Cancer cells utilise glycolysis to produce lactate, even in the presence of sufficient levels of oxygen. Excess lactate is removed from cancer cells by MCT1 and MCT4, to prevent intracellular acidosis, apoptosis and to aid the continuous glycolytic flux. MCT1 and MCT4 are over-expressed in many types of human cancers, which correlates with reduced overall survival and increased treatment resistance. The potential role of MCT1 and MCT4 in two EOC cell lines (Skov3 and OV90) was investigated in this study. MCT1 was expressed at similar levels in Skov3 and OV90 cells. Therefore, stable cell lines over-expressing MCT1 were produced using both cell lines. MCT4 was expressed at high levels in Skov3 cells, but very low levels in OV90 cells. Therefore, a stable cell line with MCT4 silencing under the inducible control of doxycycline was produced using Skov3 cells, and a stable cell line to over-express MCT4 was produced using OV90 cells. The consequences of these genetic modifications on the metabolic phenotype, metastatic abilities and the sensitivity of cell lines to treatment with Carboplatin and Paclitaxel, were assessed in normoxia and 1 % hypoxia and 0.1 % hypoxia. Over-expressing MCT1 in Skov3 cells, had no effect on their metabolic phenotype or the sensitivity to treatment with Carboplatin and Paclitaxel. However, over-expressing MCT1 in Skov3 cells significantly enhanced their metastatic abilities, which correlated with reduced focal adhesion size. Silencing MCT4 in Skov3 cells, had no effect on the use of glycolysis, sensitivity to treatment with Carboplatin and Paclitaxel, or their metastatic abilities. However, following MCT4 silencing there was a significant increase in the levels of intracellular ROS. Over-expressing MCT1 in OV90 cells, had no effect on lactate levels or intracellular ROS. However, there was a significant reduction in both their glycolytic activity and mitochondrial mass. Furthermore, over-expressing MCT1 in OV90 cells, increased their resistance to treatment with Paclitaxel, which correlated with increased Pgp and LDHA expression. Over-expressing MCT4 in OV90 cells, caused an increase in the use of glycolysis and increased cell survival in hypoxia. There was also a significant enhancement in the metastatic abilities of these cells following the over- expression of MCT4, which correlated with reduced focal adhesion size. Furthermore, over-expressing MCT4 in OV90 cells, increased their resistance to treatment with Paclitaxel, which correlated with an increase in the expression of Pgp and LDHA.In summary, the findings of this study revealed that MCT1 and MCT4 play a significant role in the biological function of Skov3 and OV90 cells. High expression levels of MCT4 correlated with an increase in glycolysis and cell survival in hypoxia. Whereas high expression levels of MCT1 and MCT4 in correlated with an increase in the metastatic abilities, as well as with Paclitaxel resistance and increased Pgp expression.

No description available.

<p>Selective therapeutic targeting of tumors requires identification of differences between the homeostatic requirements of cancer and host cells. One such difference is the manner in which cancer cells acquire energy. Cancer cells often grow in an environment of local hypoxia; under these conditions tumor cells depend on glycolysis for energy, but are unable to perform oxidative phosphorylation. Many tumor cells, despite normoxic conditions, continue to perform glycolysis without oxidative phosphorylation. The net result of glycolysis without oxidative phosphorylation is twofold: the need to consume a greater amount of glucose than a non-cancerous host cell, and the burden of increased intracellular lactic acid. The proteins responsible for the transport of lactic acid in and out of cells are known as the monocarboxylate transporters (MCTs). Monocarboxylate Transporter 1 (MCT1) and Monocarboxylate Transporter 4 (MCT4) are the MCTs that play a major role in the transport of lactic acid. Tumor cells depend on MCT1 and MCT4 activity to excrete excess intracellular lactic acid to maintain neutral intracellular pH and homeostasis. Using human neuroblastoma and prostate cancer cell lines this work demonstrates that tumor cells can be selectively targeted tumor under conditions of hypoxia or acidosis in vitro with the drug lonidamine, with a small molecule inhibitor selective for MCT1, or with RNA interference of MCT1. Inhibition of MCT1 activity in neuroblastoma cells under acidic extracellular conditions results in intracellular acidification and cell death. MCT1 mRNA is expressed in human neuroblastoma and positively correlated with clinical risk profile. Inhibition of MCT1 activity in hypoxic prostate cancer cells results in a reduction of lactate excretion, decreased intracellular pH, inhibition of ATP production, and subsequent cell death. MCT1 expression in sections of human prostate tumors has been demonstrated to validate MCT1 as a target in prostate cancer.</p> <p>Through the Pasteur and Warburg effects, tumors have an increased demand for glucose. Some cancers store glycogen, but the reasons for this are largely unknown. It is hypothesized that tumor glycogen is used to promote tumor survival during transient hypoxia or low glucose, and that the mechanisms by which glycogen is stored is a potential therapeutic target in cancer. Tumors from human cell lines (WiDr, PC3, FaDu) have been grown in nude mice, sectioned and stained to measure glycogen storage. Using consecutive frozen sections, levels of hypoxia, glucose, lactate, ATP, and CD31, an endothelial cell marker, have been determined. These sections have been employed to elucidate the "architecture" of tumor metabolism in terms of vessel distance. Additionally, PAS-stained EF5 labeled human tumor samples were used to obtain calibrated hypoxia measurements to correlate with PAS. These studies demonstrate a correlation between hypoxia and the formation of glycogen deposits in human tumors and nude mouse xenografts. In cell culture, formation of glycogen deposits after exposure to hypoxia has been demonstrated, in addition to expression of glycogen synthase in human cancer cell lines.</p> <p>The development of novel selective cancer chemotherapeutics will require the identification of differences between cancerous cells and normal host cells to exploit as targets. Several differences in metabolism, including the need to excrete excess lactic acid and store glycogen under hypoxic conditions, are such targets. Novel therapeutics exploiting these targets should be effective against cancer cells and minimally toxic to host cells.</p> / Dissertation

It has been shown that tumour cells are capable of switching to glycolytic metabolism for the production of ATP even in the presence of oxygen, this is known as aerobic glycolysis or the 'Warburg effect'. The glycolytic phenotype has been associated with tumour aggressiveness and poor outcome in several cancer types. This makes the area of cancer metabolism an attractive area for the potential identification of new therapeutic targets. One key component, required for cells to maintain the glycolytic phenotype, is the presence of monocarboxylate transporters that are capable of exporting lactate. These transporters are vital for the maintenance of the intracellular pH of cells under these conditions. This study was centred around the hypothesis that altering expression of MCTs would impact on the metabolism of tumour cells and, therefore, other key characteristics of cells relating to metastatic capabilities and survival following treatment. For the purpose of this work, prostate cancer cell lines were transfected with lentiviral particles targeting overexpression of MCT1 or MCT4, or knockdown of MCT4. Following transfection, cellular metabolic profiles were assessed under normoxic and hypoxic conditions and the metastatic phenotype of each cell line was investigated. Additionally, the effect of MCT expression on response to chemotherapy and radiation therapy was explored, and a siRNA metabolome screen was performed to identify combinations of targets that may produce synthetic lethality in prostate cancer cell lines. It was shown that changes in the expression of MCT1 or MCT4 did not cause significant changes in the metastatic phenotypes of the prostate cancer cell lines investigated. Some differences were observed in the metabolic pathways used by these prostate cancer cells following alterations in MCT expression. For example, overexpression of MCT1 in DU145 cells resulted in an increase in intracellular lactate. Additionally, MCT4 knockdown in PC3 cells was able to reduce OXPHOS under reduced oxygen. MCT1 overexpression was able to sensitise androgen-independent prostate cancer cells to treatment with chemotherapy and radiation therapy. Furthermore, combinations of siRNA treatments were identified that may be capable of producing synthetic lethality. In summary, findings in this study indicated that targeting MCT1 and MCT4 expression could offer therapeutic benefit in prostate cancer. However, it was also highlighted that the roles of these transporters are specific to cancer type, and even cell line.

Mastitis is often bacterial in origin. Lipoteichoic acid (LTA) and lipopolysaccharide (LPS), endotoxins from gram-positive and gram-negative bacteria, respectively, are potent inducers of mammary gland inflammation. Inflammation can alter expression of transporters responsible for transport of substrates important in synthesis of milk constituents and cellular metabolic energy. Since, gram-positive and gram-negative bacterial infections cause a different clinical course of mastitis, I investigated whether LTA and LPS differentially alter proton-coupled (MCT1) and sodium-coupled monocarboxylate transporter (SMCT1, SMCT2) expression and functional outcomes of altered expression. Human mammary epithelial cells (MCF-12A) were incubated with 1 microgram/mL LPS or LTA for 6, 12 and 24 hours and mRNA expression of TNF-alpha, IL-1β, IL-6, MCT1, SMCT1, and SMCT2 were measured using Quantitative RT-PCR. LPS decreased SMCT1, but increased SMCT2 expression after 6 h, while LTA increased MCT1 expression at 6 h, followed by gradual decrease in expression until 24 h. To know whether such differential changes in transporter expression by LPS and LTA could cause changes in cellular energy production, I quantified creatine (Cr) and high-energy phosphate substrates (CrP, ATP, ADP, AMP) and oxygen consumption rates using HPLC and Hansatech oxygen electrode, respectively. At 12 h, LPS increased concentrations of Cr, CrP, ATP and ADP, whereas LTA caused changes in CrP and ADP concentrations relative to control. Both LPS and LTA decreased oxygen consumption rates after 12 h. Furthermore, to know whether changes in transporter expression lead to differences in substrate availability, I performed uptake studies for carnitine using radiolabelled tritium L-carnitine. LPS and LTA challenge did not affect the affinity, but caused a 2-3-fold increase in maximal activity (Vmax) of carnitine transport. Although increases in Vmax were not significant, the increase in Vmax after 12 h exposure by LPS and LTA corresponds to changes in mRNA expression of the OCTN2 transporter (previously reported in the laboratory). In conclusion, LPS and LTA differentially alter mRNA expression of transporters, which leads to changes in cellular energy levels and oxygen consumption rates and possibly to changes in the functional activity of transporters. Whether such differences contribute to the different clinical course of mastitis warrants further investigation.

ABSTRACT The purpose of this project was to investigate monocarboxylate (i.e. pyruvate and lactate) transport in the preimplantation stage of embryo development. Much progress has been made over the last 15 years towards understanding preimplantation and peri-implantation embryo physiology, including metabolic preferences during this period. It is known that as the cells (blastomeres) of an embryo compact via tight junctions and the embryo differentiates into a blastocyst, a metabolic “switch” occurs to allow the blastocyst to take up glucose at a rapid rate, obtaining energy derived from glycolysis. Glucose transporter molecules have been identified and characterized during this period of development and a paradigm for glucose transport has been described. However, during the early cleavage stages (days 1-3 post-fertilization), the embryo preferentially derives its metabolic energy from the monocarboxylate pyruvate. Evidence for the expression of pyruvate transporter molecules (a family of proton-coupled monocarboxylate co-transporters, MCT) has only been indicated via some kinetic studies on pH homeostasis and PCR analysis for MCT expression, and results have been conflicting (Gibb et al., 1997, Harding et al., 1999, Herubel et al., 2002). This project aimed to clarify discrepancies in reports for mRNA expression of MCT and to enhance the understanding of monocarboxylate transport processes during preimplantation development by pioneering investigations into protein expression for various MCT isoforms. Transport kinetics for monocarboxylate, DL-lactate, were examined by measuring the uptake of radioactive [3H]-DL-lactate from the medium by two-cell embryos and blastocysts. It was discovered that blastocysts demonstrate significantly higher affinity for DL-lactate compared to zygotes (Km 20 + 10 v 87 + 35 mM lactate; p=0.03), which suggested that alterations in the expression of various MCT isoforms might be expected as the embryo developed to a blastocyst. The rate of transport showed a trend towards a decrease from the zygote to blastocyst stages, although this could not be confirmed as significant within the limitations of this experiment. Mouse embryos, both in vivo and in vitro-derived, were collected and pooled at the zygote, two-cell, morula and blastocyst stages of development. RNA purification, reverse-transcription and PCR were used to analyze the expression of the four best-characterized MCT isoforms. MCT1, MCT2 and MCT4 were all found to be expressed in oocytes and mouse embryos from the zygote through to the preimplantation blastocyst. MCT3, an isoform uniquely expressed in the retina, was not detected at any stage in embryos. Since glucose has been implicated in regulatory processes involving glucose transporter expression in mouse embryos (Pantaleon et al., 2005, Pantaleon et al., 2001), mRNA expression was examined in the presence or absence of glucose in the culture media to determine whether the same phenomena applied to MCT. It was discovered that MCT1 and MCT4 isoforms were responsive to glucose-deprivation as evidenced by a reduction in mRNA expression in compacted morula cultured from the zygote stage without glucose. When glucose-deprived embryos were exposed to a brief high concentration of glucose during the 4-cell stage of development and continued in culture without glucose, the expression of mRNA for MCT1 and MCT4 persisted post-compaction, demonstrating that glucose exposure is necessary for the continued expression of these two isoforms in the mouse blastocyst. MCT2 mRNA did not respond to the absence of glucose in this way, and mRNA expression persisted in either the presence or absence of glucose. To follow these analyses of MCT gene transcription during early embryo development, confocal laser scanning immunofluorescence and western blotting were used to identify the expression of MCT proteins at various stages of development. Culture in the presence or absence of glucose was again employed to determine whether the changes seen in mRNA expression were conveyed at the protein level. All three proteins were identified throughout preimplantation development, though their locations were uniquely different. MCT1 was notably absent from plasma membranes at all stages, and was detected diffusely within the cytoplasm. In expanding blastocysts MCT1 tended to concentrate in the cortical cytoplasm of blastomeres and staining was more intense in the polar trophectoderm. In this cytoplasmic location its function is unclear. MCT1 does not appear to be a key transporter of monocarboxylates into and out of the embryo, but it may have a role in shuttling pyruvate and lactate within the cytoplasm to maintain metabolic and redox homeostasis. In embryos cultured without glucose, the immunostaining intensity for MCT1 gradually decreased as morulae degenerated and died. Protein loss occurred from the morula stage onwards, whilst mRNA was already undetectable at this stage. This would indicate that glucose signals which maintain mRNA expression most likely operate at the level of gene activation/transcription with latent effects on protein expression. MCT4 appeared to be located on the plasma membranes of oocytes and 2-cell embryos and nuclear staining was evident throughout preimplantation development, however plasma membrane expression was not apparent in morulae and blastocysts. This is consistent with earlier kinetic evidence of a low affinity lactate transporter (Km 87 + 35 mM lactate) operating at the early preimplantation stages. MCT4 has the lowest affinity for lactate of all the characterized MCT to date. Kinetic data also suggests that a change might occur in MCT protein expression as the embryo progresses to a blastocyst with a higher affinity lactate transporter taking precedence, and the loss of MCT4 from the plasma membrane at these later stages supports this view. Similarly to MCT1, MCT4 mRNA expression was also found to be dependent on glucose exposure during the early preimplantation period, and embryos cultured entirely without glucose demonstrated a loss of MCT4 mRNA expression at the morula stage. MCT4 typically exists as a lactate exporter in glycolytic tissues and it most likely exports lactate from the embryo for pH and redox homeostasis during this period of development. Protein localization studies found MCT2 to be located on the plasma membranes of oocytes, zygotes, 2-cell embryos, and polarized to the surface of the outer blastomeres of morulae and blastocyst trophectodermal cells. Throughout preimplantation development, MCT2 protein co-localized with peroxisomal catalase in peroxisome-sized granules throughout the cells. Known to be a high affinity pyruvate transporter, given its location in embryos it was proposed here that MCT2 most likely imports pyruvate to fuel early embryos, and later works as a bifunctional pyruvate/lactate importer/exporter on the transporting epithelium (trophectoderm) of blastocysts to maintain the pH, redox and metabolic status of the embryo. MCT2 was an enigma to the other MCT. Its expression in the absence of glucose behaved in an opposite way to that of MCT1 and MCT4, with mRNA expression persisting in the absence of glucose. In fact, MCT2 and catalase proteins demonstrated a quantitative increase in embryos lacking glucose, and the increase in staining was noticed as an increase in the density of peroxisome-like structures (or peroxisome proliferation) within the embryo. As such, it was decided to investigate the possibility that peroxisome proliferators (Peroxisome Proliferator Activated Receptors, PPARs) were involved in the control of MCT expression in the same way that they are known to control the expression of catalase and other peroxisomal proteins. At this stage, no MCT isoforms had been identified as being under the control of PPARs, although it was known that their expression was most likely controlled at the level of transcription, with no translational or post-translational controlling elements. PPARα, one of three isoforms (α, γ and β/δ) was selected as a likely candidate given that it controls peroxisomal proliferation and fatty acid β-oxidation processes at the level of transcription in other tissues, and it was known to be upregulated in conditions of starvation and oxidative stress. PPARα mRNA was shown to be expressed in early cleavage preimplantation mouse embryos, but its expression was reduced in morulae and blastocysts. Further, lack of glucose led to persistence of PPARα mRNA expression at the morula stage. PPARα protein was also demonstrated to stain more brightly in early preimplantation embryos compared to later stages. Further experimentation demonstrated that the phenomenon of increased catalase and MCT2 expression in embryos cultured without glucose could be mimicked in the presence of glucose by treating these embryos with the PPARα-selective agonist, WY14,643. The timing and quantitative nature of this upregulation were very similar, suggesting that PPARα was in some way involved in the glucose-deprived upregulation pathway for catalase and MCT2. To further investigate this pathway, oxidative stress was investigated in embryos cultured in the presence and absence of glucose to test whether the generation of reactive oxygen species contributed to the PPARα/MCT2 phenomenon. It was demonstrated that within 2 h of culture in the absence of glucose, hydrogen peroxide levels were significantly elevated in zygotes. Amelioration of increased peroxide generation in glucose-deprived embryos using a non-selective flavoenzyme inhibitor diphenyleneiodonium (DPI) eliminated any increases in PPARα and MCT2 protein expression that were earlier noted in the absence of glucose. To summarize, MCT1, MCT2 and MCT4 mRNA and protein expression were successfully demonstrated in mouse preimplantation embryos and all were confirmed to be in some way regulated by glucose in the culture medium. In the absence of glucose, mRNA expression for MCT1 and MCT4 were reduced to undetectable levels in morulae indicating that their expression was glucose-dependent. Paradoxically, glucose deprivation caused an increase in PPARα, catalase and MCT2 protein expression. PPARα-selective agonism in the presence of glucose induced similar timing and effects on catalase and MCT2 upregulation, implicating PPARα in this pathway. Hydrogen peroxide levels were significantly elevated within 2 h of culture in the absence of glucose. This peroxide elevation could be quenched to control levels by treating these embryos with DPI, and reducing hydrogen peroxide to control levels also eliminated the upregulation of PPARα and MCT2, implicating oxidative stress as an important component in the glucose-deprivation induced upregulation of MCT2. The experimental data presented in this thesis demonstrate that from its very conception, the embryo interacts with, adapts to, and is indeed affected by the external environment in which it develops. Even components like glucose, once considered simply as metabolic substrates, have profound effects on gene transcription and protein expression within the embryo which may impact on later its developmental competence, a reality we need to consider more deeply in light of the implementation of artificial reproductive technologies widely used today in zoology, agriculture and clinically, in humans.

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No. of bitstreams: 1 regatieri_ic_dr_jabo.pdf: 1026482 bytes, checksum: 93ce299c664eb44473c4cdf6c6496fb4 (MD5) Previous issue date: 2016-10-19 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O transportador de monocarboxilato isoforma 1 (MCT1), presente na membrana das hemácias, e sua proteína auxiliar CD147 têm como função transportar H+ e lactato do plasma para dentro das hemácias, mantendo assim, a homeostase ácido-base e retardando a acidose sistêmica e fadiga muscular. Dessa forma, o objetivo desse estudo foi comparar as quantidades das proteínas MCT1 e CD147 em hemácias de cavalos Árabes e Quartos de Milha com diferentes níveis de desempenho atlético. Além disso, objetivou-se buscar por polimorfismos para os genes MCT1, CD147, DMRT3 e PDK4, a fim de checar associações entre os polimorfismos e o desempenho nas raças. Cavalos Árabes e Quartos de Milha foram divididos em dois grupos de acordo com o desempenho em provas de enduro e provas de corridas, respectivamente. A quantidade de MCT1 e CD147 na membrana plasmática das hemácias foi determinada por western blotting com unidades arbitrárias de densidade óptica (OD) e anticorpos reagentes à espécie humana anti-MCT1 e anti-CD147. Os dados para as quantidades de proteínas foram analisados pelo PROC MIXED do SAS. O modelo incluiu a idade como covariável e os efeitos fixos de sexo, raça e grupo de desempenho dentro de raça. As correlações foram analisadas pelo teste de Pearson pelo procedimento PROC CORR. P-valores <0,01 foram considerados estatisticamente significantes. Os polimorfismos dos genes foram analisados por sequenciamento (MCT1 e CD147), PCR-RFLP (DMRT3) e ARMS-PCR (PDK4). Os pacotes estatísticos Genetics, Lattice e GenABEL foram utilizados para comparar as frequências dos grupos de desempenho no software R, com o teste exato de Fisher a 5% de significância. As proteínas MCT1 e CD147 foram encontradas nas hemácias de todos os animais. A quantidade de MCT1 foi significativamente (p<0,0001) maior em Quartos de Milha (2,99 ± 0,35 OD) do que em Árabes (1,04 ± 0,08 OD). Quartos de Milha (3,23 ± 0,38 OD) também apresentaram maior conteúdo de CD147 do que Árabes (0,88 ± 0,06 OD). Não houve diferença estatística nas quantidades de proteínas para os grupos de desempenho de ambas as raças. Correlação positiva foi encontrada entre as quantidades de MCT1 e CD147 (r=0,95; p<0,0001). O Alelo A dos polimorfismos Lys457Gln:1573A>C do gene MCT1 e Ile51Val:168A>G do gene CD147 estavam fixados em ambas as raças. Um novo polimorfismo (AY457175.1:c1498G>A) foi encontrado na sequência do gene MCT1. Para o DMRT3, todos os animais apresentaram o alelo C fixado para o polimorfismo. Árabes mostraram maior frequência para o alelo G do que Quartos de Milha (p<0,01) para o polimorfismo no gene PDK4. Entretanto, não houve diferença entre os grupos de desempenho para as duas raças. Dessa forma, conclui-se que Quartos de Milha têm maiores quantidades de MCT1 e CD147 do que Árabes. Não foi possível determinar a influência dos polimorfismos nos genes MCT1, CD147 e DMRT3 no desempenho atlético das duas raças visto que seus alelos estavam fixados. Além disso, houve diferença significativa nas frequências do polimorfismo no gene PDK4 entre Árabes e Quartos de Milha, mas não houve diferença entre os grupos de desempenho. / Monocarboxylate transporter isoform 1 (MCT1), present in the red blood cell membranes and its ancillary protein CD147 have as function transport H+ and lactate ions from the plasma into the red blood cells, thereby maintaining acid/base homeostasis and retarding systemic acidosis and muscular fatigue. Thereby, the aim of this study was to compare the amount of MCT1 and CD147 proteins in the red blood cells of Arabian and Quarter Horses with different levels of athletic ability. Furthermore, we investigated polymorphisms for MCT1, CD147, DMRT3, and PDK4 genes in Arabian and Quarter Horses in order to check associations between the polymorphisms and the performance in these breeds. Arabian horses were divided into two groups according to their performance in endurance competition and Quarter Horses were separated by its performance in races, determined by Speed Index. The amount of MCT1 and CD147 proteins in the plasma membrane of red blood cells was determined by western blotting analysis with arbitrary optical density units (OD), using a human specific anti-MCT1 and anti- CD147 antibody. Data for the amounts of proteins were analyzed using the PROC MIXED procedure of SAS software. The model for the analysis included the effects of sex, breed and performance group within breed as fixed effect and age as covariate. The correlations were analyzed by Pearson correlation test using the PROC CORR procedure of SAS software. P values <0.01 were considered statistically significant. Polymorphisms of the genes were analyzed by sequencing (MCT1 and CD147), PCR-RFLP (DMRT3) and ARMS-PCR (PDK4) techniques. The statistical packages Genetics, Lattice and GenABEL were used to compare the frequencies of the groups using the software R, with the Fisher's exact test being performed with significance level of 5%. MCT1 and CD147 proteins were found in the red blood cell membranes of all studied animals. The amount of MCT1 was significantly (p<0.0001) higher in Quarter Horses (2.99 ± 0.35 OD) than in Arabians (1.04 ± 0.08 OD). Quarter Horses (3.23 ± 0.38 OD) also showed bigger contents of CD147 than Arabians (0.88 ± 0.06 OD). There was not statistical difference in the amounts of MCT1 and CD147 between the performance groups of both breeds. Positive correlation was found between the amounts of MCT1 and CD147 (r=0.95; p<0.0001). The A allele from the polymorphisms Lys457Gln:1573A>C of MCT1 and Ile51Val:168A>G of CD147 gene, were fixed in both breeds. A new polymorphism (AY457175.1:c1498G>A) was found in the MCT1 gene sequence. For DMRT3 mutation, all the animals shown to have the C allele fixed for the polymorphism. Arabians showed significant greater frequency of the G allele than Quarter Horses (p<0.01) for the PDK4 polymorphism. However, there was not difference between the groups of performance for both breeds. In summary, it follows that the Quarter Horses have greater amount of MCT1 and CD147 proteins than Arabian. It was not possible to determine the influence of polymorphisms in MCT1, CD147 and DMRT3 genes in the athletic performance of these breeds since they had alleles fixed. There was a significant difference in the frequencies of the PDK4 polymorphism between Arabians and Quarter Horses, but there was not difference between the performance groups. / FAPESP: 2012/24193-0 / FAPESP: 2012/20697-9

Proton-coupled and sodium-dependent monocarboxylate transporters are encoded by the SLC16A and SLC5A gene family of solute carriers, and are responsible for the transport of essential nutrients such as L-lactate, pyruvate, and ketone bodies. Basigin, or CD147, acts as an ancillary protein for MCT1 and MCT4, and is involved in membrane surface expression of transporters. MCT's are also involved in the shuttling of monocarboxylic xenobiotics across cell membranes, including the drugs valproate and gamma hydroxybutyrate. MCT’s are also important for normal mammalian development, particularly during embryogenesis and early neonatal life. Previous studies have shown that ketogenic diets increase MCT expression in the brain, and the obesity biomarker leptin increases MCT1 and CD147 expression and colocalization in colonocytes. Clinical studies in post-mortem tissue demonstrated that hepatic MCT1 expression changes nonlinearly from birth to adulthood. We hypothesize that age and high fat dietary intake regulate monocarboxylate transporter and ancillary protein expression in the liver, and other organs of drug disposition during childhood obesity. The purpose of this study was to elucidate just how diet and ontogeny regulate MCT1, MCT4, CD147, and SMCT1 mRNA and protein expression in the liver, kidney, and ileum. Timed-pregnant rats were fed either normal or high fat diet, and tissue was harvested from the progeny of both cohorts at predetermined postnatal timepoints. Serum leptin levels were measured, and MCT1, MCT4, CD147, and SMCT1 transcripts were evaluated using real time quantitative PCR. Whole cell and total membrane proteins were extracted and transporter expression was analyzed via western blot. In summary, we have demonstrated age, diet, and sex dependent regulation of MCT1, MCT4, CD147, and SMCT1 expression in the liver, kidneys, and intestine, and that these effects are tissue specific. Pediatric drug-dosing is both a pressing and understudied clinical field, with the possibility of altered pharmacokinetics in obese children. Changes in hepatic, renal, and intestinal monocarboxylate transporter expressions during mammalian development may affect functional activity of these transporters and lead to altered metabolism and drug disposition. Further studies of this animal model can shine new light on the dynamic and highly-variable nature of drug pharmacokinetics in pediatric obesity.

Proton- and sodium-dependent monocarboxylate transporters (MCTs (SLC16A) and SMCTs (SLC5A)) transport monocarboxylates such as ketone bodies, lactate and pyruvate, as well as drugs such as gamma-hydroxybutyric acid. CD147 acts as an ancillary protein for MCT1 and MCT4, and is involved in membrane trafficking. Previously, it has been shown that MCT expression changes under different sex hormone conditions in skeletal muscle and Sertoli cells. However, it is unknown if MCTs, SMCTs or CD147 demonstrate sex differences in tissues where they play an important role in drug disposition. Monocarboxylate transporter substrates GHB and valproic acid have demonstrated sex differences in pharmacokinetic profiles. We hypothesize that sex hormones regulate monocarboxylate transporters and CD147 expression in drug disposition tissues, including the liver, intestine and kidney. The purpose of the current study is to evaluate sex and sex hormone dependent regulation of MCT1, MCT4, SMCT1 and CD147 mRNA and protein expression in drug disposition tissues. Liver, kidney and intestinal segments (duodenum, jejunum and ileum) were harvested from estrus cycle staged female rats, ovariectomized (OVX) females, males and castrated (CST) male rats. Hormone replacement experiments were performed to investigate testosterone and 17β-estradiol dependent regulation of renal MCTs, SMCT1 and CD147 in OVX females and CST males. mRNA of MCT1, MCT4, SMCT1 and CD147 was evaluated by real time quantitative PCR. Whole cell protein and membrane protein was extracted, target protein expression was evaluated by western blot. We have demonstrated sex and sex hormone dependent regulation of MCT1, MCT4, SMCT1 and CD147 in the liver, intestine regions and kidney occurs in a tissue specific manner. mRNA, protein expression and membrane localization of monocarboxylate transporters and CD147 were regulated differently by sex hormones. Sex differences in MCTs and SMCTs expression are important determinants of drug disposition in the body and sex differences in their regulation may contribute to differences in drug pharmacokinetics.

Le catabolisme exacerbé du glucose et de la glutamine est actuellement reconnu comme une caractéristique des cellules cancéreuses, qui leur procure un avantage prolifératif via la production et l’accumulation de plusieurs métabolites au niveau du microenvironnement. Parmi ces métabolites, l’acide lactique représente une molécule de signalisation clé, favorisant la migration et les métastases. Mon projet de thèse s’inscrit dans le contexte d’une étude du métabolisme glycolytique associé aux cellules tumorales à division rapide. Durant ce projet, nous nous sommes intéressés à la caractérisation génétique et fonctionnelle des transporteurs MCT (MonoCarboxylate Transporters) 1 et 4, qui sont des symporteurs H+/lactate dont l’expression membranaire et la fonctionnalité requièrent la liaison avec une protéine chaperonne : CD147/BASIGINE (BSG). Afin de mieux explorer la physiologie des complexes MCT/BSG, et valider le ciblage de l’export d’acide lactique comme une nouvelle approche anti-cancer, nous avons développé une stratégie visant à invalider le gène BSG et/ou MCT4, en utilisant la technologie des Zinc Finger Nucleases (ZFN), dans des lignées cellulaires cancéreuses humaines de côlon, poumon et glioblastome. D’abord, nous avons démontré, que l’effet pro-tumoral majeur de BSG est lié à son action directe sur la stabilisation des MCTs au niveau des tumeurs glycolytiques et non pas à la production des metalloprotéases. Ensuite, nous avons démontré pour la première fois que l’inhibition concomitante de MCT1 et MCT4 est nécessaire pour induire une baisse significative de la tumorigénécité in vivo. / Enhanced glucose and glutamine catabolism has become a recognized feature of cancer cells, leading to accumulation of metabolites in the tumour microenvironment, which offers growth advantages to tumours. Among these metabolites is emerging as a key signalling molecule that plays a pivotal role in cancer cell migration and metastasis. In this thesis, we focused on the genetic and functional characterization of monocarboxylate transporters (MCT) 1 and 4, which are H+/lactate symporters that require an interaction with an ancillary protein, CD147/BASIGIN (BSG), for their plasma membrane expression and function. To further explore the physiology of MCT/BSG complexes and validate the blockade of lactic acid export as an anti-cancer strategy, we designed experiments using Zinc Finger Nuclease mediated BSG and/or MCT4 gene knockouts in human colon adenocarcinoma, lung carcinoma and glioblastoma cell lines. First of all, we demonstrated that the major protumoural action of BSG is to control the energetics of glycolytic tumours via MCT1/4 activity and not to produce matrix metalloproteases. Second, we showed for the first time that combined inhibition of both MCT1 and MCT4 transporters is required to achieve a significant reduction in the tumour growth in vivo. Moreover, our findings reported that disruption of the BSG gene dramatically reduced the plasma membrane expression and lactate transport activity of both MCT1 and MCT4, leading to increased accumulation of intracellular pools of lactic and pyruvic acids, decreased intracellular pH and reduced rate of glycolysis.