Expression Of Lactate Dehydrogenase Research Articles

Acclimation to warm temperatures modulates lactate and malate dehydrogenase isozymes in juvenile Horabagrus brachysoma (Günther)

Differential expression of isozymes enables fish to tolerate temperature fluctuations in their environment. The present study explores the modulation of lactate dehydrogenase (LDH) and cytoplasmic malate dehydrogenase (sMDH) isozyme expression in the heart, muscle, brain, liver, gill, and kidney of juvenile Horabagrus brachysoma after 30 days of acclimation at 26, 31, 33, and 36°C. LDH and sMDH zymography were performed using native polyacrylamide gel electrophoresis. The zymography revealed five distinct bands of LDH isoenzymes (labelled from cathode to anode as LDH-A4, LDH-A3B1, LDH-A2B2, LDH-A1B3, and LDH-B4) and three distinct bands of sMDH isoenzymes (labelled from cathode to anode as sMDH-A2, sMDH-AB, and sMDH-B2), with considerable variation in their expression in the tissues. Acclimation to the test temperatures did not influence the expression patterns of LDH or sMDH isozymes. Densitometric analysis of individual isozyme bands revealed a reduction in the densities of bands containing the LDH-B and sMDH-B molecules, while the densities of bands containing the LDH-A and sMDH-A molecules increased in the gills and muscle, indicating the role of these organs in adaptive responses to thermal acclimation. However, the total densities of the LDH and sMDH isozymes increased with higher acclimation temperatures, indicating that adaptation to increased temperatures in H. brachysoma is primarily characterised by quantitative changes in isozyme expression.

Agomelatine protects hippocampal neurons against doxorubicin induced toxicity

Doxorubicin (DOX) is a powerful chemotherapy drug used in numerous cancer treatments, known to causes neurotoxicity and impair cognitive function in cancer patients. The main mechanism thought to be responsible for the neuronal damage related to chemotherapy is oxidative stress. Agomelatine (AGM) has recently been identified as a neuroprotective agent against toxicity and the effect of AGM on DOX-induced neurotoxicity was investigated in this study. In order to evaluate the effect of AGM on oxidative stress and apoptosis, malondialdehyde (MDA), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), and Caspase-3 expression were analyzed in the HT-22 cell line. AGM considerably reduced oxidative damage due to DOX in vitro. MDA and Caspase-3 were considerably higher in the DOX group. These values were dramatically decreased by AGM. AGM greatly increased the CAT and SOD levels when compared to the DOX group. These results suggest that DOX therapy alters antioxidant status in hippocampus, a critical cognition-related region. So if we are aware of the functional effects of DOX, it is possible to avoid some of the disabling side effects of chemotherapy in cancer patients and may prevent these effects with antioxidant agents such as AGM

Warburg effect enhanced by AKR1B10 promotes acquired resistance to pemetrexed in lung cancer-derived brain metastasis

BackgroundResistance to pemetrexed (PEM), a rare chemotherapeutic agent that can efficiently cross the blood-brain barrier, limits the therapeutic efficacy for patients with lung cancer brain metastasis (BM). Aldo-keto reductase family 1 B10 (AKR1B10) was recently found to be elevated in lung cancer BM. The link between AKR1B10 and BM-acquired PEM is unknown.MethodsPEM drug-sensitivity was assessed in the preclinical BM model of PC9 lung adenocarcinoma cells and the BM cells with or without AKR1B10 interference in vitro and in vivo. Metabolic reprogramming of BM attributed to AKR1B10 was identified by chromatography-mass spectrometry (GC-MS) metabolomics, and the mechanism of how AKR1B10 mediates PEM chemoresistance via a way of modified metabolism was revealed by RNA sequencing as well as further molecular biology experimental approaches.ResultsThe lung cancer brain metastatic subpopulation cells (PC9-BrM3) exhibited significant resistance to PEM and silencing AKR1B10 in PC9-BrM3 increased the PEM sensitivity in vitro and in vivo. Metabolic profiling revealed that AKR1B10 prominently facilitated the Warburg metabolism characterized by the overproduction of lactate. Glycolysis regulated by AKR1B10 is vital for the resistance to PEM. In mechanism, AKR1B10 promoted glycolysis by regulating the expression of lactate dehydrogenase (LDHA) and the increased lactate, acts as a precursor that stimulates histone lactylation (H4K12la), activated the transcription of CCNB1 and accelerated the DNA replication and cell cycle.ConclusionsOur finding demonstrates that AKR1B10/glycolysis/H4K12la/CCNB1 promotes acquired PEM chemoresistance in lung cancer BM, providing novel strategies to sensitize PEM response in the treatment of lung cancer patients suffering from BM.

β-HB treatment reverses sorafenib resistance by shifting glycolysis–lactate metabolism in HCC

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor. Although sorafenib and regorafenib have been approved for first-line and second-line treatment, respectively, of patients with advanced HCC, long-term treatment often results in acquired resistance. Given that glycolysis-mediated lactate production can contribute to drug resistance and impair HCC treatment efficacy, we investigated the effects of ketone body treatment on the metabolic shift in sorafenib-resistant HCC cells. We discovered differential expression of 3-hydroxymethyl glutaryl-CoA synthase 2 (HMGCS2) and the ketone body D-β-hydroxybutyrate (β-HB) in four sorafenib-resistant HCC cell lines. In sorafenib-resistant HCC cells, lower HMGCS2 and β-HB levels were correlated with more glycolytic alterations and higher lactate production. β-HB treatment enhanced pyruvate dehydrogenase (PDH) expression and decreased lactate dehydrogenase (LDHA) expression and lactate production in sorafenib-resistant HCC cells. Additionally, β-HB combined with sorafenib or regorafenib promoted the antiproliferative and antimigratory abilities of sorafenib-resistant HCC cells by inhibiting the B-raf/mitogen-activated protein kinase pathway and mesenchymal N-cadherin-vimentin axis. Although the in vivo β-HB administration did not affect tumor growth, the expression of proliferative and glycolytic proteins was inhibited in subcutaneous sorafenib-resistant tumors. In conclusion, exogenous β-HB treatment can reduce lactate production and reverse sorafenib resistance by inducing a glycolytic shift; it can also synergize with regorafenib for treating sorafenib-resistant HCC.

IRF5 promotes glycolysis in the progression of hepatocellular carcinoma and is regulated by TRIM35.

The interferon regulatory factor (IRF) family of proteins are involved in tumor progression. However, the role of IRF5 in tumorigenesis remains unknown. In this study we aimed to elucidate the functions of IRF5 in the progression of hepatocellular carcinoma (HCC). IRF5 expression in HCC was analyzed through quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemistry (IHC), etc. The Cell Counting Kit 8 (CCK8) assay, anchorage-independent assay, and EdU assay were used to evaluate the role of IRF5. The molecular mechanisms were studied by analyzing the metabolites with mass spectrum and immunoprecipitation. IRF5 was upregulated in HCC. Interfering with IRF5 inhibited the proliferation and tumorigenic potential of HCC cells. When studying the molecular mechanism, IRF5 was found to upregulate the expression of lactate dehydrogenase A (LDHA) and promoted glycolysis. Additionally, tripartite motif containing 35 (TRIM35) interacted with IRF5, promoting its ubiquitination and degradation. In the clinically obtained HCC samples, TRIM35 was negatively correlated with the expression of IRF5. These findings reveal the oncogenic function of IRF5 in the progression of HCC by enhancing glycolysis, further supporting the potential of IRF5 as a viable target for HCC therapy.

Glycosides of Buyang Huanwu decoction inhibits pyroptosis associated with cerebral ischemia-reperfusion through Nrf2-mediated antioxidant signaling pathway both in vivo and in vitro

BackgroundGlycosides are the pharmacodynamic substances of Buyang Huanwu Decoction (BYHWD) and they exert a protective effect in the brain by inhibiting neuronal pyroptosis of cerebral ischemia-reperfusion (CIR). However, the mechanism by which glycosides regulate neuronal pyroptosis of CIR is still unclear. PurposeA significant part of this study aimed to demonstrate whether glycosides have an anti-pyroptotic effect on CIR by nuclear factor erythroid 2-related factor (Nrf2)-mediated antioxidative mechanism. MethodsRats were used in vivo models of middle cerebral artery occlusion and reperfusion (MCAO/R). Neuroprotective effect of glycosides after Nrf2 inhibiting was observed by nerve function score, Nissl staining, Nrf2 fluorescence staining and pyroptotic proteins detection. SH-SY5Y cells were used in vitro models of oxygen-glucose deprivation/reperfusion (OGD/R). Glycosides was evaluated for their effects by measuring cell morphology, survival rate, lactate dehydrogenase (LDH) rate and expression of pyroptotic proteins. Nrf2 si-RNA 54–1 interference with lentivirus was used to create silenced Nrf2 SH-SY5Y cells (si-Nrf2-SH-SY5Y). Glycosides were evaluated on si-Con-SH-SY5Y and si-Nrf2-SH-SY5Y cells based on the expression of Nrf2 signaling pathway, pyroptotic proteins and cell damage manifestation. ResultsIn vivo, glycosides significantly promoted the fluorescence level of nuclear Nrf2, added more Nissl bodies, reduced neurological function scores and inhibited the pyroptotic proteins level. In vitro, glycosides significantly repaired the morphological damage of cells, promoted the survival rate, reduced the LDH rate, inhibited the pyroptosis. Moreover, antioxidant activity of glycosides was enhanced via Nrf2 activation. Both Nrf2 blocking in vivo and Nrf2 silencing in vitro significantly weakened the pyroptosis inhibitory and neuroprotective effects of glycosides. ConclusionThese results suggested for the first time that glycosides inhibited neuronal pyroptosis by regulating the Nrf2-mediated antioxidant stress pathway, thereby exerting brain protection of CIR. As a result of this study, This study improved understanding of the pharmacodynamics and mechanism of BYHWD, as well as providing a Traditional Chinese Medicine (TCM) treatment strategy for CIR .

Cyclovirobuxine D pretreatment ameliorates septic heart injury through mitigation of ferroptosis.

Myocardial dysfunction is a frequent complication in patients with severe sepsis. However, effective drugs for the prevention of myocardial dysfunction and the molecular mechanisms of the disease remain elusive. The present study demonstrated that Cyclovirobuxine D (CVB-D) could improve cardiac dysfunction in a cecal ligation and puncture (CLP) model in rodents and in a lipopolysaccharide (LPS) model in vitro. Echocardiography and histopathological examination were used to detect changes in cardiac structure and function. Kits were used to detect indicators of cardiac injury, transmission electron microscopy to detect structural changes in mitochondria and reverse transcription-quantitative PCR to detect prostaglandin-endoperoxide synthase 2 and hamp expression levels. L-Glutathione and malondialdehyde levels and superoxide dismutase activity were measured using kits. Cell viability was measured with the Cell Counting Kit-8. Iron metabolism-related proteins, inflammatory factor levels and related pathway proteins were detected using western blot analysis. Changes in L-type calcium currents were detected by membrane clamp, and contractility of cardiomyocytes was measured by Ion Optix. CVB-D attenuated CLP-induced cardiac malfunction in septic rats, with changes observed in myocardial pathological structure, creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin I (cTnI). CVB-D attenuated sepsis-induced lipid peroxidation and iron overload. In addition, CVB-D decreased the expression of CK-MB, LDH and cTnI, suppressed oxidative stress index levels and reduced the production of reactive oxygen species. CVB-D decreased LPS-induced cytoplasmic iron overload by increasing upregulation of iron uptake molecules. Conversely, CVB-D significantly increased the upregulation of ferroportin 1. CVB-D pretreatment significantly reduced the levels of hamp mRNA compared with the LPS-treated group. CVB-D pretreatment significantly reduced inflammatory factor levels and the ratio of phosphorylated vs. total signal transducer and activator of transcription 3. The expression of SLC7A11 and GPX4 was upregulated in septic cells pretreated with CVB-D, however treatment with ML385 largely decreased this upregulation. Of note, CVB-D inhibited the inward flow of calcium ions through the LTCC. In conclusion, these findings suggest that CVB-D alleviated sepsis-induced cardiac iron toxicity by alleviating iron metabolism.

Expression, Purification, and Characterization of Plasmodium vivax Lactate Dehydrogenase from Bacteria without Codon Optimization.

Plasmodium vivax is the most widespread cause of malaria, especially in subtropical and temperate regions such as Asia-Pacific and America. P. vivax lactate dehydrogenase (PvLDH), an essential enzyme in the glycolytic pathway, is required for the development and reproduction of the parasite. Thus, LDH from these parasites has garnered attention as a diagnostic biomarker for malaria and as a potential molecular target for developing antimalarial drugs. In this study, we prepared a transformed Escherichia coli strain for the overexpression of PvLDH without codon optimization. We introduced this recombinant plasmid DNA prepared by insertion of the PvLDH gene in the pET-21a(+) expression vector, into the Rosetta(DE3), an E. coli strain suitable for eukaryotic protein expression. The time, temperature, and inducer concentration for PvLDH expression from this E. coli Rosetta(DE3), containing the original PvLDH gene, were optimized. We obtained PvLDH with a 31.0 mg/L yield and high purity (>95%) from this Rosetta(DE3) strain. The purified protein was characterized structurally and functionally. The PvLDH expressed and purified from transformed bacteria without codon optimization was successfully demonstrated to exhibit its potential tetramer structure and enzyme activity. These findings are expected to provide valuable insights for research on infectious diseases, metabolism, diagnostics, and therapeutics for malaria caused by P. vivax.

Serum and Saliva LDH Levels in Patients with Habit, Oral Potentially Malignant Disorders (OPMDs), and Oral Squamous Cell Carcinoma (OSCC): A Spectrophotometry Study.

Oral potentially malignant disorders (OPMDs) are chronic lesions or conditions characterized by a potential for malignant transformation. One of the hallmarks of cancer is aerobic glycolysis which confers immortality to cancer cells, correlates with the belligerent behavior of various malignancies, and shows an increase in the expression of lactate dehydrogenase (LDH). The present study was conducted to measure and compare serum and salivary LDH levels in patients with habit, OPMDs and oral squamous cell carcinoma (OSCC) patients, and to evaluate if LDH levels can be used as a biomarker in the progression into potentially malignant disorders (PMDs), the prognosis of OSCC, and to assess if saliva is a better biomarker. Thirty patients of both genders were divided into three groups. Group I had patients with habits, group II had OPMDs, and group III had OSCC with 10 subjects from each group. Saliva and serum of patients were collected according to the standard protocol and measured for LDH using spectrophotometry of 340 nm. Group I had patients with habits, group II had OPMDs, and group III had OSCC. Group I had 6 males and 4 females, group II had 8 males and 2 females, and group III had 7 males and 3 females. The mean serum LDH level in group I was 265.50, in group II was 194.10, and in group III was 224.22. The difference was non-significant (P > 0.05). The mean salivary LDH level in group I was 345.68, in group II was 532.72, and in group III was 1.105. The difference was significant (P < 0.05). Salivary LDH is a non-invasive, cost-effective technique, which can be used as an effective modality for the diagnosis and prognosis of oral cancer as well as for monitoring tobacco users and OPMDs.

239-LB: Erythropoietin Alters Fat Mass and Gut Microbial Composition

Erythropoietin is an essential hormone for promoting erythropoiesis. Erythropoietin also stimulates angiogenesis, affects fat metabolism and can promote survival in cells expressing erythropoietin receptor. Here, we found that erythropoietin reduces fat mass, decreasing epididymis fat pad mass and subcutaneous fat pad mass of Tg6 male mice that express high transgenic erythropoietin. However, how erythropoietin activity in the gut improves fat mass and glucose metabolism is not known. Tg6 male mice have increased microvilli height and intestine length. High erythropoietin changes the gut microbiota by altering β-diversity that includes increasing the abundance of Bifidobacterium and reducing the Lactobacillaceae in Tg6 male mice. EPO decreased lactate dehydrogenase (LDHa) expression and reduces lactate production from intestinal endothelial cells that affects the gut microbiota components. Accompanying the altered microbiome are increased intestinal short-chain fatty acid levels that can affect fat metabolism. Transplantation of fecal pellet from Tg6 male mice into antibiotic treated male wild type mice reduces fat mass and mimics the lean phenotype of Tg6 mice. Conversely, transplanting feces from WT mice into Tg6 mice resulted in increased fat mass in Tg6 mice. Wild type male mice treated with erythropoietin show improved glucose tolerance and also exhibit increased intestine length and changes in the gut microbiota with increase in Bifidobacterium and decrease in Lactobacillaceae, analogous to changes in Tg6 male mice. Our results on erythropoietin activity in the gut to change gut morphology and remodel the gut microbiome provide a new mechanism by which EPO affects fat metabolism. Key words: EPO; EPOR; gut microbial composition; fat mass Disclosure W. Yin: None. H. Rogers: None. X. An: None. M. Gassmann: None. C. T. Noguchi: None.

Use of tumoral LDH to predict residual tumor burden after neoadjuvant chemotherapy in breast cancer.

e12566 Background: The Residual Cancer Burden (RCB) score has been validated as an independent prognostic factor in all subtype of breast cancer after neoadjuvant chemotherapy (NAC). Early prediction of NAC response enable better stratification for selecting chemotherapy. Glucose metabolism in cancer cells produces various glycolytic metabolites which are related to breast cancer prognosis. In this study, we evaluated the association of glucose transporters and enzymes involved in glucose metabolism with clinic-pathological factors for early prediction of NAC response. Methods: RCB scores were retrospectively evaluated from patients with stage I-III breast cancer treated with neoadjuvant chemotherapy. We selected 7 target transporters and enzymes [GLUT1, hexokinase 2 (HK2), lactate dehydrogenase (LDH), MCT1, MCT4, IGF1RA, and IGF1RB] involved in glucose metabolism and evaluated in relationships with pretreatment clinical/pathological characteristics and treatment outcomes including RCB. Results: 236 breast cancer patients who underwent NAC followed by surgery between 2010 and 2016 in Kyungpook National University Chilgok Hospital were included. Among them, 145 (61.4%), 44 (18.6%), and 47 (19.9%) were luminal, HER2-enriched, and TNBC subtypes. RCB 0 was determined 50 (21.2%), 35 (14.8%) RCB I, 91 (38.6%) RCB II and 60 (25.4%) RCB III. After a median follow-up of 98 months, 60 (25.4%) relapses, 43 (18.2%) distant relapses, and 34 (14.4%) deaths were observed. RCB score was significantly associated with disease specific survival (DSS) and overall survival (OS). RCB III were significantly increased risk of relapse (HR=13.517, CI [1.809-101.010], P&lt;0.001). The tumoral expression rates of the target molecules were 76.0, 31.1, 42.1, 54.9, 67.9, 78.4, and 49.8% for GLUT1, HK2, LDH, MCT1, MCT4, IGF1RA, and IGF1RB, respectively. Among them, LDH was significantly associated with RCB III (OR=4.533, P=0.014) and worse disease specific survival (HR=2.29 P=0.019). Conclusions: Tumoral expression of LDH could be considered as an independent biomarker in predicting high risk of relapse (RBC III) and survival in all subtype of breast cancer.

Anti-Warburg effect via generation of ROS and inhibition of PKM2/β-catenin mediates apoptosis of lambertianic acid in prostate cancer cells.

To elucidate the underlying antitumor mechanism of lambertianic acid (LA) derived from Pinus koraiensis, the role of cancer metabolism related molecules was investigated in the apoptotic effect of LA in DU145 and PC3 prostate cancer cells. MTT assay for cytotoxicity, RNA interference, cell cycle analysis for sub G1 population, nuclear and cytoplasmic extraction, lactate, Glucose and ATP assay by ELISA, Measurement of reactive oxygen species (ROS) generation, Western blotting, and immunoprecipitation assay were conducted in DU145 and PC3 prostate cancer cells. Herein LA exerted cytotoxicity, increased sub G1 population and attenuated the expression of pro-Caspase3 and pro-poly (ADP-ribose) polymerase (pro-PARP) in DU145 and PC3 cells. Also, LA reduced the expression of lactate dehydrogenase A (LDHA), glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2 (PKM2) with reduced production of lactate in DU145 and PC3 cells. Notably, LA decreased phosphorylation of PKM2 on Tyr105 and inhibited the expression of p-STAT3, cyclin D1, C-Myc, β-catenin, and p-GSK3β with the decrease of nuclear translocation of p-PKM2. Furthermore, LA disturbed the binding of p-PKM2 and β-catenin in DU145 cells, which was supported by Spearman coefficient (0.0463) of cBioportal database. Furthermore, LA generated ROS in DU145 and PC3 cells, while ROS scavenger NAC (N-acetyl L-cysteine) blocked the ability of LA to reduce p-PKM2, PKM2, β-catenin, LDHA, and pro-caspase3 in DU145 cells. Taken together, these findings provide evidence that LA induces apoptosis via ROS generation and inhibition of PKM2/β-catenin signaling in prostate cancer cells.

Mild hypoxia as therapy for post-ischemic mitochondrial injury

Introduction: Mitochondrial injury occurs following cellular ischemia in the setting of myocardial infarction, stroke, and cardiac arrest (CA). Effective strategies for reducing injury are limited. Hypoxia has shown benefit in reversing mitochondrial mediated neurodegenerative disease but is little studied in the setting of post-ischemic injury and is regarded as potentially harmful. In this study, we investigated the effects of mild hypoxia on global post-ischemic injury following CA. We hypothesized that brief mild hypoxia following CA would improve mitochondrial function resulting in improved cellular metabolism and physiological recovery. Methods and Results: Anesthetized C57BL6 mice underwent brief asystolic CA (12 min) followed bycardiopulmonary resuscitation(CPR). One hour following successful CPR, mice were randomized to receive a brief episode (6 hours) of normoxia (21% O2) or hypoxia (10% O2) in an environmental chamber. Post intervention, the mice were returned to room air (21% O2). Mild hypoxia improved cardiac mitochondrial complex 1 respiration in the heart by 28% and ATP-related oxygen consumption by 49% (n=3, P&lt;0.05 vs normoxia, respectively) compared to normoxia treated mice. Hypoxia was further associated with improved glucose oxidation in heart as evidenced by decreases in pyruvate dehydrogenase kinase 4 (PDK4) gene and protein expression (n=4, P&lt;0.05 vs normoxia, respectively) as well as decreases in pyruvate dehydrogenase phosphorylation (n=4, P&lt;0.05, respectively) in heart. Hypoxia decreased lactate dehydrogenase (LDH) expression and lactate concentration in heart compared to normoxia treated controls (n=4, P&lt;0.05 vs normoxia, respectively). ROS production in heart by 20% (n=6, P&lt;0.05) was also decreased by mild hypoxia when compared to normoxia post CA. These changes were associated with improved recovery of myocardial function, neurological recovery, and 10-day survival following CA (n=13, P&lt;0.05 vs normoxia, respectively). Conclusion: Brief mild hypoxia treatment following global ischemic injury paradoxically improved recovery of myocardial mitochondrial respiration and metabolism and was associated with improved physiological recovery and survival. Post-ischemia hypoxia is a potential therapeutic strategy for mitochondrial ischemic injury and requires further study. No financial disclosures. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 25: MIF and NR3C2 interactively regulate glucose metabolism in pancreatic cancer

Abstract Inflammation and altered cellular metabolism are described as hallmarks of cancer. Although inflammatory signaling and metabolic reprogramming are highly integrated and play critical role in the development and progression of pancreatic ductal adenocarcinoma (PDAC). The role of inflammatory mediators in the regulation of metabolic reprogramming is not adequately studied in pancreatic cancer and remains to be explored. Here we show that macrophage migration inhibitory factor (MIF) collaborates with its downstream transcriptional factor nuclear-receptor-subfamily-3, group-C, member-2 (NR3C2) regulate metabolic reprogramming to support cancer growth and progression in PDAC. MIF enhances glucose uptake and lactate efflux through enhancing HK1 (hexokinase 1), HK2 (hexokinase 2) and LDHA (lactate dehydrogenase) expression in pancreatic cancer cell in vitro and in an orthotopic mouse model. Furthermore, a decreased HK1, HK2, LDHA expression, pyruvate and lactate production are found in patient tumors with lower MIF expression and MIF-deficiency tumors from genetically engineered KPC mouse model. Conversely, NR3C2 suppresses HK1, HK2, and LDHA expression and decreases glucose uptake and lactate efflux in pancreatic cancer. Mechanistically, MIF-mediated regulation of glycolytic metabolism involves MAPK-ERK Kinase pathway while NR3C2 interacts with AP-1 transcriptional factor in regulation of glycolysis metabolism. Taken together, our result demonstrates an interactive role of MIF and NR3C2 signaling in regulation cancer metabolism and therapeutic approaches target this axis may improve disease outcome in patients with PDAC. Citation Format: Shouhui Yang, Wei Tang, Azadeh Azizian, Philipp Ströbel, Yuuki Ohara, Helen Cawley, Nader Hanna, B. Michael Ghadimi, Nagireddy Putluri, S. Perwez Hussain. MIF and NR3C2 interactively regulate glucose metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 25.

RNA Sequencing in Hypoxia-Adapted T98G Glioblastoma Cells Provides Supportive Evidence for IRE1 as a Potential Therapeutic Target.

Glioblastoma (GBM) is an aggressive brain cancer with a median survival time of 14.6 months after diagnosis. GBM cells have altered metabolism and exhibit the Warburg effect, preferentially producing lactate under aerobic conditions. After standard-of-care treatment for GBM, there is an almost 100% recurrence rate. Hypoxia-adapted, treatment-resistant GBM stem-like cells are thought to drive this high recurrence rate. We used human T98G GBM cells as a model to identify differential gene expression induced by hypoxia and to search for potential therapeutic targets of hypoxia adapted GBM cells. RNA sequencing (RNAseq) and bioinformatics were used to identify differentially expressed genes (DEGs) and cellular pathways affected by hypoxia. We also examined expression of lactate dehydrogenase (LDH) genes using qRT-PCR and zymography as LDH dysregulation is a feature of many cancers. We found 2630 DEGs significantly altered by hypoxia (p < 0.05), 1241 upregulated in hypoxia and 1389 upregulated in normoxia. Hypoxia DEGs were highest in pathways related to glycolysis, hypoxia response, cell adhesion and notably the endoplasmic reticulum, including the inositol-requiring enzyme 1 (IRE1)-mediated unfolded protein response (UPR). These results, paired with numerous published preclinical data, provide additional evidence that inhibition of the IRE1-mediated UPR may have therapeutic potential in treating GBM. We propose a possible drug repurposing strategy to simultaneously target IRE1 and the spleen tyrosine kinase (SYK) in patients with GBM.

Metformin and thymoquinone co-treatment enhance 5-fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells.

This study investigated the chemotherapeutic effects of 5-fluorouracil (5-FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome-C/Caspase-3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative-PCR and Western blot. Markers of oxidative stress were also measured by colorimetric assays. Although all treatments induced anti-cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro-apoptotic actions that coincided with the lowest expression of CCND1/CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cytochrome-C/Caspase-3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase-1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti-cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple therapy approach showed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhancing oxidative glycolysis and augmenting oxidative stress through stronger modulation of the PI3K/mTOR/HIF1α oncogenic network.

The myokine decorin improves the cardiac function in a rat model of isoprenaline-induced myocardial infarction.

Myocardial infarction is a common cause of disability. Decorin is a myokine that has anti-inflammatory, anti-apoptotic effects. Some studies stated that decorin protects myocardium from ischemia. Other studies stated that decorin levels are associated with acute coronary syndrome. The study aimed to investigate the therapeutic role of decorin on cardiac function in a rat model of myocardial infarction. Thirty adult male Wistar rats were divided into control group-rats were subcutaneously injected with normal saline, isoprenaline-injected group-rats were subcutaneously injected with isoprenaline (85 mg/kg) once daily for 2 days to induce myocardial infarction, and decorin ± isoprenaline-injected group-rats were injected as the previous group, followed by decorin injection (0.1 mg/kg) once daily for 7 days. Cardiac hemodynamics, serum lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), oxidative stress markers, gene expression for myocardial-transforming growth factor beta 1 (TGF-β1), interleukin 1 b (IL-1β), tumor necrosis factor alpha (TNF-α), and cardiac caspase-3 immunohistochemical analysis were done. Isoprenaline + decorin group had significant improvement in cardiac hemodynamics and oxidative stress markers; significant decrease in serum CK-MB, LDH, and myocardial gene expression for TNF-α, IL-1β, and TGF-β1; and decreased cardiac caspase-3 immunoreactivity was present. Therefore, decorin can be used as a therapeutic agent after myocardial infarction as it improved the cardiac function.

Target integration of an exogenous β-glucosidase enhances cellulose degradation and ethanol production in Clostridium cellulolyticum

The bacteria Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing (CBP). However, genetic engineering is necessary to improve this organism’s cellulose degradation and bioconversion efficiencies to meet standard industrial requirements. In this study, CRISPR-Cas9n was used to integrate an efficient β-glucosidase into the genome of C. cellulolyticum, disrupting lactate dehydrogenase (ldh) expression and reducing lactate production. The engineered strain showed a 7.4-fold increase in β-glucosidase activity, a 70% decrease in ldh expression, a 12% increase in cellulose degradation, and a 32% increase in ethanol production compared to wild type. Additionally, ldh was identified as a potential site for heterologous expression. These results demonstrate that simultaneous β-glucosidase integration and lactate dehydrogenase disruption is an effective strategy for increasing cellulose to ethanol bioconversion rates in C. cellulolyticum.

Enhancing Solubility of Recombinant Plasmodium Lactate Dehydrogenase (pLDH) Using Combination of Cold-Inducible Expression System and Cold-Stirred Bioreactor

A major drawback associated with an expression of a high-level Plasmodium Lactate Dehydrogenase (pLDH) using Escherichia coli is the low solubility due to the formation of an inclusion body (IB). This study aimed to develop a suitable protocol for enhancing the solubility of pLDH expressed in E. coli. Firstly, a pLDH-encoding gene was amplified from the blood sample of malaria-infected patients and ligated into pBlueScript II KS+ for sequencing. Afterward, the pLDH gene was digested and cloned into pColdTF for expression. The recombinant plasmid was transformed into the E. coli BL21 (DE3) RIPL Codon Plus Strain. Then, the bacterial host was initially cultured at 37°C until reaching optical density (OD) at 600 nm: 0.5. Thereafter, the growth temperature was lowered to 15°C, followed by the addition of 0.1 mM IPTG into the culture medium for inducing pLDH expression. Thereafter, the bacterial hosts were cultured in a cold-stirred bioreactor (15°C). The result showed that a combination of the low culture conditions (15°C) and a low amount of IPTG increased the solubility of pLDH. This result suggests that this protocol can be a convenient method for generating high-quality recombinant protein using the E. coli system.

Protective effect of HDACIs in improves survival and organ injury after CLP-induced sepsis

IntroductionThe effects of isoform-specific histone deacetylase inhibitors (HDACIs) and the non-selective HDACI on sepsis have been profoundly reported. However, the best HDAC classes have not been fully evaluated. Therefore, this study aimed to determine which HDACIs are responsible for survival and beneficial for organ injury. MethodsExperiment I, SD rats were subjected to cecal ligation and puncture and randomly assigned to the no treatment, dimethyl sulfoxide (DMSO) only, MS-275, LMK-235, tubastatinA (TubA), trichostatin-A (TSA), and sirtinol groups (n = 5). Survival was monitored for 48 h. Experiment II, the animals were monitored for 12 h, then, blood and tissues sample were collected. Interleukin (IL)-6, IL-1β, tumour necrosis factor (TNF)-α, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH) expressions were evaluated using ELISA. Liver, heart and lung tissues were analysed via hematoxylin and eosin staining. Liver and heart tissue lysates were analysed for acetylated histones H3, H4, a-tubulin and nuclear factor kappa B (NF-κB), IL-6, IL-1β, and TNF-α using western blotting. Splenocytes were examined via flow cytometry to analyse the immune cell population. ResultsMS-275, TubA and TSA treatments significantly prolonged survival. MS-275, LMK-235, TubA and TSA significantly reduced the histopathological scores and AST, ALT, CK, LDH, IL-6, IL-1β and TNF-α levels, significantly increased acetylated of NF-κB and changed the immune cell proportion. ConclusionOur results indicated that HDACI classes I and IIb and non-selective HDACI can significantly prolong survival. Moreover, non-selective and isoform-specific class I and IIa/IIb HDACIs can attenuate inflammation and organ injury.