Blockade of 6-phosphogluconate dehydrogenase generates CD8+ effector T cells with enhanced anti-tumor function.

Although T cell expansion depends on glycolysis, T effector cell differentiation requires signaling via the production of reactive oxygen species (ROS). Because the pentose phosphate pathway (PPP) regulates ROS by generating nicotinamide adenine dinucleotide phosphate (NADPH), we examined how PPP blockade affects T cell differentiation and function. Here, we show that genetic ablation or pharmacologic inhibition of the PPP enzyme 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP results in the generation of superior CD8+ T effector cells. These cells have gene signatures and immunogenic markers of effector phenotype and show potent anti-tumor functions both in vitro and in vivo. In these cells, metabolic reprogramming occurs along with increased mitochondrial ROS and activated antioxidation machinery to balance ROS production against oxidative damage. Our findings reveal a role of 6PGD as a checkpoint for T cell effector differentiation/survival and evidence for 6PGD as an attractive metabolic target to improve tumor immunotherapy.

In vitro effects of lonidamine and 6-aminonicotinamide against Echinococcus granulosussensu stricto and Echinococcus multilocularis.

Echinococcosis is a zoonotic disease caused by cestode species of the genus Echinococcus, which demonstrates considerable medical and veterinary concerns. The development of novel drugs for echinococcosis treatment is urgently needed. In this study, we demonstrated that lonidamine (LND) and 6-aminonicotinamide (6-AN) exhibited considerable in vitro effects against both larval- and adult-stage of E. granulosussensu stricto (s. s.) and E. multilocularis. The combination of LND and 6-AN exhibited a significantly higher activity than the single drug treatment. These results highlight the therapeutic potential of LND, 6-AN and the combination of LND and 6-AN for the treatment of echinococcosis.

Targeting the pentose phosphate pathway increases reactive oxygen species and induces apoptosis in thyroid cancer cells.

The pentose phosphate pathway (PPP) plays an important role in the biosynthesis of ribonucleotide precursor and NADPH. Cancer cells frequently increase the flux of glucose into the PPP to support the anabolic demands and regulate oxidative stress. Consistently, metabolomic analyses indicate an upregulation of the PPP in thyroid cancer. In the present study, we found that the combination of glucose-6-phosphate dehydrogenase (G6PD) and transketolase inhibitors (6-aminonicotinamide and oxythiamine) exerted an additive or synergistic effect on cell growth inhibition in thyroid cancer cells. Targeting PPP significantly increased cellular reactive oxygen species (ROS) and induced endoplasmic reticulum (ER) stress and apoptosis. Suppressed cell viability could be partially rescued with treatment with the ROS scavenger or apoptosis inhibitor but not ER-stress inhibitor. Taken together, dual PPP blockade leads to pharmacologic additivity or synergism and causes ROS-mediated apoptosis in thyroid cancer cells.

Niacin analogue, 6-Aminonicotinamide, a novel inhibitor of hepatitis B virus replication and HBsAg production.

BACKGROUND: Hepatitis B surface antigen (HBsAg) is one of the important clinical indexes for hepatitis B virus (HBV) infection diagnosis and sustained seroconversion of HBsAg is an indicator for functional cure. However, the level of HBsAg could not be reduced by interferons and nucleoside analogs effectively. Therefore, identification of a new drug targeting HBsAg is urgently needed. METHODS: In this study, 6-AN was screened out from 1500 compounds due to its low cytotoxicity and high antiviral activity. The effect of 6-AN on HBV was examined in HepAD38, HepG2-NTCP and PHHs cells. In addition, the antivirus effect of 6-AN was also identified in mouse model. FINDINGS: 6-AN treatment resulted in a significant decrease of HBsAg and other viral markers both in vitro and in vivo. Furthermore, we found that 6-AN inhibited the activities of HBV SpI, SpII and core promoter by decreasing transcription factor PPARα, subsequently reduced HBV RNAs transcription and HBsAg production. INTERPRETATION: We have identified a novel small molecule to inhibit HBV core DNA, HBV RNAs, HBsAg production, as well as cccDNA to a minor degree both in vitro and in vivo. This study may shed light on the development of a novel class of anti-HBV agent.

Influence of 6-aminonicotinamide (6AN) on Leishmania promastigotes evaluated by metabolomics: Beyond the pentose phosphate pathway.

6-Aminonicotinamide (6AN) is an antimetabolite used to inhibit the NADPH-producing pentose phosphate pathway (PPP) in many cellular systems, making them more susceptible to oxidative stress. It is converted by a NAD(P)+ glycohydrolase to 6-aminoNAD and 6-aminoNADP, causing the accumulation of PPP intermediates, due to their inability to participate in redox reactions. Some parasites like Plasmodium falciparum and Coccidia are highly sensitive but not all cell types showed a strong responsiveness to 6AN, probably due to the different targeted pathway. For instance, in bacteria the main target is the Preiss-Handler salvage pathway for NAD+ biosynthesis. We were interested in testing 6AN on the kinetoplastid protozoan Leishmania as another model to clarify the mechanisms of action of 6AN, by using metabolomics. Leishmania promastigotes, the life-cycle stage residing in the sandfly, demonstrated a three order of magnitude higher EC50 (mM) compared to P. falciparum and mammalian cells (µM), although pre-treatment with 100 µM 6AN prior to sub-lethal oxidative challenge induced a supra-additive cell kill in L. infantum. By metabolomics, we did not detect 6ANAD/P suggesting that NAD+ glycohydrolases in Leishmania may not be highly efficient in catalysing transglycosidation as happens in other microorganisms. Contrariwise to the reported effect on 6AN-treated cancer cells, we did not detect 6-phosphogluconate (6 PG) accumulation, indicating that 6ANADP cannot bind with high affinity to the PPP enzyme 6 PG dehydrogenase. By contrast, 6AN caused a profound phosphoribosylpyrophosphate (PRPP) decrease and nucleobases accumulation confirming that PPP is somehow affected. More importantly, we found a decrease in nicotinate production, evidencing the interference with the Preiss-Handler salvage pathway for NAD+ biosynthesis, most probably by inhibiting the reaction catalysed by nicotinamidase. Therefore, our combined data from Leishmania strains, though confirming the interference with PPP, also showed that 6AN impairs the Preiss-Handler pathway, underlining the importance to develop compounds targeting this last route.

PMA treated THP-1-derived-IL-6 promotes EMT of SW48 through STAT3/ERK-dependent activation of Wnt/ß-catenin signaling pathway.

Colon cancer is one of the most common digestive malignant tumors that leads to high mortality worldwide, and metastasis is the primary cause of cancer-related death. It is well accepted that the epithelial-mesenchymal transition (EMT) plays a key role in the process of metastasis. As a cytokine that macrophage secretes, IL-6 is involved in the progression of tumors, including the invasion and metastasis via kinds of signaling pathways. However, the mechanism of interactions between IL-6, macrophage, EMT and colon cancer is not fully understood. Increased CD68+ macrophages and IL-6 level were found in colon tumor as compared to normal colon tissue. Metastatic lymph node showed even more CD68+ macrophages and higher IL-6 level than the primary tumor. These results suggested that macrophages and IL-6 play an important role in EMT of colon cancer. In order to investigate the effect of macrophage and IL-6 on EMT of colon cancer, we cultured human colon carcinoma cell line SW48 with conditioned medium (CM) from PMA-stimulated monocyte THP-1 cells and tested for IL-6 dependent EMT pathways. Wound healing assay and Transwell assay were used to analyze cell migration and invasion. Results showed that CM-treated SW48 cells increased IL-6 production and displayed elevated capacity of migration and invasion compared to untreated cells. Increased expressions of EMT markers (N-cadherin, Vimentin and ß-catenin) and decreased expression of EMT marker(E-cadherin) were found in CM-treated SW48 cells by Western Blot. The addition of an anti-IL-6 antibody significantly inhibited the increase of EMT markers (Vimentin and ß-catenin) as well as cell migration and invasion, suggesting that IL-6 played a critical role in promoting EMT of CM-treated SW48 cells. In addition, we found that the levels of p-STAT3 and p-ERK increased in CM-treated SW48 compared to untreated cells, which can be reversed by AG490, an inhibitor of JAK. In the meantime, the suppression of JAK-associated signaling pathways caused a decrease of ß-catenin. In summary, our study suggested that macrophage-induced IL-6 promotes migration and invasion of colon cancer cell via Wnt/ß-catenin pathway in STAT3/ERK-dependent way.

Modulation of G6PD affects bladder cancer via ROS accumulation and the AKT pathway in vitro.

Glucose-6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme of the pentose phosphate pathway. Multiple studies have previously revealed that elevated G6PD levels promote cancer progression in numerous tumor types; however, the underlying mechanism remains unclear. In the present study, it was demonstrated that high G6PD expression is a poor prognostic factor in bladder cancer, and the levels of G6PD expression increase with increasing tumor stage. Patients with bladder cancer with high G6PD expression had worse survival rates compared with those with lower G6PD expression in resected tumors. In vitro experiments revealed that knockdown of G6PD suppressed cell viability and growth in Cell Counting Kit-8 and colony formation assays, and increased apoptosis in bladder cancer cell lines compared with normal cells. Further experiments indicated that the weakening of the survival ability in G6PD-knockdown bladder cancer cells may be explained by intracellular reactive oxygen species accumulation and protein kinase B pathway suppression. Furthermore, it was additionally revealed that 6-aminonicotinamide (6-AN), a competitive G6PD inhibitor, may be a potential therapy for bladder cancer, particularly in cases with high G6PD expression, and that the combination of cisplatin and 6-AN may optimize the clinical dose or minimize the side effects of cisplatin.

Inhibition of glucose-6-phosphate dehydrogenase protects hepatocytes from aluminum phosphide-induced toxicity.

Aluminum phosphide (AlP) poisoning is a severe toxicity with 30-70% mortality rate. However, several case reports presented AlP-poisoned patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency and extensive hemolysis who survived the toxicity. This brought to our mind that maybe G6PD deficiency could protect the patients from severe fatal poisoning by this pesticide. In this research, we investigated the protective effect of 6-aminonicotinamide (6-AN)- as a well-established inhibitor of the NADP+- dependent enzyme 6-phosphogluconate dehydrogenase- on isolated rat hepatocytes in AlP poisoning. Hepatocytes were isolated by collagenase perfusion method and incubated into three different flasks: control, AlP, and 6-AN+ALP. Cellar parameters such as cell viability, reactive oxygen species (ROS) formation, mitochondria membrane potential collapse (MMP), lysosomal integrity, content of reduced (GSH) and oxidized glutathione (GSSG) and lipid peroxidation were assayed at intervals. All analyzed cellular parameters significantly decreased in the third group (6-AN+AlP) compared to the second group (AlP), showing the fact that G6PD deficiency induced by 6-AN had a significant protective effect on the hepatocytes. It was concluded that G6PD deficiency significantly reduced the hepatotoxicity of AlP. Future drugs with the power to induce such deficiency may be promising in treatment of AlP poisoning.

Real-time quantification of subcellular H2O2 and glutathione redox potential in living cardiovascular tissues.

Detecting and measuring the dynamic redox events that occur in vivo is a prerequisite for understanding the impact of oxidants and redox events in normal and pathological conditions. These aspects are particularly relevant in cardiovascular tissues wherein alterations of the redox balance are associated with stroke, aging, and pharmacological intervention. An ambiguous aspect of redox biology is how redox events occur in subcellular organelles including mitochondria, and nuclei. Genetically-encoded Rogfp2 fluorescent probes have become powerful tools for real-time detection of redox events. These probes detect hydrogen peroxide (H2O2) levels and glutathione redox potential (EGSH), both with high spatiotemporal resolution. By generating novel transgenic (Tg) zebrafish lines that express compartment-specific Rogfp2-Orp1 and Grx1-Rogfp2 sensors we analyzed cytosolic, mitochondrial, and the nuclear redox state of endothelial cells and cardiomyocytes of living zebrafish embryos. We provide evidence for the usefulness of these Tg lines for pharmacological compounds screening by addressing the blocking of pentose phosphate pathways (PPP) and glutathione synthesis, thus altering subcellular redox state in vivo. Rogfp2-based transgenic zebrafish lines represent valuable tools to characterize the impact of redox changes in living tissues and offer new opportunities for studying metabolic driven antioxidant response in biomedical research.

Design, synthesis, and evaluation of 4,6-diaminonicotinamide derivatives as novel and potent immunomodulators targeting JAK3.

In organ transplantation, T cell-mediated immune responses play a key role in the rejection of allografts. Janus kinase 3 (JAK3) is specifically expressed in hematopoietic cells and associated with regulation of T cell development via interleukin-2 signaling pathway. Here, we designed novel 4,6-diaminonicotinamide derivatives as immunomodulators targeting JAK3 for prevention of transplant rejection. Our optimization of C4- and C6-substituents and docking calculations to JAK3 protein confirmed that the 4,6-diaminonicotinamide scaffold resulted in potent inhibition of JAK3. We also investigated avoidance of human ether-a-go-go related gene (hERG) inhibitory activity. Selected compound 28 in combination with tacrolimus prevented allograft rejection in a rat heterotopic cardiac transplantation model.

Inhibition of pentose phosphate pathway suppresses acute myelogenous leukemia.

Pentose phosphate pathway (PPP) is a metabolic pathway that generates NADPH and pentose. PPP genes have been reported to be primarily or secondarily upregulated in many cancers. We aimed to study the general alteration of PPP in acute myelogenous leukemia (AML). We performed data mining and analysis of the Cancer Genome Atlas (TCGA) AML dataset for genetic alteration of the PPP gene set. In vitro studies including proliferation, migration, and invasion assays, together with metabolite consumption and oxidation assays, were performed. PPP genes were upregulated in 61 % of patients with AML. The majority of altered cases were expression changes measured by RNA sequencing. Expressions of critical PPP genes such as G6PD, PFKL, PFKP, and PGLS were consistently upregulated in all altered cases. Altered PPP is not associated with survival or disease relapse. PPP inhibition using 6-aminonicotinamide (6AN) increases glucose oxidative metabolism in AML. 6AN decreased the glucose oxidation and increased fatty acid oxidation. Here, we showed that PPP inhibition increased glucose oxidative metabolism in AML. PPP inhibition suppressed growth, migration, and invasion of AML, but not colony formation. PPP plays an important role in AML. Our results could contribute to the development of novel targeted treatment.

Multiple myeloma acquires resistance to EGFR inhibitor via induction of pentose phosphate pathway.

Multiple myeloma (MM) was characterized by frequent mutations in KRAS/NRAS/BRAF within the EGFR pathway that could induce resistance to EGFR inhibitors. We here report that EGFR inhibition solely exhibited moderate inhibition in KRAS/NRAS/BRAF wildtype (triple-WT) MM cells, whilst had no effect in myeloma cells with any of the mutated genes. The moderate inhibitory effect was conferred by induction of pentose phosphate pathway (PPP) when cells were treated with Gefitinib, the EGFR inhibitor. Combination of Gefitinib with PPP inhibitor 6AN effected synergistically in triple-WT cells. The inhibition could be restored by addition of NADPH. Dual EGFR/ERBB2 inhibitor Afatinib also exhibited similar effects. Further genetic silencing of EGFR, ERBB2 and mTOR indicated that major effect conferred by ERBB2 was via convergence to EGFR pathway in MM. Our results contributed to the individualized targeted therapy with EGFR inhibitors in MM.

Degradation of AF1Q by chaperone-mediated autophagy.

AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA.

Autophagy-dependent metabolic reprogramming sensitizes TSC2-deficient cells to the antimetabolite 6-aminonicotinamide.

UNLABELLED: The mammalian target of rapamycin complex 1 (mTORC1) is hyperactive in many human cancers and in tuberous sclerosis complex (TSC). Autophagy, a key mTORC1-targeted process, is a critical determinant of metabolic homeostasis. Metabolomic profiling was performed to elucidate the cellular consequences of autophagy dysregulation under conditions of hyperactive mTORC1. It was discovered that TSC2-null cells have distinctive autophagy-dependent pentose phosphate pathway (PPP) alterations. This was accompanied by enhanced glucose uptake and utilization, decreased mitochondrial oxygen consumption, and increased mitochondrial reactive oxygen species (ROS) production. Importantly, these findings revealed that the PPP is a key autophagy-dependent compensatory metabolic mechanism. Furthermore, PPP inhibition with 6-aminonicotinamide (6-AN) in combination with autophagy inhibition suppressed proliferation and prompted the activation of NF-κB and CASP1 in TSC2-deficient, but not TSC2-proficient cells. These data demonstrate that TSC2-deficient cells can be therapeutically targeted, without mTORC1 inhibitors, by focusing on their metabolic vulnerabilities. IMPLICATIONS: This study provides proof-of-concept that therapeutic targeting of diseases with hyperactive mTORC1 can be achieved without the application of mTORC1 inhibitors.

Identification and characterization of novel human glucose-6-phosphate dehydrogenase inhibitors.

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP(+). Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC(50) values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC(50) values. The most potent hG6PD inhibitors presented IC(50) values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC(50) ~25 µM) more strongly than that of normal MCF10-A cells (IC(50) >50 µM).

Modulation of glycolysis and the pentose phosphate pathway influences porcine oocyte in vitro maturation.

Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose-dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6-AN) and the physiological (NADPH) inhibitors of PPP induced a dose-dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.

2-Deoxy-D-glucose and 6-aminonicotinamide-mediated Nrf2 down regulation leads to radiosensitization of malignant cells via abrogation of GSH-mediated defense.

Enhanced level of nuclear erythroid-related factor-2 (Nrf2) has been associated with cancer chemo/radioresistance. Therefore, the role of Nrf2 in radiosensitization of malignant cells induced by a combination of 2-deoxy-D-Glucose (2-DG) and 6-aminonicotinamide (6-AN) was investigated. Two established human malignant cells lines namely KB (head and neck squamous carcinoma) and BMG-1 (cerebral glioma) were used. Following treatment with a combination of 2-DG (5 mM) and 6-AN (5 µM), irradiated (2Gy) KB and BMG-1 cells were assessed for protein level of Nrf2, Keap1 and γ-glutamylcysteine synthetase (γ-GCS) by western blotting and mRNA expression of γ-GCS, glutathione reductase (GR) and glutathione peroxidase (GPx1) by RT-PCR at 24 hours post treatment. A significant decrease in the level of Nrf2 with a concomitant increase in Keap1 was observed in both the irradiated malignant cells at 24 hours following treatment with combination (2-DG + 6-AN). Down regulation of γ-GCS, GR and GPx1 at 24 hours following treatment with combination (2-DG + 6-AN) resulted in abrogation of glutathione (GSH)-mediated defense in both the irradiated malignant cells. Eventual accumulation of ROS led to radiosensitization of both the malignant cells. These results indicate that deregulated Nrf2-Keap1 signalling leads to the radiosensitization of malignant cells due to abrogated glutathione defense. Metabolic modification-mediated down regulation of Nfr2 and its downstream signalling may have a potential of improving tumour radiotherapy.

Radiosensitization by 2-deoxy-D-glucose and 6-aminonicotinamide involves activation of redox sensitive ASK1-JNK/p38MAPK signaling in head and neck cancer cells.

Our previous studies on simultaneous inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) and pentose phosphate activity by 6-aminonicotinamide (6-AN) have been shown to induce oxidative stress mediated selective radiosensitization in wide range of human malignant cells. However, the mechanism of radiosensitization induced by this combination (2-DG+6-AN) is not completely understood. Since activation of apoptotic signal regulating kinase (ASK1) and subsequent apoptosis are implicated in oxidative stress response, the role of ASK1 activation in radiosensitization by this combination was investigated in the present study. Our results demonstrated that redox alterations induced by this combination activated ASK1 and subsequent apoptosis during radiosensitization of head and neck carcinoma cells (KB). In addition, mRNA and protein expression of thioredoxin and thioredoxin reductase decreased significantly under similar treatment conditions. Further, the downstream targets such as JNK and p38MAPK were also activated by this combination, and their pharmacological inhibition by SP600125 and SB201291 respectively resulted in suppression of 2-DG+6-AN mediated apoptosis in irradiated KB cells. Interestingly, the activation of ASK1 was mediated by hydrogen peroxide rather than superoxide anions as PEG-catalase but not PEG-SOD suppressed its activation. Our observations clearly suggest that redox alterations by inhibition of glucose metabolism serves as a molecular switch that activate ASK1-JNK/p38MAPK signaling in malignant cells during radiosensitization by 2-DG+6-AN. The present study emphasizes the importance of redox alterations in determining radiosensitivity of tumor cells that may greatly influence the outcome of radiation therapy.

JC virus T-antigen regulates glucose metabolic pathways in brain tumor cells.

Recent studies have reported the detection of the human neurotropic virus, JCV, in a significant population of brain tumors, including medulloblastomas. Accordingly, expression of the JCV early protein, T-antigen, which has transforming activity in cell culture and in transgenic mice, results in the development of a broad range of tumors of neural crest and glial origin. Evidently, the association of T-antigen with a range of tumor-suppressor proteins, including p53 and pRb, and signaling molecules, such as ß-catenin and IRS-1, plays a role in the oncogenic function of JCV T-antigen. We demonstrate that T-antigen expression is suppressed by glucose deprivation in medulloblastoma cells and in glioblastoma xenografts that both endogenously express T-antigen. Mechanistic studies indicate that glucose deprivation-mediated suppression of T-antigen is partly influenced by 5'-activated AMP kinase (AMPK), an important sensor of the AMP/ATP ratio in cells. In addition, glucose deprivation-induced cell cycle arrest in the G1 phase is blocked with AMPK inhibition, which also prevents T-antigen downregulation. Furthermore, T-antigen prevents G1 arrest and sustains cells in the G2 phase during glucose deprivation. On a functional level, T-antigen downregulation is partially dependent on reactive oxygen species (ROS) production during glucose deprivation, and T-antigen prevents ROS induction, loss of ATP production, and cytotoxicity induced by glucose deprivation. Additionally, we have found that T-antigen is downregulated by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the pentose phosphate inhibitors, 6-aminonicotinamide and oxythiamine, and that T-antigen modulates expression of the glycolytic enzyme, hexokinase 2 (HK2), and the pentose phosphate enzyme, transaldolase-1 (TALDO1), indicating a potential link between T-antigen and metabolic regulation. These studies point to the possible involvement of JCV T-antigen in medulloblastoma proliferation and the metabolic phenotype and may enhance our understanding of the role of viral proteins in glycolytic tumor metabolism, thus providing useful targets for the treatment of virus-induced tumors.

A combination of 2-deoxy-D-glucose and 6-aminonicotinamide induces cell cycle arrest and apoptosis selectively in irradiated human malignant cells.

Previously, we have shown that a combination of metabolic modifiers 2-deoxy-D-glucose (2-DG) and 6-aminonicotinamide (6-AN) results in oxidative stress mediated radiosensitization of malignant cells via mitochondrial dysfunction and non-coordinated expression of antioxidant defense, besides inhibition of repair and recovery. In the present study, our objective was to study, in a panel of human malignant cells of various origins (lung carcinoma, squamous carcinoma, oral carcinoma, and glioblastoma), if the inhibitory activity of combination (2-DG+6-AN+2 Gy) against tumor growth could be considered a general phenomenon and to determine its effect on the cell cycle. The results revealed that combination (2-DG+6-AN+2 Gy) treatment result in significant cell growth inhibition and induced ROS generation in all cancer cells studied. The anti-proliferative effect was related to the ability of combination (2-DG+6-AN+2 Gy) to provoke growth inhibition at the G2/M arrest and apoptosis. Furthermore, combination (2-DG+6-AN+2 Gy) induced G2/M arrest is closely correlated to decreased cyclin A, cyclin B1, and cdc2 levels.