RESUMO
INTRODUCTION: Patients with cervical cancer (CC) may experience local recurrence very often after treatment; when only clinical parameters are used, most cases are diagnosed in late stages, which decreases the chance of recovery. Molecular markers can improve the prediction of clinical outcome. Glycolysis is altered in 70% of CCs, so molecular markers of this pathway associated with the aggressiveness of CC can be identified. METHODS: The expression of 14 glycolytic genes was analyzed in 97 CC and 29 healthy cervical tissue (HCT) with microarray; only LDHA and PFKP were validated at the mRNA and protein levels in 36 of those CC samples and in 109 new CC samples, and 31 HCT samples by qRT-PCR, Western blotting, or immunohistochemistry. A replica analysis was performed on 295 CC from The Cancer Genome Atlas (TCGA) database. RESULTS: The protein expression of LDHA and PFKP was associated with poor overall survival [OS: LDHA HR = 4.0 (95% CI = 1.4-11.1); p = 8.0 × 10-3 ; PFKP HR = 3.3 (95% CI = 1.1-10.5); p = 4.0 × 10-2 ] and disease-free survival [DFS: LDHA HR = 4.5 (95% CI = 1.9-10.8); p = 1.0 × 10-3 ; PFKP HR = 3.2 (95% CI = 1.2-8.2); p = 1.8 × 10-2 ] independent of FIGO clinical stage, and the results for mRNA expression were similar. The risk of death was greater in patients with overexpression of both biomarkers than in patients with advanced FIGO stage [HR = 8.1 (95% CI = 2.6-26.1; p = 4.3 × 10-4 ) versus HR = 7 (95% CI 1.6-31.1, p = 1.0 × 10-2 )] and increased exponentially as the expression of LDHA and PFKP increased. CONCLUSIONS: LDHA and PFKP overexpression at the mRNA and protein levels was associated with poor OS and DFS and increased risk of death in CC patients regardless of FIGO stage. The measurement of these two markers could be very useful for evaluating clinical evolution and the risk of death from CC and could facilitate better treatment decision making.
Assuntos
Fosfofrutoquinases , Neoplasias do Colo do Útero , Feminino , Humanos , Biomarcadores/metabolismo , Glicólise/genética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5/metabolismo , Fosfofrutoquinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias do Colo do Útero/genéticaRESUMO
PURPOSE: This project aimed to survey the clinical characteristics and survivals of hyperprogressive disease (HPD) mediated by immune checkpoint inhibitors (ICIs) in an attempt to explore the potential predictors. METHODS: After searching PubMed, MEDLINE, Google Scholar and Cochrane Library databases, 12 studies incorporating 1766 individuals were enrolled. The data were analyzed with Review manager 5.3 software. RESULTS: The results revealed HPD correlated with previous metastatic sites > 2 (OR = 1.86, 95% CI 1.33-2.59, P = 0.0003), liver metastasis (OR = 3.35, 95% CI 2.09-5.35, P < 0.00001), Royal Marsden Hospital (RMH) score ≥ 2 (OR = 2.80, 95% CI 1.85-4.23, P < 0.00001), higher ECOG PS (OR = 1.60, 95% CI 1.13-2.27, P = 0.008) and LDH > upper limits of normal (ULN) (OR = 2.32, 95% CI 1.51-3.58, P = 0.0001). Instead, HPD was unrelated to gender, age, smoking status, PD-L1 expression, therapy, neutrophil-to-lymphocyte ratio, the histology, the status of EGFR, ALK and KRAS in non-small cell lung cancer and HER-2 expression in advanced gastric cancer. Moreover, HPD was evidently correlated with a shorter OS (HR = 2.92, 95% CI 1.79-4.76, P < 0.0001) and PFS (HR = 3.62, 95% CI 2.79-4.68, P < 0.00001). The same phenomena existed in stratified studies based on study regions and tumor types. CONCLUSIONS: This study demonstrated that HPD was related to the number of prior metastatic sites > 2, liver metastasis, RMH score ≥ 2, higher ECOG PS score and LDH > ULN. Moreover, HPD was correlated with a poor OS and PFS in patients following ICI therapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Progressão da Doença , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/patologia , Neoplasias Gástricas/patologia , Fatores Etários , Quinase do Linfoma Anaplásico/metabolismo , Antígeno B7-H1/metabolismo , Antígeno CTLA-4/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Intervalos de Confiança , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Contagem de Leucócitos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Masculino , Mutação , Razão de Chances , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores Sexuais , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Falha de Tratamento , Carga TumoralRESUMO
The enzymes D- and L-lactate dehydrogenase are involved in the reduction of pyruvate to D(+)- and L(-)-lactate, respectively. The fig-origin strain Fructobacillus tropaeoli CRL 2034 produces D- and L-lactic acids in a 9:1 ratio. In this work, two D-ldh (ldh1 and ldh2) and one L-ldh (ldh3) genes were found in the CRL 2034 genome. ldh1 and ldh2 are homologous (79% identity) and organized as contiguous operons, each gene containing 996 base pair (bp) and encoding for a 331-amino acid (aa) protein (74% identity). In contrast, ldh3 is a 927-bp gene coding for a 308-aa protein. The identity between ldh1/ldh2 and ldh3 was lower than 48%. To elucidate the role of these genes in the synthesis of lactic acid by the Fructobacillus strain, plasmid insertion mutants in each gene were generated and characterized. The growth kinetic parameters were affected only in CRL2034 ldh1::pRV300 cells, this mutant showing the lowest total lactic acid production (4.50 ± 0.15 versus 6.36 ± 0.67 g/L of wild-type strain), with a D/L ratio of 7.1:2.9. These results showed that the ldh1 gene is primarily responsible for lactic acid production by the studied strain. A comparative analysis among strains of the five Fructobacillus species revealed that the identity of D-LDH proteins was higher than 70%, while the identity of L-LDH was over 60%. Finally, phylogenetic analysis of D- and L-LDHs revealed that only D-LDH phylogeny was consistent to the phylogenetic evolution among Fructobacillus and evolutionarily related genera. Key Points â¢F. tropaeoli CRL 2034 harbors three ldh genes in its genome. â¢ldh1 and ldh2 encode D-lactate dehydrogenase; ldh3 encodes L-lactate dehydrogenase. â¢Gene ldh1 plays the major role in lactic acid production by strain CRL 2034. â¢Fructobacillus D-LDH phylogeny was consistent to phylogenetic evolution.
Assuntos
L-Lactato Desidrogenase , Ácido Láctico , Isoenzimas , L-Lactato Desidrogenase/genética , Leuconostocaceae , FilogeniaRESUMO
INTRODUCTION: Real-time polymerase chain reaction (RT-qPCR) is an important tool for analyzing gene expression. However, before analyzing the expression of target genes, it is crucial to normalize the reference genes, in order to find the most stable gene to be used as an endogenous control. A gene that remains stable in all samples under different treatments is considered a suitable normalizer. In this sense, we aimed to identify stable reference genes for normalization of target genes in the heart and liver tissues from two genetically divergent groups of chickens (Cobb 500® commercial line and Peloco backyard chickens) under comfort and acute heat stress environmental conditions. Eight reference genes (ACTB, HPRT1, RPL5, EEF1, MRPS27, MRPS30, TFRC and LDHA) were analyzed for expression stability. The samples were obtained from 24 chickens, 12 from the backyard Peloco and 12 from the Cobb 500® line, exposed to two environmental conditions (comfort and heat stress). Comfort temperature was 23°C and heat stress temperature was 39.5°C for one hour. Subsequently, the animals were euthanized, and heart and liver tissue fragments were collected for RNA extraction and amplification. To determine the stability rate of gene expression, three different statistical algorithms were applied: BestKeeper, geNorm and NormFinder, and to obtain an aggregated stability list, the RankAgregg package of R software was used. RESULTS: The most stable genes using BestKeeper tool, including the two factors (genetic group and environmental condition), were LDHA, RPL5 and MRPS27 for heart tissue, and TFRC, RPL5 and EEF1 for liver tissue. Applying geNorm algorithm, the best reference genes were RPL5, EEF1 and MRPS30 for heart tissue and LDHA, EEF1 and RPL5 for liver. Using the NormFinder algorithm, the best normalizer genes were EEF1, RPL5 and LDHA in heart, and EEF1, RPL5 and ACTB in liver tissue. In the overall ranking obtained by RankAggreg package, considering the three algorithms, the RPL5, EEF1 and LDHA genes were the most stable for heart tissue, whereas RPL5, EEF1 and ACTB were the most stable for liver tissue. CONCLUSION: According to the RankAggreg tool classification based on the three different algorithms (BestKeeper, geNorm and NormFinder), the most stable genes were RPL5, EEF1 and LDHA for heart tissue and RPL5, EEF1 and ACTB for liver tissue of chickens subjected to comfort and acute heat stress environmental conditions. However, the best reference genes may vary depending on the experimental conditions of each study, such as different breeds, environmental stressors, and tissues analyzed. Therefore, the need to perform priori studies to assay the best reference genes at the outset of each study is emphasized.
Assuntos
Galinhas/genética , Resposta ao Choque Térmico/genética , Fígado/metabolismo , Miocárdio/metabolismo , Algoritmos , Animais , Feminino , Genótipo , L-Lactato Desidrogenase/genética , Masculino , Fator 1 de Elongação de Peptídeos/genética , RNA/metabolismo , Receptores da Transferrina/genética , TemperaturaRESUMO
Lactate dehydrogenase (LDH) is a key enzyme to produce energy during hypoxia by anaerobic glycolysis. In the white shrimp Litopenaeus vannamei, two protein subunits (LDH-1 and LDH-2) were previously identified, deduced from two different transcripts that come from the same LDH gene by processing via mutually exclusive alternative splicing. LDH-1 contains exon five and LDH-2 contains exon six and the two proteins differ only in 15â¯amino acid residues. Both subunits were independently cloned and overexpressed in E. coli as a fusion protein containing a chitin binding domain. Previously, recombinant LDH-2 was successfully purified and characterized, but LDH-1 was insoluble and aggregated forming inclusion bodies. We report the production of soluble LDH-1 by testing different pHs in the buffers used to lyse the bacterial cells before the purification step and the characterization of the purified protein to show that the cDNA indeed codes for a functional and active protein. The recombinant native protein is a homotetramer of approximately 140â¯kDa composed by 36â¯kDa subunits and has higher affinity for pyruvate than for lactate. LDH-1 has an optimum pH of 7.5 and is stable between pH 8.0 and 9.0; pH data analysis showed two pKa values of 6.1⯱â¯0.15 and 8.8⯱â¯0.15 suggesting a histidine and asparagine, respectively, involved in the active site. The enzyme optimal temperature was 44⯰C and it was stable between 20 and 60⯰C. LDH-1 was slightly activated by NaCl, KCl and MgCl2 and fully inhibited by ZnCl2.
Assuntos
L-Lactato Desidrogenase/metabolismo , Penaeidae/enzimologia , Animais , Clonagem Molecular , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/isolamento & purificação , Ácido Láctico/metabolismo , Penaeidae/química , Penaeidae/genética , Penaeidae/metabolismo , Multimerização Proteica , Ácido Pirúvico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Cervical cancer continues to be a public health problem in developing countries. Previous studies have shown that cervical cancer cells display markers of aerobic glycolysis, indicating that these tumors are likely to secrete lactate. Mostly, lactate is recognized as a molecule capable of suppressing immune responses, through inhibition of T cells, MÏs, and dendritic cells. We and others have previously shown that MÏs are frequent cells infiltrating cervical cancers with the ability to inhibit antitumor immune responses and promote tumor growth through angiogenesis. Here, we have tested the hypothesis that lactate, secreted by cervical cancer cells, can modulate MÏ phenotype. First, we showed higher lactate plasma concentrations in patients with increasing cervical lesion grades, with maximum concentration in the plasma of cancer patients, which supported our hypothesis. We then inhibited lactate production in tumor cell spheroids established from cervical cancer derived cell lines, using the lactate dehydrogenase inhibitor, oxamate, prior to co-culture with monocytes. Lactate mediated part of the crosstalk between tumor cells and MÏs, promoting secretion of IL-1ß, IL-10, IL-6, and up-regulation of hypoxia induced factor-1α expression, and down-regulation of p65-NFκB phosphorylation in MÏs. We also showed that MÏs from co-cultures treated with oxamate were better inducers of T cell activation. Of note, experiments performed with inhibition of the monocarboxylate transporters rendered similar results. Our data confirms the hypothesis that lactate, secreted by cervical tumor cells, influences the phenotype of tumor MÏs, promoting a suppressive phenotype.
Assuntos
Adenocarcinoma/metabolismo , Carcinoma de Células Escamosas/metabolismo , Ácido Láctico/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Neoplasias do Colo do Útero/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica , Glicólise/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/patologia , Gradação de Tumores , Fosforilação Oxidativa/efeitos dos fármacos , Fenótipo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
The interaction between ocean warming, hypoxia and hypercapnia, suggested by climate projections, may push an organism earlier to the limits of its thermal tolerance window. In a previous study on juveniles of green abalone (Haliotis fulgens), combined exposure to hypoxia and hypercapnia during heat stress induced a lowered critical thermal maximum (CTmax), indicated by constrained oxygen consumption, muscular spams and loss of attachment. Thus, the present study investigated the cell physiology in foot muscle of H. fulgens juveniles exposed to acute warming (18⯰C to 32⯰C at +3⯰Câ¯day-1) under hypoxia (50% air saturation) and hypercapnia (~1000 µatm PCO2), alone and in combination, to decipher the mechanisms leading to functional loss in this tissue. Under exposure to either hypoxia or hypercapnia, citrate synthase (CS) activity decreased with initial warming, in line with thermal compensation, but returned to control levels at 32⯰C. The anaerobic enzymes lactate and tauropine dehydrogenase increased only under hypoxia at 32⯰C. Under the combined treatment, CS overcame thermal compensation and remained stable overall, indicating active mitochondrial regulation under these conditions. Limited accumulation of anaerobic metabolites indicates unchanged mode of energy production. In all treatments, upregulation of Hsp70 mRNA was observed already at 30⯰C. However, lack of evidence for Hsp70 protein accumulation provides only limited support to thermal denaturation of proteins. We conclude that under combined hypoxia and hypercapnia, metabolic depression allowed the H. fulgens musculature to retain an aerobic mode of metabolism in response to warming but may have contributed to functional loss.
Assuntos
Metabolismo Energético , Gastrópodes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Aquecimento Global , Resposta ao Choque Térmico , Modelos Biológicos , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Animais , Aquicultura , Dióxido de Carbono/intoxicação , Hipóxia Celular , Citrato (si)-Sintase/química , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Gastrópodes/classificação , Gastrópodes/crescimento & desenvolvimento , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Temperatura Alta/efeitos adversos , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , México , Músculos/fisiologia , Filogenia , Estabilidade Proteica , Distribuição AleatóriaRESUMO
Background: Lactate dehydrogenase (LDH) is an enzyme of glycolytic pathway, ubiquitously found in living organisms. Increased glycolysis and LDH activity are associated with many pathologic conditions including inflammation and cancer, thereby making the enzyme a suitable drug target. Studies on conserved structural and functional domains of LDH from various species reveal novel inhibitory molecules. Our study describes Escherichia coli production and characterization of a moderately thermostable LDH (LDH-GT) from Geobacillus thermodenitrificans DSM-465. An in silico 3D model of recombinant enzyme and molecular docking with a set of potential inhibitors are also described. Results: The recombinant enzyme was overexpressed in E. coli and purified to electrophoretic homogeneity. The molecular weight of the enzyme determined by MALDI-TOF was 34,798.96 Da. It exhibited maximum activity at 65°C and pH 7.5 with a KM value for pyruvate as 45 µM. LDH-GT and human LDH-A have only 35.6% identity in the amino acid sequence. On the contrary, comparison by in silico structural alignment reveals that LDH-GT monomer has approximately 80% identity to that of truncated LDH-A. The amino acids "GEHGD" as well as His179 and His193 in the active site are conserved. Docking studies have shown the binding free energy changes of potential inhibitors with LDH-A and LDH-GT ranging from −407.11 to −127.31 kJ mol−1 . Conclusions: By highlighting the conserved structural and functional domains of LDH from two entirely different species, this study has graded potential inhibitory molecules on the basis of their binding affinities so that they can be applied for in vivo anticancer studies
Assuntos
Geobacillus/enzimologia , L-Lactato Desidrogenase/metabolismo , Simulação por Computador , Estabilidade Enzimática , Reação em Cadeia da Polimerase , Clonagem Molecular , Escherichia coli/metabolismo , Simulação de Acoplamento Molecular , Glicólise , L-Lactato Desidrogenase/genéticaRESUMO
Lactate dehydrogenase (LDH) is key for anaerobic glycolysis. LDH is induced by the hypoxia inducible factor -1 (HIF-1). HIF-1 induces genes involved in glucose metabolism and regulates cellular oxygen homeostasis. HIF-1 is formed by a regulatory α-subunit (HIF-1α) and a constitutive ß-subunit (HIF-1ß). The white spot syndrome virus (WSSV) induces anaerobic glycolysis in shrimp hemocytes, associated with lactate accumulation. Although infection and lactate production are associated, the LDH role in WSSV-infected shrimp has not been examined. In this work, the effects of HIF-1 silencing on the expression of two LDH subunits (LDHvan-1 and LDHvan-2) in shrimp infected with the WSSV were studied. HIF-1α transcripts increased in gills, hepatopancreas, and muscle after WSSV infection, while HIF-1ß remained constitutively expressed. The expression for both LDH subunits increased in each tissue evaluated during the WSSV infection, translating into increased enzyme activity. Glucose concentration increased in each tissue evaluated, while lactate increased in gills and hepatopancreas, but not in muscle. Silencing of HIF-1α blocked the increase of LDH expression and enzyme activity, along with glucose (all tissues) and lactate (gills and hepatopancreas) concentrations produced by WSSV infection. These results demonstrate that HIF-1 up regulates the expression of LDH subunits during WSSV infection, and that this induction contributes to substrate metabolism in energetically active tissues of infected shrimp.
Assuntos
Regulação da Expressão Gênica/imunologia , Fator 1 Induzível por Hipóxia/genética , Imunidade Inata/genética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/imunologia , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Fator 1 Induzível por Hipóxia/metabolismo , L-Lactato Desidrogenase/química , Filogenia , Alinhamento de Sequência , Vírus da Síndrome da Mancha Branca 1/fisiologiaRESUMO
The estuarine crab Neohelice granulata was maintained under control condition or exposed to sublethal concentrations of dissolved Ni (measured: 128 and 1010µg/L) for 96h at different salinities (2 and 30ppt). After metal exposure, whole-body oxygen consumption was measured and tissue (hemolymph, gills, hepatopancreas and muscle) samples were collected. Control crabs acclimated to 2ppt salinity showed lower hemolymph concentrations of Na+ (33%), Mg2+ (19%) and K+ (30%), as well as increased LPO levels in anterior gills (379%), posterior gills (457%) and hepatopancreas (35%) with respect to those acclimated to 30ppt salinity. In crabs acclimated to 2ppt salinity, Ni exposure increased whole-body oxygen consumption (75%), hemolymph K+ concentration (52%), hemolymph (135%) and hepatopancreas (62%) LDH activity. Also, it reduced hemolymph Cl- concentration (16%) and muscle LDH activity (33%). In crabs acclimated to 30ppt salinity, Ni exposure increased LDH activity in hemolymph (195%), hepatopancreas (126%) and muscle (53%), as well as hemolymph osmolality (10%), Cl- (26%) and Ca2+ (20%) concentration. It also reduced hepatopancreas lipid peroxidation (20%) and hemolymph Mg2+ (29%) and K+ (31%) concentration. These findings indicate that N. granulata is hyper-osmoregulating in 2ppt salinity and hypo-regulating in 30ppt salinity, showing adjustments of hemolymph ionic composition and metabolic rates, with consequent higher oxidative damage to lipids in low salinity (2ppt). Ni effects are associated with metabolic (aerobic and anaerobic) disturbances in crabs acclimated to 2ppt salinity, while osmotic and ionoregulatory disturbances were more evident in crabs acclimated to 30ppt salinity.