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Altered glycolytic metabolism has been associated with chemoresistance in acute myeloid leukemia (AML). However, there are still aspects that need clarification, as well as how to explore these metabolic alterations in therapy. In the present study, we aimed to elucidate the role of glucose metabolism in the acquired resistance of AML cells to cytarabine (Ara-C) and to explore it as a therapeutic target. Resistance was induced by stepwise exposure of AML cells to increasing concentrations of Ara-C. Ara-C-resistant cells were characterized for their growth capacity, genetic alterations, metabolic profile, and sensitivity to different metabolic inhibitors. Ara-C-resistant AML cell lines, KG-1 Ara-R, and MOLM13 Ara-R presented different metabolic profiles. KG-1 Ara-R cells exhibited a more pronounced glycolytic phenotype than parental cells, with a weaker acute response to 3-bromopyruvate (3-BP) but higher sensitivity after 48 h. KG-1 Ara-R cells also display increased respiration rates and are more sensitive to phenformin than parental cells. On the other hand, MOLM13 Ara-R cells display a glucose metabolism profile similar to parental cells, as well as sensitivity to glycolytic inhibitors. These results indicate that acquired resistance to Ara-C in AML may involve metabolic adaptations, which can be explored therapeutically in the AML patient setting who developed resistance to therapy.

Cancer is a complex disease characterized by several alterations, which confer, to the cells, the capacity to proliferate uncontrollably and to resist cellular death. Multiresistance to conventional chemotherapy drugs is often the cause of treatment failure; thus, the search for natural products or their derivatives with therapeutic action is essential. Chiral derivatives of xanthones (CDXs) have shown potential inhibitory activity against the growth of some human tumor cell lines. This work reports the screening of a library of CDXs, through viability assays, in different cancer cell lines: A375-C5, MCF-7, NCI-H460, and HCT-15. CDXs' effect was analyzed based on several parameters of cancer cells, and it was also verified if these compounds were substrates of glycoprotein-P (Pgp), one of the main mechanisms of resistance in cancer therapy. Pgp expression was evaluated in all cell lines, but no expression was observed, except for HCT-15. Also, when a humanized yeast expressing the human gene MDR1 was used, no conclusions could be drawn about CDXs as Pgp substrates. The selected CDXs did not induce significant differences in the metabolic parameters analyzed. These results show that some CDXs present promising antitumor activity, but other mechanisms should be triggered by these compounds.

The "Warburg effect" consists of a metabolic shift in energy production from oxidative phosphorylation to glycolysis. The continuous activation of glycolysis in cancer cells causes rapid energy production and an increase in lactate, leading to the acidification of the tumour microenvironment, chemo- and radioresistance, as well as poor patient survival. Nevertheless, the mitochondrial metabolism can be also involved in aggressive cancer characteristics. The metabolic differences between cancer and normal tissues can be considered the Achilles heel of cancer, offering a strategy for new therapies. One of the main causes of treatment resistance consists of the increased expression of efflux pumps, and multidrug resistance (MDR) proteins, which are able to export chemotherapeutics out of the cell. Cells expressing MDR proteins require ATP to mediate the efflux of their drug substrates. Thus, inhibition of the main energy-producing pathways in cancer cells, not only induces cancer cell death per se, but also overcomes multidrug resistance. Given that most anticancer drugs do not have the ability to distinguish normal cells from cancer cells, a number of drug delivery systems have been developed. These nanodrug delivery systems provide flexible and effective methods to overcome MDR by facilitating cellular uptake, increasing drug accumulation, reducing drug efflux, improving targeted drug delivery, co-administering synergistic agents, and increasing the half-life of drugs in circulation.

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 10 percent largely due to the intense fibrotic desmoplastic reaction, characterized by high levels of extracellular matrix (ECM) collagen I that constitutes a niche for a subset of cancer cells, the cancer stem cells (CSCs). Cancer cells undergo a complex metabolic adaptation characterized by changes in metabolic pathways and biosynthetic processes. The use of the 3D organotypic model in this study allowed us to manipulate the ECM constituents and mimic the progression of PDAC from an early tumor to an ever more advanced tumor stage. To understand the role of desmoplasia on the metabolism of PDAC parenchymal (CPC) and CSC populations, we studied their basic metabolic parameters in organotypic cultures of increasing collagen content to mimic in vivo conditions. We further measured the ability of the bioenergetic modulators (BMs), 2-deoxyglucose, dichloroacetate and phenformin, to modify their metabolic dependence and the therapeutic activity of paclitaxel albumin nanoparticles (NAB-PTX). While all the BMs decreased cell viability and increased cell death in all ECM types, a distinct, collagen I-dependent profile was observed in CSCs. As ECM collagen I content increased (e.g., more aggressive conditions), the CSCs switched from glucose to mostly glutamine metabolism. All three BMs synergistically potentiated the cytotoxicity of NAB-PTX in both cell lines, which, in CSCs, was collagen I-dependent and the strongest when treated with phenformin + NAB-PTX. Metabolic disruption in PDAC can be useful both as monotherapy or combined with conventional drugs to more efficiently block tumor growth.

Antiglycolytic agents inhibit cell metabolism and modify the tumor's microenvironment, affecting chemotherapy resistance mechanisms. In this work, we studied the effect of the glycolytic inhibitors 3-bromopyruvate (3BP), dichloroacetate (DCA) and 2-deoxyglucose (2DG) on cancer cell properties and on the multidrug resistance phenotype, using lung cancer cells as a model. All compounds led to the loss of cell viability, with different effects on the cell metabolism, migration and proliferation, depending on the drug and cell line assayed. DCA was the most promising compound, presenting the highest inhibitory effect on cell metabolism and proliferation. DCA treatment led to decreased glucose consumption and ATP and lactate production in both A549 and NCI-H460 cell lines. Furthermore, the DCA pretreatment sensitized the cancer cells to Paclitaxel (PTX), a conventional chemotherapeutic drug, with a 2.7-fold and a 10-fold decrease in PTX IC 50 values in A549 and NCI-H460 cell lines, respectively. To increase the intracellular concentration of DCA, thereby potentiating its effect, DCA-loaded poly(lactic- co -glycolic acid) nanoparticles were produced. At higher DCA concentrations, encapsulation was found to increase its toxicity. These results may help find a new treatment strategy through combined therapy, which could open doors to new treatment approaches.

The reverse pH gradient is a major feature associated with cancer cell reprogrammed metabolism. This phenotype is supported by increased activity of pH regulators like ATPases, carbonic anhydrases (CAs), monocarboxylate transporters (MCTs) and sodium-proton exchangers (NHEs) that induce an acidic tumor microenvironment, responsible for the cancer acid-resistant phenotype. In this work, we analyzed the expression of these pH regulators and explored their inhibition in breast cancer cells as a strategy to enhance the sensitivity to chemotherapy. Expression of the different pH regulators was evaluated by immunofluorescence and Western blot in two breast cancer cell lines (MDA-MB-231 and MCF-7) and by immunohistochemistry in human breast cancer tissues. Cell viability, migration and invasion were evaluated upon exposure to the pH regulator inhibitors (PRIs) concanamycin-A, cariporide, acetazolamide and cyano-4-hydroxycinnamate. Additionally, PRIs were combined with doxorubicin to analyze the effect of cell pH dynamic disruption on doxorubicin sensitivity. Both cancer cell lines expressed all pH regulators, except for MCT1 and CAXII, only expressed in MCF-7 cells. There was higher plasma membrane expression of the pH regulators in human breast cancer tissues than in normal breast epithelium. Additionally, pH regulator expression was significantly associated with different molecular subtypes of breast cancer. pH regulator inhibition decreased cancer cell aggressiveness, with a higher effect in MDA-MB-231. A synergistic inhibitory effect was observed when PRIs were combined with doxorubicin in the breast cancer cell line viability. Our results support proton dynamic disruption as a breast cancer antitumor strategy and the use of PRIs to boost the activity of conventional therapy.

Tramadol and tapentadol, two structurally related synthetic opioid analgesics, are widely prescribed due to the enhanced therapeutic profiles resulting from the synergistic combination between μ-opioid receptor (MOR) activation and monoamine reuptake inhibition. However, the number of adverse reactions has been growing along with their increasing use and misuse. The potential toxicological mechanisms for these drugs are not completely understood, especially for tapentadol, owing to its shorter market history. Therefore, in the present study, we aimed to comparatively assess the putative lung, cardiac, and brain cortex toxicological damage elicited by the repeated exposure to therapeutic doses of both prescription opioids. To this purpose, male Wistar rats were intraperitoneally injected with single daily doses of 10, 25, and 50 mg/kg tramadol or tapentadol, corresponding to a standard analgesic dose, an intermediate dose, and the maximum recommended daily dose, respectively, for 14 consecutive days. Such treatment was found to lead mainly to lipid peroxidation and inflammation in lung and brain cortex tissues, as shown through augmented thiobarbituric acid reactive substances (TBARS), as well as to increased serum inflammation biomarkers, such as C reactive protein (CRP) and tumor necrosis factor-α (TNF-α). Cardiomyocyte integrity was also shown to be affected, since both opioids incremented serum lactate dehydrogenase (LDH) and α-hydroxybutyrate dehydrogenase (α-HBDH) activities, while tapentadol was associated with increased serum creatine kinase muscle brain (CK-MB) isoform activity. In turn, the analysis of metabolic parameters in brain cortex tissue revealed increased lactate concentration upon exposure to both drugs, as well as augmented LDH and creatine kinase (CK) activities following tapentadol treatment. In addition, pneumo- and cardiotoxicity biomarkers were quantified at the gene level, while neurotoxicity biomarkers were quantified both at the gene and protein levels; changes in their expression correlate with the oxidative stress, inflammatory, metabolic, and histopathological changes that were detected. Hematoxylin and eosin (H & E) staining revealed several histopathological alterations, including alveolar collapse and destruction in lung sections, inflammatory infiltrates, altered cardiomyocytes and loss of striation in heart sections, degenerated neurons, and accumulation of glial and microglial cells in brain cortex sections. In turn, Masson's trichrome staining confirmed fibrous tissue deposition in cardiac tissue. Taken as a whole, these results show that the repeated administration of both prescription opioids extends the dose range for which toxicological injury is observed to lower therapeutic doses. They also reinforce previous assumptions that tramadol and tapentadol are not devoid of toxicological risk even at clinical doses.

Tramadol and tapentadol are fully synthetic and extensively used analgesic opioids, presenting enhanced therapeutic and safety profiles as compared with their peers. However, reports of adverse reactions, intoxications and fatalities have been increasing. Information regarding the molecular, biochemical, and histological alterations underlying their toxicological potential is missing, particularly for tapentadol, owing to its more recent market authorization. Considering the paramount importance of liver and kidney for the metabolism and excretion of both opioids, these organs are especially susceptible to toxicological damage. In the present study, we aimed to characterize the putative hepatic and renal deleterious effects of repeated exposure to therapeutic doses of tramadol and tapentadol, using an in vivo animal model. Male Wistar rats were randomly divided into six experimental groups, composed of six animals each, which received daily single intraperitoneal injections of 10, 25 or 50 mg/kg tramadol or tapentadol (a low, standard analgesic dose, an intermediate dose and the maximum recommended daily dose, respectively). An additional control group was injected with normal saline. Following 14 consecutive days of administration, serum, urine and liver and kidney tissue samples were processed for biochemical, metabolic and histological analysis. Repeated administration of therapeutic doses of both opioids led to: (i) increased lipid and protein oxidation in liver and kidney, as well as to decreased total liver antioxidant capacity; (ii) decreased serum albumin, urea, butyrylcholinesterase and complement C3 and C4 levels, denoting liver synthesis impairment; (iii) elevated serum activity of liver enzymes, such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and γ-glutamyl transpeptidase, as well as lipid profile alterations, also reflecting hepatobiliary commitment; (iv) derangement of iron metabolism, as shown through increases in serum iron, ferritin, haptoglobin and heme oxygenase-1 levels. In turn, elevated serum cystatin C, decreased urine creatinine output and increased urine microalbumin levels were detected upon exposure to tapentadol only, while increased serum amylase and urine N -acetyl-β-D-glucosaminidase activities were observed for both opioids. Collectively, these results are compatible with kidney injury. Changes were also found in the expression levels of liver- and kidney-specific toxicity biomarker genes, upon exposure to tramadol and tapentadol, correlating well with alterations in lipid profile, iron metabolism and glomerular and tubular function. Histopathological analysis evidenced sinusoidal dilatation, microsteatosis, mononuclear cell infiltrates, glomerular and tubular disorganization, and increased Bowman's spaces. Although some findings are more pronounced upon tapentadol exposure, our study shows that, when compared with acute exposure, prolonged administration of both opioids smooths the differences between their toxicological effects, and that these occur at lower doses within the therapeutic range.

Monocarboxylate transporters (MCTs) inhibition leads to disruption in glycolysis, induces cell death and decreases cell invasion, revealing the importance of MCT activity in intracellular pH homeostasis and tumor aggressiveness. 3-Bromopyruvate (3BP) is an anti-tumor agent, whose uptake occurs via MCTs. It was the aim of this work to unravel the importance of extracellular conditions on the regulation of MCTs and in 3BP activity. HCT-15 was found to be the most sensitive cell line, and also the one that presented the highest basal expression of both MCT1 and of its chaperone CD147. Glucose starvation and hypoxia induced an increased resistance to 3BP in HCT-15 cells, in contrast to what happens with an extracellular acidic pH, where no alterations in 3BP cytotoxicity was observed. However, no association with MCT1, MCT4 and CD147 expression was observed, except for glucose starvation, where a decrease in CD147 (but not of MCT1 and MCT4) was detected. These results show that 3BP cytotoxicity might include other factors beyond MCTs. Nevertheless, treatment with short-chain fatty acids (SCFAs) increased the expression of MCT4 and CD147 as well as the sensitivity of HCT-15 cells to 3BP. The overall results suggest that MCTs influence the 3BP effect, although they are not the only players in its mechanism of action.

Gliomas are characterized by a marked glycolytic metabolism with a consequent production of massive amounts of lactate, even in the presence of normal levels of oxygen, associated to increased invasion capacity and to higher resistance to conventional treatment. This work aimed to understand how the metabolic modulation can influence tumour aggressive features and its potential to be used as complementary therapy. We assessed the effect of bioenergetic modulators (BMs) targeting different metabolic pathways in glioma cell characteristics. The in vivo effect of BMs was evaluated using the chicken chorioallantoic membrane model. Additionally, the effect of pre-treatment with BMs in the response to the antitumour drug temozolomide (TMZ) was analysed in vitro. Cell treatment with the BMs induced a decrease in cell viability and in migratory/invasion abilities, as well as modifications in metabolic parameters (glucose, lactate and ATP) and increased the cytotoxicity of the conventional drug TMZ. Furthermore, all BMs decreased the tumour growth and the number of blood vessels in an in vivo model. Our results demonstrate that metabolic modulation has the potential to be used as therapy to decrease the aggressiveness of the tumours or to be combined with conventional drugs used in glioma treatment., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Introduction: Most mental disorders have a chronic evolution and therefore a certain amount of psychiatric readmissions are inevitable. Several studies indicate that over 25% of child and adolescent inpatients were readmitted within one year of discharge. Several risk factors for psychiatric readmissions have been reported in the literature, but the history of repeated readmissions is the most consistent risk factor. Our aim is to calculate the readmission rates at 30 days and 12 months after discharge and to identify associated risk factors., Material and Methods: The authors consulted the clinical files of patients admitted to the Inpatient Unit between 2010 and 2013, in order to calculate the readmission rates at 30 days and at 12 months. The demographic and clinical characteristics of the readmitted patients were analyzed and compared with a second group of patients with no hospital readmissions, in order to investigate possible predictors of readmission., Results: A total of 445 patients were admitted to our inpatient unit between 2010 and 2013. Six adolescents were readmitted in a 30 days period (1.3%) and 52 were readmitted in a 12 month period after discharge (11.5%). Duration of the hospitalization and the previous number of mental health admissions were significant predictors of future hospital readmissions (p = 0.04 and p = 0.014)., Discussion: The low readmission rates may reflect the positive clinical and sociofamilial support being provided after discharge., Conclusion: Rehospitalisation is considered a fundamental target for intervention concerning prevention and intervention in mental healthcare. Thus, knowledge regarding their minimisation is crucial.

Tramadol and tapentadol are two atypical synthetic opioid analgesics, with monoamine reuptake inhibition properties. Mainly aimed at the treatment of moderate to severe pain, these drugs are extensively prescribed for multiple clinical applications. Along with the increase in their use, there has been an increment in their abuse, and consequently in the reported number of adverse reactions and intoxications. However, little is known about their mechanisms of toxicity. In this study, we have analyzed the in vivo toxicological effects in liver and kidney resulting from an acute exposure of a rodent animal model to both opioids. Male Wistar rats were intraperitoneally administered with 10, 25 and 50mg/kg tramadol and tapentadol, corresponding to a low, effective analgesic dose, an intermediate dose and the maximum recommended daily dose, respectively, for 24h. Toxicological effects were assessed in terms of oxidative stress, biochemical and metabolic parameters and histopathology, using serum and urine samples, liver and kidney homogenates and tissue specimens. The acute exposure to tapentadol caused a dose-dependent increase in protein oxidation in liver and kidney. Additionally, exposure to both opioids led to hepatic commitment, as shown by increased serum lipid levels, decreased urea concentration, increased alanine aminotransferase and decreased butyrylcholinesterase activities. It also led to renal impairment, as reflected by proteinuria and decreased glomerular filtration rate. Histopathological findings included sinusoidal dilatation, microsteatosis, vacuolization, cell infiltrates and cell degeneration, indicating metabolic changes, inflammation and cell damage. In conclusion, a single effective analgesic dose or the maximum recommended daily dose of both opioids leads to hepatotoxicity and nephrotoxicity, with tapentadol inducing comparatively more toxicity. Whether these effects reflect risks during the therapeutic use or human overdoses requires focused attention by the medical community., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Tramadol and tapentadol are extensively prescribed for the treatment of moderate to severe pain. Although these drugs are very effective in pain treatment, the number of intoxications and deaths due to both opioids is increasing, and the underlying toxic mechanisms are not fully understood. The present work aimed to study the potential biochemical and histopathological alterations induced by acute effective (analgesic) doses of tramadol and tapentadol, in Wistar rats. Forty-two male Wistar rats were divided into different groups: a control, administered with normal saline solution, and tramadol- or tapentadol-treated groups (10, 25 or 50mg/kg - typical effective analgesic dose, intermediate and maximum recommended doses, respectively). 24h after intraperitoneal administration, biochemical and oxidative stress analyses were performed in blood, and specimens from brain, lung and heart were taken for histopathological and oxidative stress studies. Both drugs caused an increase in the AST/ALT ratio, in LDH, CK and CK-MB activities in serum samples, and an increase in lactate levels in serum and brain samples. Oxidative damage, namely protein oxidation, was found in heart and lung tissues. In histological analyses, tramadol and tapentadol were found to cause alterations in cell morphology, inflammatory cell infiltrates and cell death in all tissues under study, although tapentadol caused more damage than tramadol. Our results confirmed the risks of tramadol exposure, and demonstrated the higher risk of tapentadol, especially at high doses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Altered metabolism, associated with acidification of the extracellular milieu, is one of the major features of cancer. As pH regulation is crucial for the maintenance of all biological functions, cancer cells rely on the activity of lactate exporters and proton transporters to regulate their intracellular pH. The major players in cancer pH regulation are proton pump ATPases, sodium-proton exchangers (NHEs), monocarboxylate transporters (MCTs), carbonic anhydrases (CAs) and anion exchangers (AEs), which have been shown to be upregulated in several human malignancies. Thanks to the activity of the proton pumps and transporters, tumours acidify their microenvironment, becoming more aggressive and resistant to therapy. Thus, targeting tumour pH may contribute to more effective anticancer strategies for controlling tumour progression and therapeutic resistance. In the present study, we review the role of the main pH regulators expressed in human cancer cells, including their diagnostic and prognostic value, as well as their usefulness as therapeutic targets., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tramadol and tapentadol are centrally acting, synthetic opioid analgesics used in the treatment of moderate to severe pain. Main metabolic patterns for these drugs in humans are well characterized. Tramadol is mainly metabolized by cytochrome P450 CYP2D6 to O-desmethyltramadol (M1), its main active metabolite. M1 and tapentadol undergo mainly glucuronidation reactions. On the other hand, the pharmacokinetics of tramadol and tapentadol are dependent on multiple factors, such as the route of administration, genetic variability in pharmacokinetic components and concurrent consumption of other drugs. This review aims to comparatively discuss the metabolomics of tramadol and tapentadol, namely by presenting all their known metabolites. An exhaustive literature search was performed using textual and structural queries for tramadol and tapentadol, and associated known metabolizing enzymes and metabolites. A thorough knowledge about tramadol and tapentadol metabolomics is expected to provide additional insights to better understand the interindividual variability in their pharmacokinetics and dose-responsiveness, and contribute to the establishment of personalized therapeutic approaches, minimizing side effects and optimizing analgesic efficacy.

Opioid therapy and abuse are increasing, justifying the need to study their toxicity and underlying mechanisms. Given opioid pharmacodynamics at the central nervous system, the analysis of toxic effects in neuronal models gains particular relevance. The aim of this study was to compare the toxicological effects of acute exposure to tramadol and tapentadol in the undifferentiated human SH-SY5Y neuroblastoma cell line. Upon exposure to tramadol and tapentadol concentrations up to 600μM, cell toxicity was assessed through evaluation of oxidative stress, mitochondrial and metabolic alterations, as well as cell viability and death mechanisms through necrosis or apoptosis, and related signalling. Tapentadol was observed to trigger much more prominent toxic effects than tramadol, ultimately leading to energy deficit and cell death. Cell death was shown to predominantly occur through necrosis, with no alterations in membrane potential or in cytochrome c release. Both drugs were shown to stimulate glucose uptake and to cause ATP depletion, due to changes in the expression of energy metabolism enzymes. The toxicity mechanisms in such a neuronal model are relevant to understand adverse reactions to these opioids and to contribute to dose adjustment in order to avoid neurological damage., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

This chapter covers the functionally characterized plasma membrane carboxylic acids transporters Jen1, Ady2, Fps1 and Pdr12 in the yeast Saccharomyces cerevisiae, addressing also their homologues in other microorganisms, as filamentous fungi and bacteria. Carboxylic acids can either be transported into the cells, to be used as nutrients, or extruded in response to acid stress conditions. The secondary active transporters Jen1 and Ady2 can mediate the uptake of the anionic form of these substrates by a H(+)-symport mechanism. The undissociated form of carboxylic acids is lipid-soluble, crossing the plasma membrane by simple diffusion. Furthermore, acetic acid can also be transported by facilitated diffusion via Fps1 channel. At the cytoplasmic physiological pH, the anionic form of the acid prevails and it can be exported by the Pdr12 pump. This review will highlight the mechanisms involving carboxylic acids transporters, and the way they operate according to the yeast cell response to environmental changes, as carbon source availability, extracellular pH and acid stress conditions.

We have functionally characterized the four Saccharomyces cerevisiae (Sc) Jen1 homologues of Debaryomyces hansenii (Dh) by heterologous expression in S. cerevisiae. Debaryomyces hansenii cells display mediated transport for the uptake of lactate, acetate, succinate and malate. DHJEN genes expression was detected by RT-PCR in all carbon sources assayed, namely lactate, succinate, citrate, glycerol and glucose. The heterologous expression in the S. cerevisiae W303-1A jen1Δ ady2Δ strain demonstrated that the D. hansenii JEN genes encode four carboxylate transporters. DH27 gene encodes an acetate transporter (Km 0.94 ± 0.17 mM; Vmax 0.43 ± 0.03 nmol s(-1) mg(-1)), DH17 encodes a malate transporter (Km 0.27 ± 0.04 mM; Vmax 0.11 ± 0.01 nmol s(-1) mg(-1)) and both DH18 and DH24 encode succinate transporters with the following kinetic parameters, respectively, Km 0.31 ± 0.06 mM; Vmax 0.83 ± 0.04 nmol s(-1) mg(-1)and Km 0.16 ± 0.02 mM; Vmax 0.19 ± 0.02 nmol s(-1) mg(-1). Surprisingly, no lactate transporter was found, although D. hansenii presents a mediated transport for this acid. This work advanced the current knowledge on yeast carboxylate transporters by characterizing four new plasma membrane transporters in D. hansenii., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Although the anti-cancer properties of 3BP (3-bromopyruvate) have been described previously, its selectivity for cancer cells still needs to be explained [Ko et al. (2001) Cancer Lett. 173, 83-91]. In the present study, we characterized the kinetic parameters of radiolabelled [14C] 3BP uptake in three breast cancer cell lines that display different levels of resistance to 3BP: ZR-75-1 < MCF-7 < SK-BR-3. At pH 6.0, the affinity of cancer cells for 3BP transport correlates with their sensitivity, a pattern that does not occur at pH 7.4. In the three cell lines, the uptake of 3BP is dependent on the protonmotive force and is decreased by MCTs (monocarboxylate transporters) inhibitors. In the SK-BR-3 cell line, a sodium-dependent transport also occurs. Butyrate promotes the localization of MCT-1 at the plasma membrane and increases the level of MCT-4 expression, leading to a higher sensitivity for 3BP. In the present study, we demonstrate that this phenotype is accompanied by an increase in affinity for 3BP uptake. Our results confirm the role of MCTs, especially MCT-1, in 3BP uptake and the importance of cluster of differentiation (CD) 147 glycosylation in this process. We find that the affinity for 3BP transport is higher when the extracellular milieu is acidic. This is a typical phenotype of tumour microenvironment and explains the lack of secondary effects of 3BP already described in in vivo studies [Ko et al. (2004) Biochem. Biophys. Res. Commun. 324, 269-275].

Ethanol is the most abused psychoactive substance. Accordingly to World Health Organization ethanol ranks among the top five risk factors for disease, disability and death (3.3 million/year) throughout the world. This manuscript highlights and critically analyses clinical and forensic signs related to hepatoxicity of ethanol that may lead to suspected of abuse. Namely, steatosis, jaundice, cirrhosis, hemorrhoids, esophageal varices caput medusae, ascites, petechiae, ecchymoses, splenomegaly, hemochromatosis, xanthelasma, nutritional deficiency, testicular atrophy, gynecomastia and dilated congestive cardiomyopathy are discussed and related to the toxic mechanism of ethanol.

Monocarboxylate transporters (MCTs) belong to a family of transporters, encoded by the SLC16 gene family, which is presently composed by 14 members, but only MCT1 to 4 have been biochemically characterized. They have important functions in healthy tissues, being involved in the transmembrane transport of lactic acid and other monocarboxylic acids in human cells. One of the recently recognized hallmarks of cancer is altered metabolism, with high rates of glucose consumption and consequent lactate production. To maintain this metabolic phenotype, cancer cells upregulate a series of plasma membrane proteins, including MCTs. MCT1 and MCT4, in particular, play a dual role in the maintenance of the metabolic phenotype of tumour cells. On one hand, they facilitate the efflux of lactate and, on the other hand, they contribute to the preservation of the intracellular pH, by co-transporting a proton. Thus, MCTs are attractive targets in cancer therapy, especially in cancers with a hyper-glycolytic and acid-resistant phenotype. Recent evidence demonstrates that MCTs are involved in cancer cell uptake of chemotherapeutic agents, including 3-bromopyruvate. In this way MCTs can act as "Trojan horses", as their elevated expression in cancer cells can mediate the entry of this chemotherapeutic agent into the cells and selectively kill cancer cells. As a result, MCTs will be mediators of chemotherapeutic response, and their expression can be used as a molecular marker to predict response to chemotherapy.

The multidrug resistance (MDR) phenotype, frequently observed during cancer treatment, is often associated with drug efflux pump activity. However, many other factors are also known to be involved. Cancer cells often rely on aerobic glycolysis for energy production; this is known as the "Warburg effect" and is used as a survival mechanism. Associated to this event, a reverse pH gradient across the cell membrane occurs, leading to cytosol alkalinization and extracellular acidification. In the present study, we investigated the role of different mechanisms involved in MDR, such as altered tumor microenvironment and energetic metabolism. The breast cancer cell line MCF-7, used as model, was exposed to two widely used antitumor drugs, paclitaxel (antimitotic agent) and doxorubicin (alkylating agent). Cancer pH regulation was shown to be crucial for malignant characteristics such as cell migration and drug resistance. Our results showed that a lower extracellular pH induced a higher migratory capacity and higher resistance to the studied chemotherapeutical compounds in MCF-7 cells. Besides the influence of the extracellular pH, the role of the tumor metabolism in the MDR phenotype was also investigated. Pre-treatment with different bioenergetic modulators led to cell ATP depletion and altered lactic acid production and glucose consumption, resulting in increased sensitivity to paclitaxel and doxorubicin. Overall, this study supports the potential use of compounds targeting cell metabolism and tumor microenvironment factors such as pH, as co-adjuvants in conventional chemotherapy.

Alternative matrices are steadily gaining recognition as biological samples for toxicological analyses. Hair presents many advantages over traditional matrices, such as urine and blood, since it provides retrospective information regarding drug exposure, can distinguish between chronic and acute or recent drug use by segmental analysis, is easy to obtain, and has considerable stability for long periods of time. For this reason, it has been employed in a wide variety of contexts, namely to evaluate workplace drug exposure, drug-facilitated sexual assault, pre-natal drug exposure, anti-doping control, pharmacological monitoring and alcohol abuse. In this article, issues concerning hair structure, collection, storage and analysis are reviewed. The mechanisms of drug incorporation into hair are briefly discussed. Analytical techniques for simultaneous drug quantification in hair are addressed. Finally, representative examples of drug quantification using hair are summarized, emphasizing its potentialities and limitations as an alternative biological matrix for toxicological analyses.

Gap repair is a fast and efficient method for assembling recombinant DNA molecules in Saccharomyces cerevisiae. This method produces a circular DNA molecule by homologous recombination between two or more linear DNA fragments, one of which is typically a vector carrying replicative sequences and a selective marker. This technique avoids laborious and costly in vitro purification and ligation of DNA. The DNA repair machinery can also close and ligate the linear vector by mechanisms other than homologous recombination, resulting in an empty vector. The frequency of these unwanted events can be lowered by removing the 5'-phosphate groups using phosphatase, which is the standard method used for in vitro ligation. However, phosphatase treatment is less effective for gap repair cloning than for in vitro ligation, presumably due to the ability of the S. cerevisiae DNA repair machinery to efficiently repair the missing phosphate group to allow religation. We have developed a more efficient method to prevent vector religation, based on treatment of the vector fragment with Taq DNA polymerase and dATP. This procedure prevents vector recircularization almost completely, facilitating the screening for true recombinant clones., (2012 John Wiley & Sons, Ltd)

We aimed to manipulate the metabolism of Saccharomyces cerevisiae to produce lactic acid and search for the potential influence of acid transport across the plasma membrane in this process. Saccharomyces cerevisiae W303-1A is able to use l-lactic acid but its production in our laboratory has not previously been detected. When the l-LDH gene from Lactobacillus casei was expressed in S. cerevisiae W303-1A and in the isogenic mutants jen1∆, ady2∆ and jen1∆ ady2∆, all strains were able to produce lactic acid, but higher titres were achieved in the mutant strains. In strains constitutively expressing both LDH and JEN1 or ADY2, a higher external lactic acid concentration was found when glucose was present in the medium, but when glucose was exhausted, its consumption was more pronounced. These results demonstrate that expression of monocarboxylate permeases influences lactic acid production. Ady2 has been previously characterized as an acetate permease but our results demonstrated its additional role in lactate uptake. Overall, we demonstrate that monocarboxylate transporters Jen1 and Ady2 are modulators of lactic acid production and may well be used to manipulate lactic acid export in yeast cells., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Most malignant tumors exhibit the Warburg effect, which consists in increased glycolysis rates with production of lactate, even in the presence of oxygen. Monocarboxylate transporters (MCTs), maintain these glycolytic rates, by mediating the influx and/or efflux of lactate and are overexpressed in several cancer cell types. The lactate and pyruvate analogue 3-bromopyruvate (3-BP) is an inhibitor of the energy metabolism, which has been proposed as a specific antitumor agent. In the present study, we aimed at determining the effect of 3-BP in breast cancer cells and evaluated the putative role of MCTs on this effect. Our results showed that the three breast cancer cell lines used presented different sensitivities to 3-BP: ZR-75-1 ER (+)>MCF-7 ER (+)>SK-BR-3 ER (-). We also demonstrated that 3-BP reduced lactate production, induced cell morphological alterations and increased apoptosis. The effect of 3-BP appears to be cytotoxic rather than cytostatic, as a continued decrease in cell viability was observed after removal of 3-BP. We showed that pre-incubation with butyrate enhanced significantly 3-BP cytotoxicity, especially in the most resistant breast cancer cell line, SK-BR-3. We observed that butyrate treatment induced localization of MCT1 in the plasma membrane as well as overexpression of MCT4 and its chaperone CD147. Our results thus indicate that butyrate pre-treatment potentiates the effect of 3-BP, most probably by increasing the rates of 3-BP transport through MCT1/4. This study supports the potential use of butyrate as adjuvant of 3-BP in the treatment of breast cancer resistant cells, namely ER (-).