Current Pharmaceutical Design - Volume 18, Issue 10, 2012

Decades of research on the mechanisms and processes leading to the development of cancer have provided a plethora of genes, proteins and physiological steps that may contribute to the generation and metastasis of the disease. However, despite many efforts, the success in chemotherapeutic intervention have not followed suit [1]. The causes of this are probably manifold and, among them, the difficulty to target specific proteins that are very similar to unrelated ones, (e.g. kinases), and the few cancer types or even subtypes for which they may contribute effectively in their promotion (breast cancer HER2 gene may be an example), could be discussed as potential reasons. Be that as it may, the most effective chemotherapeutic treatments to date and some of the most promising ones aim at gross differences between tumour and healthy cells: e. g. increased DNA synthesis (related to increased cell division and proliferation) and dependence on protein chaperones like Hsp90. In this sense, to target metabolic differences between neoplasms and normal tissues is an attractive approach that is currently being explored from different angles. The Warburg Effect in cancer, or glycolytic phenotype, is a feature shared by nearly all tumours, although to different degrees. Under this phenotype, tumour cells obtain their energy from glycolysis and lactic fermentation despite the presence of appropriate oxygen tensions, whereas somatic cells rely on mitochondrial respiration. Although this phenomenon was first described by Otto Warburg in the 19207apos; [2], it is in the last ten that it is receiving increasing attention, as attested by more than 30 reviews related to cancer cell metabolism published between 2010 and 2011 that deal with this phenomenon. It is beyond the scope of this issue, an in depth analysis of the glycolytic phenotype; for greater details on this, the reader may refer to some excellent works like [3, 4]. However, since this is at the base of the phenomena the present issue revisits, a short overview is imperative in order to better understand the intimate imbrication of pH homoeostasis, the proteins responsible and the glycolytic phenotype. A high rate of glucose uptake is a characteristic feature of tumour cells. This abnormal uptake is due to tumour cells metabolising glucose less efficiently than healthy cells, along with a greater demand for energy and metabolites to fuel cell proliferation. For reasons still obscure, tumour cells shut their respiratory metabolism and break glucose down to pyruvate only. In this process, the cell obtains only two ATP molecules per glucose consumed, while respiration is able to provide ca 30 under physiological conditions [5]. The advantage of using such an apparently inefficient sugar catabolism may lie on a greater availability of carbon backbones that reduce the energy expenditure necessary for their synthesis from scratch [4]. Strikingly, this same metabolic approach is chosen by micro-organisms like baker's yeast to produce high rates of growth [6]. In any event, an important issue in this type of metabolism is redox balance of pyridine nucleotide coenzymes: two NADH molecules are produced from cytosolic NAD+ per glucose molecule brought down to pyruvate. If this NADH would not be reoxidised, coenzyme pools would soon be depleted and all cellular metabolism should halt. Under respiratory conditions, these NADH molecules are re-oxidised at the mitochondria, but in tumour cells NADH is used to reduce pyruvate to lactic acid and the latter compound is, for the most part, metabolised no further. Since the dissociation constant for lactic acid is very low (pKa=3.85), one of the net results then is the release into the cytosol of two H+ per glucose molecule consumed. In addition, there may be other sources of H+ in tumours that can contribute significantly to the final count [3]. In particular, CO2 may well be a source of H+ in tumour cells, as assessed using cell lines with impaired glycolysis [7, 8]; these increased levels in CO2 have glutaminolysis at their base [8, 9]. Accumulation of protons in the cytoplasm promotes cell death, while maintenance of slightly alkaline conditions promotes proliferation [10]. Therefore, excess H+ needs to be neutralised from the cytoplasm if a tumour cell is to thrive. Keeping this in mind, it is easy to understand that proton dynamics and homoeostasis is a paramount physiological processes for a tumour cell. Moreover, it is also one of the major differential characteristics of neoplasias compared with healthy tissue. Consequently, a group of both clinical and basic cancer researchers realised the potential of targeting proton dynamics as a therapeutic strategy and gathered in Madrid in 2009. The success of this meeting led to the formation of the “International Society for Proton Dynamics in Cancer” (ISPDC) in January 2010 [1]. The present issue is inspired by the first meeting of the ISPDC held in Rome in 2010. It aims to give the reader an overview of the importance of pH and proton homoeostasis in cancer and the opportunities that proton dynamics may offer as a target for chemotherapeutic intervention.....

High rate of glycolysis is a metabolic hallmark of cancer. While anaerobic glycolysis promotes energy production under hypoxia, aerobic glycolysis, the Warburg effect, offers a proliferative advantage through redirecting carbohydrate fluxes from energy production to biosynthetic pathways. To fulfill tumor cell needs, the glycolytic switch is associated with elevated glucose uptake and lactic acid release. Altered glucose metabolism is the basis of positron emission tomography using the glucose analogue tracer [18F]- fluorodeoxyglucose, a widely used clinical application for tumor diagnosis and monitoring. On the other hand, high levels of lactate have been associated with poor clinical outcome in several types of human cancers. Although lactic acid was initially considered merely as an indicator of the glycolytic flux, many evidences originally from the study of normal tissue physiology and more recently transposed to the tumor situation indicate that lactic acid, i.e. the lactate anion and protons, directly contributes to tumor growth and progression. Here, we briefly review the current knowledge pertaining to lactic acidosis and metastasis, lactate shuttles, the influence of lactate on redox homeostasis, lactate signaling and lactate-induced angiogenesis in the cancer context. The monocarboxylate transporters MCT1 and MCT4 have now been confirmed as prominent facilitators of lactate exchanges between cancer cells with different metabolic behaviors and between cancer and stromal cells. We therefore address the function and regulation of MCTs, highlighting MCT1 as a novel anticancer target. MCT1 inhibition allows to simultaneously disrupt metabolic cooperativity and angiogenesis in cancer with a same agent, opening a new path for novel anticancer therapies.

The acid-base balance of cells is related to the concentration of free H+ ions. These are highly reactive, and their intracellular concentration must be regulated to avoid detrimental effects to the cell. H+ ion dynamics are influenced by binding to chelator substances (‘buffering’), and by the production, diffusion and membrane-transport of free H+ ions or of the H+-bound chelators. Intracellular pH (pHi) regulation aims to balance this system of diffusion-reaction-transport processes at a favourable steady-state pHi. The ability of cells to regulate pHi may set a limit to tissue growth and can be subject to selection pressures. Cancer cells have been postulated to respond favourably to such selection pressures by evolving a better means of pHi regulation. A particularly important feature of tumour pHi regulation is acid-extrusion, which involves H+-extrusion and HCO3 ¯-uptake by membrane-bound transporter-proteins. Extracellular CO2/HCO3 ¯ buffer facilitates these membrane-transport processes. As a mobile pH-buffer, CO2/HCO3 ¯ protects the extracellular space from excessive acidification that could otherwise inhibit further acid-extrusion. CO2/HCO3 ¯ also provides substrate for HCO3 ¯- transporters. However, the inherently slow reaction kinetics of CO2/HCO3 ¯ can be rate-limiting for acid-extrusion. To circumvent this, cells can express extracellular-facing carbonic anhydrase enzymes to accelerate the attainment of equilibrium between CO2, HCO3 ¯ and H+. The acid-extrusion apparatus has been proposed as a target for anti-cancer therapy. The major targets include H+ pumps, Na+/H+ exchangers and carbonic anhydrases. The effectiveness of such therapy will depend on the correct identification of rate-limiting steps in pHi regulation in a specific type of cancer.

Transformed cells suffer several changes leading to the increase of protective mechanisms and show a metabolic profile in accordance with higher proliferative capacity. In these mechanisms, changes in mitochondrial activity cause a higher glycolytic metabolism in detriment of oxidative phosphorylation. In these changes, H+-ATPase regulation seems to be importantly involved. During the last years, polyphenols and specially the stilbene resveratrol and related members of its family have been studied because they are able to affect tumour cell growth and cancer progression. Among the different effects induced by resveratrol, inhibition of H+-ATPase seems to be one important mechanism in its effect on cancer progression. Further, an ectopic H+-ATPase located in the outer surface of plasma membrane has been recently involved in cancer progression and angiogenesis. In this article we review the latest findings about resveratrol inhibition of H+-ATPase and its importance in tumour cell growth and cancer progression.

The Na+/H+-exchanger 1, NHE1 (SLC9A1) and the electroneutral Na+,HCO3 --cotransporter NBCn1 (SLC4A7) are coexpressed in a wide range of tissues. Under normal physiological conditions these transporters play an ostensibly similar role, namely that of net acid extrusion after cellular acidification. In addition, they have been implicated in multiple other cellular processes, including regulation of transepithelial transport, cell volume, cell death/survival balance, and cell motility. In spite of their apparent functional similarity, the two transporters also serve distinctly different functions and are differentially regulated. Here, we provide an update on the basic structure, function, regulation, physiology and pharmacology of NHE1 and NBCn1, with particular focus on the factors responsible for their functional similarities and differences. Finally, we highlight recent findings implicating these transporters in cancer development, and discuss issues relating to NHE1 and NBCn1 as potential targets in cancer treatment.

Among the many factors involved in the maintenance of homeostatic growth is the tight regulation of cellular pH. Intracellular pH of normal cells is maintained within a physiological range thanks to the activity of a number of pH regulators that respond to the acidbase shifts associated with normal cellular metabolic processes. Interestingly, there is a preponderance of evidence that dysregulation of intracellular pH is associated with processes that favor cell transformation such as cell cycle progression, enhanced proliferation, insensitivity to growth inhibitory stimuli, resistance to apoptosis, genomic instability and angiogenesis. Among the strategies employed by the cells to regulate intracellular pH, the Na+/H+ exchanger 1 (NHE1) protein from the Na+/H+ exchanger (NHE) family has been directly associated with cellular transformation, invasion and metastasis. These observations have heightened the interest in NHE1 as a promising novel drug target for more effective and selective anti-cancer therapeutics. Here we present a review of the basic biology of this remarkable protein and present evidence to support targeting NHE1 as a potential anti-cancer strategy.

Cancer cells show a metabolic shift that makes them overproduce protons; this has the potential to disturb the cellular acidbase homeostasis. However, these cells show cytoplasmic alkalinisation, increased acid extrusion and endosome-dependent drug resistance. Vacuolar type ATPases (V-ATPases), together with other transporters, are responsible to a great extent for these symptoms. These multi-subunit proton pumps are involved in the control of cytosolic pH and the generation of proton gradients (positive inside) across endocellular membrane systems like Golgi, endosomes or lysosomes. In addition, in tumours, they have been shown to play an important role in the acidification of the intercellular medium. This importance makes them an attractive target to control tumour cell proliferation. In the present review we present the major characteristics of this kind of proton pumps and we provide some recent insights on their in vivo regulation. Also, we review some of the consequences that V-ATPase inhibition carries for the tumour cell, such as cell cycle arrest or cell death, and provide a brief summary of the studies related to cancer made recently with commercially available inhibitors. In the light of recent knowledge on the regulation of this proton pump, some new approaches to impair V-ATPase function are also suggested.

It is becoming increasingly acknowledged that tumorigenesis is not simply characterized by the accumulation of rapidly proliferating, genetically mutated cells. Microenvironmental biophysical factors like hypoxia and acidity dramatically condition cancer cells and act as selective forces for malignant cells, adapting through metabolic reprogramming towards aerobic glycolysis. Avoiding intracellular accumulation of lactic acid and protons, otherwise detrimental to cell survival is crucial for malignant cells to maintain cellular pH homeostasis. As a consequence of the upregulated expression and/or function of several pH-regulating systems, cancer cells display an alkaline intracellular pH (pHi) and an acidic extracellular pH (pHe). Among the pH-regulating proteins, proton pumps play an important role in both drug-resistance and metastatic spread, thus representing a suitable therapeutic target. Proton pump inhibitors (PPI) have been reported as cytotoxic drugs active against several human tumor cells and preclinical data have prompted the investigation of PPI as anticancer agents in humans. This review will update the current knowledge on the antitumor activities of PPI and their potential applications.

Two of the key proteins involved in tumor acidification are the V-ATPase and the tumor-associated carbonic anhydrases (CAs), such as CA IX and XII. Although there are many chemical classes of V-ATPase inhibitors, most of them are toxic for mammals and their potential use as antitumor drugs is limited. The proton pump inhibitors (PPIs), a class of antiulcer agents in clinical use for more than 30 years, have been proven to be useful in modulating tumor acidification, presumably by inactivating V-ATPase, through modification of Cys residues essential for the catalytic activity of the ATPase. This mechanism of action has yet to be demonstrated, but several recent clinical trials showed the efficacity of this approach for inhibiting the growth of tumors and their re-sensitivization to anticancer drugs such as cisplatin, or doxorubicin. Further studies are anyhow warranted to better understand the role of PPIs in the management of cancer. The monoclonal antibodies (mAbs) girentuximab, and its 124I -radiolabelled variant targeting CA IX are in advanced clinical trials both for the treatment and imaging of hypoxic tumors overexpressing CA IX. Small molecule CA IX inhibitors, of sulfonamide and coumarin type are in advanced preclinical evaluation, both for imaging and treatment of solid tumors and metastases in which CA IX/XII are present. As cancer is still a big clinical problem and most of the hypoxic tumors do not respond to classical anticancer drugs or to radiotherapy, the development of alternative anticancer approaches, such as interference with tumor acidification through inhibition of VATPase and CAs, represents an interesting avenue for future research.

During the past 20 years, we have found that acridine orange (AO) selectively accumulates in musculoskeletal sarcomas in vivo or exerts selective cytocidal effects against sarcoma cells in vitro after illumination of the tumor cells with visible light or irradiation of the cells with low-dose X-rays. Based on the data obtained from basic research, we have employed reduction surgery followed by photo- or radiodynamic therapy using AO (AO-PDT & RDT) in 71 patients with musculoskeletal sarcomas, in an attempt to maintain excellent limb function in the patients. We have obtained good local control rates and remarkably better limb functions with this approach as compared to the results obtained with the conventional wide resection surgery. Our basic research demonstrated that AO accumulates densely in intracellular acidic vesicles, especially lysosomes, in an acidity-dependent manner. In cancer cells that proliferate under hypoxic conditions or with Warburg's effect, active glycolysis produces an enormous number of protons, which are released by the cells via proton pumps into the extracellular fluid or lysosomes to maintain a neutral pH of the cytosolic fluid. Cancer cells contain many strongly acidic lysosomes of large sizes; therefore, AO shows marked and prolonged accumulation in the acidic lysosomes of cancer cells. Photon energy excites the AO resulting in the production of activated oxygen species, which oxidize the fatty acids of the lysosomal membrane, resulting in the leakage of lysosomal enzymes and protons, followed by apoptosis of the cancer cells. Based on these observations, we conclude that AO-PDT & RDT target acidic vesicles, especially the lysosomes, in cancer cells, to exhibit selective anti-cancer cell activity. Therefore, it is suggested that AO excited by photon energy has excellent potential as an anticancer “Magic Bullet”.

Wound healing and anti-inflammatory effects of aerial parts and roots of S. acuminata, S. cana var. alpina, S. cana var. jacquiniana, S. cana var. radicosa, S. eriophora, S. laciniata ssp. laciniata, S. suberosa ssp. suberosa and S. sublanata were investigated in current study to clarify the traditional usage of Scorzonera species growing in Turkey. It is well known that some species of the Scorzonera genus are used for wound healing in Turkish and European traditional medicine. Therefore, wound healing effect of the plants was assessed by using linear incision and circular excision experimental wound models and subsequently histopathological analysis. Hydroxyproline content of the treated tissues was also assessed. Furthermore, the extracts were screened for anti-hyaluronidase activity. For the evaluation of anti-inflammatory activity, acetic acid-induced increase in capillary permeability test was used. 20% aqueous methanol extracts of the aerial parts of Scorzonera species, S. cana var. jacquiniana and S. eriophora were found to be effective on the wound and anti-inflammatory activity test models. The results of histopathological examination supported the outcome of linear incision and circular excision wound models. Phytochemical analyses of the tested extracts were also performed by using developed and validated HPLC method. Among the tested standard compounds, varying amounts of the chlorogenic acid, rutin, hyperoside and luteolin-7-glycoside were determined in Scorzonera species.

Non-systemic drugs act within the intestinal lumen without reaching the systemic circulation. The first generation included polymeric resins that sequester phosphate ions, potassium ions, or bile acids for the treatment of electrolyte imbalances or hypercholesteremia. The field has evolved towards non-absorbable small molecules or peptides targeting luminal enzymes or transporters for the treatment of mineral metabolism disorders, diabetes, gastrointestinal (GI) disorders, and enteric infections. From a drug design and development perspective, non-systemic agents offer novel opportunities to address unmet medical needs while minimizing toxicity risks, but also present new challenges, including developing a better understanding and control of non-transcellular leakage pathways into the systemic circulation. The pharmacokinetic-pharmacodynamic relationship of drugs acting in the GI tract can be complex due to the variability of intestinal transit, interaction with chyme, and the complex environment of the surface epithelia. We review the main classes of nonabsorbable agents at various stages of development, and their therapeutic potential and limitations. The rapid progress in the identification of intestinal receptors and transporters, their functional characterization and role in metabolic and inflammatory disorders, will undoubtedly renew interest in the development of novel, safe, non-systemic therapeutics.