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[Cancer Research 61, 1439-1444, February 15, 2001]
© 2001 American Association for Cancer Research

Endocrinology

Vitamin D Is a Prooxidant in Breast Cancer Cells1

Ruth Koren2, Irit Hadari-Naor, Efrat Zuck, Carmela Rotem, Uri A. Liberman and Amiram Ravid

Basil and Gerald Felsenstein Medical Research Center, Rabin Medical Center, Beilinson Campus, Petah Tikva, 49100 [R. K., C. R., U. A. L., A. R.], and Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv [R. K., I. H-N., E. Z., U. A. L.], Israel


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The anticancer activity of the hormonal form of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)2D], is associated with inhibition of cell cycle progression, induction of differentiation, and apoptosis. In addition, 1,25(OH)2D3 augments the activity of anticancer agents that induce excessive reactive oxygen species generation in their target cells. This study aimed to find out whether 1,25(OH)2D3, acting as a single agent, is a prooxidant in cancer cells. The ratio between oxidized and reduced glutathione and the oxidation-dependent inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are considered independent markers of cellular reactive oxygen species homeostasis and redox state. Treatment of MCF-7 breast cancer cells with 1,25(OH)2D3 (10–100 nM for 24–48 h) brought about a maximal increase of 41 ± 13% (mean ± SE) in the oxidized/reduced glutathione ratio without affecting total glutathione levels. The in situ activity of glutathione peroxidase and catalase were not affected by 1,25(OH)2D3, as assessed by the rate of H2O2 degradation by MCF-7 cell cultures. Neither did treatment with 1,25(OH)2D3 affect the levels of glutathione reductase or glutathione S-transferase as assayed in cell extracts. The hormone did not affect overall glutathione consumption and efflux as reflected in the rate of decline of total cellular glutathione after inhibition of its synthesis by buthionine sulfoximine. The extent of reversible oxidation-dependent inactivation of GAPDH in situ was determined by comparing the enzyme activity before and after reduction of cell extracts with DTT. The oxidized fraction was 0.13 ± 0.02 of total GAPDH in control cultures and increased by 56 ± 5.3% after treatment with 1,25(OH)2D3, which did not affect the total reduced enzyme activity. Treatment with 1,25(OH)2D3 resulted in a ~40% increase in glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the generation of NADPH. This enzyme is induced in response to various modes of oxidative challenge in mammalian cells. Taken together, these findings indicate that 1,25(OH)2D3 causes an increase in the overall cellular redox potential that could translate into modulation of redox-sensitive enzymes and transcription factors that regulate cell cycle progression, differentiation, and apoptosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Various lines of evidence, supported by pharmacological studies in animal models and a number of epidemiological studies, indicate that the hormonal form of vitamin D, 1,25(OH)2D,3 acts as an anticancer agent in vivo (1) . Vitamin D is biotransformed by two consecutive hydroxylation steps in the liver and kidney to 1,25(OH)2D, the hormone responsible for calcium and phosphate homeostasis. It is now recognized that 1,25(OH)2D3 also acts in a paracrine manner and is produced extrarenally by various cells including activated macrophages and some tumor cells (2, 3, 4) . Such local production may result in accumulation of the hormone within the tumor milieu to levels that exceed those in the circulation.

The in vivo anticancer activity of 1,25(OH)2D3 has been commonly attributed to its direct cytostatic and cytotoxic effects on cancer cells (1) . In addition, 1,25(OH)2D3 may exert some of its activity by cooperating with other anticancer agents. We and others have found that 1,25(OH)2D3 and its synthetic analogues increased the susceptibility of cancer cells to the cytotoxic/cytostatic action of tumor necrosis factor (5, 6, 7) , interleukin 1, interleukin 6 (8) , doxorubicin, menadione (9) , and radiation (10) . A feature shared by the agents whose potency is increased by 1,25(OH)2D3 is their ability to bring about excessive ROS generation in their target cells (11, 12, 13) . This common feature suggests the involvement of ROS in the interaction between 1,25(OH)2D3 and these agents. Indeed, we found that the potentiation of the cytotoxic/cytostatic action of doxorubicin or the immune cytokines by 1,25(OH)2D3 is markedly inhibited by the addition of the thiol antioxidant, N-acetylcysteine (8 , 9) . Moreover, the oxidative stress experienced by cells exposed to TNF, reflected in the reduction in soluble thiol levels late in the death process, was exacerbated when in the presence of 1,25(OH)2D3 (8) . Cells are continuously exposed to ROS produced intracellularly by normal aerobic metabolism. It is commonly accepted that, under normal conditions, the cellular level of ROS is tightly controlled and may even serve a physiological function (14 , 15) . It seems that ROS can and do function in signal transduction in mammalian cells, and that the activity of several transcription factors is regulated by the cellular redox state (16, 17, 18) .

If, indeed, 1,25(OH)2D3 increases the damaging effect of ROS produced in excess, it is possible that it will also enhance the oxidative impact of ROS produced in the course of normal aerobic metabolism. Such an effect may result in perturbation of the cellular redox state and in modulation of the activity of redox-sensitive proteins. Modulation of key redox-sensitive proteins is, in turn, likely to affect signal transduction networks and transcriptional activity (14, 15, 16, 17, 18) and thus may contribute to the cytostatic and cytotoxic actions of the hormone on cancer cells. We addressed this possibility by studying the effect of 1,25(OH)2D3 on the glutathione redox state in MCF-7 human breast cancer cells and on the redox-sensitive enzyme GAPDH. The rationale for this approach derives from several well-accepted notions: (a) thiols, and particularly glutathione, provide one of the major cellular protective mechanisms against ROS, and an increase in the ratio between the disulfide oxidation product (GSSG) of glutathione and its reduced form (GSH) is a useful marker of cell oxidative stress (19) ; (b) the glutathione pair is coupled to other major redox pairs within the cell, and changes in the ratio between GSSG and GSH reflect changes in the overall cellular redox state; and (c) the activity of the glycolytic enzyme GAPDH is extremely sensitive to thiol oxidation because of an essential cysteine residue in its active site. The extent of this oxidation is considered a sensitive marker of cellular ROS homeostasis (20, 21, 22, 23) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Tissue culture media were purchased from Biological Industries (Beit Haemek, Israel). Tissue culture dishes were from Corning Glass Work (Corning, NY). 1,25(OH)2D3 was obtained from Hoffmann-LaRoche Co. (Nutley, NJ; a generous gift from Dr. M. Uskokovic). "Baker analyzed" H2O2 was from J. T. Baker (Philipsburg, NJ). Aminotriazole, mercaptosuccinic acid, BSO, and glutathione (reduced and oxidized forms) were purchased from Sigma Chemical Co. (St. Louis, MO). ICN Pharmaceuticals, Inc. (Costa Mesa, CA) supplied 2-vinylpyridine. All other reagents were of analytical grade.

Cell Culture.
MCF-7 human breast cancer cells were cultured in Dulbecco’s modified Eagle medium containing 4.5 g/liter glucose and supplemented with 10% FCS and antibiotics. Cells were subcultured twice weekly. Experiments were initiated by seeding 300,000 cells/60-mm Petri dish. Cultures were propagated for 5 days and treated with 1,25(OH)2D3 or with the vehicle ethanol for the last 24–72 h of culture. The concentration of ethanol never exceeded 0.06%.

Determination of Glutathione.
Cells were washed with ice cold PBS [phosphate buffer, 0.01 M; KCl, 2.7 mM; and NaCl 0.137 MM (pH 7.4)], scraped into cold PBS containing EDTA (5 mM) by gentle pipetting, and centrifuged for 20 s at 16,000 x g to obtain cell pellets, which were then resuspended in PBS containing EDTA (1 mM). Twenty mg/ml 5-sulfosalicylic acid was added to the cell suspensions, and after 15 min on ice, the protein precipitates were centrifuged for 1 min at 16,000 x g. Total and oxidized glutathione were determined in the supernatants by the glutathione reductase recycling assay as described by Griffith (24) and adapted to microtiter plates (25) . GSSG was determined in the presence of 2-vinylpyridine to derivatize reduced glutathione (24) . GSH was calculated as the difference between total and oxidized glutathione. Protein content in the sulfosalicylic acid precipitates was determined by the method of Lowry et al. (26) .

Determination of Enzyme Activities.
Cells were washed, scraped, and centrifuged as described above for glutathione determination. For GAPDH, cell pellets were resuspended in PBS containing sucrose (0.25 M) and immediately frozen [the addition of sucrose increases the stability of GAPDH in cell extracts (21) ]. Before enzyme assay, the cells were thawed and pulse-sonicated (5 x 5 s; intensity 4) at 0°C by a Heat System Ultrasonics, Inc., sonicator (model no. W385). An aliquot of the sonicate was incubated with DTT (40 mM) for 1 h on ice. GAPDH activity was determined by following the reduction of NAD+ with glyceraldehyde-3-phosphate as substrate in arsenate buffer (27) . For glutathione reductase, cell suspensions in potassium phosphate buffer [12 mM (pH 7)] were sonicated, and enzyme activity was determined by following the oxidation of NADPH using GSSG as substrate (27) . For glutathione S-transferase, cell suspensions in potassium phosphate buffer [12 mM (pH 7)] were sonicated, and enzyme activity was determined using 1-chloro-2,4-dinitrobenzene as substrate (28) . For G6PD, cell suspensions in Tris-HCl buffer [15 mM (pH 8)] were sonicated, and enzyme activity was determined by following the reduction of NADP+ to NADPH with glucose-6-phosphate as substrate (27) .

H2O2 Degradation.
Degradation of H2O2 by cells in culture was followed essentially as described by Makino et al. (29) . In brief, the medium of cells cultured in 60-mm Petri dishes was replaced with 3 ml of H2O2 (0.2 mM) in PBS containing CaCl2 (1 mM), MgCl2 (0.5 mM), and glucose (4.5 g/liter). Cells were maintained at 37°C. Fifty-µl aliquots from the same Petri dish were collected at 5-min intervals up to 60 min, and H2O2 concentration was determined by the method of Thurman et al. (30) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The effect of 1,25(OH)2D3 on glutathione homeostasis in MCF-7 human breast cancer cells is shown in Table 1Citation . Treatment with 1,25(OH)2D3 (10 or 100 nM) for 24 or 48 h brought about a highly significant increase in the ratio of GSSG:GSH (P = 3.6 x 10-5, by ANOVA of the results from four independent experiments presented in Table 1Citation in which 3–4 replicate cultures were treated with 100 nM 1,25(OH)2D3 for 48 h). The average increase in the GSSG:GSH ratio in these experiments was 41 ± 13% (mean ± SE); that is equivalent to an increase of 4.3 ± 1.1 mV in the redox potential of glutathione. The increase in the GSSG:GSH ratio is attributable to both a slight, but significant, reduction in GSH levels (P = 0.05; ANOVA) and a marked, significant increase in GSSG (P = 0.004). There was no detectable change in the cellular content of total glutathione. The effect of the hormone was time-dependent, apparent after 24 h of treatment, but more pronounced after 48 h (experiment 4). Such a pattern is consistent with the genomic mode of action of the vitamin D receptor. The results of experiment 3 indicate that the effect is dose-dependent and already significant at a concentration of 10 nM 1,25(OH)2D3. For the sake of comparison, cultures of MCF-7 cells, prepared as described in Table 1Citation , were exposed for 1 h to H2O2 (1 mM) and the GSSG:GSH ratio was determined. The average increase in the glutathione redox potential in four independent experiments was 4.7 ± 1.1 mV (mean ± SE). It is noteworthy that the increase in redox potential after chronic exposure to 1,25(OH)2D3 was comparable with that after an acute exposure to a cytotoxic concentration of H2O2.


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  		Table 1 The effect of 1,25(OH)2D3 on glutathione level and redox potential

MCF-7 cells were plated and cultured as described in "Materials and Methods" and treated with 1,25(OH)2D3 for the indicated times. The data are presented as the mean ± SD of 3–4 replicate cultures.

 
The increase in the GSSG:GSH ratio could be attributable to two conceptually different mechanisms. One possibility is that 1,25(OH)2D3 modulates intracellular ROS production, and/or ROS handling, and thus increases the oxidative capacity of ROS produced in the course of normal aerobic metabolism. In that case, the change in glutathione redox potential can be considered a reflection of an overall change in the cellular redox state. Another possibility is that the change in the glutathione redox state is attributable to a direct and primary effect of 1,25(OH)2D3 on the activity of the enzymatic machinery responsible for glutathione homeostasis.

We first addressed this question by assessing the effect of 1,25(OH)2D3 on the in situ activity of the major enzyme responsible for glutathione oxidation, GPx. This was done by assaying the rate of GPx-dependent degradation by MCF-7 cell cultures of exogenously added H2O2. As described previously (29) , the degradation of H2O2 followed first-order kinetics for at least 60 min. The relative contribution of catalase and GPx to the degradation of H2O2 was determined by the use of their specific inhibitors, aminotriazole (31) and mercaptosuccinate (32) , respectively. The inhibitors were used at saturating concentrations as determined in preliminary experiments and were added to the cultures 1 h before and during exposure to H2O2. Table 2Citation gives the first-order rate constants for H2O2 degradation as obtained by regression analysis of the exponential decay curves. The rate of degradation in the presence of both inhibitors was indistinguishable from the spontaneous degradation of H2O2 in the absence of cells. It may thus be inferred that the combined activity of catalase and GPx fully accounts for the degradation of H2O2 by MCF-7 cells. Therefore, the degradation in the presence of aminotriazole provides a faithful measure to the in situ activity of GPx. Table 3Citation illustrates that 72-h treatment with 1,25(OH)2D3 (100 nM) had no effect on H2O2 degradation in the presence or absence of aminotriazole. These results imply that the capacity of GPx is unaffected by the hormone. The same is true also for catalase, the other enzyme responsible for H2O2 degradation. The in situ activity of GPx depends on both the enzyme level and on the concentration of GSH. GSH is continuously consumed during the degradation of H2O2, and its replenishment depends on the recycling of GSSG by glutathione reductase using the reducing power of NADPH. Our results imply that 1,25(OH)2D3 affects neither the level of GPx nor the overall capacity of the GSH regenerating system.


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  		Table 2 The contribution of catalase and GPx to H2O2 degradation by MCF-7 cells

MCF-7 cells were plated and cultured for 5 days, and the degradation of H2O2 was monitored as described in "Materials and Methods." Aminotriazole (50 mM) and mercaptosuccinate (0.2 mM) were present 1 h before and during exposure to H2O2. The data are presented as the first order rate constants of H2O2 degradation.

 

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  		Table 3 The effect of 1,25(OH)2D3 on the degradation of H2O2 by MCF-7 cells

MCF-7 cells were plated and cultured, treated with 1,25(OH)2D3 (100 nM) or vehicle for 72 h, and the degradation of H2O2 was monitored in the presence or the absence of aminotriazole (50 mM) as described in Table 2Citation . The data are presented as the first order rate constants of H2O2 degradation.

 
These conclusions were reinforced by directly assaying the activity of glutathione reductase and G6PD. The latter is the key and rate-limiting enzyme in the pentose phosphate pathway that is responsible for the generation of NADPH. Enzyme activities were assayed in cell extracts with saturating concentrations of substrates and cofactors. The results in Table 4Citation show that the same treatment that increased the GSSG:GSH ratio [48 h exposure to 1,25(OH)2D3 (100 nM)] did not affect the activity of glutathione reductase. We did, however, in agreement with a previous report (33) , find a significant increase in the activity of G6PD in extracts of 1,25(OH)2D3-treated cells. Another glutathione-dependent enzyme that can affect the GSSG:GSH ratio is glutathione transferase. The peroxidase activity of this enzyme against organic peroxides contributes to the generation of GSSG, and its transferase activity causes GSH depletion (34) . Table 4Citation shows no detectable effect of 1,25(OH)2D3 on glutathione transferase activity in MCF-7 cell extracts.


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  		Table 4 Effect of 1,25(OH)2D3 on glutathione reductase, glutathione S-transferase, and G6PD

MCF-7 cells were plated and cultured as described in Table 1Citation and treated with 1,25(OH)2D3 (100 nM) for the last 48 h of culture. Cells were harvested, cell extracts were prepared, and enzyme activities were assayed as described in "Materials and Methods."

 
Modulation of glutathione consumption and efflux could also affect the glutathione redox state. Fig. 1ACitation shows the effect of a 48-h exposure to the inhibitor of glutathione synthesis, BSO, on total glutathione levels in MCF-7 cell cultures. In accordance with previous reports in various experimental systems (35) , exposure to BSO brings about a ~90% decrease in cellular glutathione levels. This effect is dose-dependent and saturates at a concentration of 20 µM (Fig. 1A)Citation . 1,25(OH)2D3-treated and untreated cells were exposed to this saturating concentration of BSO and the decline in glutathione levels was monitored as a function of time (Fig. 1B)Citation . It is evident that the rate of decline in glutathione is not affected by 1,25(OH)2D3. Assuming that the decline in the cellular glutathione level is the result of glutathione consumption and efflux, this finding indicates that these processes are not affected by the hormone.



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  		Fig. 1. Effect of 1,25(OH)2D3 on cellular glutathione levels in the presence of BSO. A, MCF-7 cell cultures were treated with BSO at various concentrations for 48 h. B, cells were treated with 1,25(OH)2D3 (100 nM) for 48 h and then with BSO (20 µM) for the indicated times. The data are presented as the mean ± SD of triplicate cultures (in some cases, the error bars are smaller than the symbols and cannot be seen).

 
Taken together, the results of Tables 3Citation and 4Citation and Fig. 1Citation lead to the conclusion that treatment with 1,25(OH)2D3 does not have a direct effect on the capacity of the major enzymatic systems responsible for glutathione homeostasis. It is thus possible that the change in the GSSG:GSH ratio reflects an overall increase in the cellular redox potential. We further assessed this notion by examining the effect of the hormone on the redox-sensitive enzyme, GAPDH. This household enzyme, known for its role in glycolysis, has an essential cysteine residue in its active site that is extremely sensitive to thiol oxidation (20 , 22 , 36 , 37) . Our first objective was to determine the fraction of cellular GAPDH that is oxidized under normal culture conditions. To this end we assayed the activity of GAPDH immediately after the preparation of cell extracts and after a 60-min incubation at 0°C with DTT (40 mM). (Preliminary experiments indicated that there was no further recovery of GAPDH activity at higher DTT concentrations or after longer incubation periods). We assume that GAPDH activity after treatment with DTT represents the total level of cellular GAPDH. We found that reduction with DTT increased GAPDH activity in MCF-7 cell extracts. This increase (13.2 ± 1.5%, mean ± SE; P = 4.7 x 10-4; paired t test of six independent experiments) represents the in situ reversibly oxidized GAPDH. Using this experimental set up, we examined the effect of 1,25(OH)2D3 on total GAPDH levels and the ratio between the oxidized and reduced fraction of the enzyme (GAPDHox;GAPDHred). The data depicted in Fig. 2Citation clearly show that treatment with the hormone (72 h; 100 nM), significantly increased the GAPDHox;GAPDHred ratio in MCF-7 cells. The average increase in the GAPDH redox ratio was 56 ± 5.3% (mean ± SE; P = 0.014; paired t test). In the same experiments, there was no difference in total GAPDH activity (assayed after reduction with DTT) as a result of treatment with 1,25(OH)2D3 (1.27 ± 0.13, mean ± SE; in control versus 1.33 ± 0.15 µmol/min x mg protein in 1,25(OH)2D3-treated cultures). This finding is in agreement with a previous report that the hormone has no effect on GAPDH gene expression in MCF-7 cells (38) . The effect of 1,25(OH)2D3 on the extent of thiol oxidation of the redox-sensitive enzyme, GAPDH, is compatible with the notion that 1,25(OH)2D3 increases the cellular redox potential in MCF-7 cells.



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  		Fig. 2. Effect of 1,25(OH)2D3 on the oxidized fraction of GAPDH in MCF-7 cells. Cells were treated with 1,25(OH)2D3 (100 nM) for 3 days. GAPDH activity in cell extracts was determined without or after treatment with DTT (40 mM, 1 h). Oxidized GAPDH is the increment in GAPDH activity after DTT treatment. Each bar represents one independent experiment. The data are presented as the mean of 2–4 replicate cultures. Right: bars, ± SE of the six experiments.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major finding of this study is that treatment of MCF-7 human breast cancer cells with 1,25(OH)2D3 brings about an increase in the redox potential of two unrelated thiol redox couples: GSSG-GSH and the oxidized and reduced forms of GAPDH. These changes in redox potential are not accompanied by changes in total glutathione or GAPDH levels. The increase in the GSSG:GSH ratio is not associated with modulations of the major GSH-consuming or -extrusion systems or failure of the GSH regenerating system. The latter is evidenced by the observation that 1,25(OH)2D3 does not affect the rate of degradation of exogenous H2O2 under conditions when GPx is rate-limiting. This in situ activity of the cultured cells depends on the combined and coordinated activities of GPx and glutathione reductase, which in turn depend upon the availability of GSH and NADPH, respectively. A simple stoichiometric calculation shows that the amount of GSH consumed during the degradation of H2O2 in these experiments (Tables 2Citation and 3)Citation equals ~14-fold the total glutathione pool (Table 1)Citation . Because 1,25(OH)2D3 did not decrease the ability of the GSH regenerating systems to cope with such severe oxidative challenge, it seems safe to conclude that the same holds true under normal aerobic metabolism. The reversible oxidation of cys149 in GAPDH may be attributable to S-nitrosylation, S-thiolation (formed via an intermediate thyil radical), or the formation of sulfenic acid (37 , 39, 40, 41) . Various lines of evidence implicate protein disulfide isomerase, thioredoxin, glutaredoxin, and GSH in the reduction of the oxidized cysteine in GAPDH (20 , 42 , 43) . Because the change in GSH levels after 1,25(OH)2D3 treatment is marginal (Table 1)Citation , and GSSG is not involved in GAPDH deactivation (44 , 45) , it is unlikely that the increase in GAPDH redox potential is secondary to the effect on glutathione redox potential. An inference compatible with our findings is that the 1,25(OH)2D3-induced increase in the redox potential of these two independent thiol redox couples is a manifestation of an overall shift in the cellular redox potential. In line with this mechanism is the comparable increase in the redox ratios of the two couples caused by treatment with the hormone, 41 ± 13% for GSSG:GSH as compared with 56 ± 5% for GAPDHox:GAPDHred. Treatment with 1,25(OH)2D3 also increased the cellular level of G6PD (Ref. 33, and Table 4Citation ). The level of this enzyme, which has a key role in the generation of NADPH via the pentose phosphate pathway, increases in response to various modes of oxidative challenge in mammalian cells (46) . It is conceivable therefore, that its elevation in 1,25(OH)2D3-treated cells is an additional consequence of the prooxidant action of the hormone.

The prooxidant action of 1,25(OH)2D3 in MCF-7 cells could result from increased intracellular ROS production in the course of aerobic metabolism, and/or from changes in ROS handling. We show here (Table 3)Citation that the ability to degrade H2O2 either by GPx or catalase or to handle organic peroxides by glutathione transferase (Table 4)Citation is not impaired in 1,25(OH)2D3-treated cells. However, we have reported previously that treatment with 1,25(OH)2D3 inhibited the expression of one of the major constituents of the cellular defense system against ROS, the enzyme Cu/Zn SOD (9) . This decrease could be one of the mechanisms underlying the prooxidant action of 1,25(OH)2D3. Indeed, it was previously shown that overexpression of SOD protects MCF-7 cells from injury caused by various ROS generating agents (47 , 48) . Decrease in SOD levels would cause a shift in the balance between superoxides and H2O2. Increased levels of superoxides can, in turn, cause increased oxidative damage attributable to interaction with NO to form the highly toxic peroxynitrite (49) and to increased availability of free iron that supports hydroxyl radical formation via the Fenton reaction (50) .

Changes in the redox state could translate into reversible oxidation of cysteines in major proteins that determine cell fate, such as protein kinases, protein tyrosine phosphatases, and transcription factors (e.g., Sp1, activator protein-1, nuclear factor {kappa}B, and p53; Refs. 14, 15, 16, 17, 18 , 51 , 52 ). Key components of the apoptotic process, such as mitochondrial permeability transition pores and caspases, are also subject to redox regulation (53 , 54) . Oxidation of the cysteine in the active site of GAPDH may be considered a sensitive, easily accessible marker for these processes. It is noteworthy that the increase in the cellular redox potential reported here (3–8 mV) is of the same order of magnitude as the redox change (15 mV) that was shown to abolish the DNA-binding ability of the transcription factors activator protein-1 and nuclear factor {kappa}B (55) . It was also shown that, at a GSSG:GSH ratio of 0.05 (similar to the values presented in this study), 50% of the cysteine residues in the DNA-binding domain of c-Jun were reversibly glutathionylated (56) and thus sensitive to small changes in the cellular redox potential. These and similar findings indicate that perturbations in the cellular redox state similar to those found in the present study can indeed affect the function of redox-sensitive proteins. Our finding that the effect of 1,25(OH)2D3 on the cellular redox state is similar to that caused by an hour of exposure to the potent oxidant H2O2, provides additional evidence of the biological significance of the action of this hormone.

The anticancer activity of 1,25(OH)2D3 is associated with induction of differentiation, cell cycle arrest, and apoptosis, and with sensitization of cancer cells to the cytotoxic action of some host anticancer agents and therapeutic modalities. The data of this study lead us to present the notion that an increase in the cellular redox potential plays a role in the various anticancer actions of active vitamin D metabolites and analogues.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by the Israel Science Foundation administered by the Israel Academy of Sciences and Humanities (Research Grant No. 601/99). Back

2 To whom requests for reprints should be addressed, at Felsenstein Medical Research Center, Beilinson Campus, Rabin Medical Center, Petah Tikva 49100, Israel. Phone: 972-3-9377394; Fax: 972-3-9211478; E-mail: rkoren{at}post.tau.ac.il Back

3 The abbreviations used are: 1,25(OH)2D, 1,25-dihydroxyvitamin D; ROS, reactive oxygen species; aminotriazole, 3-amino-1,2,4-triazole; BSO, buthionine sulfoximine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6PD, glucose-6-phosphate dehydrogenase; GPx, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; SOD, superoxide dismutase. Back

Received 9/11/00. Accepted 12/13/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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