Combination Of Metabolic Inhibitors Research Articles

The metabolic landscape of urological cancers: New therapeutic perspectives.

Deregulation of cell metabolism is an established cancer hallmark that contributes to tumor initiation and progression, as well as tumor heterogeneity. In solid tumors, alterations in different metabolic pathways, including glycolysis, pentose phosphate pathway, glutaminolysis and fatty acid metabolism, support the high proliferative rates and macromolecule biosynthesis of cancer cells. Despite advances in therapy, urothelial tumors still exhibit high recurrence and mortality rates, especially in advanced stages of disease. These tumors harbor gene mutations and expression patterns which play an important role in metabolic reprogramming. Taking into account the unique metabolic features underlying carcinogenesis in these cancers, new and promising therapeutic targets based on metabolic alterations must be considered. Furthermore, the combination of metabolic inhibitors with conventional targeted therapies may improve effectiveness of treatments. This review will summarize the metabolic alterations present in urological tumors and the results with metabolic inhibitors currently available.

Abstract P5-05-03: Targeting hypoxia-induced cancer cell metabolism in breast cancer

Abstract Rational: Hypoxia contributes to high tumor grade in human breast cancer and is associated with resistance to radiation and chemotherapy. Hypoxia is generally encountered in perinecrotic regions throughout the heterogeneous tumor microenvironment and may be due to rapid tumor grow, insufficient neovascularization or as a result of antiangiogenic and chemoradiation therapies which cause acute vascular thrombosis. Cancer stem-like cells residing within these hypoxic zones compensate for the low concentration of oxygen through alterations in metabolism that favor aerobic glycolysis and de novo fatty acid synthesis. These metabolic derangements are known to enhance proliferation and survival, although the contributing molecular mechanisms are not fully understood. Here we evaluate a panel of FDA approved azole drugs for alternative use as glycolytic inhibitors with and without the fatty acid synthase (FASN) inhibitor, TBV-3166. TVB-3166 is currently in phase II clinical trials and is the first FASNi approved for human testing. METHODS: IC50 values of single or combined agents were determined by MTT under conditions of normoxia and hypoxia using sister plates of estrogen receptor positive or triple negative breast cancer cells, MCF-7, SKBR3 and MDA-MB-231. RESULTS: Because the combination of Itraconazole and TVB-3166 displayed a synergistic enhancement in cell death at a low nanomolar concentrations in MCF-7 cells, we next assessed the efficacy of these drugs in an in vivo xenograft model of breast cancer. Here, 3.6 million MCF-7 cells were implanted into the right flanks of (n=16) athymic nude mice, which were then randomized by tumor volume into 4 groups. Itraconazole and TVB-3166 were administered at 60 mg/kg daily by oral gavage as single agents or combined throughout the duration of the study. After three weeks, mice treated with combined inhibitors exhibited best response with a statistically significant decrease in tumor volume compared to the vehicle control group. CONCLUSION: Given the significant tumor response to the combination of metabolic inhibitors, further studies that evaluate the radiosensitizing effects under conditions of hypoxia are warranted. By targeting hypoxia-induced metabolic pathways during radiotherapy, we may enhance cancer stem-like cell death to dramatically improve patient outcome. Citation Format: Cavazos DA, Gruslova A, Garcia M, Brenner AJ. Targeting hypoxia-induced cancer cell metabolism in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-05-03.

METB-03TARGETING CANCER CELL METABOLISM IN GLIOBLASTOMA

RATIONALE: Hypoxia contributes to high tumor grade in human glioblastoma and is associated with resistance to radiation and chemotherapy. Hypoxia is generally encountered in perinecrotic regions throughout the heterogeneous tumor microenvironment and may be due to rapid tumor grow, insufficient neovascularization or as a result of antiangiogenic and chemoradiation therapies which cause acute vascular thrombosis. Cancer stem-like cells residing within these hypoxic zones compensate for the low concentration of oxygen through alterations in metabolism that favor aerobic glycolysis and de novo fatty acid synthesis. These metabolic derangements are known to enhance proliferation and survival, although the contributing molecular mechanisms are not fully understood. Here we evaluate a panel of FDA approved azole drugs for alternative use as glycolytic hexoinase 2 inhibitors with and without the fatty acid synthase (FASN) inhibitor, TBV-3166. TVB-3166 is currently in phase II clinical trials and is the first FASNi approved for human testing. METHODS: IC50 values of single or combined agents were determined by MTT under conditions of normoxia and hypoxia using sister plates of the commercial GBM cell lines, U87 and T98. Because the combination of Itraconazole and TVB-3166 displayed a synergistic enhancement in cell death at low nanomolar concentrations in GBM cells, we next assessed the efficacy of these drugs on clonogenic potential. Here, 100 cells were seeded onto 10 cm2 plates and grown under normoxic or hypoxic conditions for 10 days after exposure to single or combined agents. After 10 days post-treatment the plates treated with the combination of metabolic inhibitors exhibited best response. CONCLUSION: Given the significant clonogenic response to the combination of metabolic inhibitors, additional studies that further evaluate the chemotherapeutic effects of these metabolic inhibitors are warranted. By targeting hypoxia-induced metabolic pathways, we may enhance cancer stem-like cell death and eventually improve patient outcome.

Manipulation of tumor metabolism for therapeutic approaches: ovarian cancer-derived cell lines as a model system.

Malignant transformation of cells is often accompanied by up-regulation of glycolysis-related enzymes and transporters, as well as a distortion of mitochondrial respiration. As a consequence, most malignant tumors utilize high amounts of glucose and produce and accumulate high concentrations of lactate, even in the presence of oxygen. This phenomenon has been termed 'Warburg Effect'. Here, we aimed at resolving the interrelation between tumor metabolism, reactive oxygen species, double strand DNA breaks and radio-resistance in ovarian cancer-derived cells. As a model system two ovarian cancer-derived cell lines, OC316 and IGROV-1, and its corresponding xenografts were used. First, the metabolic properties of the xenografts were tested to ensure that initial in vitro data might later be transferred to in vivo data. In parallel, three inhibitors of tumor cell metabolism, 2-deoxy-D-glucose, an inhibitor of glycolysis, oxamate, a pyruvate analogue and inhibitor of lactate dehydrogenase, and rotenone, a specific inhibitor of mitochondrial electron complex I, were tested for their effect on the metabolism and radio-sensitivity of the respective ovarian cancer-derived cell lines. We found that all three inhibitors tested led to significant changes in the tumor cell energy metabolism at non-cytotoxic concentrations. Furthermore, we found that inhibition of tumor glycolysis by 2-deoxy-D-glucose in combination with rotenone decreased the radio-resistance at a clinically relevant radiation dose. This apparent radio-sensitizing effect appears to be based on an increased level of double strand DNA breaks 1 h and 24 h after gamma irradiation. Both cancer-derived cell lines maintained their metabolic properties, as well as their protein expression profiles and levels of reactive oxygen species in xenografts, thus providing a suitable model system for further in vivo investigations. A combination of metabolic inhibitors and reactive oxygen species-generating therapies, such as irradiation, may effectively enhance the therapeutic response in particularly metabolically highly active (ovarian) tumors.

Leukemia cells demonstrate a different metabolic perturbation provoked by 2-deoxyglucose

The shift in energy metabolism from oxidative phosphorylation to glycolysis can serve as a target for the inhibition of cancer growth. Here, we examined the metabolic changes induced by 2-deoxyglucose (2-DG), a glycolysis inhibitor, in leukemia cells by metabolome analysis. NB4 cells mainly utilized glucose as an energy source by glycolysis and oxidative phosphorylation in mitochondria, since metabolites in the glycolytic pathway and in the tricarboxylic acid (TCA) cycle were significantly decreased by 2-DG. In THP-1 cells, metabolites in the TCA cycle were not decreased to the same extent by 2-DG as in NB4 cells, which indicates that THP-1 utilizes energy sources other than glucose. TCA cycle metabolites in THP-1 cells may be derived from acetyl-CoA by fatty acid β-oxidation, which was supported by abundant detection of carnitine and acetylcarnitine in THP-1 cells. 2-DG treatment increased the levels of pentose phosphate pathway (PPP) metabolites and augmented the generation of NADPH by glucose-6-phosphate dehydrogenase. An increase in NADPH and upregulation of glutathione synthetase expression resulted in the increase in the reduced form of glutathione by 2-DG in NB4 cells. We demonstrated that a combination of 2-DG and inhibition of PPP by dehydroepiandrosterone (DHEA) effectively suppressed the growth of NB4 cells. The replenishment of the TCA cycle by fatty acid oxidation by carnitine palmitoyltransferase in THP-1 cells, treated by 2-DG, might be regulated by AMPK, as the combination of 2-DG and inhibition of AMPK by compound C potently suppressed the growth of THP-1 cells. Although 2-DG has been effective in preclinical and clinical studies, this treatment has not been fully explored due to concerns related to potential toxicities such as brain toxicity at high doses. We demonstrated that a combination of 2-DG and DHEA or compound C at a relatively low concentration effectively inhibits the growth of NB4 and THP-1 cells, respectively. These observations may aid in the identification of appropriate combinations of metabolic inhibitors at low concentrations which do not cause toxicities.

Probing the Impact of Valency on the Routing of Arginine-Rich Peptides into Eukaryotic Cells

Multivalency represents a critical parameter in cell biology responsible for the overall avidity of low-affinity interactions and the triggering of cellular events. Functions such as catalytic activity, cellular uptake, or localization are frequently linked to the oligomeric state of a protein. This study explores the impact of multivalency on the import and routing of peptides into cells. Specifically, cationic import sequences such as decaarginine, decalysine, and the HIV Tat peptide (GRKKRRQRRRAP, residues 48-59) as well as the nuclear localization sequence from SV40 large T-antigen were assembled into defined peptide oligomers by fusing them to the tetramerization domain of human p53 (residues 325-355, hp53(tet) domain). The resulting tetravalent peptides typically displayed between 10- and 100-fold enhancements in cellular import and intracellular routing properties in relation to their monomeric homologues. These peptides were not toxic to cells. Flow cytometry results and transfection assays indicated that tetravalent decaarginyl peptides (10R-p53(tet) and NLS-10R-p53(tet)) were the peptides most efficiently routed into cells. Their mechanism of import was subsequently examined on unfixed, viable cells using a combination of metabolic inhibitors, flow cytometry, and microscopy techniques. These studies revealed that tetravalent arginine-rich peptides bind to heparan sulfate on the cell surface, are internalized at 37 degrees C, but not at 4 degrees C, via a clathrin-mediated pathway, and accumulate into endosome-like acidic compartments. A fraction of these tetravalent peptides access the cytosol and accumulate in the nucleus of cells. This study concludes that the oligomerization of proteins harboring arginine-rich peptide chains may profoundly influence their ability to enter and be routed into cells.

Reversible connexin 43 dephosphorylation during hypoxia and reoxygenation is linked to cellular ATP levels.

Altered gap junction coupling of cardiac myocytes during ischemia may contribute to development of lethal arrhythmias. The phosphoprotein connexin 43 (Cx43) is the major constituent of gap junctions. Dephosphorylation of Cx43 and uncoupling of gap junctions occur during ischemia, but the significance of Cx43 phosphorylation in this setting is unknown. Here we show that Cx43 dephosphorylation in synchronously contracting myocytes during ischemia is reversible, independent of hypoxia, and closely associated with cellular ATP levels. Cx43 became profoundly dephosphorylated during hypoxia only when glucose supplies were limited and was completely rephosphorylated within 30 minutes of reoxygenation. Similarly, direct reduction of ATP by various combinations of metabolic inhibitors and by ouabain was closely paralleled by loss of phosphoCx43 and recovery of phosphoCx43 accompanied restoration of ATP. Dephosphorylation of Cx43 could not be attributed to hypoxia, acid pH or secreted metabolites, or to AMP-activated protein kinase; moreover, the process was selective for Cx43 because levels of phospho-extracellular signal regulated kinase (ERK)1/2 were increased throughout. Rephosphorylation of Cx43 was not dependent on new protein synthesis, or on activation of protein kinases A or G, ERK1/2, p38 mitogen-activated protein kinase, or Jun kinase; however, broad-spectrum protein kinase C inhibitors prevented Cx43 rephosphorylation while also sensitizing myocytes to reoxygenation-mediated cell death. We conclude that Cx43 is reversibly dephosphorylated and rephosphorylated during hypoxia and reoxygenation by a novel mechanism that is sensitive to nonlethal fluctuations in cellular ATP. The role of this regulated phosphorylation in the adaptation to ischemia remains to be determined.

EGF and TGFα modulate structural and functional differentiation of the mammary gland from pregnant mice in vitro: Possible role of the arachidonic acid pathway

Epidermal growth factor (EGF) has been suggested to be involved in mammary gland development by mitogenic stimulation of the ductal and alveolar epithelium in virgin mice. The present studies demonstrate that also in late-pregnant mice EGF leads to proliferation of the ductal, ductular, and alveolar epithelium. The mitogenic effect is associated with structural and functional dedifferentiation of alveolar cells as revealed by analysis of morphology, expression of cytosolic and secretory proteins, and fatty acid synthesis. Using a combination of metabolic inhibitors, the dedifferentiating effect of EGF could be blocked while the mitogenic action was not influenced. This finding demonstrates that the signal transduction pathway leading to dedifferentiation and mitosis can be separated, and that the dedifferentiating effect of EGF is independent of its mitogenic properties, but is probably mediated by activation of the arachidonic acid-dependent pathways (cyclo- and lipoxygenase pathways). Release of arachidonic acid from the endogenous phospholipid pool was found to be an early response of the explants to EGF. Accordingly, arachidonic acid itself proved to be capable of inducing epithelial dedifferentiation but failed to stimulate proliferation. TGF alpha showed qualitatively similar effects as EGF but was generally a stronger agonist. It is suggested that EGF and TGF alpha also play a role in mammary gland physiology during pregnancy by final developing and maintenance of the lobulo-alveolar structure in the mammary gland and prevention of premature onset of lactation, and that this is mediated through the PLA2-arachidonic acid signalling cascade.

Mechanisms of Organophosphate, Pyrethroid, and DDT Resistance in Citrus Thrips (Thysanoptera: Thripidae)

Synergism with metabolic inhibitors showed that resistance to fluvalinate was polyfactorial in a strain of citrus thrips, Scirtothrips citri (Moulton). Mixed-function oxidases were responsible for a major portion of the resistance (synergism as much as 61-fold at LC90), followed by est erases (synergism as much as 14-fold at Le90) Glutathione S-transferases may or may not be involved in fluvalinate resistance. Lack of total fluvalinate synergism with combinations of metabolic inhibitors, as well as limited synergism of DDT with piperonyl butoxide + dichlorophenyl ethanol, suggests a knock-down resistance ( kdr ) component. Sesame oil synergized fluvalinate up to 48-fold at LC90, suggesting the possible use of this product in the field. Resistance to dimethoate that was apparent in the parental TB84 strain before selection with fluvalinate may be due to enhanced activity of mixed-function oxidases.

Adenosine triphosphate depletion induces a rise in cytosolic free calcium in canine renal epithelial cells.

An elevation in cytosolic free calcium (Cai) produced by cellular ATP depletion may contribute to the initiation of cytotoxic events in renal ischemia. To evaluate whether ATP depletion results in a rise in Cai we examined the effect of cyanide and 2-deoxy-D-glucose on the Cai of Madin-Darby canine kidney cells. Exposure to the metabolic inhibitors resulted in a rise in Cai from 112 +/- 11 to 649 +/- 99 nM in 15 min. This combination of metabolic inhibitors also resulted in a decrement of cell ATP to 11 +/- 2% of control by 15 min. Experiments that were performed with other metabolic inhibitors confirm that the increment in Cai is due to inhibition of ATP synthesis. With the removal of cyanide and 2-deoxy-D-glucose, Cai recovered to 101 +/- 16 nM. In the absence of extracellular calcium activity (Ca0), Cai declined from 127 +/- 7 to 38 +/- 6 nM, whereas with cyanide plus 2-deoxy-D-glucose in the absence of Ca0 the Cai rose from 108 +/- 21 to 151 +/- 28 nM. Because the rise in Cai produced by ATP depletion in the absence of Ca0 is significantly less than that which occurs in the presence of Ca0, influx of Ca0 is necessary for the maximal rise of Cai. The rise in Cai that occurred in the absence of Ca0 suggests that the release of calcium from intracellular stores contributes to the increment in Cai seen with ATP depletion. TMB-8, an inhibitor of calcium release from intracellular stores, blunted the rise in Cai by nearly 50%. Neither verapamil nor nifedipine inhibited the rise in Cai. This study demonstrates that ATP depletion induced by the metabolic inhibitors cyanide and 2-deoxy-D-glucose is associated with a rapid and reversible increase in Cai. Both Ca0 influx and Cai redistribution contribute to this rise.

Inhibition of concanavalin A-induced agglutination of Novikoff tumor cells by cytochalasins and metabolic inhibitors: Role of cell-surface morphology and the distribution of concanavalin A receptors

Previous drug studies have suggested that concanavalin A (ConA)-induced cytoagglutination may be influenced by a system of contractile microfilaments. The present study was undertaken to determine the effects of microfilament-active drugs (cytochalasins B and D) (CB and CD) and inhibitors of ATP formation (NaN 3 and 2,4-dinitrophenol (DNP)) on ConA-induced agglutination of Novikoff cells and to investigate the mechanism(s) whereby these agents alter the surface properties of cells. The study described herein demonstrated that 1. 1. CB or CD inhibit cytoagglutination at concentrations above 1 or 0.1 μg/ml, respectively. 2. 2. NaN 3 and DNP both inhibit cytoagglutination, but DNP is more effective and specific. 3. 3. Combinations of metabolic inhibitors (NaN 3 or DNP) with CB lead to a greater reduction of cytoagglutination than with either agent alone. 4. 4. Maximal (>95%) cytoagglutination is still achieved in the presence of CB, CD, NaN 3, DNP, or combinations of these drugs. 5. 5. None of the drugs tested induced or allowed the redistribution of ConA receptors as measured by ferritin-ConA labeling. 6. 6. Both CB and CD induced the appearance of numerous blebs (zeioses) at the cell periphery, whereas metabolic inhibitors did not. 7. 7. The concentration of either CB or CD required to alter cell-surface morphology paralleled the concentration necessary to inhibit cytoagglutination. These results suggest that the cytochalasins inhibit ConA-induced agglutination of Novikoff cells by their effect on cell-surface morphology, rather than by their effects on the topographical distribution of cell-surface lectin receptors, and that metabolic inhibitors reduce agglutinability by a different mechanism than the cytochalasins.