Contemporary Perspectives on the Warburg Effect Inhibition in Cancer Therapy.

In the 1920s, Otto Warburg observed the phenomenon of altered glucose metabolism in cancer cells. Although the initial hypothesis suggested that the alteration resulted from mitochondrial damage, multiple studies of the subject revealed a precise, multistage process rather than a random pattern. The phenomenon of aerobic glycolysis emerges not only from mitochondrial abnormalities common in cancer cells, but also results from metabolic reprogramming beneficial for their sustenance. The Warburg effect enables metabolic adaptation of cancer cells to grow and proliferate, simultaneously enabling their survival in hypoxic conditions. Altered glucose metabolism of cancer cells includes, inter alia, qualitative and quantitative changes within glucose transporters, enzymes of the glycolytic pathway, such as hexokinases and pyruvate kinase, hypoxia-inducible factor, monocarboxylate transporters, and lactate dehydrogenase. This review summarizes the current state of knowledge regarding inhibitors of cancer glucose metabolism with a focus on their clinical potential. The altered metabolic phenotype of cancer cells allows for targeting of specific mechanisms, which might improve conventional methods in anti-cancer therapy. However, several problems such as drug bioavailability, specificity, toxicity, the plasticity of cancer cells, and heterogeneity of cells in tumors have to be overcome when designing therapies based on compounds targeted in cancer cell energy metabolism.

Different continuous exercise training intensities induced effect on sertoli-germ cells metabolic interaction; implication on GLUT-1, GLUT-3 and MCT-4 transporting proteins expression level.

Despite other tissues, the effect of different exercise training protocols (ETPs) on the expression levels of metabolic substrates transmembrane transporters in the testicular tissue, remains completely unexplored. Thus, the effects of low, moderate and high-intensity ETPs on the SCs and germ cells potentials in GLUT-1, GLUT-3 and MCT-4 expression levels was investigated in this study. The animals were assigned into 4 groups, including sedentary control, low-intensity continuous (LICT), moderate-intensity (MICT) and high-intensity (HICT) ETPs-induced groups (n = 6/group). The GLUT-1, GLUT-3 and MCT-4 expressions, cytoplasmic carbohydrate storages of SCs and germ cells, the SCs survival and the spermatogenesis and spermiogenesis rates were assessed. The LICT and MICT did not significantly alter the protein expression levels of GLUT-3 and MCT-4 in the SCs and germ cells, while decreased the GLUT-1 protein content versus the sedentary control animals. In contrast, the HICT remarkably suppressed the GLUT-1 and MCT-4 in both SCs, and germ cells and diminished GLUT-3 in SCs and increased in the germ cells. No significant changes were revealed in the cytoplasmic carbohydrate storage in the LICT and MICT groups, while significantly diminished in the HICT group. The HICT group showed a failed spermatogenesis and spermiogenesis, which were not demonstrated in the sedentary control, LICT and MICT groups. In conclusion, the HICT, by reducing the GLUT-1, GLUT-3 and MCT-4 protein contents in the SCs and reducing the SCs survival, can suppress the glucose transmembrane transport and inhibit the lactate export from SCs, which in turn, ends with failed spermatogenesis and spermiogenesis.

Mitochondrial pyruvate carrier abundance mediates pathological cardiac hypertrophy.

Cardiomyocytes rely on metabolic substrates, not only to fuel cardiac output, but also for growth and remodelling during stress. Here we show that mitochondrial pyruvate carrier (MPC) abundance mediates pathological cardiac hypertrophy. MPC abundance was reduced in failing hypertrophic human hearts, as well as in the myocardium of mice induced to fail by angiotensin II or through transverse aortic constriction. Constitutive knockout of cardiomyocyte MPC1/2 in mice resulted in cardiac hypertrophy and reduced survival, while tamoxifen-induced cardiomyocyte-specific reduction of MPC1/2 to the attenuated levels observed during pressure overload was sufficient to induce hypertrophy with impaired cardiac function. Failing hearts from cardiomyocyte-restricted knockout mice displayed increased abundance of anabolic metabolites, including amino acids and pentose phosphate pathway intermediates and reducing cofactors. These hearts showed a concomitant decrease in carbon flux into mitochondrial tricarboxylic acid cycle intermediates, as corroborated by complementary 1,2-[13C2]glucose tracer studies. In contrast, inducible cardiomyocyte overexpression of MPC1/2 resulted in increased tricarboxylic acid cycle intermediates, and sustained carrier expression during transverse aortic constriction protected against cardiac hypertrophy and failure. Collectively, our findings demonstrate that loss of the MPC1/2 causally mediates adverse cardiac remodelling.

SLC5A8 regulates the biological behaviors of cervical cancer cells through mediating the Wnt signaling pathway.

OBJECTIVE: This study aims to investigate the role of solute transport family 5 member 8 (SLC5A8) in the progress of cervical cancer (CC) to provide a theoretical basis for the treatment of CC. PATIENTS AND METHODS: Tissues were obtained from 58 patients diagnosed with CC in our hospital. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) analysis was used to detect the expression level of SLC5A8 in CC tissues and cell lines. SLC5A8 level was up-regulated by transfection of SLC5A8 overexpression plasmid. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and flow analysis were designed to measure the cell proliferation, cell cycle, and apoptosis of CC cells. RESULTS: The mRNA expression of SLC5A8 was down-regulated in CC tissues and cell lines. Transfection of SLC5A8 overexpression plasmid successfully over-expressed SLC5A8. In addition, an inhibited activation of Wnt signaling pathway was detected in CC cells after over-expression of SLC5A8. Besides, decreased proliferation activity and increased apoptosis were also observed in CC cells overexpressing SLC5A8 plasmid. Moreover, the impaired proliferation activity and increased apoptosis proportion of CC cells induced by SLC5A8 over-expression could be counteracted by the Wnt signaling pathway activator LiCl. CONCLUSIONS: SLC5A8 alleviates the progression of CC by regulating the Wnt signaling pathway.

Spatiotemporal Changes of Cerebral Monocarboxylate Transporter 8 Expression.

Background: Mutations of monocarboxylate transporter 8 (MCT8), a thyroid hormone (TH)-specific transmembrane transporter, cause a severe neurodevelopmental disorder, the Allan-Herndon-Dudley syndrome. In MCT8 deficiency, TH is not able to reach those areas of the brain where TH uptake depends on MCT8. Currently, therapeutic options for MCT8-deficient patients are missing, as TH treatment is not successful in improving neurological deficits. Available data on MCT8 protein and transcript levels indicate complex expression patterns in neural tissue depending on species, brain region, sex, and age. However, information on human MCT8 expression is still scattered and additional efforts are needed to map sites of MCT8 expression in neurovascular units and neural tissue. This is of importance because new therapeutic strategies for this disease are urgently needed. Methods: To identify regions and time windows of MCT8 expression, we used highly specific antibodies against MCT8 to perform immunofluorescence labeling of postnatal murine brains, adult human brain tissue, and human cerebral organoids. Results: Qualitative and quantitative analyses of murine brain samples revealed stable levels of MCT8 protein expression in endothelial cells of the blood-brain barrier (BBB), choroid plexus epithelial cells, and tanycytes during postnatal development. Conversely, the neuronal MCT8 protein expression that was robustly detectable in specific brain regions of young mice strongly declined with age. Similarly, MCT8 immunoreactivity in adult human brain tissue was largely confined to endothelial cells of the BBB. Recently, cerebral organoids emerged as promising models of human neural development and our first analyses of forebrain-like organoids revealed MCT8 expression in early neuronal progenitor cell populations. Conclusions: With respect to MCT8-deficient conditions, our analyses not only strongly support the contention that the BBB presents a lifelong barrier to TH uptake but also highlight the need to decipher the TH transport role of MCT8 in early neuronal cell populations in more detail. Improving the understanding of the spatiotemporal expression in latter barriers will be critical for therapeutic strategies addressing MCT8 deficiency in the future.

CAIX is a predictor of pathological complete response and is associated with higher survival in locally advanced breast cancer submitted to neoadjuvant chemotherapy.

BACKGROUND: Locally advanced breast cancer often undergoes neoadjuvant chemotherapy (NAC), which allows in vivo evaluation of the therapeutic response. The determination of the pathological complete response (pCR) is one way to evaluate the response to neoadjuvant chemotherapy. However, the rate of pCR differs significantly between molecular subtypes and the cause is not yet determined. Recently, the metabolic reprogramming of cancer cells and its implications for tumor growth and dissemination has gained increasing prominence and could contribute to a better understanding of NAC. Thus, this study proposed to evaluate the expression of metabolism-related proteins and its association with pCR and survival rates. METHODS: The expression of monocarboxylate transporters 1 and 4 (MCT1 and MCT4, respectively), cluster of differentiation 147 (CD147), glucose transporter-1 (GLUT1) and carbonic anhydrase IX (CAIX) was analyzed in 196 locally advanced breast cancer samples prior to NAC. The results were associated with clinical-pathological characteristics, occurrence of pCR, disease-free survival (DFS), disease-specific survival (DSS) and overall survival (OS). RESULTS: The occurrence of pCR was higher in the group of patients whith tumors expressing GLUT1 and CAIX than in the group without expression (27.8% versus 13.1%, p = 0.030 and 46.2% versus 13.5%, p = 0.007, respectively). Together with regional lymph nodes staging and mitotic staging, CAIX expression was considered an independent predictor of pCR. In addition, CAIX expression was associated with DFS and DSS (p = 0.005 and p = 0.012, respectively). CONCLUSIONS: CAIX expression was a predictor of pCR and was associated with higher DFS and DSS in locally advanced breast cancer patients subjected to NAC.

Differential expression and immunoreactivity of thyroid hormone transporters MCT8 and OATP1C1 in rat ovary.

Thyroid hormones (THs) regulate several physiological processes in female mammals, many of which are related to reproduction such as steroidogenesis in the ovary, oocyte and granulosa cells maturation, follicular development and differentiation, and ovulation. THs actions require the presence of THs transporters to facilitate their cellular uptake and efflux. MCT8 and OATP1C1 are the principal THs transporters. The aim of the present study was to determine the gene expression and cellular localization of MCT8 and OATP1C1 in the rat ovary during the diestrus-II cycle phase. Ovaries of virgin adult rats were histologically processed. Reverse Transcription-PCR and immunohistochemistry analyses for MCT8 and OATP1C1 were done. MCT8 gene expression level was significantly higher (P ≤ 0.01) than that of OATP1C1 in the rat ovary. MCT8 and OATP1C1 were found in all types of ovarian cells but with different immunoreactivity. MCT8 showed stronger immunoreactivity in tertiary and Graafian follicles, corpus luteum and blood vessels, whereas OATP1C1's immunoreactivity was stronger in stroma cells, tunica albuginea, and blood vessels. Our results provide evidence that THs and their transporters are both necessary for ovarian function and that any alteration in these transporters could interfere with reproductive processes such as ovulation and steroidogenesis, compromising fertility.

Expression of Monocarboxylate Transporter 1 Is Associated With Better Prognosis and Reduced Nodal Metastasis in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Because lactate is believed to support tumor growth, monocarboxylate transporters (MCTs), which transport lactate, have been investigated in multiple tumors. However, the significance of MCTs in pancreatic cancer is unclear. METHODS: A retrospective survey was conducted on 240 patients who underwent surgical resection for pancreatic ductal adenocarcinoma without preoperative treatment. The expression of MCT1, MCT2, MCT3, MCT4, and the glucose transporter 1 (GLUT1) was assessed in tumor cells and cancer-associated fibroblasts (CAFs) by tissue microarrays and immunohistochemistry. The impact of their expression on patient outcome and clinicopathological characteristics was also analyzed. RESULTS: In tumor cells, MCT1, MCT2, MCT3, MCT4, and GLUT1 were detected in 52 (22%), 31 (13%), 149 (62%), 204 (85%), and 235 (98%) cases, respectively. In CAFs, MCT2, MCT4, and GLUT1 were detected in 9 (3.8%), 178 (74%), and 36 (15%) cases, respectively. In tumor cells, MCT1 expression was associated with extended overall and progression-free survival and decreased nodal metastasis. Conversely, MCT4 expression in CAFs was associated with shortened survival. CONCLUSIONS: In tumor cells, MCT1 expression is associated with better prognosis and reduced nodal metastasis in pancreatic cancer, contrary to findings of past in vitro studies. Conversely, MCT4 expression in CAFs is indicative of worse prognosis.

Decreased expression of SLC16A12 mRNA predicts poor prognosis of patients with clear cell renal cell carcinoma.

Solute carrier family 16, member 12 (SLC16A12) is a highly -expressed protein in the kidney and has been reported to participate in the transport of creatine. However, the clinical values of SLC16A12 in clear cell renal cell carcinoma (ccRCC) have not been explored.SLC16A12 RNA-seq data and clinical information were downloaded from the Cancer Genome Atlas (TCGA) database. We compared its expression in ccRCC and paracancerous tissues, then the result was further validated with our cohort. The impact on the clinical significance of SLC16A12 in ccRCC was also assessed.Compared with paracancerous tissue, SLC16A12 was significantly downregulated in the tumor tissues both in mRNA and protein level. In TCGA cohort, SLC16A12 mRNA expression was associated with several clinicopathological parameters, including T stages (P < .001), M stages (P = .009), TNM stages (P < .001), and differentiated grades (P = .001). Kaplan-Meier analysis showed that the overall survival of patients with low expression of SLC16A12 mRNA was significantly worse than that of patients with high expression (P < .001). Furthermore, both univariate (HR = 0.371, 95%CI: 0.269-0.513, P < .001) and multivariate (HR = 0.485, 95%CI: 0.297-0.793, P = .004) Cox regression analyses suggested that low expression of SLC16A12 mRNA was an independent prognostic factor for patients with ccRCC.Overall, we uncovered that decreased expression of SLC16A12 is a poor prognostic factor for patients with ccRCC. SLC16A12 might be a potential biomarker and therapeutic target in ccRCC.

Monocarboxylate Transporter 4 (MCT4) Overexpression Is Correlated with Poor Prognosis of Osteosarcoma.

BACKGROUND Monocarboxylate transporter 4 (MCT4) is a critical element for glycolytic metabolism and malignant behaviors in many tumor cells. This study aimed to determine the expression level of MCT4 protein and its prognostic value in osteosarcoma. MATERIAL AND METHODS MCT4 expression was detected via immunohistochemical and Western blot analysis for 100 osteosarcoma patients. The correlation between MCT4 expression and clinical factors among the patients was analyzed using the chi-square test. Overall survival of osteosarcoma patients was estimated by Kaplan-Meier analysis. The prognostic value of MCT4 was evaluated using Cox regression analysis with adjustments for clinicopathological variables. RESULTS MCT4 expression was significantly upregulated in osteosarcoma tissues compared with that in adjacent normal ones, detected via both immunohistochemical and Western blot analyses. High MCT4 expression showed a positive association with distant metastasis (P=0.000) and recurrence (P=0.000) of osteosarcoma. Kaplan-Meier analysis indicated that overall survival of osteosarcoma patients was significantly higher in the low MCT4 expression group than in the high expression group (log-rank test, P<0.001). Multivariate analysis indicated that MCT4 expression and clinical stage, which are tightly related to the prognosis of osteosarcoma, might be independent predictors of osteosarcoma prognosis. CONCLUSIONS High MCT4 expression appears to contribute to osteosarcoma progression and the upregulation of MCT4 may predict poor prognosis among osteosarcoma patients.

Changes in thyroid hormone activity disrupt photomotor behavior of larval zebrafish.

High throughput in vitro, in silico, and computational approaches have identified numerous environmental chemicals that interfere with thyroid hormone (TH) activity, and it is posited that human exposures to such chemicals are a contributing factor to neurodevelopmental disorders. However, whether hits in screens of TH activity are predictive of developmental neurotoxicity (DNT) has yet to be systematically addressed. The zebrafish has been proposed as a second tier model for assessing the in vivo DNT potential of TH active chemicals. As an initial evaluation of the feasibility of this proposal, we determined whether an endpoint often used to assess DNT in larval zebrafish, specifically photomotor behavior, is altered by experimentally induced hyper- and hypothyroidism. Developmental hyperthyroidism was simulated by static waterborne exposure of zebrafish to varying concentrations (3-300 nM) of thyroxine (T4) or triiodothyronine (T3) beginning at 6 h post-fertilization (hpf) and continuing through 5 days post-fertilization (dpf). Teratogenic effects and lethality were observed at 4 and 5 dpf in fish exposed to T4 or T3 at concentrations >30 nM. However, as early as 3 dpf, T4 (> 3 nM) and T3 (> 10 nM) significantly increased swimming activity triggered by sudden changes from light to dark, particularly during the second dark period (Dark 2). Conversely, developmental hypothyroidism, which was induced by treatment with 6-propyl-2-thiouracil (PTU), morpholino knockdown of the TH transporter mct8, or ablation of thyroid follicles in adult females prior to spawning, generally decreased swimming activity during dark periods, although effects did vary across test days. All effects of developmental hypothyroidism on photomotor behavior occurred independent of teratogenic effects and were most robust during Dark 2. Treatment with the T4 analog, Tetrac, restored photomotor response in mct8 morphants to control levels. Collectively, these findings suggest that while the sensitivity of photomotor behavior in larval zebrafish to detect TH disruption is influenced by test parameters, this test can distinguish between TH promoting and TH blocking activity and may be useful for assessing the DNT potential of TH-active chemicals.

The association between markers of tumour cell metabolism, the tumour microenvironment and outcomes in patients with colorectal cancer.

Tumour cell anaerobic metabolism has been reported to be a prognostic factor in colorectal cancer. The present study investigated the association between monocarboxylate transporter (MCT) 1, MCT 2, lactate dehydrogenase (LDH) 1 and LDH 5, the tumour microenvironment, and outcome in patients with colorectal cancer. A cohort of 150 patients with stage I-III CRC were utilised to assess tumour cell expression of MCT-1, MCT-2, LDH-1 and LDH-5 by immunohistochemistry. Expression levels were dichotomised and associations with tumour factors, the tumour microenvironment and survival analysed. Nuclear LDH-5 associates with poor prognosis (HR 1.68 95% CI 0.99-2.84, p = 0.050) and trends toward increased tumour stroma percentage (TSP, p = 0.125). Cytoplasmic MCT-2 also trends toward increased TSP (p = 0.081). When combined into a single score; nuclear LDH-5 + TSP significantly associated with decreased survival independent of stage (HR 2.61 95% CI 1.27-5.35, p = 0.009), increased tumour budding (p = 0.002) and decreased stromal T-lymphocytes (p = 0.014). Similarly, cytoplasmic MCT-2 + TSP significantly associated with decreased survival (HR 2.32 95% CI 1.31-4.11, p = 0.003), decreased necrosis (p = 0.039), and increased tumour budding (p = 0.004). The present study reports that the combination of TSP and nuclear LDH-5 was significantly associated with survival, increased tumour budding, and decreased stromal T-lymphocytes. This supports the hypothesis that increased stromal invasion promotes tumour progression via modulation of tumour metabolism. Moreover, MCT-2 and LDH-5 may provide promising therapeutic targets for patients with stromal-rich CRC.

Involvement of monocarboxylate transporter 1 (SLC16A1) in the uptake of l-lactate in human astrocytes.

PURPOSE: Astrocytes, the most abundant glial cells in the central nervous system (CNS), help neurons survive. Monocarboxylate transporters (MCTs) are reported to transport l-lactate, which is important for CNS physiology and cognitive function. However, it remains unclear which MCT isoform is functionally expressed by human astrocytes. The aim of this study was to establish the contribution of each MCT isoform to l-lactate transport in human astrocytes. METHODS: The function of l-lactate transport was studied using NHA cells as a human astrocyte model and radiolabeled l-lactate. The expression of MCT in human astrocytes was detected by immunohistochemistry staining. RESULTS: The cellular uptake of l-lactate was found to be pH- and concentration-dependent with a Km value for l-lactate uptake of 0.64mM. This Km was similar to what has been previously established for MCT1-mediated l-lactate uptake. α-Cyano-4- hydroxycinnamate (CHC) and 5-oxoproline, which are both MCT1 inhibitors, were found to significantly inhibit the uptake of l-lactate, suggesting MCT1 is primarily responsible for l-lactate transport. Moreover, MCT1 protein was expressed in human astrocytes. CONCLUSION: pH-dependent l-lactate transport is mediated by MCT1 in human astrocytes.

Arginine vasopressin altered the expression of monocarboxylate transporters in cultured astrocytes isolated from stroke-prone spontaneously hypertensive rats and congenic SHRpch1_18 rats.

BACKGROUND: Astrocytes support a range of brain functions as well as neuronal survival, but their detailed relationship with stroke-related edema is not well understood. We previously demonstrated that the release of lactate from astrocytes isolated from stroke-prone spontaneously hypertensive rats (SHRSP/Izm) was attenuated under stroke conditions. The supply of lactate to neurons is regulated by astrocytic monocarboxylate transporters (MCTs). The purpose of this study was to examine the contributions of arginine vasopressin (AVP) and/or hypoxia and reoxygenation (H/R) to the regulation of MCTs and neurotrophic factor in astrocytes obtained from SHRSP/Izm and congenic SHRpch1_18 rats. METHODS: We compared AVP-induced lactate levels, MCTs, and brain-derived neurotrophic factor (BDNF) in astrocytes isolated from SHRSP/Izm, SHRpch1_18, and Wistar Kyoto rats (WKY/Izm). The expression levels of genes and proteins were determined by PCR and Western blotting (WB). RESULTS: The production of lactate induced by AVP was increased in astrocytes from all three strains. However, the levels of lactate were lower in SHRSP/Izm and SHRpch1_18 animals compared with the WKY/Izm strain. Gene expression levels of Slc16a1, Slc16a4, and Bdnf were lowered by AVP in SHRSP/Izm and SHRpch1_18 rats compared with WKY/Izm. The increase of MCT4 that was induced by AVP was blocked by the addition of a specific nitric oxide (NO) chelator, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO). Furthermore, AVP increased the expression of iNOS and eNOS proteins in WKY/Izm and SHRSP/Izm rat astrocytes. However, the iNOS expression levels in SHRSP astrocytes differed from those of WKY/Izm astrocytes. The increase of MCT4 protein expression during AVP treatment was blocked by the addition of a specific NF-kB inhibitor, pyrrolidine dithiocarbamate (PDTC). The induction of MCT4 by AVP may be regulated by NO through NF-kB. CONCLUSIONS: These results suggest that the expression of MCTs mediated by AVP may be regulated by NO. The data suggest that AVP attenuated the expression of MCTs in SHRSP/Izm and SHRpch1_18 astrocytes. Reduced expression of MCTs may be associated with decreased lactate production in SHRSP.

Differential regulation of monocarboxylate transporter 8 expression in thyroid cancer and hyperthyroidism.

OBJECTIVE: Thyroid hormone (TH) transporters are expressed in thyrocytes and most play a role in TH release. We asked whether expression of the monocarboxylate transporter 8 (MCT8) and the L-type amino acid transporters LAT2 and LAT4 is changed with thyrocyte dedifferentiation and in hyperfunctioning thyroid tissues. DESIGN AND METHODS: Protein expression and localization of transporters was determined by immunohistochemistry in human thyroid specimen including normal thyroid tissue (NT, n = 19), follicular adenoma (FA, n = 44), follicular thyroid carcinoma (FTC, n = 45), papillary thyroid carcinoma (PTC, n = 40), anaplastic thyroid carcinoma (ATC, n = 40) and Graves' disease (GD, n = 50) by calculating the 'hybrid' (H) score. Regulation of transporter expression was investigated in the rat follicular thyroid cell line PCCL3 under basal and thyroid stimulating hormone (TSH) conditions. RESULTS: MCT8 and LAT4 were localized at the plasma membrane, while LAT2 transporter showed cytoplasmic localization. MCT8 expression was downregulated in benign and malignant thyroid tumours as compared to NT. In contrast, significant upregulation of MCT8, LAT2 and LAT4 was found in GD. Furthermore, a stronger expression of MCT8 was demonstrated in PCCL3 cells after TSH stimulation. CONCLUSIONS: Downregulation of MCT8 in thyroid cancers qualifies MCT8 as a marker of thyroid differentiation. The more variable expression of LATs in distinct thyroid malignancies may be linked with other transporter properties relevant to altered metabolism in cancer cells, i.e. amino acid transport. Consistent upregulation of MCT8 in GD is in line with increased TH release in hyperthyroidism, an assumption supported by our in vitro results showing TSH-dependent upregulation of MCT8.

Effect of chronic γ-hydroxybutyrate (GHB) administration on GHB toxicokinetics and GHB-induced respiratory depression.

BACKGROUND: γ-hydroxybutyrate (GHB) has a high potential for illicit use; overdose of this compound results in sedation, respiratory depression and death. Tolerance to the hypnotic/sedative and electroencephalogram effects of GHB occurs with chronic GHB administration; however, tolerance to respiratory depression has not been evaluated. GHB toxicodynamic effects are mediated predominantly by GABAB receptors. Chronic treatment may affect monocarboxylate transporters (MCTs) and alter the absorption, renal clearance and brain uptake of GHB. OBJECTIVES: To determine effects of chronic GHB dosing on GHB toxicokinetics, GHB-induced respiratory depression, and MCT expression. METHODS: Rats were administered GHB 600 mg/kg intravenously daily for 5 days. Plasma, urine and tissue samples and respiratory measurements were obtained on days 1 and 5. Plasma and urine were analyzed for GHB by LC/MS/MS and tissue samples for expression of MCT1, 2 and 4 and their accessory proteins by QRT-PCR. RESULTS: No differences in GHB pharmacokinetics or respiratory depression were observed between days 1 and 5. Opposing changes in MCT1 and MCT4 mRNA expression were observed in kidney samples on day 5 compared to GHB-naïve animals, and MCT4 expression was increased in the intestine. CONCLUSIONS: The lack of tolerance observed with GHB-induced respiratory depression, in contrast to the tolerance reported for the sedative/hypnotic and electroencephalogram effects, suggests that different GABAB receptor subtypes may be involved in different GABAB-mediated toxicodynamic effects of GHB. Chronic or binge users of GHB may be at no less risk for fatality from respiratory arrest with a GHB overdose than with a single dose of GHB.

Glycolysis-related protein expression in thyroid cancer.

We aimed to demonstrate the differences in the expression of glucose metabolism-related proteins according to the thyroid cancer subtypes and investigate the implications of these differences. A total of 566 thyroid cancer patients, including 342 cases of papillary thyroid carcinoma, 112 cases of follicular carcinoma, 70 cases of medullary carcinoma, 23 cases of poorly differentiated carcinoma, 19 cases of anaplastic carcinoma, and 152 cases of follicular adenoma, were enrolled in the study. Immunohistochemical staining for glucose transporter 1, hexokinase II, carbonic anhydrase IX, and monocarbonylate transporter 4 was performed, and the relationship between immunoreactivity and clinicopathologic parameters was analyzed. Glucose transporter 1 and tumoral monocarbonylate transporter 4 expression levels were shown to be the highest in anaplastic carcinoma, and medullary carcinoma showed the highest carbonic anhydrase IX and lowest hexokinase II levels compared with other subtypes. Stromal expression of monocarbonylate transporter 4 was observed in papillary thyroid carcinoma and anaplastic carcinoma samples. Conventional papillary thyroid carcinoma tumors expressed higher levels of glucose transporter 1, and tumoral and stromal monocarbonylate transporter 4, than the follicular variant, which showed a higher expression of carbonic anhydrase IX. Papillary thyroid carcinoma samples with BRAF V600E mutation were shown to have higher glucose transporter 1, hexokinase II, carbonic anhydrase IX, and tumoral monocarbonylate transporter 4 expression levels. Univariate analysis showed that papillary thyroid carcinoma cases with glucose transporter 1 positivity had shorter overall survival, patients with medullary carcinoma and hexokinase II positivity were shown to have a shorter disease-free survival and overall survival, and tumoral monocarbonylate transporter 4 positivity was associated with shorter overall survival compared with papillary thyroid carcinoma patients with negativity for each marker. Disease-free survival and overall survival of patients with poorly differentiated carcinoma were shown to be significantly decreased when glucose transporter 1 and tumoral monocarbonylate transporter 4 are expressed. We demonstrated that the expression levels of glycolysis-related proteins differ between thyroid cancer subtypes and are correlated with poorer prognosis, depending on the subtype.

GLUT1, MCT1/4 and CD147 overexpression supports the metabolic reprogramming in papillary renal cell carcinoma.

Papillary Renal Cell carcinoma (pRCC) is the second most common type of RCC, accounting for about 15% of all RCCs. Surgical excision is the main treatment option. Still, 10 - 15 % of clinically localized tumours will recur and/or develop metastasis early after surgery, and no reliable prognostic biomarkers are available to identify them. It is known that pRCC cells rely on high rates of aerobic glycolysis, characterized by the up-regulation of many proteins and enzymes related with the glycolytic pathway. However, a metabolic signature enabling the identification of advanced pRCC tumours remains to be discovered. The aim of this study was to characterize the metabolic phenotype of pRCCs (subtypes 1-pRCC1 and 2-pRCC2) by evaluating the expression pattern of the glucose transporters (GLUTs) 1 and 4 and the monocarboxylate transporters (MCTs) 1 and 4, as well as their chaperon CD147. We analysed the clinico-pathological data and the protein and mRNA expression of GLUT1, GLUT4 and MCT1, MCT4 and CD147 in tumours from Porto and TCGA series (http://cancergenome.nih.gov/), respectively. With the exception of GLUT4, plasma membrane expression of all proteins was frequently observed in pRCCs. GLUT1 and MCT1 membrane overexpression was significantly higher in pRCC2 and significantly associated with higher pN-stage and higher Fuhrman grade. Overexpression of GLUT1, MCT1/4 and CD147, supports the metabolic reprograming in pRCCs. MCT1 expression was associated with pRCC aggressiveness, regardless of the tumour histotype.

Differential expression of endothelial nutrient transporters (MCT1 and GLUT1) in the developing eyes of mice.

The blood-brain barrier in the neonatal brain expresses the monocarboxylate transporter (MCT)-1 rather than the glucose transporter (GLUT)-1, due to the special energy supply during the suckling period. The hyaloid vascular system, consisting of the vasa hyaloidea propria and tunica vasculosa lentis, is a temporary vasculature present only during the early development of mammalian eyes and later regresses. Although the ocular vasculature manifests such a unique developmental process, no information is available concerning the expression of endothelial nutrient transporters in the developing eye. The present immunohistochemical study using whole mount preparations of murine eyes found that the hyaloid vascular system predominantly expressed GLUT1 in the endothelium, in contrast to the brain endothelium. Characteristically, the endothelium in peripheral regions of the neonatal hyaloid vessels displayed a mosaic pattern of MCT1-immunoreactive cells scattered within the GLUT1-expressing endothelium. The proper retinal vessels first developed by sprouting angiogenesis endowed with filopodia, which were absolutely free from the immunoreactivities of GLUT1 and MCT1. The remodeling retinal capillary networks and veins in the surface layer of the retina mainly expressed MCT1 until the weaning period. Immunostaining of MCT1 in the retina revealed fine radicular processes projecting from the endothelium, differing from the MCT1-immunonegative filopodia. These findings suggest that the expression of nutrient transporters in the ocular blood vessels is differentially regulated at a cellular level and that the neonatal eyes provide an interesting model for research on nutrient transporters in the endothelium.

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.