Glucose deprivation increases nuclear DNA repair protein Ku and resistance to radiation induced oxidative stress in human cancer cells.

Recent studies have indicated that nutrient deprivation particularly glucose may play a major role in tumor cell tolerance to a generally oxidative stress environment in solid tumors. Here, we studied the impact of glucose deprivation on the response of human colon (HT29) and prostate (DU145) cancer cells to gamma radiation. A significant decrease in intracellular glucose level was observed in glucose deprived cells as measured by bioreductive assay. The survival of HT29 and DU145 were increased by 30 and 100% respectively when these cells were exposed to gamma radiation in the absence of glucose compared to that in the presence of glucose. In glucose depleted medium, glutathione (GSH), a free radical scavenger, content remained the same, and showed no correlation with the radiation resistance induced by glucose deprivation. Glucose regulated protein78 (GRP78), a stress response survival protein, was not significantly increased in cells deprived of glucose for 4 h compared to those cells in glucose. DNA repair protein Ku, which is known to play a major role in cellular resistance to radiation, was significantly increased in glucose deprived cancer cells that showed enhanced radiation resistance. These results have demonstrated, for the first time, that glucose deprivation mediated stress increased the expression of nuclear Ku and resistance to radiation induced oxidative stress in human cancer cells. The additional resistance caused by glucose deprivation in cancer cells has clinical significance since solid tumors are known to have low level of glucose due to diffusion limited blood supply and higher metabolic activity.

DNA strand breakage by bivalent metal ions and ionizing radiation.

PURPOSE: To investigate mechanisms of DNA breakage via the interaction of bivalent metal ion, thiol reducing agent and ionizing radiation, in *OH scavenging abilities comparable to those in cells. MATERIALS AND METHODS: We measured the effects of 10 min exposure to 200 microM Fe2+ vs. Fe3+ on the induction of single (SSB) and double (DSB) strand breaks in unirradiated and oxically irradiated SV40 DNA, in aqueous solution containing 75 or 750 mM glycerol and/or 5 mM glutathione (GSH). RESULTS: Fe2+ or GSH alone produced little DNA damage. However, their combination produced a dramatic increase in the production of both SSB and DSB. Experiments with ferric ion suggest that it produces DNA damage only after partial reduction to ferrous by GSH. Induction efficiencies for SSB in the presence of Fe2+/GSH showed additivity of the effects of radiation alone with those from Fe2+/GSH. However, the corresponding induction efficiencies for DSB demonstrated a 2.5-fold enhancement. CONCLUSIONS: Our results are consistent with a model in which reduced bivalent metal ions plus thiols, in the presence of O2, produce DSB in DNA primarily via local clusters of hydroxyl radicals arising from site specific Fenton reactions. The synergism observed between DSB production by Fe/GSH and by ionizing radiation, also believed to occur via local clusters of hydroxyl radicals, is consistent with this model. Our results suggest that both normally present intracellular iron and ionizing radiation may be important sources of oxidative stress in cells.

Role of vicinal protein thiols in radiation and cytotoxic responses.

Glutathione (GSH) and more recently protein thiols (P-SH) have been found to play a major role in cellular radiation response. However, the effects of protein vicinal thiols, which are important for the functions of several major enzymes, on cellular responses to radiation have not been clearly delineated. Here we investigated the effects of depleting GSH and protein vicinal thiols (HS-P-SH) and P-SH on cell toxicity and radiation response. We used hydroxyethyldisulfide (HEDS, beta-mercaptoethanol-disulfide) alone and in combination with phenylarsine oxide (PAO) to alter P-SH, HS-P-SH and GSH. HEDS, a direct substrate for thioredoxin reductase and an indirect substrate for glutaredoxin (thioltransferase), did not alter protein vicinal thiols in cells. However, PAO, which specifically forms a covalent adduct with vicinal thiols, blocked bioreduction of HEDS; there was a concomitant and yet unexplained decrease in K1 cell GSH in the presence of HEDS and PAO. G6PD+ (K1) and G6PD- (E89) cells treated with L-buthionine sulfoximine (L-BSO) for 72 h to deplete GSH followed by PAO showed an increased cytotoxic response. However, the surviving E89 cells showed a 10,000-fold greater radiation lethality than the K1 cells. The effects of rapid depletion of GSH by a combination of L-BSO and dimethyfumarate (DMF), a glutathione-S-transferase substrate, were also investigated. Under these conditions, PAO radiosensitized the E89 cells more than 1000-fold over the K1 cells. The potential mechanisms for the altered response may be related to the inhibition of thioredoxin reductase and glutaredoxin. Both are key enzymes involved in DNA synthesis, protein homeostasis and cell survival. With GSH removed, vicinal thiols appear to play a critical role in determining cell survival and radiosensitivity. Decreasing P-SH and removing GSH and vicinal thiols is extremely toxic to K1 and E89 cells. We conclude that radiation sensitivity and cell survival are dependent on vicinal thiol and GSH. In the former and latter cases, the protein thiols are also important.

Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and gamma radiation.

Ku protein is a heterodimer (Ku70 and Ku86) known to play an important role in V(D)J recombination, apoptosis, telomere fusion, and double-strand break repair. Its role in double-strand breaks is relevant to cancer therapy because lack of Ku86 causes one of the most radiation-responsive phenotypes (hamster cells, XRS5). Although it is known that the heterodimer is necessary for the various functions of this protein, the impact of targeting Ku in human cancer cells has not been shown due to lack of appropriate approaches. It is also not known whether complete knock-out of Ku protein is required to enhance the sensitivity of human cells to gamma radiation as Ku protein is much more abundant in human cells than in hamster cells. In the current article, we have investigated the direct effect of Ku70 depletion in human cervical epithelioid (HeLa) and colon carcinoma (HCT116) cells. We specifically targeted Ku70 mRNA by use of small interfering RNA (siRNA). Of the five Ku70 siRNA synthesized, three inhibited the expression of Ku70 by up to 70% in HeLa cells. We have tested the effect of chemically synthesized siRNAs for target sequence 5 (CS #5) on the response of HeLa cells 72 hours after transfection to gamma radiation and etoposide, as this showed the maximum inhibition of Ku70 expression. Ku70 siRNA induced a decrease in the surviving fraction of irradiated HeLa cells by severalfold. Similar sensitizing effects were observed for etoposide, a topoisomerase II inhibitor. Studies with HCT116 cells using the same Ku70 siRNA (CS #5) showed a direct correlation between expression of Ku70 and sensitization to radiation and etoposide treatments.

Radiation response of cells during altered protein thiol redox.

The major focus of this work was to investigate how altered protein thiol redox homeostasis affects radiation-induced cell death. We used the cells of wild-type CHO cell line K1, the CHO cell line E89, which is null for G6PD activity, and a radiation-sensitive CHO cell line, XRS5. The protein-thiol redox status of cells was altered with cell-permeable disulfides, hydroxyethyldisulfide (HEDS) or lipoate. HEDS is primarily reduced by thioltransferase (glutaredoxin), with GSH as the electron donor. In contrast, lipoate is reduced by thioredoxin reductase. HEDS was reduced at a greater rate than lipoate by G6PD-containing K1 (wild-type) cells. Reduction of disulfides by G6PD-deficient cells was significantly slower with HEDS as substrate and was nearly absent with lipoate. The rate of reduction of HEDS by E89 cells decelerated to near zero by 30 min, whereas the reduction continued at nearly the same rate during the entire measurement period for K1 cells. HEDS treatment decreased the GSH and protein thiol (PSH) content more in G6PD-deficient cells than in G6PD-containing cells. On the other hand, lipoate did not significantly alter the protein thiol, but it increased the GSH in K1 cells. Acute depletion of GSH by l-buthionine-sulfoximine (l-BSO) in combination with dimethylfumarate significantly decreased the rate of reduction of HEDS by K1 cells close to that of G6PD-deficient cells. Prior GSH depletion by l-BSO alone significantly decreased the PSH in glucose-depleted E89 cells exposed to HEDS, but this did not occur with K1 cells. The radiation response of G6PD-deficient cells was significantly sensitized by HEDS, but HEDS did not have this effect on K1 cells. The DNA repair-deficient XRS5 CHO cells displayed the same capacity as K1 cells for HEDS reduction, and like K1 cells the XRS5 cells were not sensitized to radiation by HEDS treatment. Deprivation of glucose, which provides the substrate for G6PD in the oxidative pentose phosphate cycle, decreased the rate of bioreduction of HEDS and lipoate in G6PD-containing cells to the level in G6PD-deficient cells. In the absence of glucose, HEDS treatment diminished non-protein thiol and protein thiol to the same level as those in G6PD-deficient cells and sensitized the K1 cells to HEDS treatment. However, depletion of glucose did not alter the sensitivity of XRS5 cells in either the presence or absence of HEDS. Overall the results suggest a major role for pentose cycle control of protein redox state coupled to the activities of the thioltransferase and thioredoxin systems. The results also show that protein thiol status is a critical factor in cell survival after irradiation.

A bioactive probe for glutathione-dependent antioxidant capacity in breast cancer patients: implications in measuring biological effects of arsenic compounds.

INTRODUCTION: Glutathione, a major cellular non-protein thiol (NPSH), serves a central role in repairing damage induced by cancer drugs, pollutants and radiation and in the detoxification of several cancer chemotherapeutic drugs and toxins. Current methods measure glutathione levels only, which require cellular extraction, rather than the glutathione recycling dependent antioxidant activity in intact cells. Here, we present a novel method using a bioactive probe of the oxidative pentose phosphate cycle, termed the OxPhos™ test, to quantify glutathione recycling dependent antioxidant activity in whole blood and intact human and rodent cells without the need for the isolation and cytoplasm extraction of cells. METHODS: OxPhos™ test kit (Rockland Immunochemicals, USA), which uses hydroxyethyldisulfide (HEDS) as a probe for the oxidative pentose phosphate cycle, was used in these studies. The results with OxPhos™ test kit in human blood and intact cells were compared with total thiol and high pressure liquid chromatography/electrochemical detection of HEDS metabolism. RESULTS: The OxPhos™ test measured glutathione-dependent antioxidant activity both in intact human and rodent cells and breast cancer patient's blood with a better correlation coefficient and biological variability than the thiol assay. Additionally, human blood and mammalian cells treated with various arsenicals showed a concentration-dependent decrease in activity. DISCUSSION: The results demonstrate the application of this test for measuring the antioxidant capacity of blood and the effects of environmental pollutants/toxins. It opens up new avenues for an easy and reliable assessment of glutathione-dependent antioxidant capacity in various diseases such as stroke, blood borne diseases, infection, cardiovascular disease and other oxidative stress related diseases and as a prognostic indicator of chemotherapy response and toxicity. The use of this approach in pharmacology/toxicology including screening drugs that improve the glutathione-dependent antioxidant capacity and not just the glutathione level is clinically relevant since mammalian cells require glutathione dependent pathways for antioxidant activity.

A bioactive probe of the oxidative pentose phosphate cycle: novel strategy to reverse radioresistance in glucose deprived human colon cancer cells.

The specific effects of glucose deprivation on oxidative pentose phosphate cycle (OPPC) function, thiol homeostasis, protein function and cell survival remain unclear due to lack of a glucose-sensitive chemical probe. Using p53 wild type and mutant human colon cells, we determined the effects of hydroxyethyl disulfide (HEDS) on NADPH, GSH, GSSG, total glutathione, total non-protein and protein thiol levels, the function of the DNA repair protein Ku, and the susceptibility to radiation-induced free radicals under normal glucose or glucose-deprived conditions. HEDS is rapidly detoxified in normal glucose but triggered a p53-independent metabolic stress in glucose depleted state that caused loss of NADPH, protein and non-protein thiol homeostasis and Ku function, and enhanced sensitivity of both p53 wild type and mutant cells to radiation induced oxidative stress. Additionally, high concentration of HEDS alone induced cell death in p53 wild type cells without significant effect on p53 mutant cells. HEDS offers a useful tool to gain insights into how glucose metabolism affects OPPC dependent stress-induced cellular functions and injury, including in tumor cells, where our findings imply a novel therapeutic approach to target glucose deprived tumor. Our work introduces a novel probe to address cancer metabolism and ischemic pathology.

Development of polyamine transport ligands with improved metabolic stability and selectivity against specific human cancers.

Polyamine homeostasis is critical for life and is accomplished via a balance of polyamine biosynthesis, degradation, and transport. Rapidly dividing cancer cells have been shown to have high polyamine transport activity compared to normal cells, likely due to their high requirement for polyamine metabolites. The polyamine transport system (PTS) is a therapeutically relevant target, as it can provide selective drug delivery to cancer cells. This report describes the synthesis and biological evaluation of multimeric polyamine derivatives as efficient PTS ligands. Arylmethyl-polyamine derivatives were synthesized to address two important concerns in PTS drug design: (a) PTS selectivity and (b) stability to amine oxidases. N(1),N(1')-[Naphthalene-1,4-diylbis(methylene)]bis{N(4)-[4-(methylamino)butyl])butane-1,4-diamine}, 3b, was found to have an optimal balance between these parameters and demonstrated excellent targeting of melanoma (e.g., MALME-3M) and breast cancer cells (e.g., T47D) over other cancer cell lines. These results provide a method to selectively target cancers via their intrinsic need for polyamine metabolites.

Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro.

Cell viability assays have a variety of well known practical and technical limitations. All the available approaches have disadvantages, such as non-linearity, high background and cumbersome protocols. Several commonly used tetrazolium chemicals rely upon generation of a colored formazan product formed by mitochondrial reduction of these compounds via phenazine methosulfate (PMS). However, sensitivity is inherently limited because their reduction relies on mitochondrial bioreduction and cellular transport of PMS, as well as accessibility to tetrazolium chemicals. In this study, we identify hydroxethyldisulfide (HEDS) as an inexpensive probe that can measure cellular metabolic activity without the need of PMS. In tissue culture medium, HEDS accurately quantitated metabolically active live cells in a linear manner superior to tetrazolium based and other assays. Cell toxicity produced by chemotherapeutics (cisplatin, etoposide), oxidants (hydrogen peroxide, acetaminophen), toxins (phenyl arsine oxide, arsenite) or ionizing radiation was rapidly determined by the HEDS assay. We found that HEDS was superior to other commonly used assays for cell viability determinations in its solubility, membrane permeability, and intracellular conversion to a metabolic reporter that is readily transported into the extracellular medium. Our findings establish the use of HEDS in a simple, rapid and low cost assay to accurately quantify viable cells.

Mutation in G6PD gene leads to loss of cellular control of protein glutathionylation: mechanism and implication.

More than 400 million people are susceptible to oxidative stress due to glucose-6-phosphate dehydrogenase (G6PD) deficiency. Protein glutathionylation is believed to be responsible for loss of protein function and/or cellular signaling during oxidative stress. To elucidate the implications of G6PD deficiency specifically in cellular control of protein glutathionylation, we used hydroxyethyldisulfide (HEDS), an oxidant which undergoes disulfide exchange with existing thiols. G6PD deficient (E89) cells treated with HEDS showed a significant increase in protein glutathionylation compared to wild-type (K1) cells. In order to determine whether increase in global protein glutathionylation by HEDS leads to loss of function of an important protein, we compared the effect of HEDS on global protein glutathionylation with that of Ku protein function, a multifunctional DNA repair protein, using a novel ELISA. E89 cells treated with HEDS showed a significant loss of Ku protein binding to DNA. Cellular protein thiol and GSH, whose disulfide is involved in protein glutathionylation, were decreased by HEDS in E89 cells with no significant effect in K1 cells. E89 cells showed lower detoxification of HEDS, that is, conversion of disulfide HEDS to free sulfhydryl mercaptoethanol (ME), compared to K1 cells. K1 cells maintained their NADH level in the presence of HEDS but that of E89 cells decreased by tenfold following a similar exposure. NADPH, a cofactor required to maintain reduced form of the thiols, was decreased more in E89 than K1 cells. The specific role of G6PD in the control of such global protein glutathionylation and Ku function was further demonstrated by reintroducing the G6PD gene into E89 (A1A) cells, which showed a normal phenotype.

Detection of reactive oxygen species via endogenous oxidative pentose phosphate cycle activity in response to oxygen concentration: implications for the mechanism of HIF-1alpha stabilization under moderate hypoxia.

The oxidative pentose phosphate cycle (OPPC) is necessary to maintain cellular reducing capacity during periods of increased oxidative stress. Metabolic flux through the OPPC increases stoichiometrically in response to a broad range of chemical oxidants, including those that generate reactive oxygen species (ROS). Here we show that OPPC sensitivity is sufficient to detect low levels of ROS produced metabolically as a function of the percentage of O2. We observe a significant decrease in OPPC activity in cells incubated under severe and moderate hypoxia (ranging from <0.01 to 4% O2), whereas hyperoxia (95% O2) results in a significant increase in OPPC activity. These data indicate that metabolic ROS production is directly dependent on oxygen concentration. Moreover, we have found no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize hypoxia-inducible factor 1alpha (HIF-1alpha) under moderate hypoxia. Myxothiazol, an inhibitor of mitochondrial electron transfer, did not prevent HIF-1alpha stabilization under moderate hypoxia. Moreover, the levels of HIF-1alpha that we observed after exposure to moderate hypoxia were comparable between rho0 cells, which lack functional mitochondria, and the wild-type cells. Finally, we find no evidence for stabilization of HIF-1alpha in response to the non-toxic levels of H2O2 generated by the enzyme glucose oxidase. Therefore, we conclude that the oxygen dependence of the prolyl hydroxylase reaction is sufficient to mediate HIF-1alpha stability under moderate as well as severe hypoxia.

Mutation in the glucose-6-phosphate dehydrogenase gene leads to inactivation of Ku DNA end binding during oxidative stress.

Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the oxidative pentose phosphate cycle, regulates the NADPH/NADP(+) ratio in eukaryotic cells. G6PD deficiency is one of the most common mutations in humans and is known to cause health problems for hundreds of millions worldwide. Although it is known that decreased G6PD functionality can result in increased susceptibility to oxidative stress, the molecular targets of this stress are not known. Using a Chinese hamster ovary G6PD-null mutant, we previously demonstrated that exposure to a thiol-specific oxidant, hydroxyethyldisulfide, caused enhanced radiation sensitivity and an inability to repair DNA double strand breaks. We now demonstrate a molecular mechanism for these observations: the direct inhibition of DNA end binding activity of the Ku heterodimer, a DNA repair protein, by oxidation of its cysteine residues. Inhibition of Ku DNA end binding was found to be reversible by treatment of the nuclear extract with dithiothreitol, suggesting that the homeostatic regulation of reduced cysteine residues in Ku is a critical function of G6PD and the oxidative pentose cycle. In summary, we have discovered a new layer of DNA damage repair, that of the functional maintenance of repair proteins themselves. In view of the rapidly escalating number of roles ascribed to Ku, these results may have widespread ramifications.