Unveiling the role of PYGB in pancreatic cancer: a novel diagnostic biomarker and gene therapy target.

PURPOSE: Pancreatic cancer (PC) is a highly malignant tumor that poses a severe threat to human health. Brain glycogen phosphorylase (PYGB) breaks down glycogen and provides an energy source for tumor cells. Although PYGB has been reported in several tumors, its role in PC remains unclear. METHODS: We constructed a risk diagnostic model of PC-related genes by WGCNA and LASSO regression and found PYGB, an essential gene in PC. Then, we explored the pro-carcinogenic role of PYGB in PC by in vivo and in vitro experiments. RESULTS: We found that PYGB, SCL2A1, and SLC16A3 had a significant effect on the diagnosis and prognosis of PC, but PYGB had the most significant effect on the prognosis. Pan-cancer analysis showed that PYGB was highly expressed in most of the tumors but had the highest correlation with PC. In TCGA and GEO databases, we found that PYGB was highly expressed in PC tissues and correlated with PC's prognostic and pathological features. Through in vivo and in vitro experiments, we found that high expression of PYGB promoted the proliferation, invasion, and metastasis of PC cells. Through enrichment analysis, we found that PYGB is associated with several key cell biological processes and signaling pathways. In experiments, we validated that the MAPK/ERK pathway is involved in the pro-tumorigenic mechanism of PYGB in PC. CONCLUSION: Our results suggest that PYGB promotes PC cell proliferation, invasion, and metastasis, leading to poor patient prognosis. PYGB gene may be a novel diagnostic biomarker and gene therapy target for PC.

Glycogen phosphorylase B promotes ovarian cancer progression via Wnt/ß-catenin signaling and is regulated by miR-133a-3p.

BACKGROUND: Ovarian cancer is one of the most common gynecologic cancers with high morbidity and mortality in women. Glycogen metabolism plays a critical role in cancer development and glycogen phosphorylase B (PYGB) has reported to be involved in various tumors. Here, we explored the role of PYGB in ovarian cancer. METHODS: PYGB mRNA expression were examined in ovarian cancer tissue and also analyzed using the dataset from The Cancer Genome Atlas cohort. Correlations between PYGB expression and prognosis of ovarian cancer patients were analyzed. PYGB was silenced to evaluate the ovarian cell proliferation, invasion and migration in vitro and tumorigenesis in vivo. MiR-133a-3p targeting PYGB was identified using online tools and confirmed with luciferase reporter experiment. MiR-133a-3p overexpression using miRNA mimics was conducted to evaluate its function on ovarian cancer cells. RESULTS: We showed that PYGB was upregulated in ovarian cancer tissue and high level of PYGB expression is markedly correlated with poor prognosis of ovarian cancer patients. PYGB knockdown significantly suppressed ovarian cancer cell proliferation, invasion and migration. Xenograft tumor formation further demonstrated that knockdown PYGB inhibited ovarian tumor development. Bioinformatics analysis revealed that PYGB regulated Wnt/ß-catenin signaling pathway in ovarian cancer cells. Mechanistically, miR-133a-3p directly bound to 3'-untranslated region of PYGB and overexpression miR-133a-3p suppressed proliferation, invasion and migration in ovarian cancer cells. CONCLUSION: Our data suggest that miR-133a-3p/PYGB/Wnt-ß-catenin axis plays a critical role in human ovarian cancer, which might serve as a promising therapeutic target of ovarian cancer treatment in the future.

Silencing of PYGB suppresses growth and promotes the apoptosis of prostate cancer cells via the NF­κB/Nrf2 signaling pathway.

Brain­type glycogen phosphorylase (PYGB) is an enzyme that metabolizes glycogen, whose function is to provide energy for an organism in an emergency state. The present study purposed to investigate the role and mechanism of PYGB silencing on the growth and apoptosis of prostate cancer cells. A cell counting kit­8 assay and flow cytometry were performed to determine the cell viability, apoptosis and reactive oxygen species (ROS) content, respectively. Colorimetry was performed to analyze the activity of caspase­3. Western blotting and reverse transcription­quantitative polymerase chain reaction were used to evaluate the associated mRNA and protein expression levels. The results revealed that PYGB was upregulated in prostate cancer tissues and was associated with disease progression. In addition, PYGB silencing suppressed the cell viability of PC3 cells. PYGB silencing promoted apoptosis of PC3 cells via the regulation of the expression levels of cleaved­poly (adenosine diphosphate­ribose) polymerase, cleaved­caspase­3, B­cell lymphoma­2 (Bcl­2) and Bcl­2­associated X protein. PYGB silencing increased the ROS content in PC3 cells, and affected nuclear factor (NF)­κB/nuclear factor­erythroid 2­related factor 2 (Nrf2) signaling pathways in PC3 cells. In conclusion, PYGB silencing suppressed the growth and promoted the apoptosis of prostate cancer cells by affecting the NF­κB/Nrf2 signaling pathway. The present study provided evidence that may lead to the development of a potential therapeutic strategy for prostate cancer.

PYGB siRNA inhibits the cell proliferation of human osteosarcoma cell lines.

Osteosarcoma is the most common malignant bone carcinoma that primarily occurs between childhood to adolescence. It was suggested by recent research that the Brain type glycogen phosphorylase (PYGB) gene may serve an important role in various types of cancer. In the present study, the PYGB gene was knocked down in order to evaluate the cell viability, invasion and migration of the human osteosarcoma cell lines MG63 and HOS. The expression levels of PYGB in osteosarcoma and bone cyst tissue samples, as well as in the osteosarcoma cell lines were identified using reverse transcription­quantitative polymerase chain reaction and western blot assay. Subsequently, a Cell Counting kit 8 assay was employed to evaluate cell proliferation. Cell apoptosis rate and cell cycle distribution were measured by flow cytometry. In addition, cell invasion and migration were evaluated through a Transwell assay. The expression levels of the cell apoptosis and tumor metastasis associated proteins B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein, E­cadherin, Twist, matrix metalloproteinase (MMP)­9 and MMP2 were measured via western blotting. PYGB exhibited a higher expression level in the osteosarcoma tissue samples, particularly in the human osteosarcoma cell lines MG63 and HOS. Knockdown of PYGB resulted in a decline in cell proliferation, invasion and migration, which was coupled with induced cell apoptosis and cell cycle arrest in MG63 and HOS cells. Furthermore, alterations in the expression of apoptosis and metastasis associated proteins indicated that small interfering (si)PYGB may have regulated cell viability by targeting the Bcl/Caspase and cyclin dependent kinase (CDK)­1 signaling pathway. In conclusion, PYGB siRNA exerted an inhibitory effect on the cell viability of the human osteosarcoma cells MG63 and HOS by blocking the Caspase/Bcl and CDK1 signaling pathway, highlighting novel potential therapeutic methods for treating osteosarcoma.

Glycogen metabolism in brain and neurons - astrocytes metabolic cooperation can be altered by pre- and neonatal lead (Pb) exposure.

Lead (Pb) is an environmental neurotoxin which particularly affects the developing brain but the molecular mechanism of its neurotoxicity still needs clarification. The aim of this paper was to examine whether pre- and neonatal exposure to Pb (concentration of Pb in rat offspring blood below the "threshold level") may affect the brain's energy metabolism in neurons and astrocytes via the amount of available glycogen. We investigated the glycogen concentration in the brain, as well as the expression of the key enzymes involved in glycogen metabolism in brain: glycogen synthase 1 (Gys1), glycogen phosphorylase (PYGM, an isoform active in astrocytes; and PYGB, an isoform active in neurons) and phosphorylase kinase ß (PHKB). Moreover, the expression of connexin 43 (Cx43) was evaluated to analyze whether Pb poisoning during the early phase of life may affect the neuron-astrocytes' metabolic cooperation. This work shows for the first time that exposure to Pb in early life can impair brain energy metabolism by reducing the amount of glycogen and decreasing the rate of its metabolism. This reduction in brain glycogen level was accompanied by a decrease in Gys1 expression. We noted a reduction in the immunoreactivity and the gene expression of both PYGB and PYGM isoform, as well as an increase in the expression of PHKB in Pb-treated rats. Moreover, exposure to Pb induced decrease in connexin 43 immunoexpression in all the brain structures analyzed, both in astrocytes as well as in neurons. Our data suggests that exposure to Pb in the pre- and neonatal periods results in a decrease in the level of brain glycogen and a reduction in the rate of its metabolism, thereby reducing glucose availability, which as a further consequence may lead to the impairment of brain energy metabolism and the metabolic cooperation between neurons and astrocytes.

Molecular Mechanisms of Allosteric Inhibition of Brain Glycogen Phosphorylase by Neurotoxic Dithiocarbamate Chemicals.

Dithiocarbamates (DTCs) are important industrial chemicals used extensively as pesticides and in a variety of therapeutic applications. However, they have also been associated with neurotoxic effects and in particular with the development of Parkinson-like neuropathy. Although different pathways and enzymes (such as ubiquitin ligases or the proteasome) have been identified as potential targets of DTCs in the brain, the molecular mechanisms underlying their neurotoxicity remain poorly understood. There is increasing evidence that alteration of glycogen metabolism in the brain contributes to neurodegenerative processes. Interestingly, recent studies with N,N-diethyldithiocarbamate suggest that brain glycogen phosphorylase (bGP) and glycogen metabolism could be altered by DTCs. Here, we provide molecular and mechanistic evidence that bGP is a target of DTCs. To examine this system, we first tested thiram, a DTC pesticide known to display neurotoxic effects, observing that it can react rapidly with bGP and readily inhibits its glycogenolytic activity (kinact = 1.4 × 105 m-1 s-1). Using cysteine chemical labeling, mass spectrometry, and site-directed mutagenesis approaches, we show that thiram (and certain of its metabolites) alters the activity of bGP through the formation of an intramolecular disulfide bond (Cys318-Cys326), known to act as a redox switch that precludes the allosteric activation of bGP by AMP. Given the key role of glycogen metabolism in brain functions and neurodegeneration, impairment of the glycogenolytic activity of bGP by DTCs such as thiram may be a new mechanism by which certain DTCs exert their neurotoxic effects.

An Isozyme-specific Redox Switch in Human Brain Glycogen Phosphorylase Modulates Its Allosteric Activation by AMP.

Brain glycogen and its metabolism are increasingly recognized as major players in brain functions. Moreover, alteration of glycogen metabolism in the brain contributes to neurodegenerative processes. In the brain, both muscle and brain glycogen phosphorylase isozymes regulate glycogen mobilization. However, given their distinct regulatory features, these two isozymes could confer distinct metabolic functions of glycogen in brain. Interestingly, recent proteomics studies have identified isozyme-specific reactive cysteine residues in brain glycogen phosphorylase (bGP). In this study, we show that the activity of human bGP is redox-regulated through the formation of a disulfide bond involving a highly reactive cysteine unique to the bGP isozyme. We found that this disulfide bond acts as a redox switch that precludes the allosteric activation of the enzyme by AMP without affecting its activation by phosphorylation. This unique regulatory feature of bGP sheds new light on the isoform-specific regulation of glycogen phosphorylase and glycogen metabolism.

Insights into Brain Glycogen Metabolism: THE STRUCTURE OF HUMAN BRAIN GLYCOGEN PHOSPHORYLASE.

Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 Å) and in complex with its allosteric activator AMP (3.4 Å). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen.

Brain isoform glycogen phosphorylase as a novel hepatic progenitor cell marker.

An appropriate liver-specific progenitor cell marker is a stepping stone in liver regenerative medicine. Here, we report brain isoform glycogen phosphorylase (GPBB) as a novel liver progenitor cell marker. GPBB was identified in a protein complex precipitated by a monoclonal antibody Ligab generated from a rat liver progenitor cell line Lig-8. Immunoblotting results show that GPBB was expressed in two liver progenitor cell lines Lig-8 and WB-F344. The levels of GPBB expression decreased in the WB-F344 cells under sodium butyrate (SB)-induced cell differentiation, consistent with roles of GPBB as a liver progenitor cell marker. Short hairpin RNA (shRNA)-mediated GPBB knockdown followed by glucose deprivation test shows that GPBB aids in liver progenitor cell survival under low glucose conditions. Furthermore, shRNA-mediated GPBB knockdown followed by SB-induced cell differentiation shows that reducing GPBB expression delayed liver progenitor cell differentiation. We conclude that GPBB is a novel liver progenitor cell marker, which facilitates liver progenitor cell survival under low glucose conditions and cell differentiation.

Neurons have an active glycogen metabolism that contributes to tolerance to hypoxia.

Glycogen is present in the brain, where it has been found mainly in glial cells but not in neurons. Therefore, all physiologic roles of brain glycogen have been attributed exclusively to astrocytic glycogen. Working with primary cultured neurons, as well as with genetically modified mice and flies, here we report that-against general belief-neurons contain a low but measurable amount of glycogen. Moreover, we also show that these cells express the brain isoform of glycogen phosphorylase, allowing glycogen to be fully metabolized. Most importantly, we show an active neuronal glycogen metabolism that protects cultured neurons from hypoxia-induced death and flies from hypoxia-induced stupor. Our findings change the current view of the role of glycogen in the brain and reveal that endogenous neuronal glycogen metabolism participates in the neuronal tolerance to hypoxic stress.

The N-terminus of glycogen phosphorylase b is not required for activation by adenosine 5'-monophosphate.

The, so far unsuccessful, search for selective effective inhibitors of glycogen phosphorylase for the treatment of type II diabetes has made phosphorylase an active target of research for the past 20 years. Many crystallographic structures of phosphorylase are currently available to aid in this research. However, those structures have been interpreted, at least in part, on the basis of work conducted with a proteolytically derived form of phosphorylase that lacked the N-terminus (phosphorylase b'). It has been reported that phosphorylase b' shows no allostery, neither homotropic nor heterotropic. The original report on phosphorylase b' examined the allosteric characteristics over very narrow ranges of effector and substrate concentrations and reported the presence of proteolytic cleavages in addition to the removal of the N-terminus. We have applied molecular biological techniques to generate a truncate lacking the N-terminus with known primary structure, and we have established conditions for fully quantifying the allosteric effect of AMP on glycogen phosphorylase b. We report here for the first time the full thermodynamic effect of AMP on phosphorylase b. Our findings with a truncate lacking the N-terminus show that the effect of AMP binding does not depend on the N-terminus.

Expression of the brain and muscle isoforms of glycogen phosphorylase in rat heart.

Heart glycogen represents a store of glucosyl residues which are mobilized by the catalysis of glycogen phosphorylase (GP) and are mainly destined to serve as substrates for the generation of ATP. The brain isoform of GP (GP BB) was studied in rat heart in comparison with the muscle isoform (GP MM) to find functional analogies to the brain. Western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed that at the protein level, but not at the mRNA level, the content of GP BB is similar in heart and brain. In contrast, GP MM is more abundant in the heart than in the brain. Immunocytochemically GP BB was colocalized with GP MM in cardiomyocytes. GP MM was also detected in interstitial cells identified as fibroblasts. The physiological role of co-expression of GP BB and GP MM in cardiomyocytes and in brain astrocytes is discussed in a comparative way.

Clinicopathological significance of BGP expression in non-small-cell lung carcinoma: relationship with histological type, microvessel density and patients' survival.

AIMS: Brain-type glycogen phosphorylase (BGP) is the major isoform of glycogen phosphorylase found in fetal and neoplastic tissues, and is generally thought to induce glucose supply during an ischaemic period. This study was performed to investigate BGP expression in non-small-cell lung carcinoma (NSCLC). METHODS: A total of 119 cases of NSCLC, including 63 squamous cell carcinomas (SqCCs) and 56 adenocarcinomas (ACs), were imunohistochemically evaluated for BGP expression, and its expression was correlated with clinicopathological parameters. RESULTS: In total, 76.5% were positive, while non-neoplastic bronchial epithelial cells were weakly positive and pneumocytes were negative. High BGP expression was noted in 78.6% of ACs and 36.5% of SqCCs (p=0.001). Microvessel density was higher in the low BGP expression tumours (29.6 +/- 16.9/mm(2)) than in the high expression tumours (22.8+/-13.8/mm(2)) (p=0.017). BGP expression did not correlate with patient age or tumour stage, but was more frequent in females than males. Kaplan-Meier analysis showed that high BGP expression was associated with poorer survival (p=0.032). CONCLUSIONS: BGP is expressed in NSCLC, particularly AC, and is an independent poor prognostic factor.

Gastric and intestinal phenotypes of gastric carcinoma with reference to expression of brain (fetal)-type glycogen phosphorylase.

PURPOSE: Although reports have suggested that differentiated gastric carcinomas have different phenotypes, i.e., gastric and intestinal type, this classification is complicated and can be confusing. Our previous studies have demonstrated a close relationship between carcinogenesis in differentiated-type gastric cancer and the expression of brain (fetal)-type glycogen phosphorylase (BGP). The purpose of this study was to investigate the relationship between the mucin phenotype of gastric carcinoma and BGP expression. METHODS: Ninety-six specimens of gastric carcinoma were studied using specific anti-BGP antibody. Correlation of BGP expression with intestinal and gastric phenotypes was determined with the anti-mucin antibodies, HGM, CD10, and MUC2. RESULTS: BGP was expressed in 82.6% (38/46) of differentiated type and in 24.0% (12/50) of undifferentiated type carcinomas. The incidence of BGP positivity was significantly greater in the differentiated-type carcinoma than in the undifferentiated type (P < 0.001). The proportions of gastric, mixed and intestinal types in differentiated and undifferentiated gastric carcinomas were 13.0%, 47.8%, and 39.2%, and 56.0%, 32.0%, and 12.0%, respectively. In both differentiated and undifferentiated types, the phenotype of gastric and intestinal mucin expression corresponded very well with BGP expression, that is, more than 90% of carcinomas with gastric type did not express BGP, whereas approximately 90% of carcinomas with intestinal type did express BGP. CONCLUSIONS: The classification of gastric and intestinal phenotypes of gastric carcinoma in terms of BGP expression was simpler and clearer than such classification in terms of mucin immunohistochemistry. It is suggested that BGP is a useful biomarker for the classification of intestinal and gastric type carcinoma of the human stomach, including classification from the carcinogenetic point of view.