Oxidative stress mediates apoptotic effects of ascorbate and dehydroascorbate in human Myelodysplasia cells in vitro

Toxicology in Vitro 27 (2013) 1542–1549

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Oxidative stress mediates apoptotic effects of ascorbate and dehydroascorbate in human Myelodysplasia cells in vitro Ana Cristina Gonçalves a,b,c, Vera Alves d, Teresa Silva e, Cristina Carvalho c,f, Catarina Resende de Oliveira c,g, Ana Bela Sarmento-Ribeiro a,b,c,⇑ a

Applied Molecular Biology and Hematology University Clinic, Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal Centre of Investigation in Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal Centre for Neuroscience and Cell Biology (CNC), University of Coimbra, 3004-504 Coimbra, Portugal d Immunology Institute, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal e Hematopathology, Institute of Pathological Anatomy, Faculty of Medicine, University of Coimbra, Coimbra, Portugal f Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, 3004-504 Coimbra, Portugal g Biochemistry Institute, Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal b c

a r t i c l e

i n f o

Article history: Received 5 July 2012 Accepted 19 March 2013 Available online 27 March 2013 Keywords: Myelodysplastic Syndrome Ascorbate Dehydroascorbate Oxidative stress Apoptosis Anti-cancer therapy

a b s t r a c t The Myelodysplastic Syndromes are stem cell heterogeneous disorders characterized by peripheral cytopenias and hypercellular bone marrow, which can evolute to acute leukaemia. Vitamin C can act as an antioxidant, ascorbic acid (AA) donates two electrons and becomes oxidized to dehydroascorbic acid (DHA). Under physiological conditions, vitamin C predominantly exists in its reduced (AA) form but also exists in trace quantities in the oxidized form (DHA). This study evaluates the therapeutic potential of vitamin C in Myelodysplastic Syndromes (MDSs). F36P cells (MDS cell line) were treated with ascorbate and dehydroascorbate alone and in combination with cytarabine. Cell proliferation and viability were assessed by trypan blue assay and cell death was evaluated by optical microscopy and flow cytometry. The role of reactive oxygen species, mitochondrial membrane potential, BAX, BCL-2 and cytochrome C were also assessed. Vitamin C decreases cell proliferation and viability in a concentration, time and administration dependent-manner inducing cell death by apoptosis, which was shown to be associated to an increased in superoxide production, mitochondrial membrane depolarization. These compounds modulate BCL-2, BAX and cytochrome C release. These results suggest that vitamin C induces cell death trough apoptosis in F36P cells and may be a new therapeutic approach in Myelodysplasia. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Naturally occurring dietary agents, known to produce chemopreventive effects in experimental cancer models, have been shown to target signalling intermediates in apoptotic pathways, especially because diet and nutrition are key factors in cancer risk modulation. These dietary agents display numerous functions on genetic transcription modulation, being involved in activation or inhibition of specific genes and in induction of cell death (Aggarwald and Shishodia, 2006; Martin, 2006). In recent years, because of their low systemic toxicity, vitamins have been evaluated for their anti-tumour activities and have gained importance because of their prophylactic and therapeutic potential role in several ⇑ Corresponding author. Address: Applied Molecular Biology, Faculty of Medicine, University of Coimbra, Azinhaga de Sta Comba, Celas, 3000-548 Coimbra, Portugal. Tel.: +351 239480217; fax: +351 239480048. E-mail address: [email protected] (A.B. Sarmento-Ribeiro). 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.03.009

diseases. Antioxidants, such as vitamin C, show protective effects and, on the other hand, they can develop pro-oxidant properties, dependably on their concentration and cell type (Ratnam et al., 2006). Vitamin C is a major water-soluble vitamin that exhibits various biological functions, such as antioxidant properties, being implicated in the regeneration of a-tocopherol. Humans cannot synthesize vitamin C de novo and thus have to acquire most body storage of vitamin C through fruits and vegetables or vitamin supplements. Under physiological conditions, vitamin C predominantly exists in its reduced form but also exists in trace quantities in the oxidized form. Vitamin C is mainly transported in the dehydroascorbic acid (DHA) form and can be regenerated to ascorbic acid (AA) either enzymatically or non-enzymatically. Besides the antioxidant proprieties, vitamin C displays also a pro-oxidant function by inducing an increase in reactive oxygen species (ROS) production (McEligot et al., 2005; Verrax and Calderon, 2008). The antioxidant activity of vitamin C resides primarily in its ability to donate electrons and

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therefore by acting as a reductive agent. In fact, AA donates two electrons and becomes oxidized to DHA. However, in a concentration dependent manner, vitamin C can produce hydrogen peroxide at cytotoxic levels, which can generate hydroxyl radical in the presence of divalent cations such as iron and cooper (González et al., 2005; Bhat et al., 2006). Several studies have shown that vitamin C alone or combined with other antioxidants can potentiate the efficacy of several chemotherapeutic drugs, such 5-fluorouracil, doxorubicine, vincristin, adriamycin or gencitabin, either in vitro or in vivo. However, other authors report that vitamin C may have a negative effect decreasing the activity of new anti-tumoral drugs, such as bortezomib (Zou et al., 2006) or Trail Ligand (Perez-Cruz et al., 2007). Reactive oxygen species (ROS) are formed constantly as a consequence of metabolic and other biochemical reactions mainly in mitochondria as well as induced by external factors such as drugs exposure. Many cancer cells show an increase in ROS production as a result of oxidative metabolism. Since cells can only tolerate certain ROS levels, the increase in oxidative stress can lead to oxidative damage of lipids, nucleic acids and proteins (McEligot et al., 2005; Valko et al., 2007). On the other hand, cancer cells readily take up vitamin C in vitro and certain human tumour cells have higher vitamin C levels compared with adjacent normal cells. The higher intracellular concentration of vitamin C may have effects on tumour growth and in the tumour’s ability to respond to chemotherapy and radiation therapy (Agus et al., 1999). The Myelodysplastic Syndromes (MDSs) are a heterogeneous group of stem cell disorders characterized by ineffective haematopoiesis, cytopenia and a higher potential of evolution to myeloid leukaemia. MDS may arise both de novo and as a consequence of chemo- or radiotherapy. The natural history of these diseases ranges from a chronic course to a rapid course towards leukeamic progression, where approximately 30% of MDS cases transform in acute myeloid leukaemia (AML) (Hirai, 2003; Nimer, 2008; Mufti et al., 2008). Different treatment options are available for MDS patients ranging from supportive care, which helps relieve symptoms, to aggressive treatment that may slow or prevent progression of the disease. Cytarabine is a drug effective in MDS, producing complete response rates of 15–20% when used in a low-dose. However, virtually all patients treated with low-dose cytarabine relapse (Nimer, 2008). The pathogenesis of MDS is complex, since hematopoietic cells and the hematopoietic bone marrow microenvironment are both involved in disease establishment and progression. The presence of oxidative stress markers in MDS patients indicates a potential role for pro-oxidant pathways in the maintenance/progression of these disorders (Farquhar and Bowen, 2003). Moreover, the treatment with the antioxidant aminothiol amifostine suggests that oxidative stress could be a new strategy in MDS treatment (Farquhar and Bowen, 2003). In this study, we pretend to evaluate the therapeutic potential of vitamin C in MDS, as in monotherapy and/or as adjuvant to conventional anti-carcinogenic therapies.

2. Materials and methods 2.1. Materials Roosevelt Park Memorial Institute Medium 1640 medium (RPMI 1640, Gibco, Invitrogen, Barcelona, Spain), foetal bovine serum (FBS, Gibco, Invitrogen, Barcelona, Spain), penicillin/streptomycin (Gibco, Invitrogen, Barcelona, Spain), recombinant interleucin-3 (rh-Il-3, GIBCO, Invitrogen, Barcelona, Spain), phosphate buffered saline (PBS, Sigma, Sintra, Portugal), ascorbic acid (AA, Sigma,

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Sintra, Portugal), dehydroascorbic acid (DHA, Sigma, Sintra, Portugal), cytarabine (Ara-C, Sigma, Sintra, Portugal), annexin V-FITC (AV) and propidium iodide (PI, Immunotech kit, Beckman Coulter, Inc., Marseille, France), 20 ,70 -dichlorodihydrofluorescin diacetate (H2DCF-DA, Molecular Probes, Invitrogen, Barcelona, Spain), hydroetidine (HE, Sigma, Sintra, Portugal), JC-1 (Molecular Probes, Invitrogen, Barcelona, Spain), mercury orange (MO, Sigma, Sintra, Portugal), hydrogen peroxide (Sigma, Sintra, Portugal), culture flasks and well-plates (Sarstedt, Rio Tinto, Portugal). 2.2. Cell culture The F36P cells, a Myelodysplastic Syndrome cell line established from a patient with refractory anaemia with excess of blast in transformation (RAEB-t), was purchased from European Collection of Cell Cultures (ECACC, UK). Cell line was routinely grown in RPMI-1640 medium (L-glutamine 2 mM, HEPES-Na 25 mM, penicillin 100U/mL and streptomycin 100 lg/mL) supplemented with 10 ng/mL recombinant interleucin-3 (rh-Il-3) and 10% heat-inactivated foetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO2. All experiments were performed between 10 and 25 passage numbers and F36P cells were seeded at a density of 0.75  106 cells/mL. Ascorbic acid (AA) and dehydroascorbic acid (DHA) was always freshly prepared by dissolving these compounds in water at a final concentration of 100 mM, immediately prior to the experiments. After preparation AA and DHA were sterilised by passing through a 0.20 lm membrane. Cells were incubated in the absence and in the presence of increasing AA and DHA concentrations, ranging from 50 lM to 5 mM, and 50 nM cytarabine (Ara-C). 2.3. Cell viability and proliferation assays Cell viability and proliferation were assessed by the trypan blue exclusion test. Briefly, viable cells were identified by their ability to exclude dye, whereas dye stained nonviable cells. At each 24 h, exposure and not exposure (control) cells were harvest and the number of stained (nonviable) and unstained (viable) cells were counted using a haemocytometer (Neubauer chamber). The viability was calculated as percentage of viable cells and cell proliferation was determinate by the number of viable cells (density). 2.4. Cell death evaluation F36P cell death was evaluated under the conditions describe above by optical microscopy through morphological assessment of May–Grünwald–Giemsa stained slides and by flow cytometry using the Annexin V and Propidium Iodide double staining. For morphological assessment, cells were transferred to slides fixed, stained and evaluated under light microscopy, using a Nikon Eclipse 80i equipped with a Nikon Digital Camera DXm 1200F. For flow cytometry analysis, F36P cells were stained simultaneously with Annexin V (AV), labelled with the fluorescent probe fluorescein isothyocianate (FITC) and with PI. This assay discriminates among intact cells (AV/PI), early apoptotic cells (AV+/ PI) and late apoptotic or necrotic cells (AV+/PI+). After drug treatments in the above conditions, cells were co-stained with AV-FITC and PI using the manufacturer’s recommended protocol (Immunotech Kit). Briefly, cells were washed with ice-cold PBS (centrifuged at 500g for 5 min), resuspended in 100 lL of binding buffer and incubated with 1 lL of AV-FITC solution and 5 lL of PI solution for 10 min on ice in the dark. After incubation time, cells were diluted in 400 lL of ice-cold binding buffer, and analyzed by flow cytometry (Dourado et al., 2007). Results are expressed in % ± SD of at least three independent experiments. Flow cytometry analysis was performed using a six-parameter, four-colour FACSCalibur™

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flow cytometer (Becton Dickinson, San Jose, CA). For each assay 1  106 cells were used and at least 10,000 events were collected by acquisition using CellQuest software (Becton Dickinson, San Jose, CA) and analyzed using Paint-a-gate software (Becton Dickinson, San Jose, CA). 2.5. Evaluation of Reactive Oxygen Species production The ROS production in F36P cells was determined by oxidation of 20 ,70 -dichlorodihydrofluorescin diacetate (H2DCF-DA) and hydroetidine (HE). Cells cultured in the absence and in the presence of AA and DHA were incubated with 5 lM H2DCF-DA for 30 min and with 2 lM of HE during 15 min, at 37 °C in the dark. Cells were then washed twice with PBS, resuspended in the same solution and fluorescence was detected by flow cytometry (Almeida et al., 2008). Results are expressed in median fluorescence intensity (MFI) ± SD of at least three independent experiments. 2.6. Mitochondrial membrane potential measurement Mitochondrial membrane potential (wmit) in F36P cells treated with and without vitamin C were measured using JC1 (Molecular Probes), as described by others (Almeida et al., 2008). Briefly, after incubation in the absence or in the presence of AA or DHA, cells were washed with PBS (centrifugation at 300g during 5 min) and incubated with JC-1 at final concentration 5 lg/mL for 15 min at 37 °C, in the dark. At the end of the incubation period, the cells were washed twice in PBS, resuspended in a total volume of 500 lL and analyzed by flow cytometry. 2.7. Evaluation of apoptotic proteins expression by flow cytometry The modulation of BAX, BCL-2 and cytochrome c expression levels were analyse in cells cultured in the absence and in the presence of AA and DHA. F36P cells were centrifuged and incubated with monoclonal antibodies anti-BAX, anti-BCL-2 and anti-cytochrome c antibodies (Santa Cruz Antibodies, Heidelberg, Germany), according with manufactured protocols. The levels of cellular fluorescence, proportional to the concentration of apoptotic proteins in each cell, were measured by flow cytometry and results were plotted in Mean Fluorescence Intensity (MFI) arbitrary units. This value represents the medium fluorescence intensity detected in the cells, which is proportional to the number of molecules labelled by the antibody. 2.8. Measurement of catalase and MnSOD activity Catalase and MnSOD activity was measured according to Carvalho et al. (2010). Briefly, catalase activity was measured by oxygen production ratio after the addition of 1 lM of H2O2 to 25 lg of protein, and monitored polarographically with a Clark oxygen electrode (YSI Model 5331, Yellow Springs Inst) connected to a suitable recorder in a 1 ml thermostated, water-jacketed closed chamber with magnetic stirring. The reactions were carried out at 25 °C in 1 ml of the reaction medium (130 mM sucrose, 50 mM KCl, 2.5 mM MgCl2, 2.5 mM KH2PO4, 100 lM EGTA, 5 mM Hepes at pH 7.4). MnSOD activity was determined spectrophotometrically, at 550 nm. After the incubation of 100 lg of protein in 1.4 ml of phosphate buffer (50 mM K2HPO4 and 100 lM EDTA, pH 7.8), 0.025 mM hypoxanthine, 0.025% Triton X-100, 0.1 mM nitrobluetetrazolium (NBT) in the presence or absence of 1.33 mM KCN, the reaction was started with the addition of 0.025 U/ml xanthine oxidase, and the reaction was allowed to continue for 200 s at 25 °C, with continuous magnetic stirring. The measurements were performed in a Jasco V560 UV/VIS Spectrophotometer, against a blank, prepared in the absence of

hypoxanthine. The activity of MnSOD was calculated using a standard curve, prepared with different concentrations of superoxide dismutase commercially available. 2.9. Statistical analysis Data were expressed as mean ± SD of the number of independent experiments indicated in the figure legends, each one performed in triplicate. Student’s t-test and/or analysis of variance (ANOVA test) were used to determine the statistical significance, considering a p-value of <0.05. 3. Results 3.1. Vitamin C decreases F36P cell line proliferation and viability inducing cell death by apoptosis Initially, we investigate if ascorbic acid (AA) and dehydroascorbic acid (DHA) in pharmacological concentrations (ranging from 50 lM to 5 mM) influence the proliferation and survival of F36P cells, a Myelodysplastic Syndrome cell line (Fig. 1). The exposure of F36P cells to AA and DHA induced a decreased on cell proliferation and viability in a dose and time-dependent manner, as shown in Fig. 1A. After 48 h exposure, the effect of DHA was slightly more pronounced than that of AA in almost all ranges of the concentration used in the experiments and, at this time, a reduction in cell viability to values close to 50% was achieved with approximately 2.5 mM of AA and between 1 mM and 2.5 mM of DHA. Since, the IC50 was not found in the dose response curves, a 2 mM IC50 was estimated by non-linear regression analysis. To evaluate whether the mode of administration of vitamin C influences the cytotoxic effect, 50 lM of AA and DHA were added to cell culture, every 24 h. As shown in Fig. 1, daily administration of vitamin C induces a decreased in cell growth and viability of F36P cells. This decrease is higher than that observed in the same cells treated with higher concentrations in single administration. Moreover, Vitamin C plus cytarabine (Ara-C) in low concentrations (50 nM), has an anti-proliferative and cytotoxic effect higher than that induced by each of the drugs alone. Cell death was also analyzed by flow cytometry using annexin V/propidium iodide incorporation, as shown in Fig. 2. Vitamin C, oxidized (DHA) and reduced (AA) form, induces a decrease in F36P viable cells (DHA: 52 ± 5% with 2 mM and 44 ± 4% with 50 lM in daily administration; AA: 55 ± 6% with 2 mM and 55 ± 3% with 50 lM in daily administration) and leads preferentially to an increase in the percentage of apoptotic and apoptotic/necrotic cells. Moreover, the combination of 50 lM DHA plus 50 nM Ara-C have shown a higher cytotoxic potential (52 ± 4%) than the combination with AA (59 ± 2%). Morphological features of F36P cells treated with DHA and AA confirm the results obtain by flow cytometry (Fig. 3). Morphological characteristics of apoptotic cell death, such ‘blebbing’, chromatin condensation, nuclear fragmentation and apoptotic bodies (Fig. 3B and C) were observed as well a high number of abnormal mitosis (data not showed). Besides that, F36P cells incubated with DHA and AA loose dysplastic morphology, a characteristic of this cell line. In order to know if the ascorbate and dehydroascobate modulate apoptotic proteins, BAX, BCL-2 and cytochrome c expression levels were analyzed. As depicted in Fig. 4, a significant increase of intracellular concentration of BAX, is observed when cell are treated with 2 mM of both compounds (DHA: 1.87-fold; AA: 2.35-fold), or with 50 lM daily (AA in daily administration: 2.4-fold; DHA in daily administration: 2.1-fold). The combination

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Fig. 1. Proliferative and viability dose response curves in a F36P cell line. F36P cells were incubated in a density of 0.75  106 cells/ml, during 96 h, in the absence or in the presence of increasing concentrations of ascorbic acid (AA), dehydroascorbic acid (DHA) or cytarabine (Ara-C), as indicated in figure. Proliferative and viability dose response curves are established by counting viable and non-viable cells each 24 h, as described in Material and methods. Cell proliferation (A) represents viable cells and cell viability (B) is expressed in percentage (%) normalized to control. Data are expressed as mean ± SD obtained from 5 independent experiments. ⁄ Daily administration.

of vitamin C plus Ara-C induced an increase in BAX expression of 1.6-fold in the cells treated with DHA combination and 1.7-fold in cells treated with AA combination. Besides that, we also observe a decrease in BCL-2 expression levels when cells were treated with 2 mM of AA (0.84-fold), 50 lM of AA in daily administration (0.76-fold) and with the combination of vitamin C plus Ara-C (approximately 0.85-fold). Moreover, the changes in BAX and BCL-2 expression levels are accompanied by an increase in cytochrome c release in all conditions tested. 3.2. Vitamin C generate oxidative stress and mitochondrial dysfunction on F36P cells To clarify if oxidative stress was involved in the cytotoxicity induced by vitamin C, we analyzed ROS production, namely

hydrogen peroxide (H2O2) and superoxide anion (O 2 ). As represented in Fig. 5A, F36P cells treated with DHA and AA shown an increase in ROS production compared to untreated cells (controls). When F36P cells are treated with 2 mM AA and DHA we observe an increase in H2O2 intracellular levels, respectively, 1.4 and 1.7 times higher and 1.6 and 1.5 times higher in the combination with Ara-C. Moreover, DHA and AA, when compared to control cells, induce an increase in O 2 intracellular production of approximately 2-fold in cells treated with 2 mM and 50 lM in daily administration of DHA, and about 1.8-fold in cells cultured in the presence of AA in the same conditions. When F36P cells were treated with the combination of 50 lM of DHA or AA plus 50 nM of Ara-C, the increase observed was approximately 1.7 times highest. The increase in ROS production observed in cells treated with 2 mM and 50 lM in daily administration of AA was followed by a

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decrease in catalase and MnSOD activity (Fig. 5B). On the other hand, DHA induced a highest decrease in catalase activity and an increase in MnSOD activity, especially in cells treated with 50 lM in daily administration. To study the mitochondrial involvement in cell death induced by vitamin C mitochondrial membrane potential of treated and untreated cells was evaluated, by flow cytometry using the JC1 dye. In apoptotic cells, mitochondrial membrane potential collapses, and JC-1 cannot accumulate within the mitochondria, remaining in the monomeric form in cytosol. These cells, exhibit a higher monomer/aggregate ratio of JC1 (M/A) than viable cells. We observed an increase in M/A of JC1 proportional to vitamin C concentration in treated cells, reflecting a decrease in mitochondrial membrane potential. As we can see in Fig. 6, we observed a statistically significant increase in the M/A JC1 ratio, in agreement with the results obtained by optical microscopy referred above. However,

this increase is higher in cells treated with AA form relatively to that observed in cells treated with DHA, for the same concentrations of these compounds.

4. Discussion The consumption of fruits and vegetables is recommended for the prevention of chronic diseases, including cancer, and its preventive effect is evidenced in several epidemiological studies (Aggarwald and Shishodia, 2006; Martin, 2006). However, the effect of bioactive compounds present in fruit and vegetables at the cellular and molecular levels remains poorly understood. Therapeutic strategies currently used to treat cancer, such as chemotherapy and radiation, induce primarily cell death in an unspecific way through ROS production. On the other hand, these

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Fig. 2. Analysis of Cell death induced by vitamin C in F36P cells by flow cytometry. F36P cells were incubated in a density of 0.75  106 cells/ml, during 48 h, in the absence or in the presence of increasing concentrations of ascorbic acid (AA) or dehydroascorbic acid (DHA), as indicated in figure. Cell death was detected by annexin v and propidium iodide staining and analyzed by flow cytometry. Data are expressed as percentage (%) of viable cells (V), late apoptotic/necrotic cells (A/N), necrosis (N) and early apoptotic cells (EA) as mean ± SD obtained of three independent determinations. ⁄ Daily administration.

conventional cancer therapies have higher toxicity and, consequently, secondary effects. The possibility of natural compounds trigger apoptosis, with higher specificity to tumour cells open new perspectives in cancer therapy, especially in MDS (Nimer, 2008; Strom et al., 2008). Vitamin C has a controversial history as a cancer treatment, and vitamin C administration may produce huge differences in plasma concentrations. In this context we analyzed the effect of this vitamin in monotherapy and as adjuvant of conventional therapy. Our results suggest that both forms of vitamin C, either the reduced form (AA) or the oxidized form (DHA), in high doses induce a cytostatic and cytotoxic effects in human myelodysplastic cells, which may be related to their pro-oxidant effect. However, the effect of DHA is obtained earlier and at lower concentration than that of AA, which could be related to the cellular distribution of vitamin C. DHA rapidly enters the cells through the glucose transporters, namely GLUT1 transport system (Agus et al., 1999; Reynolds and Zhitokovich, 2007). Neoplastic cells have higher energy requirements that are offset by the increased number of membrane glucose transporters (González et al., 2005). On the other hand, AA is introduced into cell by co-transport with sodium, but only in some cell types (González et al., 2005; Wilson, 2005). These facts seem to explain the selective anti-cancer action of vitamin C observed earlier for the oxidized form of vitamin C, DHA, but also the high selectivity of this vitamin for neoplastic cells as compared to normal cells. Once inside the cell, DHA is

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Fig. 4. Apoptotic protein expression levels in F36P cells treated with vitamin C by flow cytometry. BAX, BCL-2 and cytochrome c levels were analyzed by flow cytometry using monoclonal antibodies. Results are expressed as medium fluorescence intensity (MFI) normalized to control and represents mean ± SD of fluorescence intensity detected in three independent experiments. ⁄ Daily administration. § p < 0.05; §§p < 0.01.

converted to AA by reductases with intracellular formation of ROS. Thus, the use of the oxidized form of vitamin C appears to be more effective and selective in the induction of cell death, and with lower systemic toxicity, since this form of vitamin C causes less formation of ROS in normal cells and outside the target cells, especially in blood plasma (Reynolds and Zhitokovich, 2007). The exact mechanism by which vitamin C causes injury and decreases in cell survival is unclear yet. It was described that vitamin C induces a significant increase in ROS formation which is proportional to its concentration and to cell death induction. According to Chen et al. (2005) during the process of interconversion of vitamin C occurs formation of ROS. Maramag et al. (1997) suggest that OH is formed through the Fenton reaction, in which the anion or ascorbyl radical reduce metal ions, such as iron and copper, which react with H2O2 producing the radical OH. Alternatively, the anion or ascorbyl radical can react with O2 originating O2, which then can reduce Fe3+, initiating the Haber–Weiss reaction (Maramag et al., 1997). These mechanisms may well be the cause of the observed pro-oxidant effect of vitamin C and could explain the cytotoxic effects observed in the human myelodysplastic cell line. Furthermore, some studies indicate that tumour cells have decreased antioxidant enzymes, namely catalase (Benade et al., 1969; Lamson and Brignall, 1999). Catalase is responsible for the detoxification of H2O2 and reduced levels of this enzyme may lead to accumulation of H2O2 during the oxidation of vitamin C (Drisko et al., 2003; Li and Schelihorn, 2007). Thus, the formation of H2O2 in cells treated with DHA result from dismutation of O2 mediated by the increased in MnSOD and the decreased in catalase activity. In cells treated with AA the formation of H2O2 is mediated by the reduction in catalase activity. The cytotoxicity of vitamin C in F36P cells may also be related to mitochondrial dysfunction, as suggested by the observed decrease

Fig. 3. Morphological aspects of F36P cells treated with vitamin C. Control cells (A) and cells treated with 2 mM of AA (B) and DHA (C) are stained with May–Grünwald– Giemsa, after 48 h of incubation. Cells smears show morphological features of apoptosis, such as nuclear fragmentation, blebbing and apoptotic bodies. Amplification 500.

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A

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Fig. 5. ROS expression levels and antioxidant enzymes activity in F36P cells treated with vitamin C by flow cytometry. ROS levels (A), hydrogen peroxide (H2O2) and superoxide (O2 ), and antioxidant activity (B), catalase and MnSOD, were determine as described in Material and methods. Results are expressed as medium fluorescence intensity (MFI) normalized to control and represents mean ± SD of three independent experiments. ⁄ Daily administration. §p < 0.05; §§p < 0.01; §§§p < 0.001.

compounds, is in agreement with the decrease of mitochondrial membrane potential and the increase in apoptosis/necrosis, observed in a concentration-dependent manner. Moreover, these findings were further confirmed by the involvement of ascorbate and dehydroascorbate in the modulation of BAX and BCL-2 expression. The activities of BAX, proapoptotic protein oppose of BCL-2, allow the release of cytochrome c from the mitochondria inducing apoptosis. Since the increased of BAX and decreased of BCL-2 is involved in DHA and AA-induced apoptosis, these compounds might have an important role in potential future cancer treatment and might constitute a possible chemotherapeutic agent for treatment of MDS patients, in monotherapy or as adjuvant of conventional chemotherapy.

Fig. 6. Analysis of mitochondrial membrane potential, by flow cytometry, in F36P cells treated with vitamin C. The mitochondrial membrane potential were analyzed by flow cytometry using JC1 fluorescent probes, as describe in material and methods. JC-1 probe coexist in monomeric or aggregate form depending on the mitochondrial membrane potential an increase in the monomer/aggregate ratio (M/A ratio) indicates a decreased in the mitochondrial membrane potential. Results are expressed in mean ± SD of monomer/aggregate ratio of JC-1 and this ratio was calculated as the fraction of MFI observed for each molecule. ⁄ Daily administration. § p < 0.05; §§p < 0.01; §§§p < 0.001.

in mitochondrial membrane potential, leading to ROS generation. In fact, the reduction of mitochondrial membrane potential and subsequent induction of cell death and/or the mitochondrial dysfunction induced by vitamin C can increase ROS production. The cytotoxic effect observed in cells treated with high concentrations of these

Conflict of interest statement Each author states that there is no financial or personal interest in any company or organization sponsoring the research. Acknowledgments Center of Investigation in Environment, Genetics and Oncobiology (CIMAGO) and Clinical Haematology Service, University Hospitals of Coimbra (HUC) funded this study. We thank Paula Morreira from Institute of Physiology, FMUC – Faculty of Medicine and Center for Neuroscience and Cell Biology (CNC), University of Coimbra, for help with enzymatic activity assays.

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