Predicting the safety and efficacy of butter therapy to raise tumour pHe: an integrative modelling study

Background: Clinical positron emission tomography imaging has demonstrated the vast majority of human cancers exhibit significantly increased glucose metabolism when compared with adjacent normal tissue, resulting in an acidic tumour microenvironment. Recent studies demonstrated reducing this acidity through systemic buffers significantly inhibits development and growth of metastases in mouse xenografts.\ud \ud Methods: We apply and extend a previously developed mathematical model of blood and tumour buffering to examine the impact of oral administration of bicarbonate buffer in mice, and the potential impact in humans. We recapitulate the experimentally observed tumour pHe effect of buffer therapy, testing a model prediction in vivo in mice. We parameterise the model to humans to determine the translational safety and efficacy, and predict patient subgroups who could have enhanced treatment response, and the most promising combination or alternative buffer therapies.\ud \ud Results: The model predicts a previously unseen potentially dangerous elevation in blood pHe resulting from bicarbonate therapy in mice, which is confirmed by our in vivo experiments. Simulations predict limited efficacy of bicarbonate, especially in humans with more aggressive cancers. We predict buffer therapy would be most effectual: in elderly patients or individuals with renal impairments; in combination with proton production inhibitors (such as dichloroacetate), renal glomular filtration rate inhibitors (such as non-steroidal anti-inflammatory drugs and angiotensin-converting enzyme inhibitors), or with an alternative buffer reagent possessing an optimal pK of 7.1–7.2.\ud \ud Conclusion: Our mathematical model confirms bicarbonate acts as an effective agent to raise tumour pHe, but potentially induces metabolic alkalosis at the high doses necessary for tumour pHe normalisation. We predict use in elderly patients or in combination with proton production inhibitors or buffers with a pK of 7.1–7.2 is most promising

A general reaction-diffusion model of acidity in cancer invasion

We model the metabolism and behaviour of a developing cancer tumour in the context of its microenvironment, with the aim of elucidating the consequences of altered energy metabolism. Of particular interest is the Warburg Effect, a widespread preference in tumours for cytosolic glycolysis rather than oxidative phosphorylation for glucose breakdown, as yet incompletely understood. We examine a candidate explanation for the prevalence of the Warburg Effect in tumours, the acid-mediated invasion hypothesis, by generalising a canonical non-linear reaction–diffusion model of acid-mediated tumour invasion to consider additional biological features of potential importance. We apply both numerical methods and a non-standard asymptotic analysis in a travelling wave framework to obtain an explicit understanding of the range of tumour behaviours produced by the model and how fundamental parameters govern the speed and shape of invading tumour waves. Comparison with conclusions drawn under the original system—a special case of our generalised system—allows us to comment on the structural stability and predictive power of the modelling framework

Bicarbonate and dichloroacetate: Evaluating pH altering therapies in a mouse model for metastatic breast cancer

BACKGROUND:The glycolytic nature of malignant tumors contributes to high levels of extracellular acidity in the tumor microenvironment. Tumor acidity is a driving force in invasion and metastases. Recently, it has been shown that buffering of extracellular acidity through systemic administration of oral bicarbonate can inhibit the spread of metastases in a mouse model for metastatic breast cancer. While these findings are compelling, recent assessments into the use of oral bicarbonate as a cancer intervention reveal limitations.METHODS:We posited that safety and efficacy of bicarbonate could be enhanced by dichloroacetate (DCA), a drug that selectively targets tumor cells and reduces extracellular acidity through inhibition of glycolysis. Using our mouse model for metastatic breast cancer (MDA-MB-231), we designed an interventional survival study where tumor bearing mice received bicarbonate, DCA, or DCA-bicarbonate (DB) therapies chronically.RESULTS:Dichloroacetate alone or in combination with bicarbonate did not increase systemic alkalosis in mice. Survival was longest in mice administered bicarbonate-based therapies. Primary tumor re-occurrence after surgeries is associated with survival rates. Although DB therapy did not significantly enhance oral bicarbonate, we did observe reduced pulmonary lesion diameters in this cohort. The DCA monotherapy was not effective in reducing tumor size or metastases or improving survival time. We provide in vitro evidence to suggest this outcome may be a function of hypoxia in the tumor microenvironment.CONCLUSIONS:DB combination therapy did not appear to enhance the effect of chronic oral bicarbonate. The anti-tumor effect of DCA may be dependent on the cancer model. Our studies suggest DCA efficacy is unpredictable as a cancer therapy and further studies are necessary to determine the role of this agent in the tumor microenvironment.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement

Prisoner's Dilemma in Cancer Metabolism

As tumors outgrow their blood supply and become oxygen deprived, they switch to less energetically efficient but oxygen-independent anaerobic glucose metabolism. However, cancer cells maintain glycolytic phenotype even in the areas of ample oxygen supply (Warburg effect). It has been hypothesized that the competitive advantage that glycolytic cells get over aerobic cells is achieved through secretion of lactic acid, which is a by-product of glycolysis. It creates acidic microenvironment around the tumor that can be toxic to normal somatic cells. This interaction can be seen as a prisoner's dilemma: from the point of view of metabolic payoffs, it is better for cells to cooperate and become better competitors but neither cell has an incentive to unilaterally change its metabolic strategy. In this paper a novel mathematical technique, which allows reducing an otherwise infinitely dimensional system to low dimensionality, is used to demonstrate that changing the environment can take the cells out of this equilibrium and that it is cooperation that can in fact lead to the cell population committing evolutionary suicide

Reduction of metastasis using a non-volatile buffer

The tumor microenvironment is acidic as a consequence of upregulated glycolysis and poor perfusion and this acidity, in turn, promotes invasion and metastasis. We have recently demonstrated that chronic consumption of sodium bicarbonate increased tumor pH and reduced spontaneous and experimental metastases. This occurred without affecting systemic pH, which was compensated. Additionally, these prior data did not rule out the possibility that bicarbonate was working though effects on carbonic anhydrase, and not as a buffer per se. Here, we present evidence that chronic ingestion of a non-volatile buffer, 2-imidazole-1-yl-3-ethoxycarbonylpropionic acid (IEPA) with a pKa of 6.9 also reduced metastasis in an experimental PC3M prostate cancer mouse model. Animals (n = 30) were injected with luciferase expressing PC3M prostate cancer cells either subcutaneously (s.c., n = 10) or intravenously (i.v., n = 20). Four days prior to inoculations, half of the animals for each experiment were provided drinking water containing 200 mM IEPA buffer. Animals were imaged weekly to follow metastasis, and these data showed that animals treated with IEPA had significantly fewer experimental lung metastasis compared to control groups (P < 0.04). Consistent with prior work, the pH of treated tumors was elevated compared to controls. IEPA is observable by in vivo magnetic resonance spectroscopy and this was used to measure the presence of IEPA in the bladder, confirming that it was orally available. The results of this study indicate that metastasis can be reduced by non-volatile buffers as well as bicarbonate and thus the effect appears to be due to pH buffering per se

Proton pump inhibition induces autophagy as a survival mechanism following oxidative stress in human melanoma cells

Proton pump inhibitors (PPI) target tumour acidic pH and have an antineoplastic effect in melanoma. The PPI esomeprazole (ESOM) kills melanoma cells through a caspase-dependent pathway involving cytosolic acidification and alkalinization of tumour pH. In this paper, we further investigated the mechanisms of ESOM-induced cell death in melanoma. ESOM rapidly induced accumulation of reactive oxygen species (ROS) through mitochondrial dysfunctions and involvement of NADPH oxidase. The ROS scavenger N-acetyl--cysteine (NAC) and inhibition of NADPH oxidase significantly reduced ESOM-induced cell death, consistent with inhibition of cytosolic acidification. Autophagy, a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, represents a defence mechanism in cancer cells under metabolic stress. ESOM induced the early accumulation of autophagosomes, at the same time reducing the autophagic flux, as observed by WB analysis of LC3-II accumulation and by fluorescence microscopy. Moreover, ESOM treatment decreased mammalian target of rapamycin signalling, as reduced phosphorylation of p70-S6K and 4-EBP1 was observed. Inhibition of autophagy by knockdown of Atg5 and Beclin-1 expression significantly increased ESOM cytotoxicity, suggesting a protective role for autophagy in ESOM-treated cells. The data presented suggest that autophagy represents an adaptive survival mechanism to overcome drug-induced cellular stress and cytotoxicity, including alteration of pH homeostasis mediated by proton pump inhibition

Investigation of tumor hypoxia using a two-enzyme system for in vitro generation of oxygen deficiency

<p>Abstract</p> <p>Background</p> <p>Oxygen deficiency in tumor tissue is associated with a malign phenotype, characterized by high invasiveness, increased metastatic potential and poor prognosis. Hypoxia chambers are the established standard model for <it>in vitro </it>studies on tumor hypoxia. An enzymatic hypoxia system (GOX/CAT) based on the use of glucose oxidase (GOX) and catalase (CAT) that allows induction of stable hypoxia for <it>in vitro </it>approaches more rapidly and with less operating expense has been introduced recently. Aim of this work is to compare the enzymatic system with the established technique of hypoxia chamber in respect of gene expression, glucose metabolism and radioresistance, prior to its application for <it>in vitro </it>investigation of oxygen deficiency.</p> <p>Methods</p> <p>Human head and neck squamous cell carcinoma HNO97 cells were incubated under normoxic and hypoxic conditions using both hypoxia chamber and the enzymatic model. Gene expression was investigated using Agilent microarray chips and real time PCR analysis. ¹⁴C-fluoro-deoxy-glucose uptake experiments were performed in order to evaluate cellular metabolism. Cell proliferation after photon irradiation was investigated for evaluation of radioresistance under normoxia and hypoxia using both a hypoxia chamber and the enzymatic system.</p> <p>Results</p> <p>The microarray analysis revealed a similar trend in the expression of known HIF-1 target genes between the two hypoxia systems for HNO97 cells. Quantitative RT-PCR demonstrated different kinetic patterns in the expression of carbonic anhydrase IX and lysyl oxidase, which might be due to the faster induction of hypoxia by the enzymatic system. ¹⁴C-fluoro-deoxy-glucose uptake assays showed a higher glucose metabolism under hypoxic conditions, especially for the enzymatic system. Proliferation experiments after photon irradiation revealed increased survival rates for the enzymatic model compared to hypoxia chamber and normoxia, indicating enhanced resistance to irradiation. While the GOX/CAT system allows independent investigation of hypoxia and oxidative stress, care must be taken to prevent acidification during longer incubation.</p> <p>Conclusion</p> <p>The results of our study indicate that the enzymatic model can find application for <it>in vitro </it>investigation of tumor hypoxia, despite limitations that need to be considered in the experimental design.</p

Oncogene Activation Induces Metabolic Transformation Resulting in Insulin-Independence in Human Breast Cancer Cells

Normal breast epithelial cells require insulin and EGF for growth in serum-free media. We previously demonstrated that over expression of breast cancer oncogenes transforms MCF10A cells to an insulin-independent phenotype. Additionally, most breast cancer cell lines are insulin-independent for growth. In this study, we investigated the mechanism by which oncogene over expression transforms MCF10A cells to an insulin-independent phenotype. Analysis of the effects of various concentrations of insulin and/or IGF-I on proliferation of MCF10A cells demonstrated that some of the effects of insulin were independent from those of IGF-I, suggesting that oncogene over expression drives a true insulin-independent proliferative phenotype. To test this hypothesis, we examined metabolic functions of insulin signaling in insulin-dependent and insulin-independent cells. HER2 over expression in MCF10A cells resulted in glucose uptake in the absence of insulin at a rate equal to insulin-induced glucose uptake in non-transduced cells. We found that a diverse set of oncogenes induced the same result. To gain insight into how HER2 oncogene signaling affected increased insulin-independent glucose uptake we compared HER2-regulated gene expression signatures in MCF10A and HER2 over expressing MCF10A cells by differential analysis of time series gene expression data from cells treated with a HER2 inhibitor. This analysis identified genes specifically regulated by the HER2 oncogene, including VAMP8 and PHGDH, which have known functions in glucose uptake and processing of glycolytic intermediates, respectively. Moreover, these genes specifically implicated in HER2 oncogene-driven transformation are commonly altered in human breast cancer cells. These results highlight the diversity of oncogene effects on cell regulatory pathways and the importance of oncogene-driven metabolic transformation in breast cancer

Bone Is Not Essential for Osteoclast Activation

Background: The mechanism whereby bone activates resorptive behavior in osteoclasts, the cells that resorb bone, is unknown. It is known that avb3 ligands are important, because blockade of avb3 receptor signaling inhibits bone resorption, but this might be through inhibition of adhesion or migration rather than resorption itself. Nor is it known whether avb3 ligands are sufficient for resorption the consensus is that bone mineral is essential for the recognition of bone as the substrate appropriate for resorption. Methodology/Principal Findings: Vitronectin- but not fibronectin-coated coverslips induced murine osteoclasts to secrete tartrate-resistant acid phosphatase, as they do on bone. Osteoclasts incubated on vitronectin, unlike fibronectin, formed podosome belts on glass coverslips, and these were modulated by resorption-regulating cytokines. Podosome belts formed on vitronectin-coated surfaces whether the substrates were rough or smooth, rigid or flexible. We developed a novel approach whereby the substrate-apposed surface of cells can be visualized in the scanning electron microscope. With this approach, supported by transmission electron microscopy, we found that osteoclasts on vitronectin-coated surfaces show ruffled borders and clear zones characteristic of resorbing osteoclasts. Ruffles were obscured by a film if cells were incubated in the cathepsin inhibitor E64, suggesting that removal of the film represents substrate-degrading behavior. Analogously, osteoclasts formed resorption-like trails on vitronectin-coated substrates. Like bone resorption, these trails were dependent upon resorbogenic cytokines and were inhibited by E64. Bone mineral induced actin rings and surface excavation only if first coated with vitronectin. Fibronectin could not substitute in any of these activities, despite enabling adhesion and cell spreading. Conclusions/Significance: Our results show that ligands avb3 are not only necessary but sufficient for the induction of resorptive behavior in osteoclasts; and suggest that bone is recognized through its affinity for these ligands, rather than by its mechanical or topographical attributes, or through a putative ‘mineral receptor’