Cytoprotection of vascular endotheliocytes by phosphorylated ascorbate through suppression of oxidative stress that is generated immediately after post-anoxic reoxygenation or with alkylhydroperoxides

Vascular endotheliocytes BAE-2 underwent the gradually proceeding cell death until 48 h after reoxygenation (Reox) following 3 h anoxia (Anox), but protected by pre-Anox administration with L-ascorbic acid (Asc)-2-O-phosphate (Asc2P), an autooxidation-resistant Asc derivative, but not by Asc itself. This cytoprotection with Asc2P was achieved in a glucose (Glc)-lacking buffer more advantageously than in a Glc-containing buffer where less efficiency had been demonstrated for Asc entry into BAE-2 cells than in a Glc-lacking buffer. Superoxide anion radicals were detected explosively in the extracellular space at 2-5 min after Reox following the Anox treatment of HUVE endotheliocytes, and were thereafter retained at levels as high as approximately one-half of the maximum level until 60 min after Reox, as shown by cytochrome c reduction assay. Superoxide anions at 3 and 60 min after Reox were suppressed by pre-Anox administration with Asc2P, but not with Asc or dehydro-Asc, and were not suppressed by post-Anox administration with Asc2P; the cytoprotection may need the intracellular accumulation of the ROS-scavenging effector Asc that is converted from Asc2P until 3 min after Reox. The ROS-generator tert-butylhydroperoxide (t-BuOOH) also induced both the diminished cell viability and nuclear DNA strand cleavages of BAE-2 endotheliocytes, which were also protected dose-dependently with Asc2P. The cytoprotection was attributed to reduction of intracellular ROS including hydroperoxide and hydrogen peroxide with Asc2P as shown by fluorometry with the redox indicator CDCFH-DA. Thus Anox/Reox-induced cell death can be prevented by Asc2P that suppresses ROS-generation immediately after Reox following Anox more efficiently in the intracellular sphere rather than in the extracellular space.     

维生素C衍生物对癌细胞浸润转移能力的抑制作用机理研究

前期研究表明Asc2P6Plm能够有效地抑制癌细胞的浸润转移，本文试图以Asc2P6Plm对人成纤维瘤细胞浸润转移作用探讨维生素C衍生物对癌细胞转移能力抑制的机理，对HT-1080细胞分别以50-300μmol/LAsc2P6Plm处理1h，随着Asc2P6Plm浓度的增大，细胞移动的数目明显减少，Asc2P6Plm对HT-1080细胞移动的抑制作用呈现出量效关系，Asc2P6Plm对ROS的清除作用，通过自旋捕集剂DMPO以电子自旋共振方法进行研究，HT-1080细胞经Asc2P6Plm处理后，细胞内的自由基水平与对照组相比有显著的降低，用F-actin的分子探针NBD研究表明，随处理时间延长，细胞内荧光强度与对照组相比显著降低，Western blots研究表明，细胞核内的RhoA蛋白量随Asc2P6Plm处理时间延长而逐渐增加，研究提示，Asc2P6Plm对癌细胞浸润转移能力的抑制作用是与抑制癌细胞内的ROS，提高细胞核内RhoA水平，降低细胞质内F-actin相关。     

The protective effect of ascorbic acid derivative on PC12 cells: involvement of its ROS scavenging ability

L-ascorbic acid 2-phosphate-6-palmitate (Asc2P6P) was synthesized and its effect on the damage of PC12 cells induced by H2O2 was investigated. 200 microM H2O2 in a treatment period of 4 hours in our experiment resulted in substantial cell loss. With the increasing concentration of antioxidants, such H2O2-induced cytotoxicity was significantly prevented and the corresponding intracellular and extracellular ROS levels decreased concurrently by pre-treatment with Asc2P6P and Asc. It was found that Asc2P6P was superior to L-ascorbic acid in its protective role and showed a dose-dependent manner during a 24-hour treatment. The higher potency of Asc2P6P's protective role on PC12 cells was correlated with its more effective ROS scavenging ability. HPLC assay demonstrated that Asc2P6P could easily enter the cells and be converted into Asc persistently, which contributed to its distinguished role in protecting PC12 cells against H2O2-induced cytotoxicity.     

Refolding of soluble leukemia inhibitory factor receptor fusion protein (gp 190 sol DAF) from urea

Uptake of L-[1-14C]ascorbic acid (Asc) of 12.5-200 µM for 1 h intobovine aortic endothelial BAE-2 cells grown to confluence was as low as43-64% (per cell) of uptake into the cells grown to nearly one-fourthconfluence. [14C]Asc undergoing transmembrane uptake was concentrated andaccumulated in the cell less efficiently ([Asc]in/ex = 8-13) at confluencethan at subconfluence ([Asc]in/ex = 15-24). The declined Asc uptake atconfluence is attributable to slowdown of the cell cycle, because a similardecrease in [Asc]in/ex was shown by subconfluent cells precultured inserum-insufficient medium, resulting in an increase in G1 phase andconcurrent decreases in S and G2 + M phase distributions as determined byflow cytometry. [1-14C]Dehydroascorbic acid (DehAsc) was taken up andaccumulated as Asc, after metabolic reduction, without detectable DehAsc.The [Asc]in/ex values for DehAsc at confluence were as low as 15-69%of those at subconfluence in contrast to the values as retentive as62-75% for Asc, suggesting the moderate control of Asc uptake againstslowdown of the cell cycle. At either confluence or subconfluence,dose-dependence for DehAsc uptake was more marked than for Asc uptake asshown by an uphill slope in a curve of doses versus [Asc]in/ex for DehAsc incontrast to a downhill slope for Asc, suggesting the moderate control forAsc uptake against fluctuation of the dose. Increasing of coexistent glucoseof 5 mM to 20-40 mM, plasma concentrations in diabetic patients, declinedDehAsc uptake to 46-48%, which was less moderately controlled thanAsc uptake retained to 59-73%. Asc uptake did not compete with DehAscuptake, suggesting different transporter proteins for Asc and DehAsc. Thus,Asc uptake into the aortic endothelial cells is more moderately controlledagainst slowdown of the cell cycle, decreasing of the extracellularconcentrations or increasing of coexistent glucose than DehAsc uptake,suggesting a homeostatic advantage of Asc over DehAsc in terms of retentionof intracellular Asc contents within a definite range.     

Preventive effects of phosphorylated ascorbate on ultraviolet-B induced apoptotic cell death and DNA strand cleavage through enrichment of intracellular vitamin C in skin epidermal keratinocytes

Mortality of mouse keratinocytes Pam212 that were irradiated with ultraviolet-B (UVB) was shown to be repressed by pre-irradiated administration with L-ascorbic acid (Asc) or more markedly with Asc-2-O-phosphate (Asc2P), but not with dehydroascorbic acid (DehAsc) or Asc-2-O-alpha-glucoside (Asc2G), although not repressed by post-irradiated administration. The cytoprotection by Asc2P was restricted against UVB below 5-20 mJ/cm2, and exhibited markedly by administration for a period over 2 h, which may be caused by intracellular Asc that was accumulated via dephosphorylation of Asc2P and was increased, 6-24 h after, to levels above twice as abundant as those of Asc-administration. Pre-irradiated Asc2P-administration slightly repressed a DNA ladder-like electrophoretic pattern for UVB-irradiated keratinocytes, containing the histone-bound DNA fragments as shown by ELISA assay, and appreciably repressed the DNA-3'OH cleavage terminals as shown by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stain. Thus, prevention of UVB-induced cell death by Asc2P was shown to occur concurrently with inhibition of DNA cleavages and enrichment of intracellular Asc.     

L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts

Proliferation of human skin fibroblasts was stimulated significantly by the presence of L-ascorbic acid 2-phosphate (Asc 2-P). The presence of Asc 2-P (0.1-1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2-fold as well as cell growth 4-fold. Coexistence of L-azetidine 2-carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2-P in a dose-dependent manner. Supplementation of the medium with Asc 2-P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum-rich cells surrounded by dense ECM. These results indicate that Asc 2-P is useful in culture systems as a long-acting vitamin C derivative and also that it promotes reorganization of a three-dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.     

Age-dependent telomere-shortening is repressed by phosphorylated alpha-tocopherol together with cellular longevity and intracellular oxidative-stress reduction in human brain microvascular endotheliocytes

Cellular life-span of neonatal human brain microvascular endotheliocytes (HBME) was estimated by population doubling levels (PDLs) for serial subcultivations until spontaneous proliferation stoppage, and was 2.4-fold longer for continuous administration with the 6-O-phosphorylated derivative (TocP) of alpha-tocopherol (Toc), being bio-available owing to its water-solubility, or TocP plus 2-O-phosphorylated ascorbate (Asc2P), and 1.3-fold longer with Asc2P, at a dose of 150 microM, than for the non-administered control. Enlarged cell diameters indicative of cellular aging were repressed for TocP-administered cells as analyzed with a channelizer. Age-dependent shortening of telomeric DNA length (291 bp/PDL) was slowed markedly for TocP (165 bp/PDL) or TocP plus Asc2P, but slightly for Asc2P. Telomerase activity as assessed by the PCR-based TRAP method was detectable slightly at younger ages but no longer at middle ages for the non-administered cells, but, for TocP-administered cells, was intensely detected at younger ages and appreciably until middle ages. Intracellular TocP amounts were not changed age-dependently in contrast to a marked decrease in Toc which accrued from TocP esterolysis. This may be partly attributed to age-dependent changes in the lipid peroxidation product acrolein (ACR), which was abundant at older ages in non-administered cells, but scarcely in TocP-administered cells. Furthermore, intracellular reactive oxygen species (ROS) such as H(2)O(2) and hydroperoxides as detected using the redox indicator CDCFH-DA was less abundant in TocP-administered cells than in non-administered cells. Thus the telomeric-DNA retention, concurrently with retained telomerase activity, was shown to be correlated with cellular longevity, and may be supported by diminished oxidative stress, in hydrophobic microenvironment, which can be achieved by TocP rather than AscP.     

Ascorbate stimulates monooxygenase-dependent steroidogenesis in adrenal zona glomerulosa

It is well known that ascorbic acid (Asc) is highly concentrated in the adrenal gland, but its function in the gland is not thoroughly elucidated. We therefore examined the possibility that Asc participates in steroidogenic monooxygenase systems of the adrenal cortex with the aid of the regenerating system including outer mitochondrial membrane cytochrome b (OMb). When Asc availability was limited in rat mutants unable to synthesize Asc, the increase in plasma aldosterone concentration under Na-deficiency was suppressed without effect on plasma corticosterone concentration. Aldosterone formation in the isolated mitochondrial fraction of the zona glomerulosa (zG) of the adrenal cortex was stimulated by the addition of Asc and NADH, while corticosterone formation was not. Consistently zG showed a high level of Asc regeneration activity and was rich in OMb among adrenocortical zones. Taken together, the enhanced aldosterone formation that is catalyzed by one of the steroidogenic monooxygenases, P450aldo, may be supported by Asc with its regenerating system.     

Ascorbic acid stimulates barrier function of cultured endothelial cell monolayer

The macromolecular permeability of cultured bovine aortic, bovine venous, and human umbilical vein endothelial cell monolayers was decreased significantly in culture medium containing L-ascorbic acid (Asc Acid; 0.01–0.1 mM) and L-ascorbic acid 2-phosphate (Asc 2-P). Dithiothreitol, which shows reducing activity equivalent to that of Asc Acid, did not affect endothelial permeability. Asc Acid induced a sixfold increase in collagen synthesis by the endothelial cells. The coexistence of L-azetidine 2-carboxylic acid, an inhibitor of collagen synthesis, attenuated the effect of Asc 2-P in a dose-dependent manner. Another collagen synthesis inhibitor, ethyl-3,4-dihydroxybenzoate, also inhibited collagen synthesis and increased endothelial permeability. The decrease in permeability of the endothelial monolayer was dependent on a reduction of the permeability coefficient of the endothelial monolayer. These findings indicate that endothelial barrier function is stimulated by Asc Acid via an increase in collagen synthesis. © 1995 Wiley-Liss, Inc.     

Effect of a novel antifungal peptide P852 on cell morphology and membrane permeability of Fusarium oxysporum

P852, a novel cyclic peptide isolated from Bacillus amyloliquefaciens L-H15, showed potent antifungal activity against several major plant fungal pathogens including Fusarium oxysporum. To elucidate the antifungal mechanism, the impact of P852 on the cell morphology and membrane permeabilization of F. oxysporum was studied. By applying electron microscopy and fluorescent techniques, we showed that P852 treatment caused the morphological change of F. oxysporum cells and disrupted its cell structure, including formation of blebs, broken hyphae, deformation of membrane, intracellular organization disruption, pore formation, and cell lysis. Our findings provide insights into the mode of action of P852, which laying a foundation to develop P852 as a novel antifungal agent to control plant fungal pathogens.     

Slow-down of age-dependent telomere shortening is executed in human skin keratinocytes by hormesis-like-effects of trace hydrogen peroxide or by anti-oxidative effects of pro-vitamin C in common concurrently with reduction of intracellular oxidative stress

The cellular life-span of cultivated human skin epidermis keratinocytes NHEK-F was shown to be extended up to 150% of population doubling levels (PDLs) by repetitive addition with two autooxidation-resistant derivatives of ascorbic acid (Asc), Asc-2-O-phosphate (Asc2P), and Asc-2-O-alpha-glucoside (Asc2G), respectively, but to be not extended with Asc itself. In contrast, hydrogen peroxide (H(2)O(2)) as dilute as 20 microM which was non-cytotoxic to the keratinocytes, or at 60 microM being marginally cytotoxic achieved the cellular longevity, unexpectedly, up to 160 and 120% of PDLs, respectively, being regarded as a hormesis-like stimulatory effect. The lifespan-extended cells that were administered with Asc2P, Asc2G, or 20 microM H(2)O(2) were prevented from senescence-induced symptoms such as PDL-dependent enlargement of a cell size of 14.7 microm finally up to 17.4 microm upon Hayflick's limit-called loss of proliferation ability as estimated with a channelizer, and retained young cell morphological aspects such as thick and compact shape and intense attachment to the culture substratum even upon advanced PDLs, whereas other non-extended cells looked like thin or fibrous shape and large size upon lower PDLs. The PDL-dependent shortening of telomeric DNA of 11.5 kb finally down to 9.12-8.10 kb upon Hayflick's limit was observed in common for each additive-given cells, but was decelerated in the following order: 20 microM H(2)O(2) > Asc2P = Asc2G > 60 microM H(2)O(2) > Asc = no additive, being in accord with the order of cell longevity. Intracellular reactive oxygen species (ROS) was diminished by Asc2P, Asc2G or 20 microM H(2)O(2), but not significantly by Asc or 60 microM H(2)O(2) as estimated by fluorometry using the redox indicator dye CDCFH. There was no appreciable difference among NHEK keratinocytes that were administered with or without diverse additives in terms of telomerase activity per cell, which was 1.40 x 10(4)-4.48 x 10(4) times lower for the keratinocytes than for HeLa cells which were examined as the typical tumor cells. Thus longevity of the keratinocytes was suggested to be achieved by slowdown of age-dependent shortening of telomeric DNA rather than by telomerase; telomeres may suffer from less DNA lesions due to the continuous and thorough repression of intracellular ROS, which was realized either by pro-vitamin C such as Asc2P or Asc2G that exerted an antioxidant ability more persistent than Asc itself or by 20 microM H(2)O(2) which diminished intracellular ROS assumedly through a hormesis-like effect.     

Cytoprotective effects of the lipoidic‐liquiform pro‐vitamin C tetra‐isopalmitoyl‐ascorbate (VC‐IP) against ultraviolet‐A ray‐induced injuries in human skin cells together with collagen retention,MMP inhibition and p53 gene repression

Irradiation with ultraviolet‐A (UVA) ray at doses of 20–100 J/cm² diminished the cell viability of human keratinocytes HaCaT and human melanoma cells HMV‐II, both of which were protected by pre‐irradiational administration with the ascorbic acid (Asc) derivative, VC‐IP (2,3,5,6‐O‐tetra‐2′‐hexyldecanoyl‐L‐ascorbic acid; vitamin C‐isopalmityl tetraester), which is the first lipoidic‐liquiform pro‐vitamin C by itself that is materialized by esterization of all four intramolecular hydroxyl groups of an Asc molecule with branched chain fatty groups, resulting in molecular fluidity higher than that of the corresponding straight chains. Irradiation with UVA to HaCaT keratinocytes was shown to cause the formation of 8‐hydroxydeoxyguanosine (8‐OHdG), translocation of phosphatidylserine in the inner layer into the outer layer of cell membrane, and lowering of a mitochondrial membrane potential, all of which were repressed by pre‐irradiational administration with VC‐IP. Expression of p53 gene, another hallmark of UV‐induced DNA damages, was promoted by UVA irradiation to the keratinocytes but also repressed by VC‐IP. Administration with VC‐IP of 10–50 µM to human fibroblasts NHDF achieved the enhancement of collagen synthesis, repression of matrix metalloprotease‐2/9 activity, and increasing of intracellular Asc contents more markedly than that with Asc itself of the same concentrations. Thus UVA‐induced diverse harmful effects could be prevented by VC‐IP, which was suggested to ensue intrinsically from the persistent enrichment of intracellular Asc, through esterolytic conversion of VC‐IP to a free‐form Asc molecule, resulting in relief to UVA‐caused oxidative stress. J. Cell. Biochem. 106: 589–598, 2009. © 2009 Wiley‐Liss, Inc.     

Homing of annexin-labeled stem cells to apoptotic cells

Ischemic diseases are characterized by the presence of pro-apoptotic stimuli, which initiate a cascade of processes that lead to cell injury and death. Several molecules and events represent detectable indicators of the different stages of apoptosis. Among these indicators is phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane, which can be detected by annexinV (ANXA5) conjugation. This is a widely used in vivo and in vitro assay marking the early stages of apoptosis. We report here on an original method that employs PS-ANXA5 conjugation to target stem cells to apoptotic cells. Mesenchymal stem cells (MSCs) from GFP-positive transgenic rats were biotinylated on membrane surfaces with sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHS-LC-biot) and then bound to avidin. The avidin-biotinylated MSCs were labeled with biotin conjugated ANXA5. Bovine aortic endothelial cells (BAE-1 cells) were exposed to UVC to induce caspasedependent apoptosis. Finally, we tested the ability of ANXA5-labeled MSCs to bind BAE-1 apoptotic cells: suspended ANXA5-labeled MSCs were seeded for 1 hour on a monolayer of UV-treated or control BAE-1 cells. After washing, the number of MSCs bound to BAE-1 cells was evaluated by confocal microscopy. Statistical analysis demonstrated a significant increase in the number of MSCs tagged to apoptotic BAE-1 cells. Therefore, stem cell ANXA5 tagging via biotin-avidin bridges could be a straightforward method of improving homing to apoptotic tissues. A. Gerasimou, R. Ramella and A. Brero contributed equally to this paper.     

Cytoprotection against ischemia-induced DNA cleavages and cell injuries in the rat liver by pro-vitamin C via hydrolytic conversion into ascorbate

To search a regimen for prevention of post-ischemic reperfusional (I/R) injuries, I/R in the liver was induced by 30-min clamping and subsequent unfastening of the portal vein of a rat, which underwent previous intravenous administration with ascorbic acid (Asc) of 1 mg/kg or the autooxidation-resistant pro-vitamin C, 2-O-alpha-D-glucosylated Asc (Asc2G) or 2-O-phosphorylated Asc (Asc2P) of 1 mg Asc equivalent/kg from the viewpoint of utilization of antioxidants that can promptly scavenge I/R-derived reactive oxygen species. The administration with Asc, Asc2P or Asc2G prevented some features of hepatic I/R injuries such as release of hepatic marker enzymes GOT and GPT into the blood vessel, cellular degenerative symptoms including vacuolation and cell fragmentation, and nuclear DNA strand cleavage as detected by TUNEL staining. The preventive effects on I/R injuries were in the order: Asc2G > Asc2P >> Asc. This order of preventive degrees of three anti-oxidants is partly attributable to proper efficiency of conversion to vitamin C and stability in blood stream; Asc2P was moderately converted to a free monoanion form of Asc in human serum, but, in rat serum, so efficiently converted to Asc as to undergo the resultant oxidative decomposition before reaching the liver, whereas Asc2G underwent scarce conversion to Asc in human serum but moderate conversion in rat serum, suggesting that Asc2P might be less cytoprotective against I/R injury than Asc2G in the rat liver in a way different from the human liver. In contrast Asc was so susceptible to autooxidation as to be rapidly decomposed in either rat or human serum. The concentrations of ascorbyl radicals (AscR) in serum were unchanged during I/R for sham-operated rats, but appreciably diminished time-dependently for I/R-operated rats as shown by ESR spectra. A marked increase in serum AscR occurred in rats receiving Asc, Asc2G or Asc2P, but it was time-dependently restored down to the pre-ischemic level of AscR in I/R-operated rats more rapidly than in sham-operated rats. Thus, hepatic I/R injuries were shown to be prevented more markedly by Asc2G or Asc2P than by Asc, which is attributable to efficiencies of both vitamin C conversion and subsequent AscR retention.     

The effect of ascorbic acid on arachidonic acid and prostaglandin E2 metabolism in B16 murine melanoma cells

Ascorbic acid (Asc), arachidonic acid (AA) and prostaglandin E2 (PGE2) are reported to be important in maintaining the stability of the cell matrix. Asc has also been shown to influence fatty acid (FA) and PGE2 synthesis, with the result that effects of Asc on cell growth are suggested to be mediated through the metabolism of these two compounds. This study examined the effect of Asc, supplemented over the concentration range of 0-100 micrograms/ml, on the in vitro cell growth of non-malignant LLCMK (monkey kidney) cells and malignant B16 murine melanoma cells. The effects of Asc supplementation on AA and PGE2 levels in the cell stroma and membrane fractions of the two cell types was also determined. Asc had no significant inhibitory or stimulatory effect on the growth of either the B16 or LLCMK cells. The total percentage AA composition determined in the B16 control cells (combined stroma and membrane fractions), was similar to that determined in the LLCMK control cells. Asc supplementation of the B16 cells, resulted in an inverse relationship between B16 cell growth and total percentage AA composition. PGE2 concentration in the control B16 cells (combined stroma and membrane fractions) was significantly higher than that detected in the control LLCMK cells. No PGE2 was detected in the B16 stroma fraction, with all appearing to be located in the membrane fraction. However, upon the supplementation of the B16 cells with increasing Asc concentrations, PGE2 appeared to be mobilized from the membrane fraction, resulting in increasing PGE2 levels in the stroma fraction relative to the membrane fraction. This was accompanied by a significant decrease in PGE2 concentration, in the membrane fraction. B16 cell growth and total (stroma and membrane fractions) PGE2 concentration in these cells was inversely related, when cultures were supplemented with increasing levels of Asc. Asc supplementation of the LLCMK cells did not appear to have any significant effect on AA or PGE2 metabolism in these cells.     

Effect of actin cytoskeleton disruption on electric pulse‐induced apoptosis and electroporation in tumour cells

Electric pulses are known to affect the outer membrane and intracellular structures of tumour cells. By applying electrical pulses of 450 ns duration with electric field intensity of 8 kV/cm to HepG2 cells for 30 s, electric pulse‐induced changes in the integrity of the plasma membrane, apoptosis, viability and mitochondrial transmembrane potential were investigated. Results demonstrated that electric pulses induced cell apoptosis and necrosis accompanied with the decrease of mitochondrial transmembrane potential and the formation of pores in the membrane. The role of cytoskeleton in cellular response to electric pulses was investigated. We found that the apoptotic and necrosis percentages of cells in response to electric pulses decreased after cytoskeletal disruption. The electroporation of cell was not affected by cytoskeletal disruption. The results suggest that the disruption of actin skeleton is positive in protecting cells from killing by electric pulses, and the skeleton is not involved in the electroporation directly.     

Cytoprotection by pro-vitamin C against ischemic injuries in perfused rat heart together with differential activation of MAP kinase family

The cardiac muscle cells are known to be killed by ischemia-reperfusion (I/R) treatment that produce reactive oxygen species (ROS). We analyzed the function of the autooxidation-resistant pro-vitamin C, 2-O-alpha-D-glucosylated derivative (Asc2G) of ascorbic acid (Asc), in protecting against I/R injury of the heart in rat. The serum release of the intracellular enzyme CPK due to I/R injury decreased upon injection with Asc2G. Out of the mitogen-activated protein (MAP) kinase family members, MAP kinase and JNK underwent the down-regulation in contrast to up-regulation of p38 compared with the I/R-treated control in the absence of Asc2G. These data suggest important roles for differential activation of the MAP kinase family in cytoprotection against I/R injury by Asc2G.     

Role of MAPK phosphorylation in cytoprotection by pro-vitamin C against oxidative stress-induced injuries in cultured cardiomyoblasts and perfused rat heart

The reactive oxygen species (ROS) are known to be generated upon post-ischemic reperfusion (I/R) of the heart, and to injure cardiac muscle cells. The hydrogen peroxide-induced mortality of rat cardiomyoblasts H2c9 was markedly inhibited by previous administration with auto-oxidation-resistant pro-vitamin C, the 2-O-phosphorylated derivative (Asc2P) of ascorbic acid (Asc). The cytoprotection was partially counteracted by an inhibitor of MAPK (mitogen-activated protein kinase) kinase (MEK) as shown by DNA strand cleavage assay and mitochondrial dehydrogenase assay. Immunostains indicated that phosphorylated MAPK increased in the hydrogen peroxide-treated cardiomyoblasts, and that this action was moderately inhibited by Asc2P and restored nearly to the initial, pretreatment level by combined administration of the MEK inhibitor and Asc2P. The I/R-induced cell injuries in perfused rat hearts as estimated by extracellular release of the cardiac enzyme CPK were inhibited by 2-O-alpha-glucosylascorbic acid (Asc2G) and Asc, whereas the observed cytoprotection for the cardiomyoblasts was partially counteracted by the MEK inhibitor. The increase in phosphorylated MAPK in I/R-operated hearts was moderately inhibited by pro-vitamin C, but restored nearly to the normal non-operated level by combined administration with the MEK inhibitor. This is in contrast to no alteration in levels of non-phosphorylated MAPK for all the cases examined as shown by Western blots, consistent with results of immunostains for the cardiomyoblasts. The inhibitory effect of the MEK inhibitor on MAPK phosphorylation was, therefore, suggested to counteract the cytoprotective effects of pro-vitamin C via a thorough interruption of the phosphorylated MAPK signaling pathway. This was not true of ROS-related events; the scavenging effects of Asc2G and Asc on hydroxyl radicals generated from I/R-operated heart were not affected by combined administration with the MEK inhibitor, as shown by the spin-trapping DMPO-based ESR method.     

Potentiated susceptibility of ascites tumor to acyl derivatives of ascorbate caused by balanced hydrophobicity in the molecule

Orders of susceptibility of Ehrlich ascites tumor to L-ascorbic acid (Asc), its 6-stearoyl (6S), 6-palmitoyl (6P) and 2,6-dipalmitoyl (DP) derivatives were assessed in vitro and in vivo: 6P (a 50% growth inhibitory concentration (IC50) for cultured cells, 12 microM; an increased life-span of treated mice, 283%) greater than 6S (61 microM; 240%) much greater than Asc (430 microM; 122%) greater than or equal to DP (greater than 200 microM; 89%), indicating that the enhanced susceptibility was due to acyl moiety substituted at C6-hydroxyl group of Asc, but was retracted by further substitution at C2-hydroxyl group. Equimolar mixture of Asc and palmitic acid, stearic acid or their methyl esters was much less cytotoxic than 6P or 6S. Thus the enhanced susceptibility was not primarily due to an additive cytotoxic effect of ascorbyl and acyl moieties, but to a balanced hydrophobicity introduced into the molecule by a poorly cytotoxic acyl moiety.     

Ergosterol depletion under bifonazole treatment induces cell membrane damage and triggers a ROS-mediated mitochondrial apoptosis in Penicillium expansum

Penicillium expansum is the causal agent of blue mold in harvested fruits and vegetables during storage and distribution, causing serious economic loss. In this study we seek the action modes of bifonazole against this pathogen. Bifonazole exhibited strong antifungal activity against P. expansum by inhibiting ergosterol synthesis. The ergosterol depletion caused damage to the cell structure and especially cell membrane integrity as observed by SEM and TEM. With increased unsaturated fatty acids contents, the cell membrane viscosity decreases and can no longer effectively maintain the cytoplasm, which ultimately decreases extracellular conductivity, changes intracellular pH and ion homeostasis. Exposure of hyphal cells to bifonazole shows that mitochondrial respiration is inhibited and reactive oxygen species (ROS) levels–including H₂O₂ and malondialdehyde (MDA) – are significantly increased. The functional impairment of mitochondria and cell membrane eventually cause cell death through intrinsic apoptosis and necroptosis.