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Formelli,F. and Cleris,L. (1993) Synthetic retinoid fenretinide is effective against a human ovarian carcinoma xenograft and potentiates cisplatin. VIT-C 100 µM.
Carcinogenesis vol.18 no.5 pp.943–948, 1997

Role of antioxidants and intracellular free radicals in retinamideinduced cell death

Domenico Delia3, Antonella Aiello, Luca Meroni, Marco Nicolini1, John C.Reed2 and Marco A.Pierotti Istituto Nazionale Tumori, Division of Experimental Oncology A, Milan, 1University of Milan, Department of Medical Chemistry and Biochemistry, Milan, Italy and 2The Burnham Institute, Cancer Research Center, La Jolla, CA, USA 3To

whom correspondence should be addressed at Division OSA, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milano, Italy

The cancer chemopreventive synthetic retinoid N-(4hydroxyphenyl)retinamide (HPR) possesses antiproliferative and apoptotic activity at pharmacological doses. In this study we show that addition of antioxidants to HL-60 cells cultured in the presence of 3 µM HPR, markedly suppresses the apoptopic effect of the retinoid and significantly prolongs cell survival (48–96 h). We also show, by the use of the oxidation-sensitive probe 29,79-dichlorofluorescin diacetate (DCF-DA) and in combination with flow cytometric and spectrofluorimetric analysis, that treatment of cells with 3 µM HPR results in an immediate and sustained production of intracellular free radicals, most likely hydroperoxides. Interestingly, the formation of these HPR-induced free radicals is effectively blocked by the water soluble antioxidants L-ascorbic acid and Nacetyl-L-cysteine. Neither 3–15 µM N-(4-methoxyphenyl) retinamide (MPR), the structurally similar but biologically inert analog of HPR, nor 3 µM doses of the retinoids alltrans retinoic acid, 9-cis-retinoic acid, TTNPB and SR11237 induce intracellular free radicals, thus indicating that the specificity of this phenomenon is restricted to HPR. Altogether, we provide the first direct evidence that HPR stimulates the generation of intracellular free radicals, which appear to have a causative role in the induction of apoptosis in vitro. Our findings raise the possibility that the therapeutic efficacy of HPR may, at least in part, depend on these apoptosis-inducing oxidative phenomena.

efficacy of HPR for the prevention of controlateral breast cancer in women surgically treated for node-negative breast cancer (4), and of basal cell carcinoma in patients with oral leukoplakia (5). Data from the former trial already indicate that HPR protects against the development of ovarian carcinoma (6). The in vitro activities of HPR comprise growth inhibition of a variety of tumour cell lines, including breast carcinomas (7–9), neuroblastomas (10), small cell lung carcinomas (11), leukaemias and lymphomas (12), and induction of apoptotic cell death at doses which are achievable pharmacologically (10–12). Recently, we have shown that cell killing by HPR is strongly antagonized by deregulated expression of the antiapoptotic gene bcl-2 as well as by certain antioxidants (13). This evidence, coupled with the reported role of bcl-2 in an antioxidative pathway (14), have led us to speculate that HPR induces apoptosis by eliciting oxidative stress, as in the case of apoptosis induced, for instance, by ionizing radiation or TNF-α, and which involves free radicals (15,16). Although retinoid responses involve a complex network of nuclear retinoic acid receptors (RARs and RXRs) (17), current experimental data suggest that HPR may function by receptorindependent (12,18) and/or receptor-dependent mechanisms. Regarding the latter, very recently it has been found (9,19) that HPR is capable of transactivating RAR-γ more potently than RAR-β, but has little effect on RAR-α. Nevertheless, several issues remain unresolved including whether the biological response is mediated by HPR itself or by some potential metabolite. This study was undertaken to elucidate the role of antioxidants and oxidative stress in cell death induced by HPR. We show that pharmacological doses of HPR, but not of other retinoids tested, rapidly elicit the formation of intracellular free radicals, whose detoxification by antioxidants blocks the apoptotic activity of the retinoid. These data raise the possibility that the therapeutic effectiveness of HPR may, at least in part, depend on its pro-oxidative activity.

Introduction

Materials and methods

N-(4-hydroxyphenyl)retinamide (HPR*) is a synthetic retinoic acid derivative with cancer chemopreventive and therapeutic activities in experimental systems (1). Studies in animals have shown that HPR significantly inhibits the incidence of carcinogen-induced mammary and urinary bladder lesions, induces complete regression of transplanted tumours, and even potentiates the activity of cisplatin (1–3). Randomised clinical trials supported by the US National Cancer Institute are ongoing in our Institute to establish the

Cell line and reagents The myeloid leukaemia cell line HL-60 , mycoplasma free, was grown in RPMI-1640 (Bio-Wittaker, Walkersville, MD) plus 10% heat-inactivated fetal calf serum (Irvine Scientific, Santa Ana, CA), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mmol/l glutamine. HPR and N-(4-methoxyphenyl)retinamide (MPR) were a gift of RW Johnson Pharmaceutical Research Institute, Springhouse, PA. All-trans retinoic acid (ATRA) was from Sigma (St Louis, MO), whereas the retinoids 9-cis-retinoic acid (9-cis-RA), Ro13– 7410 (TTNBP) and SR11237 were a kind gift of Arthur Levine, Hoffman-La Roche, Nutley, NJ. Stock solutions of the retinoids in ethanol, stored at 220°C, were always prediluted 1:10 in foetal calf serum prior to further dilutions in culture media. The antioxidants N-acetyl-L-cysteine (NAC), Lascorbic acid (VIT-C), α-tocopherol (VIT-E), deferoxamine (DFX) (all from Sigma), were used at maximal, non-cytotoxic doses, determined previously by a 72 h dose–response analysis. In assays involving multiple treatments, the antioxidants were added to cells 3 h before addition of other compounds. Viable cells were quantitated on trypan blue stained suspensions using a haemocytometer. Discrimination between apoptotic and necrotic cells was achieved by dual fluorescence microscopy analysis on samples stained with

*Abbreviations: HPR, N-(4-hydroxyphenyl)retinamide; MPR, N-(4-methoxyphenyl)retinamide; RAR, retinoic acid receptor; RXR, retinoid X receptor; TTNPB,(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1impropenyl] benzoic acid; ATRA, all-trans retinoic acid; 9-cis RA, 9-cis retinoic acid; DCF-DA, 29,79-dichlorofluorescin diacetate; DHR, dihydrorhodamine 123; VIT-C, L-ascorbic acid ; VIT-E, α-tocopherol; NAC, N-acetylL-cysteine; DFX, deferoxamine. © Oxford University Press

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D.Delia et al 5 µg/ml of the DNA-specific supravital dye Hoechst-33342 (Calbiochem, San Diego, CA) (30 min at 37°C) and with 10 µg/ml propidium iodide (Sigma) (permeable in necrotic but not in viable or apoptotic cells). Flow cytometric quantification of apoptotic cells was performed on propidium iodide stained samples, as described (13), using a FACSvantage instrument (BectonDickinson, San Jose, CA). In most cases, apoptotic cells were also verified on cytospin preparations by a modified TUNEL technique, as described (12). Fluorescent measurements of intracellular oxidants 29,79-dichlorofluorescin diacetate (DCF-DA, cat. D-399) and dihydrorhodamine 123 (DHR, cat. D-632), purchased from Molecular Probes (Eugene, OR), were used to detect production of free radicals. The fluorescence of these cell-permeable agents significantly increases after oxidation, particularly by hydroperoxides (20,21). Stock solutions of DCF-DA (50 mM) and DHR (1 mM) in DMSO, were stored at 220°C. Exponentially growing cells (23105/ml) were loaded with 50 µM of DCF-DA or 1 µM DHR (15 min at 37°C), hence treated (or not, for negative controls) with HPR or with H2O2. In some assays, cells were incubated for 3 h with antioxidants prior to loading with DCF-DA (or DHR) and treatment with HPR . Samples were harvested at regular intervals and immediately analysed (10 000 cells/sample) by flow cytometry using a 488 nm excitation beam. The mean fluorescence intensity (MFI) was determined by LysisII software (Becton-Dickinson) analysis of the recorded histograms. Endogenous and extracellular oxidised DCF-DA were quantitated on samples (23105 cells/ml) cultured in phenol-free Hanks balanced salt solution (HBSS) (Sigma). After labelling and treatment as reported above, the samples were centrifuged, the supernatants recovered and diluted 1:4 in phosphate buffered saline (PBS), while the pellets were disrupted by freezing in liquid nitrogen and diluted in 2 ml of cold PBS. Both fractions were analysed on a Perkin-Elmer MPF-44A spectrofluorimeter (excitation wavelength 492 6 1 nm, emission 522 6 5 nm).

Results Antioxidants delay the onset of HPR-induced cell death Treatment of HL-60 cells with 3 µM HPR results in .60% apoptotic death by 24 h (Figure 1). Remarkably, in the presence of non cytotoxic doses of the antioxidants VIT-C, NAC, VIT-E and DFX the killing activity of HPR is virtually abrogated (Table I and Figure 1). To determine whether antioxidants block or simply delay the action of HPR, we have analysed these effects over a longer period of time. As shown in Figure 2, while only 5% of the cells remained alive after 48 h of treatment with HPR, at the same time point the cells incubated with HPR plus VIT-C were mostly alive, and actually their number increased almost 3-fold, relative to the number of seeded cells. By 72 h, however, the protective effect of VIT-C against HPR was no longer effective, as evidenced by the sharp drop of cell viability. To ascertain whether this was due to the antioxidant activity of VIT-C being consumed, cells were replenished, after 48 h of treatment with HPR plus VITC, with fresh VIT-C and kept in culture for another 48 h before being analysed. Remarkably, the survival of these cells was significantly enhanced (Figure 2, empty column), resulting, at 96 h in a 6-fold higher cell number, relative to cells that had not been replenished with VIT-C. Also NAC antagonized the toxic effect of HPR, though less markedly than VIT-C, resulting in a 2-fold increase in viable cell counts at 24 h, which gradually declined thereafter. Together, these results emphasize the role of antioxidants in suppressing cell death by HPR. Intracellular oxidants are induced upon treatment with HPR The protection afforded by antioxidants against cell death by HPR suggests that free radicals may be involved in this phenomenon. To verify this possibility, we examined the levels of intracellular free radicals before and after exposure to HPR using the cell permeant dyes DCF-DA and DHR, which become highly fluorescent after oxidation. The fluorescence of these dyes, which increases proportionally with the amount 944

of intracellular oxidised compound, can also be measured in individual cells by flow cytometry (14,20,21). As both probes yielded similar results, only those obtained with DCFDA are reported. The flow cytometric analysis showed that the mean fluorescence intensity (MFI) of DCF-DA-loaded cells augmented markedly and rapidly following treatment with 3 µM HPR (Figure 3A), resulting in 3.8-fold increase by 15 min, and 5.2 by 30 min, relative to the fluorescence of untreated control samples. By 2 h, however, the MFI of both untreated and HPR-treated samples declined appreciably (Figure 3A), supposedly because of the poor intracellular retention of the fluorochrome once it is oxidized (information from manufacturer). To verify this possibility, the amount of fluorochrome present in the intracellular and extracellular compartments was quantitated separately by spectrofluorimetry. The results showed (Figure 3B) that while the intracellular fluorescence increased during the first 30 min of exposure to HPR, to progressively decline thereafter, the fluorescence present in the culture medium of the same samples steadily increased as a function of time. On the other hand, only minimal basal levels of DCF-DA fluorescence were found in the intracellular and extracellular compartments of samples that had not been treated with HPR (Figure 3B, open symbols). Collectively, these data indicated that HPR causes a sustained, and not transient, intracellular oxidation of DCF-DA, which is then subjected to leakage from cells. The production of intracellular free radicals was HPR-dosedependent (Figure 4), as evidenced by the fact that the fluorescence of cells treated for 30 min with 100 nM, 1 µM and 3 µM HPR increased 2.1-, 3.2- and 5.2-fold, respectively, relative to that of untreated controls. Doses of retinoid equal to or below 10 nM, however, did not affect the basal DCFDA signal. Similarly, the DCF-DA fluorescence of H2O2treated cells proportionally increased with increasing the dose of the oxidant above 1 µM (Figure 3A). When comparing the MFI values of these dose–response analysis, the intracellular oxidation elicited by 3 µM HPR was equal to that caused by 100 µM H2O2 (Figure 3A). To determine whether the ability to elicit intracellular oxidants was unique to HPR, other retinoids were also investigated. In particular the retinamide derivative MPR, which is also the major inert biological metabolite of HPR and is ineffective in triggering cell death (13), ATRA and 9-cis-RA, two promoters of HL-60 cell differentiation (22), TTNPB and SR11237, two retinoids highly selective for RAR and RXR, respectively (23). Unlike HPR, however, none of these retinoids modified the fluorescence of DCF-DA-loaded cells when used at 3 µM, as well as 15 µM in the case of MPR (to compensate for its lower intracellular uptake; DD, unpublished observation), clearly indicating that formation of free radicals may strictly depend on the structural features of the retinoid. Remarkably, the increment of HPR-induced DCF-DA fluorescence was completely blocked by VIT-C and NAC (Table II and Figure 4B), strongly suggesting that these antioxidants intercept and neutralise the retinoid-elicited free radicals, which would otherwise oxidise DCF-DA. However, VIT-E, an antioxidant reactive with hydrophobic radicals, did not prevent the HPR-induced intracellular oxidation of DCF-DA. DFX, which inhibits the iron redox cycling (24), partially suppressed HPR-induced oxidation of DCF-DA. Discussion The retinoic acid derivative HPR has remarkable cancer chemopreventive and therapeutic activity in animal systems

Antioxidants, free radicals and retinamide-induced cell death

Fig. 1. Flow cytometric DNA analysis. The histograms were generated from the analysis of cells pre-treated for 3 h with (or without, for controls) the antioxidants, then incubated for 24 h with 3 µM HPR and subsequently stained with propidium iodide, as reported in Materials and methods. The subdiploid peak left of G1 is generated by cells containing fragmented DNA, whose percentage is indicated.

Table I. Inhibition of HPR-induced cell death by antioxidants Compound

Apoptosis inhibition

VIT-C 100 µM NAC 10 mM VIT-E 1 mM DFX 10 µM

97 89 95 91

6 6 6 6

3% 6% 4% 6%

HL-60 cells were pre-treated for 3 h with (or without, for control) the indicated doses of antioxidants prior to addition of 3 µM HPR and incubation for 24 h. Following staining with propidium iodide, as indicated in Materials and methods, the cells were examined by flow cytometry (10 000 events per sample) to quantitate cells with subdiploid, apoptotic DNA. Apoptotic cells in samples treated with HPR alone were 75 6 12%. Numbers denote the percentage protection 6 SD afforded by the individual compounds, and were obtained from not less than three independent experiments.

(1–3), and is currently being evaluated for the prevention of breast cancer and squamous cell carcinoma in humans (4,5). Despite the promising clinical potential of HPR, its mode of action remains unclear. We and others have recently shown that HPR possesses growth-inhibitory as well as apoptosisinducing activities at doses achievable pharmacologically (10– 12), thus suggesting that the therapeutic efficacy of HPR is linked to these functions (25). More recently, we have shown that HPR-induced cell death is antagonised by deregulated levels of the anti-apoptotic gene bcl-2 and by antioxidants

Fig. 2. Effect of antioxidants on the survival of HPR-treated cells. Samples were pre-incubated (or not, for controls) for 3 h with 100 µM VIT-C or 10 mM NAC, then treated with 3 µM HPR and examined every 24 h for viability by trypan blue staining. The viability at 96 h of cells replenished with fresh VIT-C (100 µM) by 48 h of treatment with HPR plus VIT-C, is indicated by the open column. Data are average of three independent experiments 6 SD.

(13), arguing that HPR responses and ultimately apoptosis involve the activation of an oxidative pathway. In this study, we have shown that antioxidants significantly prolong the survival of cells exposed to HPR by at least 48– 72 h, thus suggesting the involvement of an oxidative pathway 945

D.Delia et al

Fig. 3. Generation of intracellular oxidants in reponse to HPR. Time-course fluorescence analysis of DCF-DA-loaded cells exposed to 3 µM HPR. (A) Typical flow cytometric fluorescence histograms of retinoid-treated and untreated samples are shown. Note the loss of fluorescence by 120 min. (B) The levels of fluorescence inside the cells and in the culture medium of the same samples, determined by spectrofluorimetry, are reported (data are average of two independent experiments 6 SD).

in this type of cell death. In this regard, it is worth noting that an oxidative pathway may be involved in cell death elicited by cytokines or by conditions of trophic factor deprivation, as demonstrated by experiments with various antioxidants (15,26). Nevertheless, not all forms of cell death involve an oxidative pathway, as in the case of FAS-induced apoptosis (27). It is important to note that the suppressed biological activity of HPR by the antioxidants is not the result of a diminished uptake of HPR, since comparable levels of retinoid in cells exposed or not to the antioxidants are detected by HPLC analysis (DD, unpublished). Taking advantage of DCF-DA and DHR, which become brightly fluorescent after oxidation (14,20,21), we have demonstrated in cells treated with HPR the formation of free radicals, detectable after 10 min of treatment, at a time when the amount of intracellular HPR accounts for 70% of the plateau level (13). Importantly, neither the structurally related retinamide MPR, which is also a biologically inert metabolite of HPR (8,13), nor ATRA, 9-cis-RA, TTNPB or SR11273, four retinoids with different selectivities for RAR and RXR molecules, and whose biological effects differ from those elicited by HPR (13,22,23), induce the formation of free radicals. Additionally, we have shown that the oxidation of DCF-DA in cells treated with HPR is blocked by the water-soluble antioxidants NAC and VIT-C, and to a less extent by DFX, strongly suggesting these molecules antagonise cell death by neutralising free radicals. It should be noted, however, that the lipophilic chain-breaking antioxidant Vit-E fails to block the intracellular oxidation of DCF-DA in response to HPR. 946

While this result presumably reflects the limited specificity of the DCF-DA indicator for water-soluble reactive oxygen species (28), nevertheless the fact that Vit-E suppresses the biological activity of HPR, most likely through its capacity to intercept peroxyl radicals and so terminate lipid peroxidation reactions, particularly damaging to membranes and recently implicated in apoptotic events (29,30), suggests the possibility that HPR-elicited free radicals cause cell death by inducing oxidative membrane damage. It is worth noting that the results described in this study, obtained by the use of the HL-60 cell line, were also observed in several other human leukemia and lymphoma cell lines, including the RAR-defective and ATRA-unresponsive cell lines HL-60R and NB4–306 (DD, not shown), thus implying that oxidative stress is a common, cell-type and retinoidreceptor independent event in HPR response. Thus far, the mechanism by which HPR evokes free radicals remains unknown, but currently we cannot exclude the possibility that HPR itself is metabolised to a free radical, or that it somehow interferes with the intracellular pathway responsible for maintaining the oxidant-antioxidant balance. In summary, we have shown, for the first time, that HPR elicits the rapid and sustained formation of intracellular oxidants, most likely water-soluble free radicals, whose detoxification by antioxidants blocks the apoptotic activity of the retinoid. This finding, together with molecular data showing that HPR is an activator of retinoid receptors (9,19), supports the view that HPR has two main modes of action, one retinoid-receptor independent and associated with induction of free radicals and

Antioxidants, free radicals and retinamide-induced cell death

Fig. 4. HPR- and H2O2-dose-dependent formation of intracellular free radicals and effect of antioxidants. Typical flow cytometric fluorescence histograms of DCF-DA loaded cells treated for 30 min with varing doses of HPR or H2O2 (A) or with 3 µM HPR after a 3 h pre-incubation with the antioxidants (B).

Table II. Inhibition of HPR-induced intracellular free radicals by antioxidants Compound

Inhibition

VIT-C 100 µM NAC 10 mM VIT-E 1 mM DFX 10 µM

100% 100% 5% 25%

After a pre-incubation for 3 h with the antioxidants, the cells were loaded with DCF-DA, treated for 30 min with 3 µM HPR, and finally analysed (10 000 events per sample) by flow cytometry. Positive and negative controls consisted of DCF-DA-loaded cells, treated or not with 3 µM HPR, respectively. The mean fluorescence intensity (MFI) values were used to calculate, for each compound, the percentage inhibition of HPR-induced DCF-DA fluorescence. Data are average of three independent experiments.

apoptotic cell death, the other retinoid-receptor dependent and associated with transcriptional regulation of as yet unidentified target genes. The contribution of each of these activities with regard to the therapeutic and chemopreventive efficacy of HPR in vivo remains to be elucidated. Acknowledgements This work was financially supported by the Associazione Italiana Ricerca Cancro (AIRC), the CNR (PF-BTBS) and the National Institutes of Health (CA60181). The artwork was by Mario Azzini.

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