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1.
Clin Biochem ; 130: 110790, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38969054

RESUMO

This study aims to investigate the alteration of salivary biomarker profiling in the development of oral submucous fibrosis (OSMF) and to explore the influence of saliva in the diagnosis of OSMF. A systematic search of published articles using the PRISMA guidelines was conducted to identify relevant studies on OSMF and saliva. All eligible studies, including case-control, cross-sectional studies, cohort, and pilot studies, contained the evaluation of salivary biomarker profiling in patients with OSMF. Salivary biomarker data from 28 selected articles were categorized into nine groups, and their mean values were determined. A three-step meta-analysis was performed by grouping salivary biomarker profiling into more heterogeneous categories based on OSMF classification, considering functional, histological, and clinical grading. The salivary biomarker profiling analysis revealed significant alterations in all markers, indicating their efficacy in OSMF diagnosis. Subgroup analyses highlighted significant associations in oxidative stress and protein with increased mean values, particularly emphasizing lipid peroxidase (LPO), malondialdehyde (MDA), and lactate dehydrogenase (LDH). Conversely, decreased mean values were observed in glutathione, glutathione peroxidase (GPx), superoxide dismutase (SOD), and vitamins. Notably, OSMF grading analysis demonstrated a significant difference in weighted effect sizes for histological grading, particularly in stage IV. The study underscores the alteration of specific salivary biomarkers, particularly those associated with LPO, MDA, LDH, glutathione, GPx, SOD, and vitamins, in diagnosing and grading OSMF.


Assuntos
Biomarcadores , Glutationa Peroxidase , Malondialdeído , Fibrose Oral Submucosa , Saliva , Superóxido Dismutase , Humanos , Biomarcadores/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , L-Lactato Desidrogenase/metabolismo , Malondialdeído/metabolismo , Fibrose Oral Submucosa/metabolismo , Fibrose Oral Submucosa/patologia , Fibrose Oral Submucosa/diagnóstico , Estresse Oxidativo , Saliva/metabolismo , Superóxido Dismutase/metabolismo , Vitaminas
2.
PeerJ ; 12: e17672, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38952967

RESUMO

Background: Mitochondrial creatine kinase (MtCK) plays a pivotal role in cellular energy metabolism, exhibiting enhanced expression in various tumors, including colorectal cancer (CRC). Creatine kinase mitochondrial 2 (CKMT2) is a subtype of MtCK; however, its clinical significance, biological functions, and underlying molecular mechanisms in CRC remain elusive. Methods: We employed immunohistochemical staining to discern the expression of CKMT2 in CRC and adjacent nontumor tissues of patients. The correlation between CKMT2 levels and clinical pathological factors was assessed. Additionally, we evaluated the association between CKMT2 and the prognosis of CRC patients using Kaplan-Meier survival curves and Cox regression analysis. Meanwhile, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression levels of CKMT2 in different CRC cell lines. Finally, we explored the biological functions and potential molecular mechanisms of CKMT2 in CRC cells through various techniques, including qRT-PCR, cell culture, cell transfection, western blot, Transwell chamber assays, flow cytometry, and co-immunoprecipitation. Results: We found that CKMT2 was significantly overexpressed in CRC tissues compared with adjacent nontumor tissues. The expression of CKMT2 is correlated with pathological types, tumor size, distant metastasis, and survival in CRC patients. Importantly, CKMT2 emerged as an independent prognostic factor through Cox regression analysis. Experimental downregulation of CKMT2 expression in CRC cell lines inhibited the migration and promoted apoptosis of these cells. Furthermore, we identified a novel role for CKMT2 in promoting aerobic glycolysis in CRC cells through interaction with lactate dehydrogenase B (LDHB). Conclusion: In this study, we found the elevated expression of CKMT2 in CRC, and it was a robust prognostic indicator in CRC patients. CKMT2 regulates glucose metabolism via amplifying the Warburg effect through interaction with LDHB, which promotes the growth and progression of CRC. These insights unveil a novel regulatory mechanism by which CKMT2 influences CRC and provide promising targets for future CRC therapeutic interventions.


Assuntos
Neoplasias Colorretais , Efeito Warburg em Oncologia , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Masculino , Feminino , Linhagem Celular Tumoral , Prognóstico , Creatina Quinase Mitocondrial/metabolismo , Creatina Quinase Mitocondrial/genética , Progressão da Doença , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , Pessoa de Meia-Idade , Proliferação de Células , Apoptose , Regulação Neoplásica da Expressão Gênica
3.
Nat Commun ; 15(1): 5980, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39013948

RESUMO

Hyperpolarised magnetic resonance imaging (HP-13C-MRI) has shown promise as a clinical tool for detecting and characterising prostate cancer. Here we use a range of spatially resolved histological techniques to identify the biological mechanisms underpinning differential [1-13C]lactate labelling between benign and malignant prostate, as well as in tumours containing cribriform and non-cribriform Gleason pattern 4 disease. Here we show that elevated hyperpolarised [1-13C]lactate signal in prostate cancer compared to the benign prostate is primarily driven by increased tumour epithelial cell density and vascularity, rather than differences in epithelial lactate concentration between tumour and normal. We also demonstrate that some tumours of the cribriform subtype may lack [1-13C]lactate labelling, which is explained by lower epithelial lactate dehydrogenase expression, higher mitochondrial pyruvate carrier density, and increased lipid abundance compared to lactate-rich non-cribriform lesions. These findings highlight the potential of combining spatial metabolic imaging tools across scales to identify clinically significant metabolic phenotypes in prostate cancer.


Assuntos
Ácido Láctico , Imageamento por Ressonância Magnética , Fenótipo , Neoplasias da Próstata , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Humanos , Ácido Láctico/metabolismo , Imageamento por Ressonância Magnética/métodos , Próstata/diagnóstico por imagem , Próstata/metabolismo , Próstata/patologia , Isótopos de Carbono , Gradação de Tumores , Mitocôndrias/metabolismo , L-Lactato Desidrogenase/metabolismo
4.
PLoS One ; 19(6): e0304526, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38857221

RESUMO

In vitro models, such as primary cells and continuous cell lines routinely used for evaluating drug candidates, have limitations in their translational relevance to human diseases. Organotypic cultures are increasingly being used to assess therapeutics for various cancers and infectious diseases. Monitoring drug cytotoxicity in cell cultures is crucial in drug development, and several commercially available kits for cytotoxicity assessment offer distinct advantages and limitations. Given the complexity of organoid cultures, including donor-driven variability, we investigated drug-treated, tissue stem cell-derived human intestinal organoid responses with commonly used cell cytotoxicity assay kits. Using seven different compounds, we compared the cytotoxicity assay performance of two different leaky membrane-based and two metabolism-based assays. Significant variability was seen in reported viability outcomes across assays and organoid lines. High baseline activity of lactate dehydrogenase (LDH) in four human intestinal organoid lines required modification of the standard LDH assay protocol. Additionally, the LDH assay reported unique resilience to damage in a genetically-modified line contrasting results compared to other assays. This study highlights factors that can impact the measurement of cell cytotoxicity in intestinal organoid models, which are emerging as valuable new tools for research and pre-clinical drug testing and suggest the need for using multiple assay types to ensure reliable cytotoxicity assessment.


Assuntos
L-Lactato Desidrogenase , Organoides , Humanos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/citologia , L-Lactato Desidrogenase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Intestinos/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo
5.
Bioorg Chem ; 149: 107503, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38823312

RESUMO

Lactate dehydrogenase (LDH), a crucial enzyme in anaerobic glycolysis, plays a pivotal role in the energy metabolism of tumor cells, positioning it as a promising target for tumor treatment. Rutin, a plant-based flavonoid, offers benefits like antioxidant, antiapoptotic, and antineoplastic effects. This study employed diverse experiments to investigate the inhibitory mechanism of rutin on LDH through a binding perspective. The outcomes revealed that rutin underwent spontaneous binding within the coenzyme binding site of LDH, leading to the formation of a stable binary complex driven by hydrophobic forces, with hydrogen bonds also contributing significantly to sustaining the stability of the LDH-rutin complex. The binding constant (Ka) for the LDH-rutin system was 2.692 ± 0.015 × 104 M-1 at 298 K. Furthermore, rutin induced the alterations in the secondary structure conformation of LDH, characterized by a decrease in α-helix and an increase in antiparallel and parallel ß-sheet, and ß-turn. Rutin augmented the stability of coenzyme binding to LDH, which could potentially hinder the conversion process among coenzymes. Specifically, Arg98 in the active site loop of LDH provided essential binding energy contribution in the binding process. These outcomes might explain the dose-dependent inhibition of the catalytic activity of LDH by rutin. Interestingly, both the food additives ascorbic acid and tetrahydrocurcumin could reduce the binding stability of LDH and rutin. Meanwhile, these food additives did not produce positive synergism or antagonism on the rutin binding to LDH. Overall, this research could offer a unique insight into the therapeutic potential and medicinal worth of rutin.


Assuntos
L-Lactato Desidrogenase , Rutina , Rutina/química , Rutina/farmacologia , Rutina/metabolismo , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/química , Humanos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Estrutura Molecular , Relação Estrutura-Atividade , Relação Dose-Resposta a Droga , Simulação de Acoplamento Molecular , Simulação por Computador , Antineoplásicos/química , Antineoplásicos/farmacologia
6.
Exp Cell Res ; 440(2): 114135, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38901791

RESUMO

Liver fibrosis is a significant health concern globally due to its association with severe liver conditions like cirrhosis and liver cancer. Histone lactylation has been implicated in the progression of hepatic fibrosis, but its specific role in liver fibrosis, particularly regarding H3K18 lactylation, remained unclear. To investigate this, we established in vivo and in vitro models of liver fibrosis using carbon tetrachloride (CCl4) injection in rats and stimulation of hepatic stellate cells (HSCs) with TGF-ß1, respectively. We found that histone lactylation, particularly H3K18 lactylation, was upregulated in both CCl4-induced rats and TGF-ß1-activated HSCs, indicating its potential involvement in liver fibrosis. Further experiments revealed that lactate dehydrogenase A (LDHA) knockdown inhibited H3K18 lactylation and had a beneficial effect on liver fibrosis by suppressing HSC proliferation, migration, and extracellular matrix (ECM) deposition. This suggests that H3K18 lactylation promotes liver fibrosis progression. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that H3K18 lactylation facilitated the transcription of SOX9, a transcription factor associated with fibrosis. Importantly, overexpression of SOX9 counteracted the effects of LDHA silencing on activated HSCs, indicating that SOX9 is downstream of H3K18 lactylation in promoting liver fibrosis. In summary, this study uncovers a novel mechanism by which H3K18 lactylation contributes to liver fibrosis by activating SOX9 transcription. This finding opens avenues for exploring new therapeutic strategies for hepatic fibrosis targeting histone lactylation pathways.


Assuntos
Progressão da Doença , Células Estreladas do Fígado , Histonas , Cirrose Hepática , Ratos Sprague-Dawley , Fatores de Transcrição SOX9 , Animais , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/genética , Cirrose Hepática/induzido quimicamente , Histonas/metabolismo , Histonas/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Ratos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Masculino , Proliferação de Células , Tetracloreto de Carbono , Fator de Crescimento Transformador beta1/metabolismo , Humanos , Transcrição Gênica , Movimento Celular/genética , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética
7.
PLoS Biol ; 22(6): e3002666, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38905316

RESUMO

Breast cancer is the most prevalent malignancy and the most significant contributor to mortality in female oncology patients. Potassium Two Pore Domain Channel Subfamily K Member 1 (KCNK1) is differentially expressed in a variety of tumors, but the mechanism of its function in breast cancer is unknown. In this study, we found for the first time that KCNK1 was significantly up-regulated in human breast cancer and was correlated with poor prognosis in breast cancer patients. KCNK1 promoted breast cancer proliferation, invasion, and metastasis in vitro and vivo. Further studies unexpectedly revealed that KCNK1 increased the glycolysis and lactate production in breast cancer cells by binding to and activating lactate dehydrogenase A (LDHA), which promoted histones lysine lactylation to induce the expression of a series of downstream genes and LDHA itself. Notably, increased expression of LDHA served as a vicious positive feedback to reduce tumor cell stiffness and adhesion, which eventually resulted in the proliferation, invasion, and metastasis of breast cancer. In conclusion, our results suggest that KCNK1 may serve as a potential breast cancer biomarker, and deeper insight into the cancer-promoting mechanism of KCNK1 may uncover a novel therapeutic target for breast cancer treatment.


Assuntos
Neoplasias da Mama , Proliferação de Células , Histonas , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Glicólise/genética , Histonas/metabolismo , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , Lactato Desidrogenase 5/metabolismo , Lactato Desidrogenase 5/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Prognóstico , Regulação para Cima/genética
8.
Eur J Med Chem ; 275: 116598, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-38925013

RESUMO

Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC50s of 46 nM and 72 nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4 h in mice.


Assuntos
Antineoplásicos , Proliferação de Células , Inibidores Enzimáticos , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Animais , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Proliferação de Células/efeitos dos fármacos , Administração Oral , Camundongos , Relação Estrutura-Atividade , Estrutura Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Disponibilidade Biológica , Relação Dose-Resposta a Droga , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , Linhagem Celular Tumoral , Modelos Moleculares
9.
Int J Mol Sci ; 25(11)2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38891805

RESUMO

Plasmodium knowlesi is the only Plasmodium that causes zoonotic disease among the Plasmodium that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, is a biomarker for diagnosing infection by Plasmodium spp. parasite. Therefore, this study aimed to efficiently produce the soluble form of P. knowlesi LDH (PkLDH) using a bacterial expression system for studying malaria caused by P. knowlesi. Recombinant pET-21a(+)-PkLDH plasmid was constructed by inserting the PkLDH gene into a pET-21a(+) expression vector. Subsequently, the recombinant plasmid was inserted into the protein-expressing Escherichia coli Rosetta(DE3) strain, and the optimal conditions for overexpression of the PkLDH protein were established using this strain. We obtained a yield of 52.0 mg/L PkLDH from the Rosetta(DE3) strain and confirmed an activity of 483.9 U/mg through experiments. This methodology for high-efficiency PkLDH production can be utilized for the development of diagnostic methods and drug candidates for distinguishing malaria caused by P. knowlesi.


Assuntos
Clonagem Molecular , L-Lactato Desidrogenase , Malária , Plasmodium knowlesi , Plasmodium knowlesi/genética , Plasmodium knowlesi/enzimologia , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Clonagem Molecular/métodos , Malária/parasitologia , Malária/diagnóstico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Animais , Humanos , Expressão Gênica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
10.
ACS Appl Mater Interfaces ; 16(26): 33246-33258, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38905518

RESUMO

Herein we report the assessment of the effects of shockwave (SW) impacts on adult rat hippocampal progenitor cell (AHPC) neurospheres (NSs), which are used as in vitro brain models, for enhancing our understanding of the mechanisms of traumatic brain injury (TBI). The assessment has been achieved by using culture dishes and a new microchip. The microchip allows the chemicals released from the brain models cultured inside the cell culture chamber under SW impacts to diffuse to the nanosensors in adjacent sensor chambers through built-in diffusion barriers, which are used to prevent the cells from entering the sensor chambers, thereby mitigating the biofouling issues of the sensor surface. Experiments showed the negative impact of the SW on the viability, proliferation, and differentiation of the cells within the NSs. A qPCR gene expression analysis was performed and appeared to confirm some of the immunocytochemistry (ICC) results. Finally, we demonstrated that the microchip can be used to monitor lactate dehydrogenase (LDH) released from the AHPC-NSs subjected to SW impacts. As expected, LDH levels changed when AHPC-NSs were injured by SW impacts, verifying this chip can be used for assessing the degrees of injuries to AHPC-NSs by monitoring LDH levels. Taken together, these results suggest the feasibility of using the chip to better understand the interactions between SW impacts and in vitro brain models, paving the way for potentially establishing in vitro TBI models on a chip.


Assuntos
Lesões Encefálicas Traumáticas , Hipocampo , Animais , Ratos , Hipocampo/metabolismo , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/metabolismo , Dispositivos Lab-On-A-Chip , Sobrevivência Celular , L-Lactato Desidrogenase/metabolismo , Proliferação de Células , Encéfalo/metabolismo , Encéfalo/patologia , Ondas de Choque de Alta Energia , Células Cultivadas , Diferenciação Celular
11.
J Pharmacol Sci ; 155(4): 131-139, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38880547

RESUMO

Elevation of the homocysteine concentration in the plasma called hyperhomocysteinemia (hHCY) during pregnancy causes a number of pre- and postnatal developmental disorders. The aim of our study was to analyze the effects of H2S donors -NaHS and N-acetylcysteine (NAC) on blood-brain barrier (BBB) permeability in rats with prenatal hHCY. In rats with mild hHCY BBB permeability assessed by Evans Blue extravasation in brain increased markedly throughout life. Administration of NaHS or NAC during pregnancy attenuated hHCY-associated damage and increased endogenous concentrations of sulfides in brain tissues. Acute application of dl-homocysteine thiolactone induced BBB leakage, which was prevented by the NMDA receptor antagonist MK-801 or H2S donors. Rats with hHCY demonstrated high levels of NO metabolite - nitrites and proinflammatory cytokines (IL-1ß, TNF-α, IL-6) in brain. Lactate dehydrogenase (LDH) activity in the serum was higher in rats with hHCY. Mitochondrial complex-I activity was lower in brain of hHCY rats. NaHS treatment during pregnancy restored levels of proinflammatory cytokines, nitrites and activity of the respiratory chain complex in brain as well as the LDH activity in serum. Our data suggest that H2S has neuroprotective effects against prenatal hHCY-associated BBB disturbance providing a potential strategy for the prevention of developmental impairments in newborns.


Assuntos
Acetilcisteína , Barreira Hematoencefálica , Citocinas , Sulfeto de Hidrogênio , Hiper-Homocisteinemia , Fármacos Neuroprotetores , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Gravidez , Hiper-Homocisteinemia/metabolismo , Feminino , Sulfeto de Hidrogênio/metabolismo , Fármacos Neuroprotetores/farmacologia , Acetilcisteína/farmacologia , Citocinas/metabolismo , Homocisteína/sangue , Homocisteína/metabolismo , Homocisteína/análogos & derivados , Ratos Wistar , Sulfetos/farmacologia , Sulfetos/administração & dosagem , Ratos , Masculino , Complicações na Gravidez , Encéfalo/metabolismo , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/sangue , Permeabilidade , Nitritos/metabolismo , Nitritos/sangue
12.
Biomolecules ; 14(5)2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38785950

RESUMO

Limited substrate availability because of the blood-brain barrier (BBB) has made the brain develop specific molecular mechanisms to survive, using lactate synthesized by astrocytes as a source of energy in neurons. To understand if lactate improves cellular viability and susceptibility to glutamate toxicity, primary cortical cells were incubated in glucose- or lactate-containing media and toxic concentrations of glutamate for 24 h. Cell death was determined by immunostaining and lactate dehydrogenase (LDH) release. Mitochondrial membrane potential and nitric oxide (NO) levels were measured using Tetramethylrhodamine, methyl ester (TMRM) and 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate (DAF-FM) live staining, respectively. LDH activity was quantified in single cells in the presence of lactate (LDH substrate) and oxamate (LDH inhibitor). Nuclei of cells were stained with DAPI and neurons with MAP2. Based on the distance between neurons and glial cells, they were classified as linked (<10 µm) and non-linked (>10 µm) neurons. Lactate increased cell death rate and the mean value of endogenous NO levels compared to glucose incubations. Mitochondrial membrane potential was lower in the cells cultured with lactate, but this effect was reversed when glutamate was added to the lactate medium. LDH activity was higher in linked neurons compared to non-linked neurons, supporting the hypothesis of the existence of the lactate shuttle between astrocytes and at least a portion of neurons. In conclusion, glucose or lactate can equally preserve primary cortical neurons, but those neurons having a low level of LDH activity and incubated with lactate cannot cover high energetic demand solely with lactate and become more susceptible to glutamate toxicity.


Assuntos
Glucose , Ácido Glutâmico , L-Lactato Desidrogenase , Ácido Láctico , Potencial da Membrana Mitocondrial , Neurônios , Animais , Ácido Glutâmico/metabolismo , Ácido Glutâmico/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Células Cultivadas , Ácido Láctico/metabolismo , Glucose/metabolismo , Metabolismo Energético/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/citologia , Óxido Nítrico/metabolismo , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ratos , Morte Celular/efeitos dos fármacos
13.
BMC Cancer ; 24(1): 615, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773429

RESUMO

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer in women. Treatment approaches that differ between estrogen-positive (ER+) and triple-negative BC cells (TNBCs) and may subsequently affect cancer biomarkers, such as H19 and telomerase, are an emanating delight in BC research. For instance, all-trans-Retinoic acid (ATRA) could represent a potent regulator of these oncogenes, regulating microRNAs, mostly let-7a microRNA (miR-let-7a), which targets the glycolysis pathway, mainly pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) enzymes. Here, we investigated the potential role of ATRA in H19, telomerase, miR-let-7a, and glycolytic enzymes modulation in ER + and TNBC cells. METHODS: MCF-7 and MDA-MB-231 cells were treated with 5 µM ATRA and/or 100 nM fulvestrant. Then, ATRA-treated or control MCF-7 cells were transfected with either H19 or hTERT siRNA. Afterward, ATRA-treated or untreated MDA-MB-231 cells were transfected with estrogen receptor alpha ER(α) or beta ER(ß) expression plasmids. RNA expression was evaluated by RT‒qPCR, and proteins were assessed by Western blot. PKM2 activity was measured using an NADH/LDH coupled enzymatic assay, and telomerase activity was evaluated with a quantitative telomeric repeat amplification protocol assay. Student's t-test or one-way ANOVA was used to analyze data from replicates. RESULTS: Our results showed that MCF-7 cells were more responsive to ATRA than MDA-MB-231 cells. In MCF-7 cells, ATRA and/or fulvestrant decreased ER(α), H19, telomerase, PKM2, and LDHA, whereas ER(ß) and miR-let-7a increased. H19 or hTERT knockdown with or without ATRA treatment showed similar results to those obtained after ATRA treatment, and a potential interconnection between H19 and hTERT was found. However, in MDA-MB-231 cells, RNA expression of the aforementioned genes was modulated after ATRA and/or fulvestrant, with no significant effect on protein and activity levels. Overexpression of ER(α) or ER(ß) in MDA-MB-231 cells induced telomerase activity, PKM2 and LDHA expression, in which ATRA treatment combined with plasmid transfection decreased glycolytic enzyme expression. CONCLUSIONS: To the best of our knowledge, our study is the first to elucidate a new potential interaction between the estrogen receptor and glycolytic enzymes in ER + BC cells through miR-let-7a.


Assuntos
Neoplasias da Mama , Glicólise , MicroRNAs , RNA Longo não Codificante , Telomerase , Tretinoína , Humanos , Tretinoína/farmacologia , Glicólise/efeitos dos fármacos , Telomerase/metabolismo , Telomerase/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Feminino , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Células MCF-7 , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética
14.
Microb Cell Fact ; 23(1): 143, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38773442

RESUMO

BACKGROUND: Zymomonas mobilis is well known for its outstanding ability to produce ethanol with both high specific productivity and with high yield close to the theoretical maximum. The key enzyme in the ethanol production pathway is the pyruvate decarboxylase (PDC) which is converting pyruvate to acetaldehyde. Since it is widely considered that its gene pdc is essential, metabolic engineering strategies aiming to produce other compounds derived from pyruvate need to find ways to reduce PDC activity. RESULTS: Here, we present a new platform strain (sGB027) of Z. mobilis in which the native promoter of pdc was replaced with the IPTG-inducible PT7A1, allowing for a controllable expression of pdc. Expression of lactate dehydrogenase from E. coli in sGB027 allowed the production of D-lactate with, to the best of our knowledge, the highest reported specific productivity of any microbial lactate producer as well as with the highest reported lactate yield for Z. mobilis so far. Additionally, by expressing the L-alanine dehydrogenase of Geobacillus stearothermophilus in sGB027 we produced L-alanine, further demonstrating the potential of sGB027 as a base for the production of compounds other than ethanol. CONCLUSION: We demonstrated that our new platform strain can be an excellent starting point for the efficient production of various compounds derived from pyruvate with Z. mobilis and can thus enhance the establishment of this organism as a workhorse for biotechnological production processes.


Assuntos
Escherichia coli , Etanol , Ácido Láctico , Engenharia Metabólica , Piruvato Descarboxilase , Zymomonas , Zymomonas/metabolismo , Zymomonas/genética , Piruvato Descarboxilase/metabolismo , Piruvato Descarboxilase/genética , Engenharia Metabólica/métodos , Etanol/metabolismo , Ácido Láctico/metabolismo , Ácido Láctico/biossíntese , Escherichia coli/metabolismo , Escherichia coli/genética , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , Alanina/metabolismo , Ácido Pirúvico/metabolismo , Fermentação
15.
Bone ; 185: 117132, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38789096

RESUMO

The mechanosensitivity of inflammation can alter cellular mechanotransduction. However, the underlying mechanism remains unclear. This study aims to investigate the metabolic mechanism of inflammation under mechanical force to guide tissue remodeling better. Herein, we found that inflammation hindered bone remodeling under mechanical force, accompanied by a simultaneous enhancement of oxidative phosphorylation (OXPHOS) and glycolysis. The control of metabolism direction through GNE-140 and Visomitin revealed that enhanced glycolysis might act as a compensatory mechanism to resist OXPHOS-induced osteoclastogenesis by promoting osteogenesis. The inhibited osteogenesis induced by inflammatory mechanical stimuli was concomitant with a reduced expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). PGC-1α knockdown impeded osteogenesis under mechanical force and facilitated osteoclastogenesis by enhancing OXPHOS. Conversely, PGC-1α overexpression attenuated the impairment of bone remodeling by inflammatory mechanical signals through promoting glycolysis. This process benefited from the PGC-1α regulation on the transcriptional and translational activity of lactate dehydrogenase A (LDHA) and the tight control of the extracellular acidic environment. Additionally, the increased binding between PGC-1α and LDHA proteins might contribute to the glycolysis promotion within the inflammatory mechanical environment. Notably, LDHA suppression effectively eliminated the bone repair effect mediated by PGC-1α overexpression within inflammatory mechanical environments. In conclusion, this study demonstrated a novel molecular mechanism illustrating how inflammation orchestrated glucose metabolism through glycolysis and OXPHOS to affect mechanically induced bone remodeling.


Assuntos
Remodelação Óssea , Glicólise , Inflamação , Osteogênese , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Transdução de Sinais , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Animais , Remodelação Óssea/fisiologia , Inflamação/metabolismo , Inflamação/patologia , Osteogênese/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , L-Lactato Desidrogenase/metabolismo , Fosforilação Oxidativa , Microambiente Celular , Masculino
16.
Cell Mol Biol (Noisy-le-grand) ; 70(5): 127-131, 2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38814226

RESUMO

This study aims to analyze the reversal of lipopolysaccharide (LPS)-induced cardiomyocyte apoptosis via α7nAChR by dexmedetomidine (Dex), so as to provide references for clinical treatment of myocardial disorders in the future. First, the research team divided cardiomyocytes (H9C2) were divided into a control group (normal culture), an LPS group (LPS-induced injury model), and an experimental group (pretreated with Dex before LPS induction). Subsequently, lactate dehydrogenase (LDH) and cell activity were detected, and the research team found that the LDH content of the control, experimental and LPS groups were in ascending order (P<0.05). The cell viability decreased and apoptosis increased in the LPS group, with cells mainly concentrating in the G2-M phase; the viability increased and apoptosis decreased in the experimental group, with blocked G1-G0 phase (P<0.05). This demonstrates that Dex can reverse LPS-induced apoptosis in cardiomyocytes. Subsequently, the research group also detected the expression of α7nAChR and NF-κB/AKT pathway, and it was seen that the expression of α7nAChR in the LPS group was higher than that in the control group, with activated NF-κB/AKT pathway; the α7nAChR expression in the experimental group was further elevated, but the NF-κB/AKT pathway was inhibited (P<0.05). The effects of Dex on cardiomyocytes were seen to be related to the α7nAChR and NF-κB/AKT pathways.


Assuntos
Apoptose , Sobrevivência Celular , Dexmedetomidina , Lipopolissacarídeos , Miócitos Cardíacos , NF-kappa B , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Receptor Nicotínico de Acetilcolina alfa7 , Dexmedetomidina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Apoptose/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Ratos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular , L-Lactato Desidrogenase/metabolismo
17.
J Ethnopharmacol ; 332: 118356, 2024 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-38763372

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Parasitic infections impose a significant burden on public health worldwide. European pharmacopoeia records and ethnopharmacological studies indicate that Hagenia abyssinica (Bruce) J.F. Gmel. has traditionally been used to treat a variety of parasitic infections, while the potential antiparasitic compounds remain ambiguous. AIM OF THE STUDY: Acetylcholinesterase (AChE), lactate dehydrogenases (LDH), and glutathione reductase (GR) are the key target enzymes in the survival of parasites. The aim of our work was to screen antiparasitic compounds targeting AChE, LDH, and GR from H. abyssinica. MATERIALS AND METHODS: Ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) combined with molecular docking was used in this study. Therein, the alamarBlue® and Ellman's methods were employed to reveal the antitrypanosomal effect and AChE inhibitory activity. Meanwhile, the UF-LC-MS was carried out to screen the potential active compounds from H. abyssinica. Subsequently, molecular docking was performed to evaluate the binding mechanisms of these active compounds with AChE, LDH, and GR. Finally, the AChE inhibitory activity of potential inhibitors was detected in vitro. RESULTS: H. abyssinica exhibited significant antitrypanosomal and AChE inhibitory activity. Corilagin, brevifolin carboxylic acid, brevifolin, quercetin, and methyl ellagic acid were recognized as potential AChE inhibitors by UF-LC-MS, while methyl brevifolin carboxylate was identified as AChE, LDH, and GR multi-target inhibitor, with binding degree ranged from 20.96% to 49.81%. Molecular docking showed that these potential inhibitors had a strong affinity with AChE, LDH, and GR, with binding energies ranging from -6.98 to -9.67 kcal/mol. These findings were further supported by the observation that corilagin, quercetin, brevifolin carboxylic acid, and methyl brevifolin carboxylate displayed significant AChE inhibitory activity compared with the positive control (gossypol, 0.42 ± 0.04 mM), with IC50 values of 0.15 ± 0.05, 0.56 ± 0.03, 0.99 ± 0.01, and 1.02 ± 0.03 mM, respectively. CONCLUSIONS: This study confirms the antiparasitic potential of H. abyssinica, supporting the traditional use of H. abyssinica in local ethnopharmacology to treat parasites. At the same time, corilagin, brevifolin carboxylic acid, brevifolin, quercetin, methyl ellagic acid, and methyl brevifolin carboxylate exert their anti-parasitic effects by inhibiting AChE, LDH, and GR, and they are expected to be natural lead compounds for the treatment of parasitic diseases.


Assuntos
Acetilcolinesterase , Inibidores da Colinesterase , Glutationa Redutase , Espectrometria de Massas , Simulação de Acoplamento Molecular , Extratos Vegetais , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/química , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/metabolismo , Acetilcolinesterase/metabolismo , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/química , Ultrafiltração , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Antiparasitários/farmacologia , Antiparasitários/química , Taninos Hidrolisáveis/farmacologia , Taninos Hidrolisáveis/química , Espectrometria de Massa com Cromatografia Líquida
18.
Cell Signal ; 120: 111200, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38719019

RESUMO

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies. Lactate dehydrogenase family genes (LDHs) play a critical role in tumor metabolism, but their functions in HNSCC have not been investigated thoroughly. Thus, we aimed to explore the value of LDHs in HNSCC. METHODS: The association between LDHs expression and mutations, methylation, copy number variations (CNVs), alternative splicing (AS) and competing endogenous RNA (ceRNA) was investigated in The Cancer Genome Atlas (TCGA). The expression level of LDHs in OSCC tissues and adjacent normal tissues was verified by qPCR. Algorithms, such as ssGSEA, ESTIMATE, xCell and TIDE were utilized to analyze the characteristics of immune infiltration. Pathway alternations were enriched by GO, GSEA and KEGG analysis. The Mantel test was employed to elucidate the correlation between metabolism and the tumor microenvironment (TME). Subsequently, MTT and colony formation assays were utilized to assess the impact of LDHB knockdown on cellular proliferation. Additionally, ATP and lactate assays were performed to examine metabolic alterations. Co-culture experiments further investigated the effect of LDHB knockdown on T cell differentiation. RESULTS: LDHs were completely analyzed in multiple databases, among which LDHB was differentially expressed in HNSCC and significantly associated with prognosis. Low LDHB expression had better clinicopathological characteristics. Downregulated LDHB expression was associated with enhanced immune cell infiltration and could influence tumor metabolism. Despite having worse cytotoxic T lymphocyte dysfunction, the LDHBlow group was predicted to respond more favorably to immune checkpoint inhibitors (ICIs) therapy. Moreover, the correlation between metabolism and TME was depicted. In vitro, LDHB knockdown resulted in inhibited cell proliferation, increased lactate levels and decreased ATP levels, while promoted the Th1 differentiation of T cells. CONCLUSIONS: Our study provided a comprehensive analysis of the LDHs and illustrated low LDHB expression could inhibit tumor cell proliferation and ATP production by influencing metabolism, with improved immune cell infiltration and better response to immunotherapy.


Assuntos
Neoplasias de Cabeça e Pescoço , Imunoterapia , L-Lactato Desidrogenase , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Microambiente Tumoral , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Diferenciação Celular , Isoenzimas
19.
Cancer Immunol Immunother ; 73(7): 127, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38739169

RESUMO

Lactate dehydrogenase B (LDHB) reversibly catalyzes the conversion of pyruvate to lactate or lactate to pyruvate and expressed in various malignancies. However, the role of LDHB in modulating immune responses against hepatocellular carcinoma (HCC) remains largely unknown. Here, we found that down-regulation of lactate dehydrogenase B (LDHB) was coupled with the promoter hypermethylation and knocking down the DNA methyltransferase 3A (DNMT 3A) restored LDHB expression levels in HCC cell lines. Bioinformatics analysis of the HCC cohort from The Cancer Genome Atlas revealed a significant positive correlation between LDHB expression and immune regulatory signaling pathways and immune cell infiltrations. Moreover, immune checkpoint inhibitors (ICIs) have shown considerable promise for HCC treatment and patients with higher LDHB expression responded better to ICIs. Finally, we found that overexpression of LDHB suppressed HCC growth in immunocompetent but not in immunodeficient mice, suggesting that the host immune system was involved in the LDHB-medicated tumor suppression. Our findings indicate that DNMT3A-mediated epigenetic silencing of LDHB may contribute to HCC progression through remodeling the tumor immune microenvironment, and LDHB may become a potential prognostic biomarker and therapeutic target for HCC immunotherapy.


Assuntos
Carcinoma Hepatocelular , DNA Metiltransferase 3A , Epigênese Genética , L-Lactato Desidrogenase , Neoplasias Hepáticas , Microambiente Tumoral , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Microambiente Tumoral/imunologia , Humanos , Animais , Camundongos , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , DNA Metiltransferase 3A/metabolismo , Regulação Neoplásica da Expressão Gênica , Metilação de DNA , Isoenzimas/genética , Isoenzimas/metabolismo , Linhagem Celular Tumoral , Inativação Gênica , Prognóstico
20.
J Transl Med ; 22(1): 474, 2024 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-38764020

RESUMO

BACKGROUND: The initiation of fibroblast growth factor 1 (FGF1) expression coincident with the decrease of FGF2 expression is a well-documented event in prostate cancer (PCa) progression. Lactate dehydrogenase A (LDHA) and LDHB are essential metabolic products that promote tumor growth. However, the relationship between FGF1/FGF2 and LDHA/B-mediated glycolysis in PCa progression is not reported. Thus, we aimed to explore whether FGF1/2 could regulate LDHA and LDHB to promote glycolysis and explored the involved signaling pathway in PCa progression. METHODS: In vitro studies used RT‒qPCR, Western blot, CCK-8 assays, and flow cytometry to analyze gene and protein expression, cell viability, apoptosis, and cell cycle in PCa cell lines. Glycolysis was assessed by measuring glucose consumption, lactate production, and extracellular acidification rate (ECAR). For in vivo studies, a xenograft mouse model of PCa was established and treated with an FGF pathway inhibitor, and tumor growth was monitored. RESULTS: FGF1, FGF2, and LDHA were expressed at high levels in PCa cells, while LDHB expression was low. FGF1/2 positively modulated LDHA and negatively modulated LDHB in PCa cells. The depletion of FGF1, FGF2, or LDHA reduced cell proliferation, induced cell cycle arrest, and inhibited glycolysis. LDHB overexpression showed similar inhibitory effect on PCa cells. Mechanistically, we found that FGF1/2 positively regulated STAT1 and STAT1 transcriptionally activated LDHA expression while suppressed LDHB expression. Furthermore, the treatment of an FGF pathway inhibitor suppressed PCa tumor growth in mice. CONCLUSION: The FGF pathway facilitates glycolysis by activating LDHA and suppressing LDHB in a STAT1-dependent manner in PCa.


Assuntos
Fatores de Crescimento de Fibroblastos , Glicólise , L-Lactato Desidrogenase , Neoplasias da Próstata , Fator de Transcrição STAT1 , Transdução de Sinais , Masculino , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Humanos , Animais , L-Lactato Desidrogenase/metabolismo , Linhagem Celular Tumoral , Fator de Transcrição STAT1/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Camundongos Nus , Proliferação de Células , Camundongos , Regulação Neoplásica da Expressão Gênica , Fator 2 de Crescimento de Fibroblastos/metabolismo , Apoptose , Lactato Desidrogenase 5/metabolismo , Isoenzimas
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