Cancer Metabolism: Fasting Reset, the Keto-Paradox and Drugs for Undoing.

In tumor cells, ketolysis "via" succinyl-CoA: 3-oxoacid-CoAtransferase (SCOT) and acetyl-CoA acetyltransferase 1 (ACAT1) is a major source of mitochondrial acetyl-CoA. Active ACAT1 tetramers stabilize by tyrosine phosphorylation, which facilitates the SCOT reaction and ketolysis. Tyrosine phosphorylation of pyruvate kinase PK M2 has the opposite effect, stabilizing inactive dimers, while pyruvate dehydrogenase (PDH), which is already inhibited by phosphorylation, is acetylated by ACAT1 and is doubly locked. This closes the glycolytic supply of acetyl-CoA. In addition, since tumor cells must synthesize fatty acids to create new membranes, they automatically turn off the degradation of fatty acids into acetyl-CoA ("via" the malonyl-CoA brake for the fatty acid carnityl transporter). Thus, inhibiting SCOT the specific ketolytic enzyme and ACAT1 should hold back tumor progression. However, tumor cells are still able to take up external acetate and convert it into acetyl-CoA in their cytosol "via" an acetyl-CoA synthetase, which feeds the lipogenic pathway; additionally, inhibiting this enzyme would make it difficult for tumor cells to form new lipid membrane and survive.

An alteration of the endocrine pancreas involved in cancer.

Tumor cells display hybrid metabolic features: some of their enzymes are phosphorylated as normally observed when catabolic hormones stimulate Gs-coupled receptors, whereas other enzymes adopt a configuration normally found in anabolic situations, mediated via tyrosine kinase receptors. Consequently, tumor cells have to rewire their metabolic pathways differently, whereas differentiated cells seem to respond preferentially to catabolic hormones. This gives mitotic cells a selective advantage since they deplete other cell reserves for their benefit. The pancreatic gamma aminobutyric acid selection switch between anabolism and catabolism explains the process, that is, a deficient release of gamma aminobutyric acid from beta cells leads to a concomitant release of catabolic glucagon and anabolic insulin and to a progressive desensitisation of insulin receptors on differentiated cells. New stem cells, with non-desensitised insulin receptors, respond to the dual anabolic and catabolic signals and rewire their metabolism in cancer mode. The aim of this letter was to discuss the causal pancreatic alteration of the anabolic-catabolic selection switch.

Low doses of arginine butyrate derivatives improve dystrophic phenotype and restore membrane integrity in DMD models.

A new approach to treating Duchenne muscular dystrophy was investigated by using the ester or amide covalent association of arginine [nitric oxide (NO) pathway] and butyrate [histone deacetylase (HDAC) inhibition] in mdx mice and patient myotubes. Two prodrugs were synthesized, and the beneficial effects on dystrophic phenotype were studied. Nerve excitability abnormalities detected in saline-treated mice were almost totally rescued in animals treated at low doses (50-100 mg/kg/d). Force and fatigue resistance were improved ≈60% and 3.5-fold, respectively, and the percentage of necrosis in heart sections was reduced ≈90% in the treated mice. A decrease of >50% in serum creatine kinase indicated an overall improvement in the muscles. Restoration of membrane integrity was studied directly by measuring the reduction (≈74%) of Evans blue incorporation in the limb muscles of the treated animals, the increase in utrophin level, and the normalization of lipid composition of the heart. In cultures of human myotubes (primary cells and cell line), both prodrugs and HDAC inhibitors increased by 2- to 4-fold the utrophin level, which was correctly localized at the membrane. ß-Dystroglycan and embryonic myosin protein levels were also increased. Finally, a 50% reduction in the number of spontaneous Ca(2+) spikes was observed after treatment with NO synthase substrate and HDAC inhibitors. Overall, the beneficial effects were obtained with doses 10 (in vivo) and 5 (in vitro) times lower than those of the salt formulation. Altogether, these data constitute proof of principle of the beneficial effects of low doses of arginine butyrate derivatives on muscular dystrophy, enhancing the NO pathway and inhibiting HDAC.

Arginine butyrate: a therapeutic candidate for Duchenne muscular dystrophy.

As a strategy to treat Duchenne muscular dystrophy, we used arginine butyrate, which combines two pharmacological activities: nitric oxide pathway activation, and histone deacetylase inhibition. Continuous intraperitoneal administration to dystrophin-deficient mdx mice resulted in a near 2-fold increase in utrophin (protein homologous to dystrophin) in skeletal muscle, heart, and brain, accompanied by an improvement of the dystrophic phenotype in both adult and newborn mice (45 and 70% decrease in creatine kinase level, respectively; 14% increase in tidal volume, 30% decrease in necrotic area in limb and 23% increase in isometric force). Intermittent administration, as performed in clinical trials, was then used to reduce the frequency of injections and to improve safety. This also enhanced utrophin level around 2-fold (EC50=284 mg/ml) and alleviated the dystrophic phenotype (inverted grid and grip test performance near to wild-type values, creatine kinase level decreased by 50%). Skin biopsies were used to monitor treatment efficacy, instead of invasive muscle biopsies, and this could be done a few days after the start of treatment. A 2-fold increase in utrophin expression was also shown in cultured human myotubes. In vivo and in vitro experiments demonstrated that the drug combination acts synergistically. Together, these data constitute a proof of principle of the beneficial effects of arginine butyrate on muscular dystrophy.

Tumor regression with a combination of drugs interfering with the tumor metabolism: efficacy of hydroxycitrate, lipoic acid and capsaicin.

Cellular metabolic alterations are now well described as implicated in cancer and some strategies are currently developed to target these different pathways. In previous papers, we demonstrated that a combination of molecules (namely alpha-lipoic acid and hydroxycitrate, i.e. Metabloc™) targeting the cancer metabolism markedly decreased tumor cell growth in mice. In this work, we demonstrate that the addition of capsaicin further delays tumor growth in mice in a dose dependant manner. This is true for the three animal model tested: lung (LLC) cancer, bladder cancer (MBT-2) and melanoma B16F10. There was no apparent side effect of this ternary combination. The addition of a fourth drug (octreotide) is even more effective resulting in tumor regression in mice bearing LLC cancer. These four compounds are all known to target the cellular metabolism not its DNA. The efficacy, the apparent lack of toxicity, the long clinical track records of these medications in human medicine, all points toward the need for a clinical trial. The dramatic efficacy of treatment suggests that cancer may simply be a disease of dysregulated cellular metabolism.

A possible primary cause of cancer: deficient cellular interactions in endocrine pancreas.

BACKGROUND: Cancer is a devastating type of disease. New and innovative ways to tackle cancers that have so far proved refractive to conventional therapies is urgently needed. It is becoming increasingly clear that, in addition to conventional therapeutics targeting by small molecules, that tumor cell metabolism presents new opportunities to target selectively specific cancer cell populations. Metabolic defects in cancer cells can be manifested in many ways that might not be readily apparent, such as altering epigenetic gene regulation for example. The complex rewiring of metabolic pathways gives tumor cells a special advantage over differentiated cells, since they deplete body stores as fuel for their growth and proliferation. Tumor metabolism looks simpler when we consider that some enzymatic switches are in a neoglucogenic direction thereby depleting body stores. However, these pathways may be inadequately switched on by catabolic hormones (glucagon, epinephrine and cortisol) in a specific situation where anabolism is activated by, for example insulin released from beta pancreatic cells or IGF, inducing mitosis and synthesis that are powered by glucose catabolism. Such a hybrid metabolic situation would be reached if a pancreatic beta cell mechanism, mediated by GABA, failed to silence neighboring alpha cells and delta cells. The inhibitory transmitter GABA hyperpolarizes alpha and delta cells via their GABA A receptors, and blocks the release of glucagon and somatostatin. Alternatively, an anomaly of alpha cell channels, would lead to a similar situation. Whatever is the alteration, anabolism fails to silence catabolism and enzymatic switches controlled by kinases and phosphatases adopt an inadequate direction, leading to a hybrid metabolic rewiring found in cancer. It is daring to formulate such a hypothesis as this. However, it is quite possible that the starting point in cancer is an alteration of the endocrine pancreas, suppressing the mechanism by which beta cells silence the neighboring alpha and delta cells, with GABA and Zn2+.

Normal human melanocytes exposed to chronic insulin and glucose supplementation undergo oncogenic changes and methyl group metabolism cellular redistribution.

Recent epidemiological studies have suggested a link between cancer and pathophysiological conditions associated with hyperinsulinemia. In this report, we address the possible role of insulin exposure in melanocyte transformation. To this aim, normal melanocytes were exposed to chronic insulin and glucose supplementation (twice the standard medium concentration) for at least 3 wk. After 3-wk treatment, melanocytes increased proliferation (doubling time: 2.7 vs. 5.6 days, P < 0.01). After 3-wk treatment or after 3-wk treatment followed by 4-wk reculture in standard medium, melanocytes were able to grow in soft agar colonies. Treated melanocytes had increased DNA content (+8%, P < 0.05), chromosomal aberrations, and modified oncoprotein profile: p-Akt expression increased (+32%, P < 0.01), Akt decreased, and c-Myc increased (+40%, P < 0.05). PP2A protein expression increased (+42, P < 0.05), while PP2A methylation decreased (-42%, P < 0.05), and PP2A activity was reduced (-27%, P < 0.05). PP2A transcription level was increased (ppp2r1a, PP2A subunit A, +44%, P < 0.05). Also, transcriptomic data revealed modifications in insr (insulin receptors, +10%, P < 0.05) and Il8 (inflammation protein, +99%, P < 0.01). Glycolysis was modified with increased transcription of Pgk1 and Hif1a (P < 0.05), decreased transcription of Pfkfb3 (P < 0.05), decreased activity of pyruvate kinase (P < 0.01), and decreased pyruvate cell content as assessed by (1)H-NMR spectroscopy. In addition, methyl group metabolism was altered with decreased global DNA methylation (-51%, P < 0.01), increased cytosolic protein methylation (+18%, P < 0.05), and consistent changes in methylated species on (1)H-NMR spectra. In conclusion, exposure to chronic insulin and glucose supplementation induces oncogenic changes and methyl group metabolism redistribution, which may be a biomarker of transformation.

Screening of well-established drugs targeting cancer metabolism: reproducibility of the efficacy of a highly effective drug combination in mice.

Alterations in metabolic pathways are known to characterize cancer. In order to suppress cancer growth, however, multiple proteins involved in these pathways have to be targeted simultaneously. We have developed a screening method to assess the best drug combination for cancer treatment based on targeting several factors implicated in tumor specific metabolism. Following a review of the literature, we identified those enzymes known to be deregulated in cancer and established a list of sixty-two drugs targeting them. These molecules are used routinely in clinical settings for diseases other than cancer. We screened a first library in vitro against four cell lines and then evaluated the most promising binary combinations in vivo against three murine syngeneic cancer models, (LL/2, Lewis lung carcinoma; B16-F10, melanoma; and MBT-2, bladder cancer). The optimum result was obtained using a combination of α-lipoic acid and hydroxycitrate (METABLOC(TM)). In this study, a third agent was added by in vivo evaluation of a large number of combinations. The addition of octreotide strongly reduced tumor development (T/C% value of 30.2 to 34.5%; P < 0.001) in the same models and prolonged animal survival (P < 0.001) as compared to cisplatin. These results were confirmed in a different laboratory setting using a human xenograft model (NCI-H69, small cell lung cancer). None of these three molecules are known to target DNA. The effectiveness of this combination in several animal models, as well as the low toxicity of these inexpensive drugs, emphasizes the necessity of rapidly setting up a clinical trial.

The metabolic advantage of tumor cells.

1- Oncogenes express proteins of "Tyrosine kinase receptor pathways", a receptor family including insulin or IGF-Growth Hormone receptors. Other oncogenes alter the PP2A phosphatase brake over these kinases. 2- Experiments on pancreatectomized animals; treated with pure insulin or total pancreatic extracts, showed that choline in the extract, preserved them from hepatomas. Since choline is a methyle donor, and since methylation regulates PP2A, the choline protection may result from PP2A methylation, which then attenuates kinases. 3- Moreover, kinases activated by the boosted signaling pathway inactivate pyruvate kinase and pyruvate dehydrogenase. In addition, demethylated PP2A would no longer dephosphorylate these enzymes. A "bottleneck" between glycolysis and the oxidative-citrate cycle interrupts the glycolytic pyruvate supply now provided via proteolysis and alanine transamination. This pyruvate forms lactate (Warburg effect) and NAD+ for glycolysis. Lipolysis and fatty acids provide acetyl CoA; the citrate condensation increases, unusual oxaloacetate sources are available. ATP citrate lyase follows, supporting aberrant transaminations with glutaminolysis and tumor lipogenesis. Truncated urea cycles, increased polyamine synthesis, consume the methyl donor SAM favoring carcinogenesis. 4- The decrease of butyrate, a histone deacetylase inhibitor, elicits epigenic changes (PETEN, P53, IGFBP decrease; hexokinase, fetal-genes-M2, increase). 5- IGFBP stops binding the IGF - IGFR complex, it is perhaps no longer inherited by a single mitotic daughter cell; leading to two daughter cells with a mitotic capability. 6- An excess of IGF induces a decrease of the major histocompatibility complex MHC1, Natural killer lymphocytes should eliminate such cells that start the tumor, unless the fever prostaglandin PGE2 or inflammation, inhibit them...

A combination of alpha lipoic acid and calcium hydroxycitrate is efficient against mouse cancer models: preliminary results.

The impact of metabolic dysregulation on tumor development has long been established. We have targeted two enzymes that are altered during carcinogenesis: pyruvate dehydrogenase (PDH), which is down-regulated, and ATP citrate lyase, which is overexpressed in cancer cells. Alpha lipoic acid is a cofactor of PDH, while hydroxycitrate is a known inhibitor of ATP citrate lyase. Our hypothesis is that a combination of these drugs may have antitumoral potential. The efficacy of these molecules was screened in vitro by treatment of different human cancer and murine cell lines. Lipoic acid reduced the cell number by 10-50% depending on concentrations (0.1-10 microM) and cell types. Calcium hydroxycitrate reduced the cell number by 5-60% at different concentrations (10-500 microM). When hydroxycitrate and lipoic acid were used together, there was a major cytotoxic effect: complete cell death was seen following 8 microM lipoic acid and 300 microM hydroxycitrate treatment for 72 h. The combination of alpha lipoic acid and hydroxycitrate was administered to healthy mice, at doses currently utilized for other indications than cancer; no demonstrable toxicity was observed. The combination was used to treat mouse syngenic cancer models: MBT-2 bladder transitional cell carcinoma, B16-F10 melanoma and LL/2 Lewis lung carcinoma. The efficacy of this combination appears similar to conventional chemotherapy (cisplatin or 5-fluorouracil) as it resulted in significant tumor growth retardation and enhanced survival. This preliminary study suggests that this combination of drugs is efficient against cancer cell proliferation both in vitro and in vivo. A clinical trial is warranted.

Hyperosmotic stress contributes to mouse colonic inflammation through the methylation of protein phosphatase 2A.

There are several reports suggesting hyperosmotic contents in the feces of patients suffering from inflammatory bowel disease (IBD). Previous works have documented that hyperosmolarity can cause inflammation attributable to methylation of the catalytic subunit of protein phosphatase 2A (PP2A) and subsequent NF-kappaB activation resulting in cytokine secretion. In this study, we demonstrate that dextran sulfate sodium (DSS) induces colitis due to hyperosmolarity and subsequent PP2A activation. Mice were randomized and fed with increased concentrations of DSS (0 mOsm, 175 mOsm, 300 mOsm, and 627 mOsm) for a duration of 3 wk or with hyperosmotic concentrations of DSS (627 mOsm) or mannitol (450 mOsm) for a duration of 12 wk. Long-term oral administration of hyposmotic DSS or mannitol had no demonstrable effect. Hyperosmotic DSS or mannitol produced a significant increase in colonic inflammation, as well as an increase in the weight of sacral lymph nodes and in serum amyloid A protein levels. Similar results were obtained through the ingestion of comparable osmolarities of mannitol. Hyperosmolarity induces the methylation of PP2A, nuclear p65 NF-kappaB activation. and cytokine secretion. The rectal instillation of okadaic acid, a well-known PP2A inhibitor, reverses the IBD. Short inhibiting RNAs (siRNAs) targeted toward PP2Ac reverse the effect of hyperosmotic DSS. The present study strongly suggests that DSS-induced chronic colitis is a consequence of the methylation of PP2Ac induced by hyperosmolarity.

L-arginine improves dystrophic phenotype in mdx mice.

A possible treatment for Duchenne muscular dystrophies would be to compensate for dystrophin loss by increasing the expression of utrophin, another cytoskeletal protein of the muscle membrane. We previously found that L-arginine, the substrate for nitric oxide synthase, significantly increased utrophin level in muscle and targeted it to the sarcolemma. Here, we have addressed the expected benefit in the mdx mice. Magnetic resonance imaging of lower limbs revealed a 35% reduction of the necrotic zones, confirmed by histological staining of muscles. This regression of the necrosis was also supported by the drastic reduction of Evans blue incorporation, a cell impermeable dye. The creatine kinase level in the serum decreased by 57%. Utrophin level increased 2- to 3-fold in muscles. Beta-dystroglycan was relocalised with utrophin to the membrane. In the diaphragm, the most affected muscle in mdx mice, the isometric tension increased by 30%, with regression of collagen and of cytoplasmic lipid overloading. Finally, molsidomine, a therapeutic agent that is converted to a NO donor, also attenuated the dystrophic phenotype. Our results suggest that pharmacological activators of the NO pathway may constitute a realistic treatment for Duchenne and Becker muscular dystrophies.

Genetic adaptation controlled by methylations and acetylations at the nuclear and cytosolic levels: a hypothetical model.

Metabolic sensors related to the maturation of metabolism seem to control a process of generic adaptation involving the silencing of genes and the expression of their copies more adapted to environmental changes. Nuclear methylases and histone deacetylases control the gene silencing process. Nuclear methylases compete with cytosolic methylases for the same methyl donnors, this will favor the expression of unmethylated more adapted gene copies, when cytosotic methylases take over. Methylated cytosolic compounds may then represent an index of this adaptation. If a more adapted gene copy is mutated, the regulatory ligand of the gene product that does not find its target may induce a reexpression of the silenced gene. The hypothetical model proposed considers that gene silencing and expression of a more adequate copy involves a non-specific gene silencer switch that depends on the histone status; the silencer switch is counteracted by the ligand of the adapted gene copy product acting like an inducer.

Muscular nitric oxide synthase (muNOS) and utrophin.

Duchenne muscular dystrophy (DMD), the severe X-linked recessive disorder which results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. Three types of treatment are envisaged: pharmacological (glucocorticoid), myoblast transplantation, and gene therapy. An alternative to the pharmacological approach is to compensate for dystrophin loss by the upregulation of another cytoskeletal protein, utrophin. Utrophin and dystrophin are part of a complex of proteins and glycoproteins, which links the basal lamina to the cytoskeleton, thus ensuring the stability of the muscle membrane. One protein of the complex, syntrophin, is associated with a muscular isoform of the neuronal nitric oxide synthase (nNOS). We have demonstrated an overexpression of utrophin, visualised by immunofluorescence and quantified by Western blotting, in normal myotubes and in mdx (the animal model of DMD) myotubes, as in normal (C57) and mdx mice, both treated with nitric oxide (NO) donor or L-arginine, the NOS substrate. There is evidence that utrophin may be capable of performing the same cellular functions as dystrophin and may functionally compensate for its lack. Thus, we propose to use NO donors, as palliative treatment of Duchenne and Becker muscular dystrophies, pending, or in combination with, gene and/or cellular therapy. Discussion has focussed on the various isoforms of NOS that could be implicated in the regeneration process. Dystrophic and healthy muscles respond to treatment, suggesting that although NOS is delocalised in the cytoplasm in the case of DMD, it conserves substantial activity. eNOS present in mitochondria and iNOS present in cytoplasm and the neuromuscular junction could also be activated. Lastly, production of NO by endothelial NOS of the capillaries would also be beneficial through increased supply of metabolites and oxygen to the muscles.

More than one way to toy with ChAT and VAChT.

Expression of choline acetyltransferase (ChAT) and of the vesicular acetylcholine transporter (VAChT) is required for the acquisition and the maintenance of the cholinergic phenotype. The ChAT and VAChT genes have been demonstrated to share a common gene locus and this suggests a coordinate regulation of their expression. In the present work, we examined the effects of several differentiating treatments on the modulation of ChAT and VAChT expression at the mRNA and protein levels in growing and differentiating NG108-15 cells. In cells grown in the presence of serum, all the agents tested-retinoic acid, dexamethasone and dibutyrylcyclicAMP (dbcAMP)-induced an increase of ChAT and VAChT mRNA levels but with different efficacy. Treatment with dbcAMP plus dexamethasone resulted in the largest increase of VAChT mRNA amount while retinoic acid mostly enhanced ChAT mRNA level. However, while ChAT activity was increased by all agents, no change in the VAChT protein level was detected. In cells differentiated by serum deprivation, only retinoic acid was effective in inducing an increase of VAChT and ChAT mRNA and ChAT activity, while we observed a downregulation by dbcAMP and dexamethasone. Treatment with the antimitotic agent cytosine arabinoside led to an increase of ChAT activity which was further largely enhanced by the addition of dbcAMP plus dexamethasone, but to only a slight change in VAChT activity. We further showed that complex glycosylation processes which might play a role in targeting and/or stability of the membrane protein VAChT are deficient in these cells. Indeed, in transient transfection assays with the reporter soluble enzyme luciferase placed under regulatory and promoter regions of the VAChT gene, we observed a modulation of luciferase expression by differentiating agents. These data illustrate the complexity of the processes which participate to the expression of the ChAT and VAChT genes, both at the transcriptional and posttranslational levels.