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.

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.

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.

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.

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.

Intrinsically lower AKT, mammalian target of rapamycin, and hypoxia-inducible factor activity correlates with increased sensitivity to 2-deoxy-D-glucose under hypoxia in lung cancer cell lines.

Down-regulation by small interfering RNA or absence of hypoxia-inducible factor (HIF-1alpha) has been shown to lead to increased sensitivity to glycolytic inhibitors in hypoxic tumor cells. In surveying a number of tumor types for differences in intrinsic levels of HIF under hypoxia, we find that the reduction of the upstream pathways of HIF, AKT, and mammalian target of rapamycin (mTOR) correlates with increased toxic effects of 2-deoxy-D-glucose (2-DG) in lung cancer cell lines when treated under hypoxia. Because HIF-1alpha translation is regulated by mTOR, we examined the effects of blocking mTOR under hypoxia with an analogue of rapamycin (CCI-779) in those cell lines that showed increased mTOR and AKT activity and found that HIF-1alpha down-regulation coincided with increased 2-DG killing. CCI-779, however, was ineffective in increasing 2-DG toxicity in cell lines that did not express HIF. These results support the hypothesis that although mTOR inhibition leads to the blockage of numerous downstream targets, CCI-779 increases the toxicity of 2-DG in hypoxic cells through down-regulation of HIF-1alpha. Overall, our findings show that CCI-779 hypersensitizes hypoxic tumor cells to 2-DG and suggests that the intrinsic expression of AKT, mTOR, and HIF in lung cancer, as well as other tumor types, may be important in dictating the decision on how best to use 2-DG alone or in combination with CCI-799 to kill hypoxic tumor cells clinically.

Hypoxia-inducible factor-1 confers resistance to the glycolytic inhibitor 2-deoxy-D-glucose.

Hypoxic regions within solid tumors harbor cells that are resistant to standard chemotherapy and radiotherapy. Because oxygen is required to produce ATP by oxidative phosphorylation, under hypoxia, cells rely more on glycolysis to generate ATP and are thereby sensitive to 2-deoxy-d-glucose (2-DG), an inhibitor of this pathway. Universally, cells respond to lowered oxygen tension by increasing the amount of glycolytic enzymes and glucose transporters via the well-characterized hypoxia-inducible factor-1 (HIF). To evaluate the effects of HIF on 2-DG sensitivity, the following three models were used: (a) cells treated with oligomycin to block mitochondrial function in the presence (HIF(+)) or absence (HIF(-)) of hypoxia, (b) cells treated with small interfering RNA specific for HIF-1alpha and control cells cultured under hypoxia, and (c) a mutant cell line unable to initiate the HIF response and its parental HIF(+) counterpart under hypoxic conditions. In all three models, HIF increased resistance to 2-DG and other glycolytic inhibitors but not to other chemotherapeutic agents. Additionally, HIF reduced the effects of 2-DG on glycolysis (as measured by ATP and lactate assays). Because HIF increases glycolytic enzymes, it follows that greater amounts of 2-DG would be required to inhibit glycolysis, thereby leading to increased resistance to it under hypoxia. Indeed, hexokinase, aldolase, and lactate dehydrogenase were found to be increased as a function of HIF under the hypoxic conditions and cell types we used; however, phosphoglucose isomerase was not. Although both hexokinase and phosphoglucose isomerase are known to interact with 2-DG, our findings of increased levels of hexokinase more likely implicate this enzyme in the mechanism of HIF-mediated resistance to 2-DG. Moreover, because 2-DG is now in phase I clinical trials, our results suggest that glycolytic inhibitors may be more effective clinically when combined with agents that inhibit HIF.

Under normoxia, 2-deoxy-D-glucose elicits cell death in select tumor types not by inhibition of glycolysis but by interfering with N-linked glycosylation.

In tumor cells growing under hypoxia, inhibiting glycolysis with 2-deoxy-d-glucose (2-DG) leads to cell death, whereas under normoxic conditions cells similarly treated survive. Surprisingly, here we find that 2-DG is toxic in select tumor cell lines growing under normal oxygen tension. In contrast, a more potent glycolytic inhibitor, 2-fluorodeoxy-d-glucose, shows little or no toxicity in these cell types, indicating that a mechanism other than inhibition of glycolysis is responsible for their sensitivity to 2-DG under normoxia. A clue to this other mechanism comes from previous studies in which it was shown that 2-DG interferes with viral N-linked glycosylation and is reversible by exogenous addition of mannose. Similarly, we find that 2-DG interferes with N-linked glycosylation more potently in the tumor cell types that are sensitive to 2-DG under normoxia, which can be reversed by exogenous mannose. Additionally, 2-DG induces an unfolded protein response, including up-regulation of GADD153 (C/EBP-homologous protein), an unfolded protein response-specific mediator of apoptosis, more effectively in 2-DG-sensitive cells. We conclude that 2-DG seems to be toxic in select tumor cell types growing under normoxia by inhibition of N-linked glycosylation and not by glycolysis. Because in a phase I study 2-DG is used in combination with an anticancer agent to target hypoxic cells, our results raise the possibility that in certain cases, 2-DG could be used as a single agent to selectively kill both the aerobic (via interference with glycosylation) and hypoxic (via inhibition of glycolysis) cells of a solid tumor.

Differential toxic mechanisms of 2-deoxy-D-glucose versus 2-fluorodeoxy-D-glucose in hypoxic and normoxic tumor cells.

The dependence of hypoxic tumor cells on glycolysis as their main means of producing ATP provides a selective target for agents that block this pathway, such as 2-deoxy-D-glucose (2-DG) and 2-fluoro-deoxy-D-glucose (2-FDG). Moreover, it was demonstrated that 2-FDG is a more potent glycolytic inhibitor with greater cytotoxic activity than 2-DG. This activity correlates with the closer structural similarity of 2-FDG to glucose than 2-DG, which makes it a better inhibitor of hexokinase, the first enzyme in the glycolytic pathway. In contrast, because of its structural similarity to mannose, 2-DG is known to be more effective than 2-FDG in interfering with N-linked glycosylation. Recently, it was reported that 2-DG, at a relatively low dose, is toxic to certain tumor cells, even under aerobic conditions, whereas 2-FDG is not. These results indicate that the toxic effects of 2-DG in selected tumor cells under aerobic conditions is through inhibition of glycosylation rather than glycolysis. The intention of this minireview is to discuss the effects and potential clinical impact of 2-DG and 2-FDG as antitumor agents and to clarify the differential mechanisms by which these two glucose analogues produce toxicity in tumor cells growing under anaerobic or aerobic conditions.

Efficacy of 2-halogen substituted D-glucose analogs in blocking glycolysis and killing "hypoxic tumor cells".

PURPOSE: Since 2-deoxy-D-glucose (2-DG) is currently in phase I clinical trials to selectively target slow-growing hypoxic tumor cells, 2-halogenated D-glucose analogs were synthesized for improved activity. Given the fact that 2-DG competes with D-glucose for binding to hexokinase, in silico modeling of molecular interactions between hexokinase I and these new analogs was used to determine whether binding energies correlate with biological effects, i.e. inhibition of glycolysis and subsequent toxicity in hypoxic tumor cells. METHODS AND RESULTS: Using a QSAR-like approach along with a flexible docking strategy, it was determined that the binding affinities of the analogs to hexokinase I decrease as a function of increasing halogen size as follows: 2-fluoro-2-deoxy-D-glucose (2-FG) > 2-chloro-2-deoxy-D-glucose (2-CG) > 2-bromo-2-deoxy-D-glucose (2-BG). Furthermore, D-glucose was found to have the highest affinity followed by 2-FG and 2-DG, respectively. Similarly, flow cytometry and trypan blue exclusion assays showed that the efficacy of the halogenated analogs in preferentially inhibiting growth and killing hypoxic vs. aerobic cells increases as a function of their relative binding affinities. These results correlate with the inhibition of glycolysis as measured by lactate inhibition, i.e. ID50 1 mM for 2-FG, 6 mM for 2-CG and > 6 mM for 2-BG. Moreover, 2-FG was found to be more potent than 2-DG for both glycolytic inhibition and cytotoxicity. CONCLUSIONS: Overall, our in vitro results suggest that 2-FG is more potent than 2-DG in killing hypoxic tumor cells, and therefore may be more clinically effective when combined with standard chemotherapeutic protocols.

2-deoxy-D-glucose increases the efficacy of adriamycin and paclitaxel in human osteosarcoma and non-small cell lung cancers in vivo.

Slow-growing cell populations located within solid tumors are difficult to target selectively because most cells in normal tissues also have low replication rates. However, a distinguishing feature between slow-growing normal and tumor cells is the hypoxic microenvironment of the latter, which makes them extraordinarily dependent on anaerobic glycolysis for survival. Previously, we have shown that hypoxic tumor cells exhibit increased sensitivity to inhibitors of glycolysis in three distinct in vitro models. Based on these results, we predicted that combination therapy of a chemotherapeutic agent to target rapidly dividing cells and a glycolytic inhibitor to target slow-growing tumor cells would have better efficacy than either agent alone. Here, we test this strategy in vivo using the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) in combination with Adriamycin (ADR) or paclitaxel in nude mouse xenograft models of human osteosarcoma and non-small cell lung cancer. Nude mice implanted with osteosarcoma cells were divided into four groups as follows: (a) untreated controls; (b) mice treated with ADR alone; (c) mice treated with 2-DG alone; or (d) mice treated with a combination of ADR + 2-DG. Treatment began when tumors were either 50 or 300 mm(3) in volume. Starting with small or large tumors, the ADR + 2-DG combination treatment resulted in significantly slower tumor growth (and therefore longer survival) than the control, 2-DG, or ADR treatments (P < 0.0001). Similar beneficial effects of combination treatment were found with 2-DG and paclitaxel in the MV522 non-small cell lung cancer xenograft model. In summary, the treatment of tumors with both the glycolytic inhibitor 2-DG and ADR or paclitaxel results in a significant reduction in tumor growth compared with either agent alone. Overall, these results, combined with our in vitro data, provide a rationale for initiating clinical trials using glycolytic inhibitors in combination with chemotherapeutic agents to increase their therapeutic effectiveness.

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.

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.

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.

Hypoxia increases tumor cell sensitivity to glycolytic inhibitors: a strategy for solid tumor therapy (Model C).

Previously, we reported that two distinct in vitro tumor cell models of hypoxia (Models A and B) are hypersensitive to glycolytic inhibitors such as 2-deoxy-D-glucose (2-DG) and oxamate [Liu et al., Biochemistry 2001;40:5542-7]. Model A consists of osteosarcoma cells (143B) treated with agents that interfere with mitochondrial oxidative phosphorylation (OxPhos), and Model B represents rho(0) cells, a variant derived from 143B cells, which, due to their deficiency in mitochondrial DNA, cannot perform OxPhos. Extending these studies, we report here on Model C, which is composed of 143B cells grown under various levels of external O(2) (0, 0.1, 0.5, 1, 5, 10, and 21%). At the lower levels of O(2) that we tested, 143B cells were hypersensitive to 2-DG and oxamate when compared with cells grown at a normal level of O(2). In contrast, 143B cells under hypoxic or aerobic conditions showed equal sensitivity to a standard chemotherapeutic agent, vinblastine. Furthermore, when treated under reduced O(2) amounts, rho(0) cells displayed no hypersensitivity to 2-DG and, in fact, were slightly more resistant than under aerobic conditions. At 0-5% O(2) levels, untreated 143B cells displayed reduced growth and elevated lactic acid levels, while rho(0) cell growth remained unaffected except at 0% O(2) where growth was inhibited by 19%. The results with Model C are in agreement with our previous data using Models A and B, and extend these studies by illustrating that within a wide range of hypoxia the growth of tumor cells is retarded and that these slow-growing cells become hypersensitized to glycolytic inhibitors. Taken together with Models A and B, the data with Model C support our hypothesis that the hypoxic environment of slow-growing cells found in the inner core of solid tumors renders them amenable to selective targeting with glycolytic inhibitors.

Multidrug resistance correlates with overexpression of Muc4 but inversely with P-glycoprotein and multidrug resistance related protein in transfected human melanoma cells.

Due to the size, glycosylation, and location in the plasma membrane of the sialomucin complex Muc4, which has been implicated in ErbB2 signaling, in the repression of apoptosis and cell adhesion, and in tumor metastasis, studies were initiated to determine whether its presence could influence cell sensitivity to anticancer drugs. Growth inhibition assays using melanoma cell lines that either express the glycoprotein (Muc4(+)) or do not (Muc4(-)) showed that Muc4 renders cells resistant to taxol, doxorubicin, vinblastine, rhodamine 123, and 2-deoxyglucose. When treated with various concentrations of doxorubicin, Muc4(+) cells were blocked less frequently in G(2) and underwent less DNA fragmentation (apoptosis and/or necrosis) than Muc4(-) cells. All of the drugs tested (except for 2-deoxyglucose) are well recognized by P-glycoprotein-mediated multidrug resistance 1 (MDR1) and to a lesser degree by multidrug resistance related protein 1 (MRP1) transporters. Therefore, transporter gene expression in these cells was assayed. Surprisingly, Muc4(+) cells expressed lower levels of both transporter genes than Muc4(-) cells. Moreover, rhodamine 123 was retained more highly in the Muc4(+) than in the Muc4(-) cells, demonstrating that these transporters are functional. Overall, these results indicate that although Muc4(+) cells express less MDR1 and MRP1, they are more resistant to drugs recognized by these transporters.

Greater cell cycle inhibition and cytotoxicity induced by 2-deoxy-D-glucose in tumor cells treated under hypoxic vs aerobic conditions.

PURPOSE: In order to investigate the hypothesis that cells found in hypoxic areas of solid tumors are more sensitive to glycolytic inhibitors than cells growing aerobically, we have previously characterized three distinct in vitro models (A, B and C) that simulate this condition. In all of the models it was shown that cells growing under hypoxic conditions are hypersensitive to the glycolytic inhibitor 2-deoxy- d-glucose (2-DG). However, in those studies cytostatic and cytotoxic effects were not distinguished from one another. Since successful treatment of cancer includes not only slowing down but also actually killing tumor cells, studies were undertaken to assess the effects of 2-DG on cell cycle progression and cell death. METHODS AND RESULTS: Using flow cytometry and cell viability assays, it was found that 2-DG caused significantly greater cell cycle inhibition and cell death in all three hypoxic models as compared to aerobically growing control cells. In model A (a chemically induced model of hypoxia in which rhodamine-123 is used to block oxidative phosphorylation), 1200 microg/ml of 2-DG was shown to induce more cell cycle arrest in late S/G(2) and more cell death than in the aerobic cell counterpart treated with 3600 microg/ml 2-DG. In rho(0) cells which are genetically constructed to be unable to perform oxidative phosphorylation (model B), an even greater window of selectivity (more than tenfold) between hypoxic and aerobic cells was found when considering 2-DG's effects on cell cycle arrest and cell death. In the environmental model (model C), where cells were grown under reduced amounts of external oxygen (0.1%), hypersensitivity to the effects of 2-DG with respect to cell cycle arrest and cell death were also observed. CONCLUSIONS: Overall, these results indicate that cells growing under anaerobic conditions respond with greater sensitivity to the effects of 2-DG on cell cycle inhibition and cell death than those growing under aerobic conditions. This supports our contention that glycolytic inhibitors added to standard chemotherapeutic protocols should increase treatment efficacy by selectively killing the slow-growing cells, which are found in the hypoxic portions of solid tumors, while sparing most of the normal cells that are also slow-growing but are living under aerobic conditions.

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.