Dehydroascorbic Acids-modified Polymer Micelles Target Cancer Cells to Enhance Anti-tumor Efficacy of Paclitaxel.

Paclitaxel (PTX), especially albumin-bound PTX in clinical, has displayed significant inhibition of tumor growth in patients. But the systemic distribution and poor water solubility of PTX often lead to severe side effects, consequently limiting the anti-tumor efficacy. In this study, we developed a novel PTX-loaded polymeric micelle drug delivery system. These self-assembled polymeric micelles from core to outside consisted of poly L-phenylalanine (pPhe), DTSSP linked poly L-lysine (pLys), poly ethylene glycol (PEG) and dehydroascorbic acids (DHA). pPhe formed the hydrophobic core to encapsulate PTX; DTSSPs on pLys covalently cross-linked and formed disulfide bond to stabilize PTX from loss in blood circulation; PEG improved solubility to lower toxicity of PTX for its high hydrophilicity; DHA targeted tumors by specifically recognizing GLUT1 mainly expressed on tumor cells. Thus, PTX would be precisely released into tumor cells with high dose of glutathione to break disulfide bond. Moreover, these PTX-loaded polymer micelles significantly suppressed tumor cell viability, proliferation, and migration in vitro, and also greatly inhibited tumor growth and prolonged survival in tumor-bearing mice without detectable side effects. Therefore, the new drug delivery system could reduce severe side effects and enhance anti-tumor efficacy of PTX via peripheral stabilization, low toxicity and tumor targeting.

Spectrophotometric method for fast quantification of ascorbic acid and dehydroascorbic acid in simple matrix for kinetics measurements.

A simple, rapid and reliable method was developed for quantifying ascorbic (AA) and dehydroascorbic (DHAA) acids and validated in 20mM malate buffer (pH 3.8). It consists in a spectrophotometric measurement of AA, either directly on the solution added with metaphosphoric acid or after reduction of DHAA into AA by dithiothreitol. This method was developed with real time measurement of reactions kinetics in bulk reactors in mind, and was checked in terms of linearity, limits of detection and quantification, fidelity and accuracy. The linearity was found satisfactory on the range of 0-6.95mM with limits of detection and quantification of 0.236mM and 0.467mM, respectively. The method was found acceptable in terms of fidelity and accuracy with a coefficient of variation for repeatability and reproducibility below 6% for AA and below 15% for DHAA, and with a recovery range of 97-102% for AA and 88-112% for DHAA.

Dehydroascorbic acid for the treatment of acute ischemic stroke.

In animal models of acute ischemic stroke, intravenous dehydroascorbic acid (DHAA), unlike ascorbic acid (AA), readily enters brain and is converted in both normal and ischemic brain into protective ascorbic acid. When given parenterally DHAA minimizes infarct volume and facilitates functional recovery. I hypothesize the same effect will occur in humans with acute ischemic stroke. Efficacy in reducing infarct volume is demonstrable in mice and rats even when DHAA is infused three hours after the experimental infarct. Moreover, there is fivefold mechanistic rational for DHA beside excellent pharmacokinetics and rapid penetration of brain and conversion to protective AA: (1) in ischemic brain, there is a precipitous decline in AA which can be reversed by intravenous DHAA; (2) after reduction of DHAA to AA in both normal and ischemic brain, AA can reduce oxidized vitamin E and glutathione, other protectors of brain against damaging reactive oxygen species which build up in ischemic brain; (3) AA itself can protect brain against damaging reactive oxygen species; (4) AA is an essential cofactor for several enzymes in brain including ten-eleven translocase-2 which upregulates production of protective molecules like brain-derived neurotrophic factor; and (5) DHAA after conversion to AA prevents both lipid oxidation and presumably oxidation of other labile substances (e.g., dopamine) in ischemic brain. In terms of safety, based on all available animal information, DHAA is safe in the proposed dosing regimen. For human clinical trials, the methodology for conducting the proposed animal safety, clinical pharmacology and phase II efficacy studies is straightforward. Finally, if DHAA preserved brain substance and function in humans, it could be employed in pre-hospital stroke patients.

L-dehydroascorbic acid can substitute l-ascorbic acid as dietary vitamin C source in guinea pigs.

Vitamin C deficiency globally affects several hundred million people and has been associated with increased morbidity and mortality in numerous studies. In this study, bioavailability of the oxidized form of vitamin C (l-dehydroascorbic acid or DHA)-commonly found in vitamin C containing food products prone to oxidation-was studied. Our aim was to compare tissue accumulation of vitamin C in guinea pigs receiving different oral doses of either ascorbate or DHA. In all tissues tested (plasma, liver, spleen, lung, adrenal glands, kidney, muscle, heart, and brain), only sporadic differences in vitamin C accumulation from ascorbate or DHA were observed except for the lowest dose of DHA (0.25mg/ml in the drinking water), where approximately half of the tissues had slightly yet significantly less vitamin C accumulation than from the ascorbate source. As these results contradicted data from rats, we continued to explore the ability to recycle DHA in blood, liver and intestine in guinea pigs, rats and mice. These investigations revealed that guinea pigs have similar recycling capacity in red blood cells as observed in humans, while rats and mice do not have near the same ability to reduce DHA in erythrocytes. In liver and intestinal homogenates, guinea pigs also showed a significantly higher ability to recycle DHA compared to rats and mice. These data demonstrate that DHA in guinea pigs-as in humans-is almost as effective as ascorbate as vitamin C source when it comes to taking up and storing vitamin C and further suggest that the guinea pig is superior to other rodents in modeling human vitamin C homeostasis.

Superoxide dictates the mode of U937 cell ascorbic acid uptake and prevents the enhancing effects of the vitamin to otherwise nontoxic levels of reactive oxygen/nitrogen species.

Exposure of U937 cells to low micromolar levels of ascorbic acid or dehydroascorbic acid, while resulting in identical ascorbic acid accumulation, is unexpectedly associated with remarkably different responses to exogenous oxidants. We observed that otherwise nontoxic levels of hydrogen peroxide, tert-butylhydroperoxide or peroxynitrite promote toxicity in cells preloaded with ascorbic acid, whereas hardly any effect was detected in cells pretreated with dehydroascorbic acid. Further experiments performed with peroxynitrite in cells preloaded with ascorbic acid provided evidence for a very rapid nonapoptotic death, preceded by early Bax mitochondrial translocation and by mitochondrial permeability transition. The notion that conversion of extracellular ascorbic acid to dehydroascorbic acid prevents the enhancing effects on oxidant toxicity and nevertheless preserves the net amount of vitamin C accumulated was also established using ascorbate oxidase as well as various sources of superoxide, namely, xanthine/xanthine oxidase or ATP-driven NADPH oxidase activation. These findings suggest that superoxide-dependent conversion of extracellular ascorbic acid to dehydroascorbic acid represents an important component of the overall survival strategy of some cell types to reactive oxygen/nitrogen species.

Hydroxyl radical generation dependent on extracellular ascorbate in rat striatum, as determined by microdialysis.

Ascorbate (AA), an antioxidant substance known as vitamin C, exists in the brain at a high concentration, although transfer into the brain after systemic administration of AA itself is limited. Intraperitoneal administration of dehydroascorbate (DHA) resulted in a rapid and progressive increase in extracellular AA in rat striatum in a dose-dependent manner. DHA administration increased 2,3- and 2,5-dihydroxybenzoate (2,3- and 2,5-DHBA) formation from salicylate in parallel with the increase in extracellular AA. Intrastriatal administration of active AA oxidase (AAO), but not the inactivated enzyme, completely suppressed the increase in 2,3- and 2,5-DHBA formation after the DHA administration. These findings suggest that extracellular AA might stimulate hydroxyl radical (OH) generation in the striatum. This is supported by the observation of dose-dependent OH generation upon intrastriatal administration of AA itself. In addition, deferoxamine, an iron chelator, decreased basal 2,3- and 2,5-DHBA formation and strongly, though not completely, suppressed the DHA-induced increase of 2,3- and 2,5-DHBA formation. Therefore, increased extracellular AA might function as a prooxidant and stimulate OH generation in cooperation with iron in rat striatum.

Vitamin C antagonizes the cytotoxic effects of antineoplastic drugs.

Vitamin C is an antioxidant vitamin that has been hypothesized to antagonize the effects of reactive oxygen species-generating antineoplastic drugs. The therapeutic efficacy of the widely used antineoplastic drugs doxorubicin, cisplatin, vincristine, methotrexate, and imatinib were compared in leukemia (K562) and lymphoma (RL) cell lines with and without pretreatment with dehydroascorbic acid, the commonly transported form of vitamin C. The effect of vitamin C on viability, clonogenicity, apoptosis, P-glycoprotein, reactive oxygen species (ROS), and mitochondrial membrane potential was determined. Pretreatment with vitamin C caused a dose-dependent attenuation of cytotoxicity, as measured by trypan blue exclusion and colony formation after treatment with all antineoplastic agents tested. Vitamin C given before doxorubicin treatment led to a substantial reduction of therapeutic efficacy in mice with RL cell-derived xenogeneic tumors. Vitamin C treatment led to a dose-dependent decrease in apoptosis in cells treated with the antineoplastic agents that was not due to up-regulation of P-glycoprotein or vitamin C retention modulated by antineoplastics. Vitamin C had only modest effects on intracellular ROS and a more general cytoprotective profile than N-acetylcysteine, suggesting a mechanism of action that is not mediated by ROS. All antineoplastic agents tested caused mitochondrial membrane depolarization that was inhibited by vitamin C. These findings indicate that vitamin C given before mechanistically dissimilar antineoplastic agents antagonizes therapeutic efficacy in a model of human hematopoietic cancers by preserving mitochondrial membrane potential. These results support the hypothesis that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response.

Genetic polymorphisms of xenobiotic enzymes affect human vitamin C excretion.

This study describes the effect of gene polymorphisms on the metabolism of vitamin C. An oral loading of 1 mmol ascorbic acid (176 mg) or dehydroascorbic (174 mg) was given to 17 healthy females volunteers who had consumed a low vitamin C diet (vitamin C < 5 mg/day) for 3 days before loading. The urinary total vitamin C was determined. The urinary excretion of vitamin C (VC) was compared between ascorbic acid (AsA) and dehydroascorbic acid (DAsA), and the 24 hour total VC excretion was same. However gene polymorphisms of glutathione S-transferases P1 (GSTP1) showed the effect on that excretion. GSTP1 is one of xenobiotic enzymes in VC metabolism. The VC excretions in 24 hour after VC loading were greater (P < .01) in AA homozygotes of GSTP1 (46.7 +/- 18.1 mg) than GA heterozygotes (28.2 +/- 14.0 mg). On the single oral administration, the type of polymorphisms of GSTP1 has stronger effect on VC metabolism than the form of VC, DAsA and AsA. This study showed that determination of nutrient requirement needs to be considered with personal genotype.

Inhibition of dehydroascorbic acid transport across the rat blood-retinal and -brain barriers in experimental diabetes.

Vitamin C is mainly transported across the blood-retinal and -brain barriers as dehydroascorbic acid (DHA) via a facilitative glucose transporter, GLUT1, and accumulates as ascorbic acid in the retina and brain. To investigate whether DHA transport to the retina and brain is changed by hyperglycemia, [14C]DHA transport across the blood-retinal and -brain barriers was examined using in vivo integration plot analysis in streptozotocin-induced diabetic rats with a 3-week duration of diabetes and in normal rats. Blood-to-retina and -brain transport of [14C]DHA was reduced by 65.5% and 84.1%, respectively, in diabetic rats compared with normal rats, whereas there was no major difference in the heart. Therefore, we propose that hyperglycemia reduces the supply of vitamin C to the retina and brain.

The effect of intracellular ascorbate on the susceptibility of HL60 and Jurkat cells to chemotherapy agents.

Chemotherapy agents initiate tumour cell apoptosis and this is thought to involve oxidative stress. In this study we have investigated the effect of the important antioxidant Vitamin C (ascorbate) on the response of HL60 and Jurkat cells to three chemotherapy drugs, namely etoposide, melphalan and arsenic trioxide (As(2)O(3)). Cells grown in routine culture media are deficient in ascorbate and to determine its effect on chemotherapy drug-induced apoptosis we supplemented the cells prior to drug exposure. We found that ascorbate had a varied effect on apoptosis and cell cycle progression. Etoposide-induced apoptosis in HL60 cells was significantly increased in ascorbate-loaded cells as measured by caspase-3 activation and DNA degradation, and this appeared to reflect a decrease in the number of necrotic cells rather than increased cytotoxicity. In contrast, ascorbate had no effect on etoposide-induced apoptosis in Jurkat cells. In both cell types melphalan-induced apoptosis was unaffected by intracellular ascorbate, whereas both apoptosis and growth arrest with low concentrations of As(2)O(3) were diminished. These results indicate that intracellular ascorbate can affect cell responses to chemotherapy drugs in a complex and somewhat unpredictable manner and that it may play an important role in the responsiveness of tumour cells to chemotherapy regimes.

Hyperglycemia inhibits the uptake of dehydroascorbate in tubular epithelial cell.

BACKGROUND/AIMS: Oxidative stress has been considered to be a common pathogenetic factor of diabetic nephropathy. But the reason why renal cells are susceptible to oxidative injury in diabetes is not clear. Vitamin C plays a central role in the antioxidant defense system and exists in two major forms. The charged form, ascorbate, is taken up into cells via sodium-dependent facilitated transport. The uncharged form, dehydroascorbate, enters cells via glucose transporter and is then converted back to ascorbate within these cells. Because dehydroascorbate and glucose compete for glucose transporters, hyperglycemia will exclude vitamin C from the cell and resulted in a decreased antioxidant capacity in some cell type that is dehydroascorbate dependent. As such, we hypothesized that some renal cells were dehydroascorbate dependent and the susceptibility of renal cells to glucose-induced injury was mediated by hyperglycemic exclusion of dehydroascorbate uptake through competing for glucose transporter. The aims of the present study were to determine whether tubular epithelial cell was dehydroascorbate dependent and the effect of dehydroascorbate on the production of reactive oxygen species in cells incubated by high glucose. METHODS: Tubular epithelial cell was cultured in RPMI-1640 medium containing 10% newborn calf serum. Intracellular ascorbate and dehydroascorbate contents were measured with vitamin C assay system. The intracellular formation of reactive oxygen species was detected with the fluorescent probe CM-H(2)DCFDA by using confocal microscopy. RESULTS: Ascorbate entry into the cells was not significantly different from background noise. In contrast, we observed a significant increase in the uptake of dehydroascorbate in tubular cell. At a dehydroascorbate concentration of 1 mM, increasing concentrations of glucose competitively inhibited dehydroascorbate entry into the cells such that the accumulation of dehydroascorbate was smaller than half maximal at about 22 mM glucose. Cytochalasin B, a kind of hexose transporter inhibitor, inhibited dehydroascorbate entry into the cells. At a glucose concentration of 25 mM, increasing concentrations of dehydroascorbate reduced reactive oxygen species generation in a dose-dependent manner when dehydroascorbate concentration was smaller than 4 mM. However, the inhibitory effect was not observed at 8 mM of dehydroascorbate. CONCLUSIONS: Tubular epithelial cells are dehydroascorbate dependent. Vitamin C exclusion from tubular epithelial cells through competition of glucose and dehydroascorbate for common transport mechanism in diabetes will deprive the cells of antioxidant ability and could lead to reactive oxygen species accumulation.

Regulation of vitamin C transport.

Ascorbic acid and dehydroascorbic acid (DHAA, oxidized vitamin C) are dietary sources of vitamin C in humans. Both nutrients are absorbed from the lumen of the intestine and renal tubules by, respectively, enterocytes and renal epithelial cells. Subsequently vitamin C circulates in the blood and enters all of the other cells of the body. Concerning flux across the plasma membrane, simple diffusion of ascorbic acid plays only a small or negligible role. More important are specific mechanisms of transport and metabolism that concentrate vitamin C intracellularly to enhance its function as an enzyme cofactor and antioxidant. The known transport mechanisms are facilitated diffusion of DHAA through glucose-sensitive and -insensitive transporters, facilitated diffusion of ascorbate through channels, exocytosis of ascorbate in secretory vesicles, and secondary active transport of ascorbate through the sodium-dependent vitamin C transporters SVCT1 and SVCT2 proteins that are encoded by the genes Slc23a1 and Slc23a2, respectively. Evidence is reviewed indicating that these transport pathways are regulated under physiological conditions and altered by aging and disease.

Facilitated glucose and dehydroascorbate transport in plant mitochondria.

Ascorbate, dehydroascorbate, and glucose transport was investigated in plant mitochondria and mitoplasts prepared from cultured BY2 tobacco cells. Using a rapid filtration method with radiolabeled ligands, we observed a specific glucose and dehydroascorbate transport, which was temperature and time dependent and saturable. Inhibition of mitochondrial respiration by KCN and the uncoupler 2,4-dinitrophenol did not influence the transport of the investigated compounds. Dehydroascorbate transport was inhibited by glucose and genistein, while glucose uptake was decreased upon 3-O-methyl-glucose, D-mannose, cytochalasin B or genistein addition. On the other hand, a low affinity low capacity ascorbate transport was found. Oxidizing agents (potassium ferricyanide or ascorbate oxidase) increased ascorbate uptake. The results demonstrate the presence of dehydroascorbate and glucose transport in plant mitochondria and suggest that it is mediated by the same or closely related transporter(s).

Vitamin C recycling is enhanced in the adaptive response to leptin-induced oxidative stress in keratinocytes.

Leptin acts on energy metabolism and plays a role in skin repair and in the modulation of cellular redox balance as well. Here, we investigated the effects of leptin on the redox homeostasis in keratinocytes, by evaluating reactive oxygen species (ROS) generation, glutathione content, antioxidant enzymes, activating protein 1 (AP-1) activity, and expression of AP-1-dependent, differentiation-specific genes. We also evaluated the systems involved in the maintenance of a positive ascorbate/dehydroascorbate ratio, i.e., transport and recycling. Leptin altered the keratinocyte redox state, as evident by enhanced ROS generation, oxidized/reduced glutathione ratio, and AP-1 activity. Still, this phenomenon was temporary. Indeed, we found an adaptive response, as demonstrated by an early induction of catalase and a late induction of specific dehydroascorbate reductase activities. In particular, leptin-treated cells showed an increased ability to reduce dehydroascorbate, both in a NADH, lipoic acid- and in a NADPH, thioredoxin-dependent manner. Our results show that leptin may induce adaptation to oxidative stress in skin, leading to an improved vitamin C homeostasis.

Ascorbic acid blunts oxidant stress due to menadione in endothelial cells.

Endothelial cells are exposed to potentially damaging reactive oxygen species generated both within the cells and in the bloodstream and underlying vessel wall. In this work, we studied the ability of ascorbic acid to protect cultured human-derived endothelial cells (EA.hy926) from oxidant stress generated by the redox cycling agent menadione. Menadione caused intracellular oxidation of dihydrofluorescein, which required the presence of D-glucose in the incubation medium, and was inhibited by intracellular ascorbate and desferrioxamine. At concentrations of 100 microM and higher, menadione depleted the cells of both GSH and ascorbate, and ascorbate loading partially prevented the decrease in GSH due to menadione. Menadione increased L-arginine uptake by the cells, but inhibited endothelial nitric oxide synthase, an effect that was prevented by acute loading with ascorbate. Ascorbate blunts menadione-induced oxidant stress in EA.hy926 cells, which may help to preserve nitric oxide synthase activity under conditions of excessive oxidant stress.

Very low vitamin C activity of orally administered L-dehydroascorbic acid.

The biological activity of L-dehydroascorbic acid (DHA), which is easily formed from L-ascorbic acid (ASC) during storage and cooking processes, has been considered to be equivalent to that of ASC on the basis of studies made several decades ago, when a specific method to determine ASC was not available. The nutritional activity of orally ingested DHA has now been evaluated by comparing ASC concentrations in 12 tissues of rats administered four different doses of ASC. Determinations were made by using the specific and sensitive method, which had been developed by us. Here it is shown that the efficiency of DHA was almost 10% of that of ASC on a molar basis, based on animal experiments using the inherently scorbutic ODS rat, which is a convenient human model animal to investigate the metabolism of vitamin C. On the basis of these findings, it is proposed that it is necessary to reevaluate the nutritional requirement of vitamin C based on both ASC and DHA contents of foods.

Intracellular flavonoids as electron donors for extracellular ferricyanide reduction in human erythrocytes.

Reduction of extracellular ferricyanide [Fe(CN)(6)](-3) to ferrocyanide by intact cells reflects the activity of a trans-plasma membrane oxidoreductase that, in human red blood cells, utilizes ascorbic acid as an electron donor. We herein report that the flavonoids quercetin and myricetin, while inhibiting dehydroascorbic acid uptake-and thus the erythrocyte ascorbic acid content-effectively stimulate the extracellular reduction of ferricyanide. Other flavonoids such as rutin, acacetin, apigenin, and genistein do not show the same effect. The notion that quercetin or myricetin may serve as an intracellular donor for a trans-plasma membrane oxidoreductase is supported by the following lines of evidence: (i) they afford direct reduction of ferricyanide; (ii) extracellular reduction of ferricyanide was not mediated by direct effects of the flavonoids released by the cells and was abolished by the sulphydryl reagent parachloromercuribenzenesulfonic acid (pCMBS); (iii) the intracellular concentrations of quercetin or myricetin well correlate with increases in ferricyanide reduction; (iv) the intracellular concentration of the flavonoids dramatically declines after ferricyanide exposure. Taken together, the results presented in this study demonstrate that myricetin and quercetin, which accumulate in large amounts in red blood cells, act as intracellular substrates of a pCMBS-sensitive trans-plasma membrane oxidoreductase. This may represent a novel mechanism whereby these flavonoids exert beneficial effects under oxidative stress conditions.