Up-regulation Of Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

The regulation of simulated artificial oro-gastrointestinal transit stress on the adhesion of Lactobacillus plantarum S7

BackgroundOro-gastrointestinal stress in the digestive tract is the main stress to which orally administered probiotics are exposed. The regulation of oro-gastrointestinal transit (OGT) stress on the adhesion and survival of probiotics under continuous exposure to simulated salivary-gastric juice-intestinal juice was researched in this study.ResultsLactobacillus plantarum S7 had a higher survival rate after exposure to simulated OGT1 (containing 0.15% bile salt) stress and OGT2 (containing 0.30% bile salt) stress. The adhesion ability of L. plantarum S7 was significantly increased by OGT1 stress (P < 0.05) but was not changed significantly by OGT2 stress (P > 0.05), and this trend was also observed in terms of the thickness of the surface material of L. plantarum S7 cells. The expression of surface proteins of L. plantarum S7, such as the 30 S ribosomal proteins, mucus-binding protein and S-layer protein, was significantly downregulated by OGT stress (P < 0.05); meanwhile, the expression of moonlight proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycorate kinase (PGK), beta-phosphoglucomutase (PGM1), GroEL and glucose-6-phosphate isomerase (PGI), was significantly upregulated (P < 0.05). However, the upregulation of GAPDH, PGK, PGM1 and PGI mediated by OGT1 stress was greater than those mediated by OGT2 stress. The quorum sensing pathway of L. plantarum S7 was changed significantly by OGT stress compared with no OGT stress cells (P < 0.05), and the expression of Luxs in the pathway was significantly upregulated by OGT1 stress (P < 0.05). The ABC transportation pathway was significantly altered by OGT1 stress (P < 0.05), of which the expression of the peptide ABC transporter substrate-binding protein and energy-coupling factor transporter ATP-binding protein EcfA was significantly upregulated by OGT stress (P < 0.05). The glycolide metabolism pathway was significantly altered by OGT1 stress compared with that in response to OGT2 stress (P < 0.05).ConclusionL. plantarum S7 had a strong ability to resist OGT stress, which was regulated by the proteins and pathways related to OGT stress. The adhesion ability of L. plantarum S7 was enhanced after continuous exposure to OGT1 stress, making it a potential probiotic with a promising future for application.

Correlation study of GAPDH, Bcl-2, and Bax protein immunoexpression in patients with colorectal adenocarcinoma.

IntroductionColorectal cancer (CRC) is the third and second most commonly diagnosed cancer worldwide in males and females, respectively. Despite prominent progress in diagnosis and treatment, the recurrence rates are still high. A tumour hypoxic environment leads to an increase in glycolytic metabolism. The crucial intermediate component of glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), could play a significant role in cancer progression. An increased level of GAPDH has been described in oncogene-induced transformation and anti-apoptotic function. In other studies, GAPDH has been involved in apoptosis induction.AimWe examined colorectal adenocarcinoma samples to assess the immunoexpression of GAPDH protein. We also evaluated the correlation between the expression of GAPDH protein and apoptotic parameters including expression of Bcl2 and Bax.Material and methodsParaffin sections were incubated for 60 min with primary antibody against GAPDH, Bcl-2, and Bax.ResultsResults of our study have shown that GAPDH expression in colorectal cancer is upregulated. We revealed significant positive correlation between expression of this protein and grade and size of tumour, and regional lymph node involvement. In the case of apoptosis-associated proteins, e.g. Bcl-2 and Bax, we found negative correlations between expression of these proteins and grade and size of tumour, lymphovascular invasion, and regional lymph node involvement. Finally, we demonstrated that GAPDH up-regulation is connected with down-regulation in Bcl-2 and Bax.ConclusionsUp-regulation of GAPDH protein and down-regulation of Bcl-2 and Bax may result in increased of cancer.

Spatial profiles of markers of glycolysis, mitochondria, and proton pumps in a rat glioma suggest coordinated programming for proliferation.

BackgroundIn cancer cells in vitro, the glycolytic pathway and the mitochondrial tricarboxylic acid (TCA) cycle are programmed to produce more precursor molecules, and relatively less ATP, than in differentiated cells. We address the questions of whether and where these changes occur in vivo in glioblastomas grown from C6 cells in rat brain. These gliomas show some spatial organization, notably in the upregulation of membrane proton transporters near the rim.ResultsWe immunolabeled pairs of proteins (as well as DNA) on sections of rat brains containing gliomas, measured the profiles of fluorescence intensity on strips 200 µm wide and at least 3 mm long running perpendicular to the tumor rim, and expressed the intensity in the glioma relative to that outside. On averaged profiles, labeling of a marker of the glycolytic pathway, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was, as expected, greater in the glioma. Over distances up to 2.5 mm into the glioma, expression of a marker of the TCA cycle, Tom20, a pre-protein receptor on the translocation complex of the mitochondrial outer membrane, was also upregulated. The ratio of upregulation of Tom20 to upregulation of GAPDH was, on average, slightly greater than one. Near the rim (0.4–0.8 mm), GAPDH was expressed less and there was a peak in the mean ratio of 1.16, SEM = 0.001, N = 16 pairs of profiles. An antibody to V-ATPase, which, by pumping protons into vacuoles contributes to cell growth, also indicated upregulation by about 40%. When compared directly with GAPDH, upregulation of V-ATPase was only 0.764, SD = 0.016 of GAPDH upregulation.ConclusionsAlthough there was considerable variation between individual measured profiles, on average, markers of the glycolytic pathway, of mitochondria, and of cell proliferation showed coherent upregulation in C6 gliomas. There is a zone, close to the rim, where mitochondrial presence is upregulated more than the glycolytic pathway, in agreement with earlier suggestions that lactate is taken up by cells near the rim.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1191-z) contains supplementary material, which is available to authorized users.

Abstract 2367: High expression of GAPDH is relevant to lung adenocarcinoma with low expression of the tumor suppressor gene Deleted in Liver Cancer-1 (DLC1)

Abstract Although glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is often considered to be a stable housekeeping marker gene, we recently determined that high levels of GAPDH mRNA expression in solid tumors was highly correlated with the expression of a group of genes, most of which are classified as cell cycle-dependent (Wang et al. PLoS One, 2013). In the current project, we have further evaluated the role of GAPDH in lung adenocarcinoma. Using data from an integrated microarray gene expression dataset and The Cancer Genome Atlas (TCGA) Project, we have now found a correlation between high mRNA expression of GAPDH and low expression of the tumor suppressor gene Deleted in Liver Cancer-1 (DLC1). DLC1 encodes a protein with strong RhoGAP (GTPase activating protein) activity and is inactivated in various human malignancies. In lung adenocarcinoma, low DLC1 expression occurred frequently together with up-regulation of GAPDH. The correlation coefficient of GAPDH and DLC1 expression in the integrated gene expression dataset was -0.6. In addition, high GAPDH and low DLC1 expression in lung cancer was associated with unfavorable prognosis. The combination of DLC1 expression level and GAPDH level improved the predictive prognostic power compared with that of the individual gene level alone. Pathway analysis indicated that low DLC1 in lung cancer was accompanied by increased expression of genes implicated in glycolysis and reduced expression of fructose-1,6-bisphosphatase (FBP1) in gluconeogenesis. Our findings suggest that GAPDH may contribute to the regulation of cell cycle genes and of tumor suppressor genes in cancer. Citation Format: Dunrui Wang, Guangxian Zhang. High expression of GAPDH is relevant to lung adenocarcinoma with low expression of the tumor suppressor gene Deleted in Liver Cancer-1 (DLC1). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2367. doi:10.1158/1538-7445.AM2014-2367

The Expression of Glyceraldehyde-3-Phosphate Dehydrogenase Associated Cell Cycle (GACC) Genes Correlates with Cancer Stage and Poor Survival in Patients with Solid Tumors

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is often used as a stable housekeeping marker for constant gene expression. However, the transcriptional levels of GAPDH may be highly up-regulated in some cancers, including non-small cell lung cancers (NSCLC). Using a publically available microarray database, we identified a group of genes whose expression levels in some cancers are highly correlated with GAPDH up-regulation. The majority of the identified genes are cell cycle-dependent (GAPDH Associated Cell Cycle, or GACC). The up-regulation pattern of GAPDH positively associated genes in NSCLC is similar to that observed in cultured fibroblasts grown under conditions that induce anti-senescence. Data analysis demonstrated that up-regulated GAPDH levels are correlated with aberrant gene expression related to both glycolysis and gluconeogenesis pathways. Down-regulation of fructose-1,6-bisphosphatase (FBP1) in gluconeogenesis in conjunction with up-regulation of most glycolytic genes is closely related to high expression of GAPDH in the tumors. The data presented demonstrate that up-regulation of GAPDH positively associated genes is proportional to the malignant stage of various tumors and is associated with an unfavourable prognosis. Thus, this work suggests that GACC genes represent a potential new signature for cancer stage identification and disease prognosis.

Glyceraldehyde-3-Phosphate Dehydrogenase: A Promising Target for Molecular Therapy in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most highly lethal malignancies ranking as the third leading-cause of cancer-related death worldwide. Although surgical resection and transplantation are effective curative therapies, very few patients qualify for such treatments due to the advanced stage of the disease at diagnosis. In this context, loco-regional therapies provide a viable therapeutic alternative with minimal systemic toxicity. However, as chemoresistance and tumor recurrence negatively impact the success of therapy resulting in poorer patient outcomes it is imperative to identify new molecular target(s) in cancer cells that could be effectively targeted by novel agents. Recent research has demonstrated that proliferation in HCC is associated with increased glucose metabolism. The glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multifunctional protein primarily recognized for its role in glucose metabolism, has already been shown to affect the proliferative potential of cancer cells. In human HCC, the increased expression of GAPDH is invariably associated with enhanced glycolytic capacity facilitating tumor progression. Though it is not yet known whether GAPDH up-regulation contributes to tumorigenesis sensu stricto, emerging evidence points to the existence of a link between GAPDH up-regulation and the promotion of survival mechanisms in cancer cells as well as chemoresistance. The involvement of GAPDH in several hepatocarcinogenic mechanisms (e.g. viral hepatitis, metabolic alterations) and its sensitivity to a new class of prospective anticancer agents prompted us to review the current understanding of the therapeutic potential of targeting GAPDH in HCC.

Human Hepatocellular Carcinoma in a Mouse Model: Assessment of Tumor Response to Percutaneous Ablation by Using Glyceraldehyde-3-Phosphate Dehydrogenase Antagonists

To characterize tumor response to percutaneous injection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antagonists in a mouse model of human hepatocellular carcinoma (HCC). Animal experiments were approved by the Johns Hopkins University Animal Care and Use Committee. Luciferase (luc) gene-expressing Hep3B tumor-bearing athymic nude mice were randomly divided into four groups of six mice each. Tumor-specific GAPDH inhibition was achieved by using percutaneous injection of GAPDH antagonists-3-bromopyruvate (3-BrPA) or GAPDH-specific short hairpin RNA (shRNA). Tumor response to treatment was assessed by using bioluminescence imaging and analysis of GAPDH function and apoptotic markers (caspase-3, caspase-9, and positive staining for terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphospate nick end labeling). HCC samples from 34 patients were obtained from the Johns Hopkins tumor bank, as approved by the Institutional Review Board, for GAPDH expression analysis. Statistical analysis was performed by using a two-sample t test or Spearman rank correlation coefficient. In vitro, 3-BrPA affected Hep3B cell viability (half maximal inhibitory concentration = 0.15 mmol/L), and GAPDH shRNA suppressed (45.5%) colony formation. In vivo, percutaneous injection of GAPDH antagonists into luc-Hep3B tumors decreased bioluminescence imaging signal and viability (3-BrPA, P < .0001; GAPDH shRNA, P = .03). The 3-BrPA treatment primarily inhibited GAPDH activity (74.5%) compared with its expression (34.3%), whereas GAPDH shRNA inhibited both activity (60.6%) and expression (44.4%). Targeted inhibition of GAPDH by using 3-BrPA or shRNA induced apoptosis. HCC samples from patients demonstrated a strong correlation between GAPDH upregulation and the proto-oncogene c-jun expression (r = 0.543, P = .003). Percutaneous injection of GAPDH antagonists induces apoptosis and blocks Hep3B tumor progression, which demonstrates the therapeutic potential of targeting GAPDH in human HCC.

Prolonged Protein Turnover of Glyceraldehyde-3-Phosphate Dehydrogenase by Phospholipase C-gamma 1 is Critical for Anchorage-Independent Growth and ATP Synthesis in Transformed Cells

Overexpression of phospholipase C-γl (PLC-γl) in rat 3Y1 fibroblasts leads to the formation of tumors in nude mice. However, the molecular mechanism for PLC-γl-mediated cellular transformation has not been studied in detail. In this study, we found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, protein levels were increased substantially in cells overexpressing PLC-γl, and that PLC-γl upregulation of GAPDH was due to a decrease in ubiquitination, followed by sustained protein turnover and subsequent accumulation. These observations suggest that regulation of the turnover rate of GAPDH is critical for anchorage-independent growth and ATP synthesis of transformed cells.

Bacterial endotoxin lipopolysaccharide induces up-regulation of glyceraldehyde-3-phosphate dehydrogenase in rat liver and lungs

Bacterial endotoxin or lipopolysaccharide (LPS) can trigger inflammatory responses and cause damage in organs such as liver and lungs when it is introduced into mammals, but the exact molecular events that mediate these responses have remained obscure. In this study, by using 2D gel electrophoresis and cDNA microarray analysis, we found that both protein and mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were significantly increased in rat liver and lungs after treatment with LPS. The results were further confirmed by Western blot and Northern blot. Given the known role of GAPDH in inducing apoptosis, our results suggest that LPS-induced GAPDH up-regulation may be an important mechanism responsible for the damage induced by Gram negative bacteria in mammalian tissue and GAPDH may be involved in the signaling pathway of LPS induced apoptosis. Our results also demonstrate that GAPDH is not a suitable internal control in gene expression studies, especially when bacterial infection is involved.

HIF-2α regulates glyceraldehyde-3-phosphate dehydrogenase expression in endothelial cells

Endothelial cells (EC) express both hypoxia inducible factor-1α (HIF-1α) and -2α (HIF-2α), yet their roles in the EC hypoxic response are unclear. Hypoxia upregulates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EC through a 5′ hypoxic regulatory element (HRE). We compared the upregulation of GAPDH in human lung microvascular EC to that in hep3B cells, another cell type known to express both HIF-1α and HIF-2α. GAPDH mRNA increased to a lesser extent in hypoxic hep3B cells than in EC, yet upregulation occurred through the same HRE that was active in EC. HIF-1α protein induction in response to hypoxia was similar in both cell types. In contrast, HIF-2α protein levels were upregulated to a greater extent and for a longer period of time by hypoxia in EC than in hep3B cells. Correspondingly, electrophoretic mobility supershift assays showed that, in EC, there was preferential binding of HIF-2α to the GAPDH HRE while, in hep3B cells, there was binding of both HIF-1α and HIF-2α. The preferential binding of HIF-2α to the GAPDH HRE in EC may account for their higher level of induction of GAPDH. These findings suggest that cell-specific patterns of HIF-1α and HIF-2α expression lead to cell-specific gene upregulation during hypoxia.

Upregulation of glyceraldehyde-3-phosphate dehydrogenase mRNA in the spleen of tumor-bearing mice

The influence of tumor implantation on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels and stability was determined in the spleen of tumor-bearing mice. While GAPDH mRNA levels were not altered in skeletal muscle, kidney and liver from tumor-bearing mice, tumor implantation led to a 5.6-fold increase in the levels of splenic GAPDH mRNA. An enhanced message stability was observed in splenocytes from tumor-bearing animals, suggesting the involvement of post-transcriptional mechanisms in the selective GAPDH mRNA accumulation after tumor implantation. The GAPDH activity/glycolytic flux ratio was 18.5 in the spleen of healthy mice. Therefore, the three-fold increase in the glycolytic flux observed after tumor implantation could hardly justify the necessity for the upregulation of GAPDH.

Identification of an oxygen responsive enhancer element in the glyceraldehyde-3-phosphate dehydrogenase gene

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced by hypoxia in endothelial cells (EC). Upregulation occurs primarily at the level of transcription and occurs to a much greater extent in EC than in other cell types. To characterize EC specific hypoxia response elements within the GAPDH gene, we performed transient transfection studies in EC, fibroblasts and smooth muscle cells using portions of the GAPDH promoter linked to a CAT reporter gene. These initial studies identified an EC specific hypoxia responsive region that was further characterized (using SV40-promoter-CAT reporter constructs) as a 19-nucleotide sequence (−130 to −112) containing both an hypoxia inducible factor-1 (HIF-1)-binding site and a novel flanking sequence. Electrophoretic mobility shift assays confirmed inducible EC protein binding to this fragment. Mutation of either the HIF-1-binding site or the flanking sequence resulted in complete loss of function and loss of inducible protein binding. Thus, a single HIF-1-binding site is necessary, but not sufficient, for hypoxic regulation of GAPDH in EC. Furthermore, the novel HIF-1 flanking sequence required for GAPDH upregulation and the protein(s) that bind to it may be EC specific.

Regulation of endothelial cell glyceraldehyde-3-phosphate dehydrogenase expression by hypoxia.

Exposure of endothelial cells (EC) to hypoxia results in the increased expression of a distinct set of proteins with molecular masses of 56, 47, 39, 36, and 34 kDa. Their induction appears to be unique to EC and the stress of decreased oxygen tension. To understand the mechanism(s) and significance of the up-regulation of these proteins we have identified the 36-kDa protein by limited amino-terminal amino acid sequencing. The 21-amino acid sequence from the bovine protein exhibited 90.5% identity with the human sequence of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Northern blot analysis showed that the time course and extent of EC GAPDH mRNA up-regulation correlated with the increase in 36-kDa protein synthesis. Nuclear runoff analysis demonstrated that this increase in GAPDH expression is regulated, in part, at the transcriptional level; however, the increase in the rate of transcription did not account for the entire mRNA accumulation, suggesting that GAPDH, like other hypoxia-regulated proteins, is posttranscriptionally regulated. Subcellular fractionation of hypoxic EC showed up-regulation of the 36-kDa protein in the cytoplasmic fraction and, to a lesser extent, in the nuclear fraction. The up-regulation of GAPDH in EC may be related to their relative hypoxia tolerance. Alternatively, the up-regulation of GAPDH in EC during hypoxia may be related to the potential nonglycolytic functions of this enzyme.