Expression of the monocarboxylate transporter MCT1 is required for virus-specific mouse CD8+ T cell memory development.

Lactate-proton symporter monocarboxylate transporter 1 (MCT1) facilitates lactic acid export from T cells. Here, we report that MCT1 is mandatory for the development of virus-specific CD8+ T cell memory. MCT1-deficient T cells were exposed to acute pneumovirus (pneumonia virus of mice, PVM) or persistent γ-herpesvirus (Murid herpesvirus 4, MuHV-4) infection. MCT1 was required for the expansion of virus-specific CD8+ T cells and the control of virus replication in the acute phase of infection. This situation prevented the subsequent development of virus-specific T cell memory, a necessary step in containing virus reactivation during γ-herpesvirus latency. Instead, persistent active infection drove virus-specific CD8+ T cells toward functional exhaustion, a phenotype typically seen in chronic viral infections. Mechanistically, MCT1 deficiency sequentially impaired lactic acid efflux from activated CD8+ T cells, caused an intracellular acidification inhibiting glycolysis, disrupted nucleotide synthesis in the upstream pentose phosphate pathway, and halted cell proliferation which, ultimately, promoted functional CD8+ T cell exhaustion instead of memory development. Taken together, our data demonstrate that MCT1 expression is mandatory for inducing T cell memory and controlling viral infection by CD8+ T cells.

Preclinical Evaluation of [18F]FACH in Healthy Mice and Piglets: An 18F-Labeled Ligand for Imaging of Monocarboxylate Transporters with PET.

The expression of monocarboxylate transporters (MCTs) is linked to pathophysiological changes in diseases, including cancer, such that MCTs could potentially serve as diagnostic markers or therapeutic targets. We recently developed [18F]FACH as a radiotracer for non-invasive molecular imaging of MCTs by positron emission tomography (PET). The aim of this study was to evaluate further the specificity, metabolic stability, and pharmacokinetics of [18F]FACH in healthy mice and piglets. We measured the [18F]FACH plasma protein binding fractions in mice and piglets and the specific binding in cryosections of murine kidney and lung. The biodistribution of [18F]FACH was evaluated by tissue sampling ex vivo and by dynamic PET/MRI in vivo, with and without pre-treatment by the MCT inhibitor α-CCA-Na or the reference compound, FACH-Na. Additionally, we performed compartmental modelling of the PET signal in kidney cortex and liver. Saturation binding studies in kidney cortex cryosections indicated a KD of 118 ± 12 nM and Bmax of 6.0 pmol/mg wet weight. The specificity of [18F]FACH uptake in the kidney cortex was confirmed in vivo by reductions in AUC0-60min after pre-treatment with α-CCA-Na in mice (-47%) and in piglets (-66%). [18F]FACH was metabolically stable in mouse, but polar radio-metabolites were present in plasma and tissues of piglets. The [18F]FACH binding potential (BPND) in the kidney cortex was approximately 1.3 in mice. The MCT1 specificity of [18F]FACH uptake was confirmed by displacement studies in 4T1 cells. [18F]FACH has suitable properties for the detection of the MCTs in kidney, and thus has potential as a molecular imaging tool for MCT-related pathologies, which should next be assessed in relevant disease models.

Development of Novel Analogs of the Monocarboxylate Transporter Ligand FACH and Biological Validation of One Potential Radiotracer for Positron Emission Tomography (PET) Imaging.

Monocarboxylate transporters 1-4 (MCT1-4) are involved in several metabolism-related diseases, especially cancer, providing the chance to be considered as relevant targets for diagnosis and therapy. [18F]FACH was recently developed and showed very promising preclinical results as a potential positron emission tomography (PET) radiotracer for imaging of MCTs. Given that [18F]FACH did not show high blood-brain barrier permeability, the current work is aimed to investigate whether more lipophilic analogs of FACH could improve brain uptake for imaging of gliomas, while retaining binding to MCTs. The 2-fluoropyridinyl-substituted analogs 1 and 2 were synthesized and their MCT1 inhibition was estimated by [14C]lactate uptake assay on rat brain endothelial-4 (RBE4) cells. While compounds 1 and 2 showed lower MCT1 inhibitory potencies than FACH (IC50 = 11 nM) by factors of 11 and 25, respectively, 1 (IC50 = 118 nM) could still be a suitable PET candidate. Therefore, 1 was selected for radiosynthesis of [18F]1 and subsequent biological evaluation for imaging of the MCT expression in mouse brain. Regarding lipophilicity, the experimental log D7.4 result for [18F]1 agrees pretty well with its predicted value. In vivo and in vitro studies revealed high uptake of the new radiotracer in kidney and other peripheral MCT-expressing organs together with significant reduction by using specific MCT1 inhibitor α-cyano-4-hydroxycinnamic acid. Despite a higher lipophilicity of [18F]1 compared to [18F]FACH, the in vivo brain uptake of [18F]1 was in a similar range, which is reflected by calculated BBB permeabilities as well through similar transport rates by MCTs on RBE4 cells. Further investigation is needed to clarify the MCT-mediated transport mechanism of these radiotracers in brain.

Development and radiosynthesis of the first 18 F-labeled inhibitor of monocarboxylate transporters (MCTs).

Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) are involved in tumor development and progression. Their expression levels are related to clinical disease prognosis. Accordingly, both MCTs are promising drug targets for treatment of a variety of human cancers. The noninvasive imaging of these MCTs in cancers is regarded to be advantageous for assessing MCT-mediated effects on chemotherapy and radiosensitization using specific MCT inhibitors. Herein, we describe a method for the radiosynthesis of [18 F]FACH ((E)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylic acid), as a novel radiolabeled MCT1/4 inhibitor for imaging with PET. A fluorinated analog of α-cyano-4-hydroxycinnamic acid (FACH) was synthesized, and the inhibition of MCT1 and MCT4 was measured via an L-[14 C]lactate uptake assay. Radiolabeling was performed by a two-step protocol comprising the radiosynthesis of the intermediate (E)/(Z)-[18 F]tert-Bu-FACH (tert-butyl (E)/(Z)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylate) followed by deprotection of the tert-butyl group. The radiofluorination was successfully implemented using either K[18 F]F-K2.2.2 -carbonate or [18 F]TBAF. The final deprotected product [18 F]FACH was only obtained when [18 F]tert-Bu-FACH was formed by the latter procedure. After optimization of the deprotection reaction, [18 F]FACH was obtained in high radiochemical yields (39.6 ± 8.3%, end of bombardment (EOB) and radiochemical purity (greater than 98%).

Semi-mechanistic kidney model incorporating physiologically-relevant fluid reabsorption and transporter-mediated renal reabsorption: pharmacokinetics of γ-hydroxybutyric acid and L-lactate in rats.

This study developed a semi-mechanistic kidney model incorporating physiologically-relevant fluid reabsorption and transporter-mediated active reabsorption. The model was applied to data for the drug of abuse γ-hydroxybutyric acid (GHB), which exhibits monocarboxylate transporter (MCT1/SMCT1)-mediated renal reabsorption. The kidney model consists of various nephron segments--proximal tubules, Loop-of-Henle, distal tubules, and collecting ducts--where the segmental fluid flow rates, volumes, and sequential reabsorption were incorporated as functions of the glomerular filtration rate. The active renal reabsorption was modeled as vectorial transport across proximal tubule cells. In addition, the model included physiological blood, liver, and remainder compartments. The population pharmacokinetic modeling was performed using ADAPT5 for GHB blood concentration-time data and cumulative amount excreted unchanged into urine data (200-1000 mg/kg IV bolus doses) from rats [Felmlee et al (PMID: 20461486)]. Simulations assessed the effects of inhibition (R = [I]/KI = 0-100) of renal reabsorption on systemic exposure (AUC) and renal clearance of GHB. Visual predictive checks and other model diagnostic plots indicated that the model reasonably captured GHB concentrations. Simulations demonstrated that the inhibition of renal reabsorption significantly increased GHB renal clearance and decreased AUC. Model validation was performed using a separate dataset. Furthermore, our model successfully evaluated the pharmacokinetics of L-lactate using data obtained from Morse et al (PMID: 24854892). In conclusion, we developed a semi-mechanistic kidney model that can be used to evaluate transporter-mediated active renal reabsorption of drugs by the kidney.

Living high - training low model applied to C57BL/6J mice: Effects on physiological parameters related to aerobic fitness and acid-base balance.

There is a scarcity of data regarding the acclimation to high altitude (hypoxic environment) accompanied by training at low altitude (normoxic conditions), the so-called "living high-training low" (LHTL) model in rodents. We aimed to investigate the effects of aerobic training on C57BL/6J mice living in normoxic (NOR) or hypoxic (HYP) environments on several parameters, including critical velocity (CV), a parameter regarded as a measure of aerobic capacity, on monocarboxylate transporters (MCTs) in muscles and hypothalamus, as well as on hematological parameters and body temperature. In each environment, mice were divided into non-trained (N) and trained (T). Forty rodents were distributed into the following experimental groups (N-NOR; T-NOR; N-HYP and T-HYP). HYP groups were in a normobaric tent where oxygen-depleted air was pumped from a hypoxia generator set an inspired oxygen fraction [FiO2] of 14.5 %. The HYP-groups were kept (18 h per day) in a normobaric tent for consecutive 8-weeks. Training sessions were conducted in normoxic conditions ([FiO2] = 19.5 %), 5 times per week (40 min per session) at intensity equivalent to 80 % of CV. In summary, eight weeks of LHTL did not promote a greater improvement in the CV, protein expression of MCTs in different tissues when compared to the application of training alone. The LHTL model increased red blood cells count, but reduced hemoglobin per erythrocyte was found in mice exposed to LHTL. Although the LHTL did not have a major effect on thermographic records, exercise-induced hyperthermia (in the head) was attenuated in HYP groups when compared to NOR groups.

A systemic analysis of monocarboxylate transporters in ovarian cancer and possible therapeutic interventions.

Monocarboxylate transporters (MCTs) play an immense role in metabolically active solid tumors by regulating concentration-dependent transport of different important monocarboxylates including pyruvate and lactate and are encoded by the SLC16A family of genes. Given the vast array of functions, these transporters play in oncogenesis, our objective was to look into the association of MCT1 (SLC16A1), MCT2 (SLC16A7), MCT3 (SLC16A8), and MCT4 (SLC16A3) with Epithelial ovarian cancer (EOC) pathophysiology by exploiting various publicly available databases and web resources. Few of the in silico findings were confirmed via in vitro experiments in EOC cell lines, SKOV3 and OAW-42. MCT1 and MCT4 were found to be upregulated at the mRNA level in OC tissues compared to normal. However, only higher level of MCT4 mRNA was found to be associated with poor patient survival. MCT4 was positively correlated with gene families responsible for invasion, migration, and immune modification, proving it to be one of the most important MCTs for therapeutic intervention. We compared the effects of MCT1/2 blocker SR13800 and a broad-spectrum MCT blocker α-Cyano Hydroxy Cinnamic Acid (α-CHCA) and discovered that α-CHCA has a greater effect on diminishing the invasive behavior of the cancer cells than MCT1/2 blocker SR13800. From our study, MCT4 has emerged as a prospective marker for predicting poor patient outcomes and a potential therapeutic target.

Unconventional roles of lactate along the tumor and immune landscape.

The tumor ecosystem evolves with dynamic interactions between cancer and normal cells, and nutrients have emerged as new regulators of cancer hallmarks. Lactate has climbed the rankings as a multifunctional molecule orchestrating many aspects of the disease onset and progression. Here, we patchwork and discuss the main recent findings conferred during the EMBO workshop titled 'Lactate: Unconventional Roles of a Nutrient Along the Tumor Landscape.'

Lactate inhibits osteogenic differentiation of human periodontal ligament stem cells via autophagy through the MCT1-mTOR signaling pathway.

BACKGROUND: Periodontal ligament stem cells (PDLSCs) play a crucial role in periodontal bone regeneration. Lactate used to be considered a waste product of glucose metabolism. In recent years, a few pieces of evidence revealed its roles in regulating the osteogenic differentiation of stem cells, but the standpoints were controversial. This study aims to investigate the effects and the mechanisms of lactate on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). METHODS: The hPDLSCs were treated with different concentrations of lactic acid and lactate to differentiate the effects of the acidic PH and ion lactate. Proliferation and cytotoxicity were measured by Cell Counting Kit-8 (CCK8) assay and Live/Dead assay. The osteogenic differentiation of hPDLSCs was analyzed by alizarin red staining, alkaline phosphatase (ALP) staining, and then osteogenic proteins and genes were measured by western blot and reverse transcription-quantitative PCR (qRT-PCR). To investigate the potential signaling pathways, MCT1 inhibitor, G-protein inhibitors, and rapamycin were used, and then autophagy-related proteins and osteogenic proteins were measured by western blot. RESULTS: The inhibition of lactic acid on the osteogenic differentiation of hPDLSCs was more significant than lactate at the same concentration. Lactate inhibited the expression of ALP which can be rescued by Gα inhibitor. Alizarin red staining, the protein expression levels of osteocalcin (OCN), osteoprotegerin (OPN), osterix (OSX), and beclin1, LC3-II/LC3-I were decreased by lactate and partly rescued by MCT1 inhibitor. Rapamycin restored the protein expression levels of beclin1, LC3-II/LC3-I and OCN, OPN, OSX under the high lactate conditions. CONCLUSIONS: Lactate inhibits the expression of ALP via Gα subunit signaling, and inhibits mineralized nodules formation and the expression of osteogenic-related proteins via reducing autophagy through the MCT1-mTOR signaling pathway.

Monocarboxylate transporters (MCTs) in skeletal muscle and hypothalamus of less or more physically active mice exposed to aerobic training.

AIMS: The synthesis of monocarboxylate transporters (MCTs) can be stimulated by aerobic training, but few is known about this effect associated or not with non-voluntary daily activities. We examined the effect of eight weeks of aerobic training in MCTs on the skeletal muscle and hypothalamus of less or more physically active mice, which can be achieved by keeping them in two different housing models, a small cage (SC) and a large cage (LC). MAIN METHODS: Forty male C57BL/6J mice were divided into four groups. In each housing condition, mice were divided into untrained (N) and trained (T). For 8 weeks, the trained animals ran on a treadmill with an intensity equivalent to 80 % of the individual critical velocity (CV), considered aerobic capacity, 40 min/day, 5 times/week. Protein expression of MCTs was determined with fluorescence Western Blot. KEY FINDINGS: T groups had higher hypothalamic MCT2 than N groups (ANOVA, P = 0.032). Significant correlations were detected between hypothalamic MCT2 and CV. There was a difference between the SC and LC groups in relation to MCT4 in the hypothalamus (LC > SC, P = 0.044). Trained mice housed in LC (but not SC-T) exhibited a reduction in MCT4 muscle (P < 0.001). SIGNIFICANCE: Our findings indicate that aerobically trained mice increased the expression of MCT2 protein in the hypothalamus, which has been related to the uptake of lactate in neurons. Changes in energy metabolism in physically active mice (kept in LC) may be related to upregulation of hypothalamic MCT4, probably participating in the regulation of satiety.

Blood-Brain Barrier Solute Carrier Transporters and Motor Neuron Disease.

Defective solute carrier (SLC) transporters are responsible for neurotransmitter dysregulation, resulting in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). We provided the role and kinetic parameters of transporters such as ASCTs, Taut, LAT1, CAT1, MCTs, OCTNs, CHT, and CTL1, which are mainly responsible for the transport of essential nutrients, acidic, and basic drugs in blood-brain barrier (BBB) and motor neuron disease. The affinity for LAT1 was higher in the BBB than in the ALS model cell line, whereas the capacity was higher in the NSC-34 cell lines than in the BBB. Affinity for MCTs was lower in the BBB than in the NSC-34 cell lines. CHT in BBB showed two affinity sites, whereas no expression was observed in ALS cell lines. CTL1 was the main transporter for choline in ALS cell lines. The half maximal inhibitory concentration (IC50) analysis of [3H]choline uptake indicated that choline is sensitive in TR-BBB cells, whereas amiloride is most sensitive in ALS cell lines. Knowledge of the transport systems in the BBB and motor neurons will help to deliver drugs to the brain and develop the therapeutic strategy for treating CNS and neurological diseases.

In Vitro and In Vivo Characterization of MCT1 Inhibitor AZD3965 Confirms Preclinical Safety Compatible with Breast Cancer Treatment.

To survive and proliferate in solid tumors, cancer cells adapt and evolve rapidly in microenvironments where oxygen and substrate bioavailability fluctuates over time and space. This creates metabolic heterogeneity. Cancer cells can further cooperate metabolically, for example by swapping glycolytic end-product lactate for blood-borne glucose. This type of cooperation can be targeted therapeutically, since transmembrane lactate exchanges are facilitated by lactate-proton symporters of the monocarboxylate (MCT) family. Among new drugs, AZD3965 is a first-in-class selective MCT1 inhibitor currently tested in Phase I/II clinical trials for patients with different types of cancers. Because MCT1 can function bidirectionally, we tested here whether and how malignant and nonmalignant cells adapt their metabolism and MCT repertoire when AZD3965 inhibits either lactate import or export. Using breast-associated malignant and nonmalignant cell lines as models, we report that AZD3965 is not directly cytotoxic. In the presence of glucose and glutamine, oxidative cells can survive when lactate uptake is blocked, and proliferating cells compensate MCT1 inhibition by overexpressing MCT4, a specialized facilitator of lactate export. Phenotypic characterization of mice focusing on metabolism, muscle and brain physiology found partial and transient memory retention defect as sole consequence of MCT1 inhibition by AZD3965. We therefore conclude that AZD3965 is compatible with anticancer therapy.

Targeting Signalling Cross-Talk between Cancer Cells and Cancer-Associated Fibroblast through Monocarboxylate Transporters in Head and Neck Cancer.

Head and Neck Squamous Cell Carcinoma (HNSCC) is an aggressive malignancy affecting more than 600,000 cases worldwide annually, associated with poor prognosis and significant morbidity. HNSCC tumors are dysplastic, with up to 80% fibroblasts. It has been reported that Cancer-Associated Fibroblasts (CAFs) facilitate HNSCC progression. Unlike normal cells, malignant cells often display increased glycolysis, even in the presence of oxygen; a phenomenon known as the Warburg effect. As a consequence, there is an increase in Lactic Acid (LA) production. Earlier, it has been reported that HNSCC tumors exhibit high LA levels that correlate with reduced survival. It has been reported that the activation of the receptor tyrosine kinase, c- MET, by CAF-secreted Hepatocyte Growth Factor (HGF) is a major contributing event in the progression of HNSCC. In nasopharyngeal carcinoma, c-MET inhibition downregulates the TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) and NADPH production resulting in apoptosis. Previously, it was demonstrated that HNSCC tumor cells are highly glycolytic. Further, CAFs show a higher capacity to utilize LA as a carbon source to fuel mitochondrial respiration than HNSCC. Earlier, we have reported that in admixed cultures, both cell types increase the expression of Monocarboxylate Transporters (MCTs) for a bidirectional LA transporter. Consequently, MCTs play an important role in signalling cross-talk between cancer cells and cancer associate fibroblast in head and neck cancer, and targeting MCTs would lead to the development of a potential therapeutic approach for head and neck cancer. In this review, we focus on the regulation of MCTs in head and neck cancer through signalling cross-talk between cancer cells and cancer-associated fibroblasts, and targeting this signalling cross talk would lead to the development of a potential therapeutic approach for head and neck cancer.

Targeting lactate production and efflux in prostate cancer.

Prostate cancer (PCa) is the most commonly diagnosed cancer in men worldwide. Screening and management of PCa remain controversial and, therefore, the discovery of novel molecular biomarkers is urgently needed. Alteration in cancer cell metabolism is a recognized hallmark of cancer, whereby cancer cells exhibit high glycolytic rates with subsequent lactate production, regardless of oxygen availability. To maintain the hyperglycolytic phenotype, cancer cells efficiently export lactate through the monocarboxylate transporters MCT1 and MCT4. The impact of inhibiting lactate production/extrusion on PCa cell survival and aggressiveness was investigated in vitro and ex vivo using primary tumor and metastatic PCa cell lines and the chicken embryo chorioallantoic membrane (CAM) model. In this study, we showed the metastatic PCa cell line (DU125) displayed higher expression levels of MCT1/4 isoforms and glycolysis-related markers than the localized prostate tumor-derived cell line (22RV1), indicating these proteins are differentially expressed throughout prostate malignant transformation. Moreover, disruption of lactate export by MCT1/4 silencing resulted in a decrease in PCa cell growth and motility. To support these results, we pharmacological inhibited lactate production (via inhibition of LDH) and release (via inhibition of MCTs) and a reduction in cancer cell growth in vitro and in vivo was observed. In summary, our data provide evidence that MCT1 and MCT4 are important players in prostate neoplastic progression and that inhibition of lactate production/export can be explored as a strategy for PCa treatment.

Monocarboxylate transporters in cancer.

BACKGROUND: Tumors are highly plastic metabolic entities composed of cancer and host cells that can adopt different metabolic phenotypes. For energy production, cancer cells may use 4 main fuels that are shuttled in 5 different metabolic pathways. Glucose fuels glycolysis that can be coupled to the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) in oxidative cancer cells or to lactic fermentation in proliferating and in hypoxic cancer cells. Lipids fuel lipolysis, glutamine fuels glutaminolysis, and lactate fuels the oxidative pathway of lactate, all of which are coupled to the TCA cycle and OXPHOS for energy production. This review focuses on the latter metabolic pathway. SCOPE OF REVIEW: Lactate, which is prominently produced by glycolytic cells in tumors, was only recently recognized as a major fuel for oxidative cancer cells and as a signaling agent. Its exchanges across membranes are gated by monocarboxylate transporters MCT1-4. This review summarizes the current knowledge about MCT structure, regulation and functions in cancer, with a specific focus on lactate metabolism, lactate-induced angiogenesis and MCT-dependent cancer metastasis. It also describes lactate signaling via cell surface lactate receptor GPR81. MAJOR CONCLUSIONS: Lactate and MCTs, especially MCT1 and MCT4, are important contributors to tumor aggressiveness. Analyses of MCT-deficient (MCT+/- and MCT-/-) animals and (MCT-mutated) humans indicate that they are druggable, with MCT1 inhibitors being in advanced development phase and MCT4 inhibitors still in the discovery phase. Imaging lactate fluxes non-invasively using a lactate tracer for positron emission tomography would further help to identify responders to the treatments.

Role of rno-miR-124-3p in regulating MCT1 expression in rat brain after permanent focal cerebral ischemia.

This study aimed to assess the role of microRNAs (miRNAs) in regulating monocarboxylate transporter-1 (MCT1) expression in rat brain after permanent focal cerebral ischemia to identify a new target for early treatment of cerebral ischemia. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats. Morphology and protein expression levels of MCT1 were assessed by immunofluorescence and Western blotting. Using bioinformatics and double luciferase reporter assays, rno-miR-124-3p was selected as a direct target for rat MCT1. Expression of rno-miR-124-3p after pMCAO was detected. Then, rats were treated with rno-miR-124-3p agomir via lateral ventricle injection, and after 6 h or 24 h ischemia, rno-miR-124-3p expression and gene and protein expression of MCT-1 were detected by qRT-PCR and Western blotting. Brain infarction was identified by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Results showed that pMCAO induced brain infarction and increased the expression of MCT1. The levels of rno-miR-124-3p after pMCAO were in contrast to those of MCT1 protein in ischemic region, while declined after 3, 6 and 12 h of pMCAO in ischemic penumbra. After administration of rno-miR-124-3p agomir, MCT1 mRNA and protein levels were increased after 6 h of pMCAO, while decreased after 24 h of pMCAO. Meanwhile, rno-miR-124-3p levels increased after both times. TTC staining showed treatment with rno-miR-124-3p agomir reduced brain infarction. The role of rno-miR-124-3p in regulating MCT1 was as a positive regulator after 6 h of pMCAO, while a negative regulator after 24 h of pMCAO, however, both activities had protective effects against cerebral ischemia.

γ-Hydroxybutyric Acid (GHB) Pharmacokinetics and Pharmacodynamics: Semi-Mechanistic and Physiologically Relevant PK/PD Model.

An overdose of γ-hydroxybutyric acid (GHB), a drug of abuse, results in fatality caused by severe respiratory depression. In this study, a semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model was developed to characterize monocarboxylate transporter 1 (MCT1)-mediated transport of GHB, as well as effects of GHB on respiration frequency, for IV doses of 200, 600, and 1500 mg/kg in rats. The proposed PK/PD model for GHB consists of nonlinear metabolism of GHB in the liver, MCT1-mediated renal reabsorption with physiologically relevant concurrent fluid reabsorption, MCT1-mediated uptake into the brain, and direct effects of binding of GHB to GABAB receptors on the PD parameter, respiration frequency. Michaelis-Menten affinity constants for metabolism, renal reabsorption, and uptake into and efflux from the brain were fixed to the observed in vitro values. The IC 50 value for the effect of GHB on respiration frequency was fixed to a reported value for binding of GHB to GABAB receptors. All physiological parameters were fixed to the reported values for a 300-g rat. The model successfully captured the GHB PK/PD data and was further validated using the data for a 600-mg/kg dose of GHB after IV bolus administration. Unbound GHB brain ECF/blood partition coefficient (Kp u,u ) values obtained from the model agreed well with values calculated using experimental ECF concentrations obtained with brain microdialysis, demonstrating the physiological relevance of this model. Sensitivity analysis indicated that the PK/PD model was stable. In conclusion, we developed a semi-mechanistic and physiologically relevant PK/PD model of GHB using in vitro drug-transporter kinetics and in vivo PK/PD data in rats.

Hypoxia-mediated upregulation of MCT1 expression supports the glycolytic phenotype of glioblastomas.

BACKGROUND: Glioblastomas (GBM) present a high cellular heterogeneity with conspicuous necrotic regions associated with hypoxia, which is related to tumor aggressiveness. GBM tumors exhibit high glycolytic metabolism with increased lactate production that is extruded to the tumor microenvironment through monocarboxylate transporters (MCTs). While hypoxia-mediated regulation of MCT4 has been characterized, the role of MCT1 is still controversial. Thus, we aimed to understand the role of hypoxia in the regulation of MCT expression and function in GBM, MCT1 in particular. METHODS: Expression of hypoxia- and glycolytic-related markers, as well as MCT1 and MCT4 isoforms was assessed in in vitro and in vivo orthotopic glioma models, and also in human GBM tissues by immunofluorescence/immunohistochemistry and Western blot. Following MCT1 inhibition, either pharmacologically with CHC (α-cyano-4-hydroxynnamic acid) or genetically with siRNAs, we assessed GBM cell viability, proliferation, metabolism, migration and invasion, under normoxia and hypoxia conditions. RESULTS: Hypoxia induced an increase in MCT1 plasma membrane expression in glioma cells, both in in vitro and in vivo models. Additionally, treatment with CHC and downregulation of MCT1 in glioma cells decreased lactate production, cell proliferation and invasion under hypoxia. Moreover, in the in vivo orthotopic model and in human GBM tissues, there was extensive co-expression of MCT1, but not MCT4, with the GBM hypoxia marker CAIX. CONCLUSION: Hypoxia-induced MCT1 supports GBM glycolytic phenotype, being responsible for lactate efflux and an important mediator of cell survival and aggressiveness. Therefore, MCT1 constitutes a promising therapeutic target in GBM.

Hypoxia optimises tumour growth by controlling nutrient import and acidic metabolite export.

In their quest for survival and successful growth, cancer cells optimise their cellular processes to enable them to outcompete normal cells in their microenvironment. In essence cancer cells: (i) enhance uptake of nutrients/metabolites, (ii) utilise nutrients more efficiently via metabolic alterations and (iii) deal with the metabolic waste products in a way that furthers their progression while hampering the survival of normal tissue. Hypoxia Inducible Factors (HIFs) act as essential drivers of these adaptations via the promotion of numerous membrane proteins including glucose transporters (GLUTs), monocarboxylate transporters (MCTs), amino-acid transporters (LAT1, xCT), and acid-base regulating carbonic anhydrases (CAs). In addition to a competitive growth advantage for tumour cells, these HIF-regulated proteins are implicated in metastasis, cancer 'stemness' and the immune response. Current research indicates that combined targeting of these HIF-regulated membrane proteins in tumour cells will provide promising therapeutic strategies in the future.