The evolutionarily conserved arginyltransferase 1 mediates a pVHL-independent oxygen-sensing pathway in mammalian cells.

The response to oxygen availability is a fundamental process concerning metabolism and survival/death in all mitochondria-containing eukaryotes. However, the known oxygen-sensing mechanism in mammalian cells depends on pVHL, which is only found among metazoans but not in other species. Here, we present an alternative oxygen-sensing pathway regulated by ATE1, an enzyme ubiquitously conserved in eukaryotes that influences protein degradation by posttranslational arginylation. We report that ATE1 centrally controls the hypoxic response and glycolysis in mammalian cells by preferentially arginylating HIF1α that is hydroxylated by PHD in the presence of oxygen. Furthermore, the degradation of arginylated HIF1α is independent of pVHL E3 ubiquitin ligase but dependent on the UBR family proteins. Bioinformatic analysis of human tumor data reveals that the ATE1/UBR and pVHL pathways jointly regulate oxygen sensing in a transcription-independent manner with different tissue specificities. Phylogenetic analysis suggests that eukaryotic ATE1 likely evolved during mitochondrial domestication, much earlier than pVHL.

2-Deoxy-d-glucose exploits increased glucose metabolism in cancer and viral-infected cells: Relevance to its use in India against SARS-CoV-2.

Mechanisms discovered to drive increased glucose metabolism in cancer cells are found to be similar to those in viral-infected cells. In this mini review, we summarize the major pathways by which the sugar analog, 2-Deoxy-d-glucose, has been shown to exploit increased glucose metabolism in cancer and how this information applies to viral-infected cells. Moreover, we highlight the relevance of these findings to the emergency approval of 2-Deoxy-d-glucose in India to be used against SARS-CoV-2, the virus responsible for COVID-19.

Regulation of Mitochondrial Respiratory Chain Complex Levels, Organization, and Function by Arginyltransferase 1.

Arginyltransferase 1 (ATE1) is an evolutionary-conserved eukaryotic protein that localizes to the cytosol and nucleus. It is the only known enzyme in metazoans and fungi that catalyzes posttranslational arginylation. Lack of arginylation has been linked to an array of human disorders, including cancer, by altering the response to stress and the regulation of metabolism and apoptosis. Although mitochondria play relevant roles in these processes in health and disease, a causal relationship between ATE1 activity and mitochondrial biology has yet to be established. Here, we report a phylogenetic analysis that traces the roots of ATE1 to alpha-proteobacteria, the mitochondrion microbial ancestor. We then demonstrate that a small fraction of ATE1 localizes within mitochondria. Furthermore, the absence of ATE1 influences the levels, organization, and function of respiratory chain complexes in mouse cells. Specifically, ATE1-KO mouse embryonic fibroblasts have increased levels of respiratory supercomplexes I+III2+IVn. However, they have decreased mitochondrial respiration owing to severely lowered complex II levels, which leads to accumulation of succinate and downstream metabolic effects. Taken together, our findings establish a novel pathway for mitochondrial function regulation that might explain ATE1-dependent effects in various disease conditions, including cancer and aging, in which metabolic shifts are part of the pathogenic or deleterious underlying mechanism.

Targeting the Kynurenine Pathway for the Treatment of Cisplatin-Resistant Lung Cancer.

Cisplatin resistance is a major barrier in the effective treatment of lung cancer. Cisplatin-resistant (CR) lung cancer cells do not primarily use glucose but rather consume amino acids such as glutamine and tryptophan (Trp) for survival. CR cells activate the kynurenine (KYN) pathway (KP) to cope with excessive reactive oxygen species (ROS) and maintain homeostasis for growth and proliferation. Consequently, indoleamine 2,3-dioxygenase-1 (IDO1) becomes an essential enzyme for CR cells' survival because it initiates and regulates the first step in the KP. Increased IDO1 activities and ROS levels are found in CR cells versus cisplatin-sensitive lung cancer. Importantly, significantly greater KYN/Trp ratio (P = 0.005) is detected in serum of patients who fail cisplatin when compared with naïve treatment. Knocking down IDO1 using shRNA or IDO1 inhibitors heightens ROS levels and results in a significant growth inhibitory effect only on CR cells and not on cisplatin-sensitive cells. Exposing CR cells to antioxidant (TIRON) results in suppression of IDO1 activity and confers resistance to IDO1 inhibition, indicating an interrelationship between ROS and IDO1. Because KYN plays a critical role in reprogramming naïve T cells to the immune-suppressive regulatory T-cell (T-reg) phenotype, we observed higher expression of TGFß, FoxP3, and CD4+CD25+ in mice bearing CR tumors compared with tumors from cisplatin-sensitive counterparts. IMPLICATIONS: Findings suggest that the enzyme-inhibitory activity and antitumor efficacy of IDO1 inhibitors rely in part on ROS levels, arguing that IDO1 expression alone may be insufficient to determine the clinical benefits for this class of experimental cancer drugs. Importantly, IDO1 inhibitors may be more suitable to treat patients with lung cancer who failed cisplatin therapy than naïve treatment patients.

Combining 2-deoxy-D-glucose with fenofibrate leads to tumor cell death mediated by simultaneous induction of energy and ER stress.

Unregulated growth and replication as well as an abnormal microenvironment, leads to elevated levels of stress which is a common trait of cancer. By inducing both energy and endoplasmic reticulum (ER) stress, 2-Deoxy-glucose (2-DG) is particularly well-suited to take advantage of the therapeutic window that heightened stress in tumors provides. Under hypoxia, blocking glycolysis with 2-DG leads to significant lowering of ATP resulting in energy stress and cell death in numerous carcinoma cell types. In contrast, under normoxia, 2-DG at a low-concentration is not toxic in most carcinomas tested, but induces growth inhibition, which is primarily due to ER stress. Here we find a synergistic toxic effect in several tumor cell lines in vitro combining 2-DG with fenofibrate (FF), a drug that has been safely used for over 40 years to lower cholesterol in patients. This combination induces much greater energy stress than either agent alone, as measured by ATP reduction, increased p-AMPK and downregulation of mTOR. Inhibition of mTOR results in blockage of GRP78 a critical component of the unfolded protein response which we speculate leads to greater ER stress as observed by increased p-eIF2α. Moreover, to avoid an insulin response and adsorption by the liver, 2-DG is delivered by slow-release pump yielding significant anti-tumor control when combined with FF. Our results provide promise for developing this combination clinically and others that combine 2-DG with agents that act synergistically to selectively increase energy and ER stress to a level that is toxic to numerous tumor cell types.

2-Deoxy-Glucose Downregulates Endothelial AKT and ERK via Interference with N-Linked Glycosylation, Induction of Endoplasmic Reticulum Stress, and GSK3ß Activation.

Interference with endothelial cell metabolism is a promising, yet unexploited strategy for angiogenesis inhibition. We reported that the glucose analogue 2-deoxy-D-glucose (2-DG) inhibits angiogenesis at significantly lower concentrations than those required for tumor cytotoxicity. Here, we found that hypersensitivity to 2-DG in endothelial cells is not associated with enhanced drug uptake compared with tumor cells, but with time-dependent, endothelial-selective inhibition of AKT and ERK phosphorylation. Downregulation of these critical survival pathways is shown to be due to 2-DG's interference with N-linked glycosylation, leading to alterations in VEGFR2 (and downstream signaling) as well as induction of endoplasmic reticulum (ER) stress, GSK3ß activation, and apoptosis. In vivo, periocular administration of 2-DG in LHBETATAG mice was associated with significant reduction of newly formed (CD105(+)) tumor capillaries, ER stress (GRP 78 expression), and endothelial apoptosis (TUNEL). These findings uniquely link N-linked glycosylation inhibition, ER stress, and ERK/AKT downregulation in endothelial cells, and provide a novel drug development strategy to overcome resistance mechanisms to currently available antiangiogenic agents.

Mcl-1 downregulation leads to the heightened sensitivity exhibited by BCR-ABL positive ALL to induction of energy and ER-stress.

BCR-ABL positive (+) acute lymphoblastic leukemia (ALL) accounts for ∼30% of cases of ALL. We recently demonstrated that 2-deoxy-d-glucose (2-DG), a dual energy (glycolysis inhibition) and ER-stress (N-linked-glycosylation inhibition) inducer, leads to cell death in ALL via ER-stress/UPR-mediated apoptosis. Among ALL subtypes, BCR-ABL+ ALL cells exhibited the highest sensitivity to 2-DG suggesting BCR-ABL expression may be linked to this increased vulnerability. To confirm the role of BCR-ABL, we constructed a NALM6/BCR-ABL stable cell line and found significant increase in 2-DG-induced apoptosis compared to control. We found that Mcl-1 was downregulated by agents inducing ER-stress and Mcl-1 levels correlated with ALL sensitivity. In addition, we showed that Mcl-1 expression is positively regulated by the MEK/ERK pathway, dependent on BCR-ABL, and further downregulated by combining ER-stressors with TKIs. We determined that energy/ER stressors led to translational repression of Mcl-1 via the AMPK/mTOR and UPR/PERK/eIF2α pathways. Taken together, our data indicate that BCR-ABL+ ALL exhibits heightened sensitivity to induction of energy and ER-stress through inhibition of the MEK/ERK pathway, and translational repression of Mcl-1 expression via AMPK/mTOR and UPR/PERK/eIF2α pathways. This study supports further consideration of strategies combining energy/ER-stress inducers with BCR-ABL TKIs for future clinical translation in BCR-ABL+ ALL patients.

ATF4 mediates necrosis induced by glucose deprivation and apoptosis induced by 2-deoxyglucose in the same cells.

Altered metabolism is a hallmark of cancer that opens new therapeutic possibilities. 2-deoxyglucose (2-DG) is a non-metabolizable glucose analog tested in clinical trials and is frequently used in experimental settings to mimic glucose starvation. However, in the present study, conducted in a rhabdomyosarcoma cell line, we find that 2-DG induces classical nuclear apoptotic morphology and caspase-dependent cell death, whereas glucose deprivation drives cells toward necrotic cell death. Necrosis induced by glucose deprivation did not resemble necroptosis or ferroptosis and was not prevented by antioxidants. Both stimuli promote endoplasmic reticulum stress. Moreover, the transcription factor ATF4 is found to mediate both the apoptosis induced by 2-DG and the glycosylation inhibitor tunicamycin, as well as the necrosis provoked by glucose withdrawal. Several hexoses partially prevented glucose deprivation-induced necrosis in rhabdomyosarcoma, although only mannose prevented apoptosis induced by 2-DG. In both cases, a reduction of cell death was associated with decreased levels of ATF4. Our results confirm previous data indicating the differential effects of these two forms with respect to inhibiting glucose metabolism, and they place endoplasmic reticulum stress as the critical mediator of glucose starvation-induced cell death.

The wonders of 2-deoxy-D-glucose.

Through the eons of time, out of all possible configurations, nature has selected glucose not only as a vital source of energy to sustain life but also as the molecule who's structure supplies the appropriate elements required for a cell to grow and multiply. This understanding, at least in part, explains the profound effects that the analog of glucose, 2-deoxy-d-glucose, has been shown to have on as common and widespread diseases as cancer, viral infection, aging-related morbidity, epilepsy, and others. This review is confined to summarizing some of the salient findings of this remarkable compound as they relate mainly to cancer.

Targeting cisplatin-resistant human tumor cells with metabolic inhibitors.

PURPOSE: Although cisplatin is the drug of choice in treating lung cancer patients, relapse and resistance is a common drawback to its clinical effectiveness. Based on cisplatin's reported ability to interfere with numerous cellular components, including mitochondria, we probed alterations in metabolism in cisplatin-resistant tumor cell lines to reveal targets for overcoming this important form of resistance. METHODS: Cisplatin-resistant lung and ovarian cancer cell lines were used to evaluate the efficacy of metabolic inhibitors for selectively targeting cisplatin-resistant cells under varying oxygen conditions. RESULTS: Three cisplatin-resistant cancer cell lines expressed lower HKII protein when compared to the respective cisplatin-sensitive cancer cell lines from which they were derived. Under anaerobic and hypoxic conditions, treatment with the glycolytic inhibitors 2-deoxyglucose (2-DG) and 2-fluorodeoxyglucose (2-FDG) correlated with increased cytotoxicity and more pronounced decreases in lactate production in cisplatin-resistant cells, indicating a greater blockage of glycolysis. Knockdown of HKI or HKII with siRNA in the parental lung cancer cell lines led to increased 2-FDG-induced cell death under anaerobic conditions. Under normal oxygen conditions, blockage of either fatty acid oxidation or deprivation of glutamine resulted in cell death in cisplatin-resistant lung cancer cell lines. CONCLUSIONS: Altered hexokinase levels in cisplatin-resistant cancer cell lines leads to increased sensitivity to glycolytic inhibition under anaerobic conditions, whereas under normoxic conditions, blockage of either fatty acid oxidation or deprivation of glutamine leads to cell death. These findings may be clinically applicable when considering cisplatin resistance.

Increased sensitivity to glucose starvation correlates with downregulation of glycogen phosphorylase isoform PYGB in tumor cell lines resistant to 2-deoxy-D-glucose.

BACKGROUND: As tumors evolve, they upregulate glucose metabolism while also encountering intermittent periods of glucose deprivation. Here, we investigate mechanisms by which pancreatic cancer cells respond to therapeutic (2-deoxy-D-glucose, 2-DG) and physiologic (glucose starvation, GS) forms of glucose restriction. METHODS: From a tumor cell line (1420) that is unusually sensitive to 2-DG under normoxia, low (14DG2)- and high (14DG5)-dose resistant cell lines were selected and used to probe the metabolic pathways involved with their response to different forms of glucose deprivation. RESULTS: Muted induction of the unfolded protein response was found to correlate with resistance to 2-DG. Additionally, 14DG2 displayed reduced 2-DG uptake, while 14DG5 was cross-resistant to tunicamycin, suggesting it has enhanced ability to manage glycosylation defects. Conversely, 2-DG-resistant cell lines were more sensitive than their parental cell line to GS, which coincided with lowered levels of glycogen phosphorylase (PYGB) and reduced breakdown of glycogen to glucose in the 2-DG-resistant cell lines. Moreover, by inhibiting PYGB in the parental cell line, sensitivity to GS was increased. CONCLUSIONS: Overall, the data demonstrate that the manner in which glucose is restricted in tumor cells, i.e., therapeutic or physiologic, leads to differential biological responses involving distinct glucose metabolic pathways. Moreover, in evolving tumors where glucose restriction occurs, the identification of PYGB as a metabolic target may have clinical application.

Conversion of 2-deoxyglucose-induced growth inhibition to cell death in normoxic tumor cells.

BACKGROUND: Inhibition of glucose metabolism has recently become an attractive target for cancer treatment. Accordingly, since 2-deoxyglucose (2-DG) competes effectively with glucose, it has come under increasing scrutiny as a therapeutic agent. The initial response of tumor cells to 2-DG is growth inhibition, which is thought to conserve energy and consequently protect cells from its ATP-lowering effects as a glycolytic inhibitor. However, since 2-DG also mimics mannose and thereby interferes with N-linked glycosylation, the question is raised of how this sugar analog inhibits tumor cell growth and whether the mechanism by which it protects cells can be manipulated to convert 2-DG-induced growth inhibition to cell death. METHODS: Cell growth and death were measured via counting viable and dead cells based on trypan blue exclusion. Markers of ATP reduction and the unfolded protein response (UPR) were detected by Western blot. Protein functions were manipulated through chemical compounds, siRNA and the use of gene-specific wild-type and knock-out mouse embryonic fibroblasts (MEFs). RESULTS: At 2-DG concentrations that can be achieved in human plasma without causing significant side effects, we find (a) It induces growth inhibition predominantly by interference with glycosylation, which leads to accumulation of unfolded proteins in the endoplasmic reticulum activating the UPR; (b) Inhibition of PERK (but not ATF6 or IRE1), a major component of the UPR, leads to conversion of 2-DG-induced growth inhibition to cell death and (c) secondarily to PERK, inhibition of GCN2, a kinase that is activated in response to low intracellular glutamine, increases 2-DG's cytotoxic effects in PERK -/- MEFs. CONCLUSIONS: Overall, these findings present a novel anticancer strategy that can be translated into therapeutic gain as they uncover the metabolic target PERK, and to a lesser degree GCN2, that when inhibited convert 2-DG's static effect to a toxic one in tumor cells growing under normoxia.

Endoplasmic reticulum stress induced by 2-deoxyglucose but not glucose starvation activates AMPK through CaMKKß leading to autophagy.

Autophagy, a well-conserved cellular self-eating process, has been shown to play a critical role in the pathophysiology of cancer. Previously, we reported that under normal O2 conditions (21% O2), the dual glucose metabolism inhibitor 2-deoxyglucose (2-DG) activates a cytoprotective autophagic response in cancer cells mainly through the induction of endoplasmic reticulum (ER) stress rather than ATP² reduction. However, the pathway(s) by which this occurs was unknown. Here, we find that ER stress induced by 2-DG as well as tunicamycin activates AMPK via Ca²âº-CaMKKß leading to stimulation of autophagy. These results suggest a new role for AMPK as a sensor of ER stress. In contrast, we find that although physiologic glucose starvation (GS) leads to ER stress which contributes to autophagy activation, it does so by a different mechanism. In addition to ER stress, GS also stimulates autophagy through lowering ATP and activating the canonical LKB1-AMPK energy sensing pathway as well as through increasing reactive oxygen species resulting in the activation of ERK. Furthermore, under hypoxia we observe that both 2-DG and GS inhibit rather than activate autophagy. This inhibition correlates with dramatically depleted ATP levels, and occurs through reduction of the PI3K III-Beclin1 complex for autophagy initiation, blockage of the conjugation of ATG12 to ATG5 for autophagosome expansion, as well as inhibition of the functional lysosomal compartment for autophagic degradation. Taken together, our data support a model where under normoxia therapeutic (2-DG) and physiologic (GS) glucose restriction differentially activate autophagy, while under hypoxia they similarly inhibit it.

Models and discovery strategies for new therapies of retinoblastoma.

INTRODUCTION: Retinoblastoma is the most common primary intraocular malignancy in children. Treatment success approaches 100% at specialized centers with globe-salvaging modalities. Primary management of retinoblastoma consists of chemoreduction with local consolidation, although newer techniques include local delivery via intra-arterial chemotherapy, periocular, or intravitreal injection. Animal models have played an integral role in the understanding of retinoblastoma tumorigenesis and have contributed to the development of globe-salvaging treatments. AREAS COVERED: This review focuses on the use of models and discovery strategies on translational research in retinoblastoma. The article reviews gene expression profiling, knockout models, and transgenic animal models. In addition, the review discusses translational applications of hyperthermia, chemotherapy (systemic and local delivery), radiation therapy, and novel targets such as hypoxia, angiogenesis, cellular metabolism, and tumor suppressor functions. EXPERT OPINION: Retinoblastoma treatment success has been driven by translational research using novel animal models and discovery strategies. Future developments require further understanding of the unique genetic events that result in tumor growth and development, as well as an understanding of the complex interactions of tumor cells with the local tumor microenvironment and how this affects tumor growth, metastasis, and response to treatment.

A phase I dose-escalation trial of 2-deoxy-D-glucose alone or combined with docetaxel in patients with advanced solid tumors.

PURPOSE: This phase I trial was initiated to evaluate the safety, pharmacokinetics (PK) and maximum tolerated dose (MTD) of the glycolytic inhibitor, 2-deoxy-D-glucose (2DG) in combination with docetaxel, in patients with advanced solid tumors. METHODS: A modified accelerated titration design was used. 2DG was administered orally once daily for 7 days every other week starting at a dose of 2 mg/kg and docetaxel was administered intravenously at 30 mg/m(2) for 3 of every 4 weeks beginning on day 1 of week 2. Following the completion of dose escalation, cohorts of patients were then treated with 2DG for 21 days or every day of each 4-week cycle for up to 12 cycles. RESULTS: Thirty-four patients were enrolled: 21 on every other week, 6 on a 21 of 28-day cycle and 7 on the continuous 2DG dosing schedule. There were no dose-limiting toxicities which met the MTD criteria. The most common adverse events were fatigue, sweating, dizziness and nausea mimicking the hypoglycemic symptoms expected from 2DG administration. Therefore, 63 mg/kg was selected as the clinically tolerable dose. The most significant adverse effects noted at 63-88 mg/kg doses were reversible hyperglycemia (100 %), gastrointestinal bleeding (6 %) and reversible grade 3 QTc prolongation (22 %). Eleven patients (32 %) had stable disease, 1 patient (3 %) partial response and 22 patients (66 %) progressive disease as their best response. There was no PK interaction between 2DG and docetaxel. CONCLUSION: The recommended dose of 2DG in combination with weekly docetaxel is 63 mg/kg/day with tolerable adverse effects.

Activation of the unfolded protein response by 2-deoxy-D-glucose inhibits Kaposi's sarcoma-associated herpesvirus replication and gene expression.

Lytic replication of the Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for the maintenance of both the infected state and characteristic angiogenic phenotype of Kaposi's sarcoma and thus represents a desirable therapeutic target. During the peak of herpesvirus lytic replication, viral glycoproteins are mass produced in the endoplasmic reticulum (ER). Normally, this leads to ER stress which, through an unfolded protein response (UPR), triggers phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), resulting in inhibition of protein synthesis to maintain ER and cellular homeostasis. However, in order to replicate, herpesviruses have acquired the ability to prevent eIF2α phosphorylation. Here we show that clinically achievable nontoxic doses of the glucose analog 2-deoxy-d-glucose (2-DG) stimulate ER stress, thereby shutting down eIF2α and inhibiting KSHV and murine herpesvirus 68 replication and KSHV reactivation from latency. Viral cascade genes that are involved in reactivation, including the master transactivator (RTA) gene, glycoprotein B, K8.1, and angiogenesis-regulating genes are markedly decreased with 2-DG treatment. Overall, our data suggest that activation of UPR by 2-DG elicits an early antiviral response via eIF2α inactivation, which impairs protein synthesis required to drive viral replication and oncogenesis. Thus, induction of ER stress by 2-DG provides a new antiherpesviral strategy that may be applicable to other viruses.

Inhibition of Akt potentiates 2-DG-induced apoptosis via downregulation of UPR in acute lymphoblastic leukemia.

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-D-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with D-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG-treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG-treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival.

Retinoblastoma treatment: impact of the glycolytic inhibitor 2-deoxy-d-glucose on molecular genomics expression in LH(BETA)T(AG) retinal tumors.

PURPOSE: The purpose of this study was to evaluate the effect of 2-deoxy-D-glucose (2-DG) on the spatial distribution of the genetic expression of key elements involved in angiogenesis, hypoxia, cellular metabolism, and apoptosis in LH(BETA)T(AG) retinal tumors. METHODS: The right eye of each LH(BETA)T(AG) transgenic mouse (n = 24) was treated with either two or six subconjunctival injections of 2-DG (500 mg/kg) or saline control at 16 weeks of age. A gene expression array analysis was performed on five different intratumoral regions (apex, center, base, anterior-lateral, and posterior-lateral) using Affymetrix GeneChip Mouse Gene 1.0 ST arrays. To test for treatment effects of each probe within each region, a two-way analysis of variance was used. RESULTS: Significant differences between treatment groups (ie, 0, 2, and 6 injections) were found as well as differences among the five retinal tumor regions evaluated (P < 0.01). More than 100 genes were observed to be dysregulated by ≥2-fold difference in expression between the three treatment groups, and their dysregulation varied across the five regions assayed. Several genes involved in pathways important for tumor cell growth (ie, angiogenesis, hypoxia, cellular metabolism, and apoptosis) were identified. CONCLUSIONS: 2-DG was found to significantly alter the gene expression in LH(BETA)T(AG) retinal tumor cells according to their location within the tumor as well as the treatment schedule. 2-DG's effects on genetic expression found here correlate with previous reported results on varied processes involved in its in vitro and in vivo activity in inhibiting tumor cell growth.

The relationship of thioredoxin-1 and cisplatin resistance: its impact on ROS and oxidative metabolism in lung cancer cells.

Elimination of cisplatin-resistant lung cancer cells remains a major obstacle. We have shown that cisplatin-resistant tumors have higher reactive oxygen species (ROS) levels and can be exploited for targeted therapy. Here, we show that increased secretion of the antioxidant thioredoxin-1 (TRX1) resulted in lowered intracellular TRX1 and contributed to higher ROS in cisplatin-resistant tumors in vivo and in vitro. By reconstituting TRX1 protein in cisplatin-resistant cells, we increased sensitivity to cisplatin but decreased sensitivity to elesclomol (ROS inducer). Conversely, decreased TRX1 protein in parental cells reduced the sensitivity to cisplatin but increased sensitivity to elesclomol. Cisplatin-resistant cells had increased endogenous oxygen consumption and mitochondrial activity but decreased lactic acid production. They also exhibited higher levels of argininosuccinate synthetase (ASS) and fumarase mRNA, which contributed to oxidative metabolism (OXMET) when compared with parental cells. Restoring intracellular TRX1 protein in cisplatin-resistant cells resulted in lowering ASS and fumarase mRNAs, which in turn sensitized them to arginine deprivation. Interestingly, cisplatin-resistant cells also had significantly higher basal levels of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Overexpressing TRX1 lowered ACC and FAS proteins expressions in cisplatin-resistant cells. Chemical inhibition and short interfering RNA of ACC resulted in significant cell death in cisplatin-resistant compared with parental cells. Conversely, TRX1 overexpressed cisplatin-resistant cells resisted 5-(tetradecyloxy)-2-furoic acid (TOFA)-induced death. Collectively, lowering TRX1 expression through increased secretion leads cisplatin-resistant cells to higher ROS production and increased dependency on OXMET. These changes raise an intriguing therapeutic potential for future therapy in cisplatin-resistant lung cancer.

2-Deoxy-D-glucose activates autophagy via endoplasmic reticulum stress rather than ATP depletion.

PURPOSE: The glucose analog and glycolytic inhibitor 2-deoxy-D-glucose (2-DG), which is currently under clinical evaluation for targeting cancer cells, not only blocks glycolysis thereby reducing cellular ATP, but also interferes with N-linked glycosylation, which leads to endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). Both bioenergetic challenge and ER stress have been shown to activate autophagy, a bulk cellular degradation process that plays either a pro- or anti-death role. Here, we investigate which pathway 2-DG interferes with that activates autophagy and the role of this process in modulating 2-DG-induced toxicity. METHODS: Pancreatic cancer cell line 1420, melanoma cell line MDA-MB-435 and breast cancer cell line SKBR3 were used to investigate the relationship between induction by 2-DG treatment of ER stress/UPR, ATP reduction and activation of autophagy. ER stress/UPR (Grp78 and CHOP) and autophagy (LC3B II) markers were assayed by immunoblotting, while ATP levels were measured using the CellTiter-Glo Luminescent Cell Viability Assay. Autophagy was also measured by immunofluorescence utilizing LC3B antibody. Cell death was detected with a Vi-Cell cell viability analyzer using trypan blue exclusion. RESULTS: In the three different cancer cell lines described earlier, we find that 2-DG upregulates autophagy, increases ER stress and lowers ATP levels. Addition of exogenous mannose reverses 2-DG-induced autophagy and ER stress but does not recover the lowered levels of ATP. Moreover, under anaerobic conditions where 2-DG severely depletes ATP, autophagy is diminished rather than activated, which correlates with lowered levels of the ER stress marker Grp78. Additionally, when autophagy is blocked by siRNA, cell sensitivity to 2-DG is increased corresponding with upregulation of ER stress-mediated apoptosis. Similar increased toxicity is observed with 3-methyladenine, a known autophagy inhibitor. In contrast, rapamycin which enhances autophagy reduces 2-DG-induced toxicity. CONCLUSIONS: Overall, these results indicate that the major mechanism by which 2-DG stimulates autophagy is through ER stress/UPR and not by lowering ATP levels. Furthermore, autophagy plays a protective role against 2-DG-elicited cell death apparently by relieving ER stress. These data suggest that combining autophagy inhibitors with 2-DG may be useful clinically.