiving any of our individual therapies alone . Nonetheless, due to the strong trends of improved response with multi-combination in vitro and in vivo, we suggest that the multi-combination of KD+KE+HBOT regimen is a promising method of administering these metabolic therapies. The KD and HBOT have been investigated as potential adjuvant cancer therapies in a number preclinical studies, demonstrating not only efficacy as individual treatments but significant synergy with other adjuvant therapies or standard of care including chemotherapy and radiation [46, 74]. Due to these promising studies, the KD is being investigated in many current or upcoming animal studies and clinical trials. Ketone supplementation is a novel therapy that would likely allow patients to more easily achieve therapeutic levels of ketosis without the need to adhere to as strict a dietary regimen than when using KD alone. Indeed, preliminary studies in our lab indicate that chronic consumption of ketone ester induces sustained therapeutic ketosis in healthy rats consuming a carbohydrate-based diet .
The effect of these metabolic therapies on proliferation and viability in vitro mimicked the effects seen in vivo. Both βHB and HBOT significantly decreased VM-M3 cell proliferation, and this effect was enhanced by decreasing blood glucose (Fig 3) . We showed previously that βHB decreased VM-M3 cell viability, even in the presence of high glucose, but this effect was enhanced by lowering glucose concentration . HBOT alone did not increase survival time in vivo, and although there was a trend of decreased viability in vitro, this was not statistically significant; however, combining HBOT with ketosis elicited potent anti-cancer effects in mice and cells (Fig 2) . Taken together, these studies suggest that combining therapeutic ketosis with HBOT creates a unique physiologic and metabolic environment that is not conducive to rapid cancer cell proliferation and survival.
Blood glucose and body weight decreased and blood βHB increased in mice receiving KD+KE therapy by day 7, prior to the onset of significant disease progression (Fig 5). As would be expected, blood glucose was lower and blood βHB was elevated in KD+KE mice compared to KD alone. KD+KE mice lost approximately 20% of their initial body weight, suggesting a potential therapeutic contribution from calorie restriction. However, we previously showed that the anti-cancer effects of KE supplementation was not due to CR in body weight matched animals . Interestingly, at day 7, KD+KE+HBOT treated mice had increased blood βHB, but blood glucose and body weight were not significantly different from controls. The discrepancy between these two groups receiving the same dietary therapy is unclear. Alteration of tissue oxygenation is known to affect metabolic capacity and blood metabolite levels . Blood glucose decreases immediately following HBOT sessions in both animals and humans [77, 78]. This may be due to an increase in insulin secretion and tissue glucose utilization which has been reported with HBOT [79–82]. In our study, blood metabolites were measured several hours after HBOT. Perhaps HBOT-induced glucose utilization by the brain and peripheral tissues stimulated gluconeogenesis or feeding behavior in the VM-M3 mice. This could possibly explain why glucose was not decreased at the time of blood measurement and why these animals did not lose weight as the KD+KE mice did. Similarly, HBOT has been shown to enhance weight gain in head and neck cancer patients following surgery and radiation . It is possible that, as in our combination treatment group, patients using HBOT would find it more difficult to reduce blood glucose to therapeutic levels. Therefore, it would be important for individuals implementing these therapeutic options to closely monitor blood glucose and ketone levels and adjust their dietary habits to maintain a state of nutritional ketosis which we propose is important for maximal therapeutic benefit. These changes would likely include increasing carbohydrate restriction or introducing caloric restriction or intermittent fasting, all of which have been reported to have anti-cancer effects on their own .
As expected, HBOT increased ROS production in VM-M3 cells when administered alone, or in combination with LG+βHB (Fig 8). Although cancer cells thrive in a state of elevated basal ROS production, they are very sensitive to modest increases or decreases in ROS or alterations in redox or antioxidant state [84, 85]. Furthermore, the differential susceptibility of cancer and normal cells to glucose deprivation has been shown to be mediated by superoxide and hydrogen peroxide production and signaling [86, 87]. This may help explain why combining HBOT with glucose restriction, such as with the KD, elicit synergistic effects in vivo . Combining ketosis with hyperbaric oxygen may specifically target cancer metabolism while supporting the health of normal tissues and preventing the many negative symptoms of disease and standard care, like metabolic syndrome and radiation necrosis [88, 89]. We and others have shown that many cancers are unable to effectively metabolize ketones; however, they are an efficient and protective energy source for healthy tissues [22, 25, 72]. Ketone metabolism decreases ROS production and increases the endogenous antioxidant profile of cells which consume them [72, 90]. Due to the abnormal mitochondrial function present in most cancers, it is unclear if ketone metabolism would reduce oxidative stress in tumors as it does in healthy tissue. While some studies have reported the ability of ketones to reduce ROS production in cancer models [13, 91], other studies have reported enhanced oxidative stress in tumors of animals treated with the KD , and in our current study, there was no change in ROS production with ketone treatment (Fig 8). This phenomenon will be important to understand as we further explore the use of the KD and ketone supplementation in cancer; however, it has not been well-studied to date. The discrepancies between the reported data are likely due to differences between cancer types and the degree to which mitochondria are dysfunctional in each particular cancer. Since many therapies, including chemotherapy, radiation, and HBOT, work in part by increasing ROS production in the tumor, simultaneous ketone metabolism by surrounding healthy tissue may work to prevent toxic side effects in these tissues. Both ketosis and HBOT have been shown to sensitize tumors to radiation and chemotherapy [20, 46, 93, 94]. Concurrent administration of these therapies may allow for the delivery of standard care at reduced doses, lessening toxic side effects and supporting the health of the individual. Importantly, βHB did not decrease ROS production in the VM-M3 cells (Fig 8), further supporting the notion that these cancer cells are not able to metabolize ketones as healthy cells do.
There is understandable concern regarding the manipulation of diet in vulnerable cancer patients who may find it difficult to consume adequate calories . If these adjuvant therapies allow for decreased yet effective doses of chemotherapy and radiation, patients will likely have an easier time consuming enough calories to support their metabolic needs. Indeed, late-stage patients given the KD report improved quality of life, and small clinical studies suggest that many patients are willing and able to implement the dietary restrictions of the ketogenic diet [11, 17, 20, 96]. Simultaneous administration of a ketone supplement such as the ketone ester could potentially provide the same benefit of a KD without the need for such restrictive food intake . Furthermore, ketone metabolism is a protein sparing process . The human brain evolved to metabolize ketones as a way to spare muscle protein during prolonged periods of food deprivation . Ketogenic diets promote weight loss in overweight patients, but they are also known to prevent muscle wasting during physical stress, energy restriction, or starvation [98–101]. In a small study on humans with advanced cancer and cachexia, patients maintained a positive nitrogen balance and gained weight when given a ketone-supplemented ketogenic diet . Indeed, an elevation in blood ketones decreases amino acid release from muscle and decreases hepatic gluconeogenesis of amino acids . Similarly, mice given a KD had significantly smaller tumors but did not lose as much body weight as control animals in a murine model of cancer cachexia . Furthermore, control animals had high levels of circulating catabolic factors which elicit triglyceride and amino acid release, but this was inhibited by βHB [15, 102]. Similar studies showed that in mice with a colon cancer that elicited a severe cachexic phenotype, the brain greatly increased expression of 3-oxoacid CoA-transferase, a ketone utilizing enzyme, and relied more heavily on βHB for energy . Enhancing ketone availability as an alternative fuel to the brain and other healthy tissues with the ketogenic diet and/or ketone supplementation will likely support their function during the cachexic state.
There is potential for synergy between these adjuvant metabolic therapies and standard care [52, 54, 96, 104]. Scheck and colleagues reported a significant synergistic effect of combining the KD with radiation therapy in a mouse model of brain cancer . Other studies have demonstrated increased efficacy by pairing the KD with chemotherapy or pharmacological agents which target metabolism [20, 94]. HBOT has been investigated as a radiosensitizer in a number of clinical studies [52, 105]. Meta-analyses conclude that HBOT appears to have a neutral or inhibiting effect on cancer progression, depending on cancer type [46, 51, 52]. When administered concurrently with radiation, HBOT potentiates efficacy, but may also increase soft tissue necrosis in nearby healthy tissue . Studies indicate that this adverse effect can be mitigated by delivering HBOT immediately prior to, rather than concurrent with, radiation therapy . Indeed, in this study, human glioma patients receiving HBOT immediately prior to radiation therapy had a median survival time of 24 months, compared to 12 months in patients receiving radiation alone, and did not experience significant side effects . As previously discussed, we hypothesize that ketone metabolism by the healthy tissue may also mitigate adverse effects and perhaps potentiate the efficacy of HBOT and/or standard care. We previously showed that HBOT alone did not affect survival in the VM-M3 model, but dramatically slowed progression when administered with a KD . Perhaps other studies that found no effect of HBOT could have revealed therapeutic potential by coupling this therapy with ketosis, either by the KD or ketone supplementation.
Despite 50 years of intensive research, cancer remains the second leading cause of death in the United States. This is largely due to a lack of therapies effective for long-term management of metastatic disease, a dilemma underlined by inadequate preclinical models. In this study, we evaluated the efficacy of a novel, non-toxic combination metabolic therapy—the ketogenic diet, ketone supplementation, and hyperbaric oxygen—in a syngeneic mouse model of metastatic disease which we believe reliably reflects the human metastatic phenotype [53, 57–59]. Our study suggests that this combination therapy presents a safe, cost-effective, adjuvant to standard care which could provide novel therapeutic options for patients with late-stage cancer. We believe that it is critically important to evaluate non-toxic adjuvant therapies like these, and to look for synergistic potential with other metabolic therapies and standard care. It is possible that we already possess therapeutic options which could significantly improve patient prognosis or outcome when delivered in the appropriate combinations.