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[Cancer Research 59, 3985-3990, August 1, 1999]
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[Cancer Research 59, 3985-3990, August 15, 1999]
© 1999 American Association for Cancer Research

Endocrinology

Anhydroretinol Induces Oxidative Stress and Cell Death1

Yanqiu Chen, Jochen Buck and Fadila Derguini2

Department of Pharmacology, Joan and Sanford I. Weill Medical College of Cornell University [Y. C., J. B.], and Department of Immunology, Memorial Sloan-Kettering Cancer Center [F. D.], New York, New York 10021


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The retro-retinoid anhydroretinol (AR), a physiological metabolite of retinol (vitamin A), induces cell death in multiple in vitro systems. AR-induced cell death is blocked by retinol and its metabolite 14-hydroxy-4,14-retro-retinol. AR has been shown also to prevent mammary cancer induced by N-methyl-N-nitrosourea in rats. We report that AR kills cells by generating reactive oxygen species. Direct measurements show that the addition of AR to lymphoblastoid cells increases the intracellular oxidative stress in a time- and dose-dependent manner. Furthermore, the amount of induced oxidative stress directly correlates with the number of dying cells. The addition of retinol, 14-hydroxy-4,14-retro-retinol, or the antioxidant, {alpha}-tocopherol (vitamin E), decreases AR-induced oxidative stress and proportionally reduces AR-induced cell death. In contrast, pretreatment with caspase inhibitors, known to inhibit apoptosis, has no effect on AR-induced cell death. This is the first demonstration of cellular reactive oxygen species production by a natural retinoid.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinol (vitamin A) is converted to different biologically active metabolites such as retinal, RA3 , and the two retro-retinoids, AR and 14-HRR (1) . AR, first isolated as a natural product from fish oils (2) , has also been found in mouse liver,4 transformed mouse fibroblasts (3) , the nematode Brugia pahangi (4) , the moth Spodoptera frugiperda, and in the Drosophila Schneider cell line S2M3 (5) . Retinol dehydratase, the enzyme that converts retinol to AR in SF-21 cells was purified and molecularly characterized (6 , 7) .

The biological functions of retinal (8) and RA (9) are well characterized, and their mechanisms of action are well defined at both cellular and molecular levels. In contrast, knowledge about the retro-retinoids AR and 14-HRR is limited to their biological activities. AR induces cell death in cultured T and B lymphocytes (5 , 10 , 11) , fibroblasts (12) , and promyelocytic leukemia cells (13) , whereas retinol and 14-HRR support the growth of these cells when cultured in serum-free medium (1 , 10 , 12 , 14 , 15) . Furthermore, AR-induced cell death is reversibly antagonized by retinol or 14-HRR (5 , 10, 11, 12) . Recently, AR was shown to prevent mammary cancer induced by N-methyl-N-nitrosourea in rats (16) . The mechanism of action is currently unknown.

Cells are constantly under attack from ROS, such as superoxide radical anion, hydrogen peroxide, and lipid peroxides generated during metabolism (17 , 18) . ROS change the cellular redox state, and damage proteins, DNA, and membrane lipids. To survive, cells rely on enzymatic and nonenzymatic antioxidant defense systems. The enzymatic system comprises mainly superoxide dismutase, catalase, and peroxidase (17) , and the nonenzymatic system is constituted of a class of small antioxidant molecules, such as vitamin E, vitamin C, and ubiquinol (19) . The antioxidant role of retinoids has also been documented (20) . Recent studies show that ROS production is tightly regulated and this regulation plays an important role in determining the survival or death of cells. ROS are downstream mediators of p53-dependent cell death, which can be prevented by antioxidants (21 , 22) . Bcl-2, the best characterized physiological cell survivor factor, down-regulates ROS (23 , 24) . ROS cause both apoptotic and necrotic cell death depending on whether their concentration is, respectively, low or high (25 , 26) . The involvement of ROS in these different forms of cell death prompted us to investigate the possible role of oxidative stress in AR-induced cell death.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Cells.
14-HRR and AR were synthesized as described (10 , 27) . Retinol was purchased from Fluka Chemical Corp. (Milwaukee, WI); {alpha}-tocopherol from Matreya Inc. (Pleasant Gap, PA); insulin, holo-transferrin, delipidated BSA from Sigma Chemical Co. (St. Louis, MO); 6-carboxy-2'7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) from Molecular Probes Inc. (Eugene, OR); caspase inhibitors Z-DEVD-FMK and Z-VAD-FMK from Calbiochem-Novabiochem Corp. (San Diego, CA); Boc-Asp-FMK was a gift from Enzyme System Products (Livermore, CA); antihuman Fas mAb (CH-11) was purchased from Upstate Biotechnology (Lake Placid, NY); [3H]thymidine (6.7 Ci/mmol) from DuPont/NEN (Boston, MA); WST-1 from Boehringer Mannheim Corp. (Indianapolis, IN). The EBV-transformed human B lymphoblastoid cell line 5/2 was established from the blood of a healthy volunteer. The human lymphoblastoid T cell line Jurkat was purchased from the American Type Culture Collection (Manassas, VA). Both cell lines were cultured in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, and 100 units/ml penicillin/streptomycin. The serum-free ITLB medium used is RPMI 1640 supplemented with 5 µg/ml insulin, 5 µg/ml holo-transferrin, 20 µM linoleic acid, and 0.2% delipidated BSA.

Flow Cytometry Analysis of Cellular Oxidative Stress and Cell Viability.
ROS production was directly measured in AR-treated cells by flow cytometry, using 6-carboxy-2'7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester), a membrane permeable and oxidant-sensitive reagent (23) . The acetate and acetoxymethyl ester groups of this reagent are enzymatically cleaved in the cell. After oxidation by cellular ROS, the resulting fluorescent product is retained inside the living cells due to its electric charges and emits light with an intensity proportional to the amount of ROS generated. Flow cytometry measures simultaneously ROS production from fluorescence intensity and cell viability from the side and forward scattering pattern, thus providing a direct correlation between oxidative stress and cell death in the same sample.

The 5/2 cells were washed once with serum-free ITLB medium, suspended in the same medium at the density of 70,000 cells/ml, and aliquoted into 6-ml sterile Falcon tubes (1 ml/tube). AR alone or AR plus retinoids or {alpha}-tocopherol were then added into each tube. At given time points, cells were treated with 5 µM 6-carboxy-2'7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) for 1 h, and fluorescence-activated cell-sorting analysis of both fluorescence and scattering pattern followed (FACSCalibur; Becton Dickinson Laboratory). For fluorescence measurement, the excitation and emission wavelengths were set at 488 and 530 nm, respectively. Six thousand cells of each sample were analyzed.

Measurement of Cell Viability by [3H]Thymidine Incorporation Assay.
Cultured 5/2 and Jurkat cells were washed once with ITLB medium and plated into 96-well microtiter plates in 100 µl/well of the same medium, at the density of 80,000 cell/ml for 5/2 cells and 160,000 cells/ml for Jurkat cells. The addition of {alpha}-tocopherol in 50 µl of ITLB medium was followed by AR in 50 µl of ITLB medium. After 2 days of incubation, cells were labeled with 1 µCi/well [3H]thymidine for 5 h.

Measurement of Cell Viability by WST-1.
Cultured Jurkat cells were washed once with ITLB medium, plated into 96-well microtiter plates in 100 µl/well of the same medium, at the density of 150,000 cells/ml. After a 30-min pretreatment with caspase inhibitors, added in 25 µl of ITLB medium, AR or antihuman Fas mAb in 25 µl of ITLB medium was added, and cells were further incubated for 9 h. WST-1 (10 µl/well) was pulsed for the last 3 h. The absorbance (A450 nm-A650 nm) was determined using a 96-well reader (Molecular Dynamics).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AR Generates Intracellular ROS.
Human B lymphoblastoid 5/2 cells were treated with 2 µM AR, and cellular oxidative stress, as well as cell viability, were measured after 3 and 5 h (Fig. 1A)Citation . After 3 h, AR-treated cells exhibited a strong ROS production, as shown by the up to 10-fold increase in fluorescence intensity in these cells. However, there was no significant difference in the light scattering pattern, thus indicating that, after 3 h, AR-treated cells were still alive. After 5 h, 50% of AR-treated cells were dead, whereas cell death was not observed in the controls. The fluorescence plot of AR-treated cells displayed two distinct peaks: R1 and R2, corresponding to dead and living cells, respectively (Fig. 1B)Citation . The fluorescence intensity in living cells decreased compared with the 3-h value, but remained higher than that measured in the controls. The low fluorescence intensity in dead cells was due to leakage of the fluorescent product through the membrane.



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  		Fig. 1. Cell death in 5/2 cells follows AR-induced oxidative stress. A, cells were treated with 0 µM or 2 µM AR. After 3 and 5 h, cellular oxidative stress was measured by fluorescence in histograms, and cell death was measured by cell scattering in dot-blots. In histograms, the X axis is the relative fluorescence intensity in log scale (FL1-H); the Y axis corresponds to the relative number of cells (Counts). In dot-plots, the X axis corresponds to the forward scattering intensity (FSC-H), and the Y axis corresponds to the side scattering intensity (SSC-H). B, fluorescence of gated cell populations in the sample treated with 2 µM AR for 5 h. Cells gated in R1 and R2 correspond to dead and living cells, respectively. Loss of membrane integrity in dead cells causes fluorescent dye to diffuse out of the cells; therefore, the cellular fluorescence intensity in dead cells is lower than in living cells.

 
Cellular Oxidative Stress and Cell Death Induced by AR Are Time- and Dose-dependent.
The 5/2 cells were treated with 0–4 µM AR, and cellular oxidative stress and cell death were followed from 1–10 h after treatment (Fig. 2)Citation . After 1 h, AR-induced oxidative stress was barely detectable. It increased significantly after 2 h in the 2 µM and 4 µM AR-treated cells and reached a maximum after 3 h for all AR doses tested (0.5–4 µM). AR-induced oxidative stress is transient. After 10 h, AR-generated ROS disappeared as the fluorescence intensity in cells that survived from AR treatment went down to the level observed in the control cells. This transient AR effect on oxidative stress may reflect a fast metabolism of AR. AR-induced cell death was first detectable at the 3-h time point in cells treated with >=2 µM AR. After 10 h, 90% of the cells treated with 4 µM AR and 50% of the cells treated with 0.5 µM AR were dead. Compared with AR-treated cells, the control cells did not exhibit any sign of cell death. The up-regulation of ROS and the induction of cell death were both dependent on the concentration of AR, and the intensity of oxidative stress correlated well with the number of dying cells. Therefore, the production of ROS may be the cause of AR-induced cell death.



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  		Fig. 2. AR-induced oxidative stress and subsequent cell death is time- and dose-dependent. Cells were treated with 0, 0.25, 0.5, 1, 2, and 4 µM AR at time 0 h. Cellular oxidative stress and cell viability were measured after 1, 2, 3, 5, and 10 h. X and Y axes are as described in Fig. 1Citation .

 
AR-generated ROS and Cell Death Are Prevented by the Antioxidant {alpha}-Tocopherol and the Retinoids Retinol and 14-HRR.
Our early studies showed that AR-induced cell death can be blocked by retinol and 14-HRR, but not by RA (5 , 10, 11, 12) . If ROS are the cause of AR-induced cell death, antioxidants should be able to reverse the AR effect. Thus, in addition to the retinoids, we tested the effect of {alpha}-tocopherol, the most potent isomer of the antioxidant vitamin E, on cellular oxidative stress and cell death in AR-treated cells. Lymphoblastoid 5/2 cells were treated with {alpha}- tocopherol (0.2 µM), retinol (2 µM), 14-HRR (2 µM), and RA (2 µM), in addition to 1 µM AR; control cells received only 1 µM AR. Oxidative stress and cell viability were measured at 3, 6, 10, and 24 h after treatment (Fig. 3A)Citation . Cells treated with AR alone showed highest oxidative stress at 3 h, all cells were dead after 24 h. The addition of {alpha}-tocopherol or retinol, to a lesser extent 14-HRR, decreased oxidative stress and blocked cell death. The lower efficacy of 14-HRR is probably due to its fast metabolism (data not shown). RA showed a negligible effect on both oxidative stress and cell death. With all compounds, the ability to suppress AR-induced oxidative stress correlated well with their ability to prevent cell death.



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  		Fig. 3. A, {alpha}-tocopherol and retinoids block both oxidative stress and cell death induced by AR. Cells incubated with or without 1 µM AR were treated with retinoids (retinol, 14-HRR, or RA at 2 µM) or {alpha}-tocopherol (0.2 µM). Cellular oxidative stress and cell viability were measured after 3, 6, 10, and 24 h, as described in Fig. 1. BCitation , {alpha}- tocopherol prevents AR-induced cell death in a dose-dependent manner. Jurkat and 5/2 cells were treated with AR (0.2–3 µM) and {alpha}-tocopherol (0–0.5 µM). [3H]thymidine incorporation was assayed after 2 days. Data represent the mean and SD of triplicate measurements.

 
The ability of {alpha}-tocopherol to suppress AR-induced cell death was further demonstrated by cell viability assays in both B lymphoblastoid 5/2 cells and T lymphoblastoid Jurkat cells. Cells were treated with different concentrations of {alpha}-tocopherol and AR, and cell viability was quantified by [3H]thymidine incorporation. As shown in Fig. 3BCitation , {alpha}-tocopherol reversed AR-induced cell death in a dose-dependent manner at different AR concentrations. Compared with retinol, {alpha}-tocopherol was about 10 times more potent.

Suppression of AR-generated ROS and Cell Death by Retinol Is Dose-dependent.
As displayed in Fig. 4Citation , cells treated with 1 µM AR and with different concentrations of retinol (0–2 µM) exhibited amounts of oxidative stress and cell death reciprocally proportional to the retinol concentration; namely, the higher the retinol concentration, the lower the amount of oxidative stress and cell death. The addition of retinol to cells not treated with AR had little effect. The same dose-dependency was observed with {alpha}-tocopherol and 14-HRR (data not shown).



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  		Fig. 4. Retinol reverses AR-induced oxidative stress and cell death in a dose-dependent manner. At time 0 h, one set of cells was treated with retinol (0–2 µM) plus 1 µM AR; the control set received retinol only. Oxidative stress and cell viability were measured at 3, 6, 10, and 24 h, as described in Fig. 1Citation .

 
Caspase Inhibitors Are Unable to Prevent AR-induced Cell Death.
We used three caspase peptide inhibitors to test the possible involvement of caspases in AR-induced cell death. Peptides Z-VAD-FMK and Boc-Asp-FMK are general inhibitors of the known caspases (28) , whereas Z-DEVD-FMK is a selective inhibitor of caspase 3 (29) . Jurkat cells pretreated with these peptides neither suppressed nor delayed cell death induced by AR, whereas all three peptides inhibited cell death by anti-Fas mAb (Fig. 5)Citation . Cytosolic extracts from AR-treated Jurkat cells did not cleave in vitro-translated caspase 3, the predominant caspase in these cells (data not shown). These results imply that caspases are not involved in AR-induced cell death.



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  		Fig. 5. Caspase inhibitors do not prevent AR-induced cell death. Cells were preincubated 30 min with Z-DEVD-FMK (0–150 µM), Z-VAD-FMK (0–200 µM), Boc-Asp-FMK (0–200 µM), then treated for 9 h with AR (0, 0.3, 1, 3 µM), or with antihuman Fas mAb (CH-11, 250 ng/ml). Cell viability was measured by absorbance (A450 nm-A650 nm) after incubation with WST-1 for the last 3 h. Data represent the mean and SD of duplicate measurements.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
AR, a metabolite of retinol, is found in micromolar amounts in mammalian liver and lung and induces cell death in different cells, such as activated T and B lymphocytes (5 , 10 , 11) , fibroblasts (12) , and promyelocytic leukemia cells (13) . The potency of AR in these cells differs; lymphoblasts are about 10 times more sensitive to AR than fibroblasts. AR-induced cell death is cell cycle independent and does not display classical signs of apoptosis (11) . Namely, the cell nuclei remain morphologically unchanged, and nuclear condensation, as well as fragmentation, are not observed. Oligonucleosomal DNA bands cannot be detected; however, DNA damage does occur, as shown in the terminal deoxynucleotidyl transferase reaction (terminal deoxynucleotidyl transferase-mediated nick end labeling assay). In addition, the cytoplasmic volume increases, cytosolic vacuoles fuse, and the ballooned cells eventually burst. AR-induced cell death can be prevented by retinol, 14-HRR, or serum, but not by RA (5 , 10, 11, 12) .

In this study, we show that AR induces cell death by up-regulating ROS production. ROS are generated within 1–5 h after AR treatment, and their production reaches a maximum around 3 h after treatment. The amount of ROS generated by AR correlates well with the number of dying cells. Both up-regulation of ROS and the subsequent AR-induced cell death can be prevented by the retinoids retinol and 14-HRR, and by {alpha}-tocopherol.

The mechanism by which AR induces ROS production is unknown; however, two hypotheses can be formulated. At relative high oxygen pressure, it is known that ß-carotene, rather than scavenging radicals, acts as a prooxidant by propagating radical chain reactions (30) . AR, a conjugated polyene molecule, shares the same structural feature as ß-carotene and, thus, may behave similarly under our assay conditions. Equally possible is that AR may up-regulate the endogenous ROS production or down-regulate the elimination of ROS by binding to a receptor. However, a receptor-mediated mechanism must be independent of transcription and translation because the addition of cycloheximide or actinomycin D neither delayed nor prevented AR-induced cell death (11) .

{alpha}-Tocopherol is known to prevent cellular lipid oxidation and to support the proliferation of various cells (31) . Its ability to prevent AR-generated ROS and subsequent cell death is, most likely, due to its antioxidant property. Thus, the effect of retinol and 14-HRR could be similarly attributed to antioxidative activity. Unlike the hydrocarbon AR, retinol and 14-HRR are allylic alcohols; this might account for their different oxidative properties. However, it remains possible that retinol and 14-HRR suppress downstream ROS production by antagonizing AR at the receptor level. The inability of RA to affect ROS production or AR-induced cell death is consistent with its weaker antioxidative activity and a possible different cellular distribution due to its COOH-terminal group.

4-HPR, a synthetic retinoid, shows promising results in the chemoprevention of breast cancer (32) . It has been suggested that its mechanism of action may not involve the nuclear RA receptors, but rather the induction of ROS (33 , 34) . Indeed, treatment of sensitive cells with micromolar concentrations of 4-HPR generates ROS and triggers both apoptotic and necrotic cell death that can be prevented by the addition of antioxidants (34 , 35 , 36) . Therefore, both AR and 4-HPR induce cell death by increasing cellular ROS concentration. However, in contrast to AR, 4-HPR induces apoptosis with DNA ladder formation. Because AR is an apolar lipophilic hydrocarbon with rigid retro-structure skeleton and 4-HPR an amphiphilic retinoid belonging to the more flexible retinoid ß-series, they may act at different subcellular sites.

We also show that AR-induced cell death is independent of caspase activation. Caspases, a family of cysteine proteases, were first linked to apoptosis through genetic studies of developmental cell death in Caenorhabditis elegans. In mammals, caspases mediate cell death induced by Fas-ligand and may be also involved in cell death due to withdrawal of cytokines and neurotrophic factors (37) . Although caspases have been demonstrated to be essential to cells undergoing apoptosis triggered by low doses of cytotoxic drugs or oxidants, high doses of these reagents induce cell death without caspase activation (26) . Because AR treatment generated a high dose of ROS, it is no surprise that caspase activation is not required for AR-induced cell death.

Natural and synthetic retinoids play an important role in cancer treatment and prevention (38) . Thus, RA is an essential component of the treatment of acute promyelocytic leukemia and by itself it induces complete remission of acute promyelocytic leukemia (39) . Retinyl acetate and 4-HPR have proven to be effective in the chemoprevention of mammary cancer induced by N-methyl-N-nitrosourea in rats (32) . Although 4-HPR presents the advantage of having low hepatotoxity compared with retinyl acetate (32) , its treatment markedly decreases the plasma concentration of retinol and causes retinoid deficiency in tissues, such as the eye pigment epithelium (33) . On the contrary, AR was shown to be remarkably nontoxic in animal studies. This low toxicity, added to the recent study on efficacy of AR in preventing mammary cancer induced by N-methyl-N-nitrosourea in rats, makes AR a candidate for chemopreventive tests against human breast cancer.


   ACKNOWLEDGMENTS
 
We thank Dr. Lonny Levin for comments on the manuscript and Dr. Bill Telford for technical assistance on flow cytometer. F. D. thanks Dr. G. Massiot for encouragement.


   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 Supported by NIH Grants DK-52797 and DK-48022. Back

2 To whom requests for reprints should be addressed, at CNRS UMR 1973, Institut de Recherche Pierre Fabre, Parc Technologique du Canal, 3, rue Ariane, 31527 Ramonville, France. Phone: 33-5-61-73-73-51; Fax: 33-5-61-73-73-73. Back

3 The abbreviations used are: RA, retinoic acid; AR, anhydroretinol; 14-HRR, 14-hydroxy-4,14-retro-retinol; ROS, reactive oxygen species; 4-HPR, N-(4-hydroxyphenyl)retinamide. Back

4 J. Buck, unpublished results. Back

Received 3/ 2/99. Accepted 6/17/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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