Fenretinide) in Combination with Retinoic Acid Enhances Differentiation and Retinoylation of Proteins

Vol. 1, 637-642, June 1995 Clinical Cancer Research 637

N-(4-Hydroxyphenyl)retinamide (Fenretinide) in Combination with Retinoic Acid Enhances Differentiation and Retinoylation of Proteins

Noriko Takahashi,’ Edward A. Sausville, whether these effects, either singly or in combination, mediate and Theodore R. Breitman the anticarcinogenic and antiproliferative activities of 4-HPR. Although 4-HPR and RA share some activities, there is Laboratory of Biological Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, evidence that they have divergent mechanisms of action. Thus, NIH, Bethesda, Maryland 20892-4255 4-HPR induces apoptosis in HL-60 and NB4 human leukemia cell lines that are resistant to RA (16), binds poorly or not at all

to the RARs or the retinoid X receptors,3 ‘ and fails to trans- ABSTRACT activate the RARs or the retinoid X receptor a in chloramphen- The synthetic retinoid, N-(4-hydroxyphenyl)retinamide icol acetyltransferase assays.5 It generally is accepted that many (4-HPR; Fenretinide), is a cancer chemopreventive and an- effects of RA are mediated by these receptors (17) which tiproliferative agent whose mechanism of action is unknown. directly activate transcription of target genes by binding to 4-HPR alone is a poor inducer of differentiation of HL-60 specific DNA sequences. cells compared to all-trans-retinoic acid (RA). Here, we RA induces the differentiation of HL-60 cells to cells with found that combinations of 4-HPR and RA synergistically many functional and morphological characteristics of mature induced differentiation of HL-60 cells. In addition, 4-HPR granulocytes (18). In addition to the retinoid nuclear receptors, increased the level of retinoylation, the covalent binding of RA acylation (retinoylation) of proteins is a candidate mecha- RA to proteins. Retinoylation occurs in many eukaryotic cell nism for the effects of RA on cells (19, 20). We showed that lines and may be involved in RA-induced differentiation. retinoylation involves the formation of a covalent bond with These results suggest that 4-HPR may be a member of a preformed protein. The extent of retinoylation is dependent on class of retinoids that are active because they displace RA the initial concentration of RA in a saturable manner. Further- from extracellular and intracellular sites or because they more, with intact HL-60 cells the EDSt) values for RA-induced inhibit RA catabolism. On the basis of these proposed mech- differentiation and for retinoylation are similar. These results anisms, retinoids that do not cause differentiation as sole suggest that retinoylation may be involved in the mechanism by agents may have utility in the clinic in combination with RA. which HL-60 cells differentiate in the presence of RA. In a previous study, we found that Am80, a synthetic INTRODUCTION retinoid which is a ligand for the nuclear retinoid receptors, is 4-HPR2 is an effective chemopreventive (1) and antipro- covalently bound to protein at a very low level compared to RA liferative agent (2-8) for a wide variety of tumor types includ- and does not induce differentiation of HL-60 cells (20). How- ing those of the breast, prostate, ovary, and bladder. 4-HPR is in ever, Am80 increases the level of retinoylation, and combina- clinical trials for cancers of the breast (9) and the bladder (10). tions of RA with Am80 synergistically induce differentiation of A mechanism for the chemopreventative and antiprolifera- HL-60 cells. These findings are consistent with a covalent tive activities of 4-HPR is unknown. 4-HPR and RA compete modification of proteins by a retinoid playing a role in differ- with retinol for binding to plasma retinol binding protein (11), entiation of HL-60 cells. inhibit the retinol-metabolizing enzymes lecithin-retinol acyl- In the present study, we have extended our earlier study transferase (12), acyl-CoA-dependent retinal reductase (12), and with Am80 by examining the modulation by 4-HPR of RA- acyl CoA-retinol acyltransferase (13), and up-regulate RAR-3 induced differentiation and retinoylation in HL-60 cells. in normal mammary epithelial cells (14). In addition, 4-HPR impairs the secretion of the retinol-plasma retinol-binding pro- MATERIALS AND METHODS tein complex from liver and other tissues (15). It is unclear Cells. Early passage (<30) human myeloid leukemia cell line HL-60 (21) and HL-60(S), an HL-60 variant with enhanced sensitivity to RA (22) (provided by Dr. K. Shudo, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan) Received 1 1/16/94; accepted 2/3/95. were maintained in RPMI 1640 medium (GIBCO, Grand Island,

1 To whom requests for reprints should be addressed, at Laboratory of NY) supplemented with 10 msi 4-(2-hydroxyethyl)-1-pipera- Biological Chemistry, Developmental Therapeutics Program, Division zinethane sulfonic acid (pH 7.3) and 10% FBS (v/v; GIBCO). of Cancer Treatment, National Cancer Institute, NIH, Building 37, Room SD-02, Bethesda, MD 20892-4255. 2 The abbreviations used are: 4-HPR, N-(4-hydroxyphenyl)retinamide (Fenretinide); RA, all-trans-retinoic acid; FBS, fetal bovine serum; CI, combination index; RAR, retinoic acid nuclear receptor; 3 A. A. Levin, personal communication. Am80, 4(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenylcarbani- 4 M. Clagett-Dame, personal communication. oyl) benzoic acid. 5 P. G. Pelicci, unpublished observations cited in Ref. 16.

The population doubling times in medium containing 10% FBS were 17 h for HL-60(S) cells and 36 h for HL-60 cells. Cell I- cultures were incubated at 37#{176}Cin a humidified atmosphere of z

5% CO2 in air. C Cell Differentiation. Cells (2 X 105/ml for HL-60 and 0 to 2 X 104/ml for HL-60(S)) were grown in RPM! 1640 medium C containing various concentrations of either RA (Sigma Chemi- cal Co., St. Louis, MO) or 4-HPR (gift from Dr. R. C. Moon, Specialized Cancer Center, University of Illinois, Chicago, IL) and either 5% or 10% FBS (serum-containing medium) or 5 at p,g/ml of both insulin and transferrmn (serum-free medium). We

estimated cell number on an electronic particle counter (Coulter .0 to Electronics, Hialeah, FL), viability by trypan blue dye exclu- 5 sion, and differentiation by the capacity of the cells to reduce nitroblue tetrazolium (23). Analysis of Combined Drug Effects. Isobologram anal- at ysis was the basis for analyzing the combined effects of 4-HPR .C

and RA on differentiation. The interaction of two inducers was 0 quantified by determining a CI value for each fixed concentra- a, tion ratio according to the classic isobologram equation (24): z

2301 = [(D)1/(Dj1] + [(D)2/(Dj2] CI Log Concentration, nM

where D is the dose required to produce an effect alone, and Fig. 1 Comparison of the effects of RA and 4-HPR on differentiation, viability, and growth of HL-60 cells. Cells (2 X 10/ml) were grown (D)1 and (D)2 are the doses of agents 1 and 2 in the mixture that with various concentrations of RA (0) or 4-HPR (El) in serum-free produce the same effect. This analysis generates the combina- medium (left panels) or in medium containing 10% FBS (right panels). tion effect as: summation (additivity or zero interaction) is Measurements were made at 92 h for cells growing in serum-free indicated when CI 1; synergism is indicated when CI < 1; medium and at 85 h for cells growing in medium containing serum. Each point is the mean of at least four measurements. The SE of each and antagonism is indicated when CI > 1. data point was 8% of the mean. Control cultures had a cell density of Retinoylation. HL-60 cells from exponentially growing 1.1 X 106/ml in serum-free medium and 1.02 X 106/ml in serum- cultures were harvested by centrifugation and resuspended in containing medium. The values for percentage of net growth were serum-free medium. RA (Sigma) and [1 1,12-3H]RA (50 Ci/ calculated with the following formula: [(cell concentration of experi- mmol; Du Pont-New England Nuclear, Boston, MA) were dis- mental culture) - (initial cell concentration)/(cell concentration of control culture) - (initial cell concentration)] X 100. solved in absolute ethanol and added to the growth medium. The final concentration of ethanol was no higher than 0.1%. HL-60 cells were grown for 24 h with 10 or 100 nt [3H]RA and various concentrations of radioinert 4-HPR or RA. greater in serum-free medium than in serum-containing medium Cells were harvested by centrifugation (200 X g for S mm) and (Fig. 1). washed extensively with phosphate-buffered NaCl solution (1.5 Differentiation induced by RA was associated with de- mM KH2PO4, 8.1 mM Na2HPO4, 136.9 mi NaC1, pH 7.2). The creased cell growth in either serum-free or serum-containing cell pellet was then extracted using the Bligh-Dyer procedure media (Fig. 1). We saw marked decreases in the percentage of X g (25) and centrifuged at 10,000 for S mm in a microcentri- viable cells only in serum-free medium containing 400 flM or 1 fuge. This extraction was repeated about five times or until there p.M RA. At 92 h, these two concentrations of RA induced 100% was <300 cpm/ml in the supernatant fraction. The delipidated differentiation, and viabilities were about 60% with 400 nM RA pellet was then dried in a centrifugal vacuum device (Savant, and 55% with 1 JiM RA (Fig. 1). At an earlier time (67 h), these Farmingdale, NY) and dissolved in 10% SDS solution. Radio- two concentrations of RA also induced 100% differentiation but activity was measured on a liquid scintillation spectrometer. with viabilities of 87% with 400 flM RA and 80% with 1 p,M RA Presentation of Results. Each experiment was per- that were much higher than at 92 h. These results indicate that formed at least twice, and most experiments were repeated at nonviable cells originated from a pool of viable mature cells and least three times with consistent results. are consistent with a report that RA-treated HL-60 cells die via apoptosis subsequent to differentiation (30). RESULTS Compared to RA, the decreased cell growth and viability Effects of RA and 4-HPR on Differentiation, Growth, by 4-HPR in serum-free medium were not coupled as closely to and Viability of HL-60 Cells. We found that 4-HPR was cell differentiation (Fig. 1). The marked decrease in viability much less active than RA in inducing differentiation of HL-60 with 4-HPR at concentrations >400 flM in serum-free medium cells (Fig. 1). These results are in agreement with previous precluded determinations of differentiation with the nitroblue reports using either serum-free (26, 27) or serum-containing tetrazolium assay, which requires live cells (23). media (16, 28, 29). The induction of differentiation, decrease in In serum-containing medium, we saw about 40% differen- viability, and inhibition of growth by either RA or 4-HPR were tiation with 4 p.M 4-HPR (Fig. 1). The percentage of viable cells

I- I- z z at at .C C 0 a) to to C C 0 a, to C 0 a, No 4-HPR

0 ;‘‘“; . 1 10 10 nM RA/’No RA RA Concentration, nM

Fig. 3 Differentiation by combinations of RA with 4-HPR of HL-60 10 100 1000 and HL-60(S) cells in medium containing serum. HL-60 cells (2 X l0 4-HPR Concentration, nM cells/ml) were grown in medium supplemented with 10% FBS. HL- Fig. 2 HL-60 cell differentiation in serum-free medium induced by 60(5) cells (2 x 104/ml) were grown in medium supplemented with 5% increasing concentrations of 4-HPR in the presence of fixed concentra- FBS. Each point is the mean of at least four measurements. The SE of tions of RA. Cells (2 X 10/ml) were grown for 92 h in serum-free each data point was 8% of the mean. medium with the indicated concentrations of RA and 4-HPR. Each point is the mean of at least four measurements. The SE of each data point was 8% of the mean. The isoboles (inset) are for combinations of 4-HPR with RA that are isoeffective (ED50) for differentiation of HL-60 cells. When there is additivity, the experimental values for a combination fall on the dashed line connecting the values for each agent alone. The ED50 value for 4-HPR alone was estimated by extrapolation. This value could not be determined experimentally because of low viability (see ‘‘Re- sults’ ‘). The viabilities were >85% for each of the other isoeffective conditions shown in the isobologram. I

was about 80%, and cell growth was inhibited about 50%. These latter results are not in agreement with those of Delia et a!. (16), 2040600 0 4 812 who found that during growth of HL-60 cells with 3 p.M 4-HPR RA Concentration, nM for 5 days there is no increase, and instead a decrease, in the Fig. 4 Isoboles for combinations of 4-HPR with RA that are isoeffec- concentration of viable cells. At 300 nt 4-HPR the level of tive for differentiation of HL-60 and HL-60(S) cells. The experimental growth inhibition seen by these workers was similar to what we points for HL-60 cells are the ED40 values calculated from Fig. 3. The saw with 4 JiM 4-HPR. Thus, one explanation for the apparent experimental points for HL-60(S) cells are the ED50 values calculated from Fig. 3, with the exception that a value for 4-HPR alone was not differences in results is that the concentrations of 4-HPR used obtained because of cytotoxicity. A ED50 value of 3 JiM for 4-HPR alone by Delia et a!. (16) are much higher than they report. Additional was estimated so that an additive isobole could be drawn. We assumed support for this explanation is that clonal proliferation of HL-60 that this value would be >1 p.M (Fig. 3) and less than the ED of 4 JiM cells is not inhibited by 1 p.M 4-HPR (29). found for HL-60 cells. Induction of Differentiation of HL-60 Cells by Combi- nations of RA and 4-HPR. Our findings that 4-HPR was a poor inducer of HL-60 cell differentiation (Fig. 1) prompted an examination of whether 4-HPR affected RA-induced differenti- cells (20). We found that 4-HPR was more toxic to HL-60(S) ation. than to HL-60 cells. Viability was 97% for HL-60(S) cultures In serum-free medium the dose-effect curves for 4-HPR exposed to 1 iM 4-HPR for 74 h and < 1% for cultures exposed were displaced to lower concentrations as a function of an to 4 LM 4-HPR. This toxicity limited to 1 p.M the maximum increase in the fixed concentration of RA (Fig. 2). The interac- concentration of 4-HPR that we could study. At this concentration between 4-HPR and RA was visualized by constructing an tion, 4-HPR did not induce differentiation of HL-60(S) cells isobole (31) (Fig. 2, inset) and quantified by calculating a CI (Fig. 3). value (24). As show in Fig. 2, inset, the two isoeffective (ED50) With either HL-60 or HL-60(S) cells the dose-effect curves combinations of 4-HPR with RA were below and to the left of for RA were displaced to lower concentrations as a function of the summation (additive or zero interaction) isobole displayed increases in the fixed concentrations of 4-HPR (Fig. 3). The by the dashed line. The CI values for these combinations were isoboles drawn from the data in Fig. 3 show that isoeffective about 0.6, indicating moderately strong synergism. combinations of RA and 4-HPR were below and to the left of the We examined also the induction of differentiation of pa- summation isoboles (Fig. 4). The extent of this synergy was rental HL-60 and HL-60(S) cells by combinations of RA and estimated by determining CI values. The CI values at the ED40 4-HPR in medium containing serum (Fig. 3). HL-60(S) is about effect level for HL-60 cells were about 0.7. The CI values for 10-fold more sensitive to RA for differentiation than HL-60 HL-60(S) cells at the ED50 effect level were about 0.5. Viability

at the ED40 effect level for HL-60 cells was 78% with 4 JiM 4-HPR alone and >92% for the other three conditions (Fig. 4). Viability at the ED50 effect level for HL-60(S) cells was >94% for each of the three conditions with RA shown in Fig. 4. 0 C Modulation of Retinoylation of HL-60 Proteins by 0 0 4-HPR. Our findings that combinations of 4-HPR and RA at synergistically induced differentiation of HL-60 cell lines (Figs. C 0 100 2-4) prompted us to see whether these increased levels of Co >. 0 differentiation correlated with changes in the level of retinoy- C lation. The levels of retinoylation by two fixed concentrations of a, [3H]RA increased as a function of the concentration of 4-HPR (Fig. 5). We did not see any cytotoxicity during this relatively short 24-h exposure. In contrast to 4-HPR, the covalent binding Concentration, nM of [3H]RA to protein decreased, because of isotope dilution, as a function of the concentration of radioinert RA (Fig. 5). Fig. 5 Changes in the level of retinoylation by increasing concentrations of RA and 4-HPR. Cells (2 X 106/ml) were grown in serum-free medium for 24 h in the presence of 10 nM (0, El) or 100 nsi (#{149},) DISCUSSION [3H]RA and the indicated concentrations of radioinert RA (0, #{149})and Although 4-HPR is an effective cancer chemopreventive 4-HPR (LI, ). Covalently bound RA was determined in the residues as described in ‘ ‘ Materials and Methods.’ ‘ Values for the levels of reti- agent and cell growth inhibitor for a wide variety oftumor types, noylated protein with either 10 or 100 nsi [3H]RA alone were set to its mechanism of action is still elusive. Patients taking 4-HPR 100%. Results shown are from one experiment. Essentially the same have markedly decreased levels of plasma retinol-binding pro- results were seen in another experiment. tein and retinol (32), and many show effects associated with hypovitaminosis A (33). The ability of 4-HPR to compete with retinol for binding to plasma retinol-binding protein (1 1), to Another explanation for the increased protein retinoylation inhibit both lecithin-retinol acyltransferase and acyl-CoA-de- seen with 4-HPR is an elevation in the levels of one or more of pendent retinal reductase (12), and to impair the secretion of the the proteins that are substrates for retinoylation. The regulatory retinol-plasma retinol-binding protein complex from liver and subunit of cyclic AMP-dependent protein type II is one of the other tissues (15) may singly or in combination be the basis for major retinoylated proteins in HL-60 and MCF-7 cells (35, 36), the side effects in vivo. It is unclear whether one or more of and its level increases about 3-fold in mammary tumors growing these specific effects is involved in the therapeutic efficacy of in animals treated with 4-HPR (37). Thus, the increased levels 4-HPR. of retinoylation in the presence of 4-HPR may, in part, reflect an The accumulating evidence that 4-HPR does not bind to the increase in the amount of this regulatory protein. nuclear retinoid receptors35 prompted us to examine whether The inability of 4-HPR to inhibit retinoylation (Fig. 5) 4-HPR influenced the ability of RA to differentiate HL-60 cells. suggests strongly that 4-HPR may not compete with RA in the Previously, we found that the synthetic retinoid Am80 does not covalent modification of protein. We could not determine di- induce differentiation of HL-60 cells in serum-free medium, rectly whether 4-HPR covalently modifies proteins because of although it is a ligand for nuclear retinoid receptors (20). How- the unavailability of highly radioactive 4-HPR. Taken together ever, Am80 increases the level of retinoylation and potentiates with our previous observations with Am80, these results offer RA-induced differentiation of HL-60 cells (20). additional evidence that covalent modification of proteins by a In this study, we saw qualitatively similar results with retinoid may play a role in inducing differentiation of HL-60 4-HPR to those seen with Am80. Combinations of 4-HPR and cells. RA synergistically induced differentiation of HL-60 cells (Figs. Our results showing synergism in the induction of differ- 2-4), and 4-HPR increased the levels of retinoylation (Fig. 5). entiation of HL-60 cells by combinations of 4-HPR with RA We saw these effects of 4-HPR in the absence of serum (Figs. may have clinical utility. Plasma concentrations of about 1 JiM 2 and 5). Thus, retinol and retinol-binding protein, which are in are seen in patients receiving p.o. doses of either 4-HPR (32) or serum, are not obligatory for some of the effects of 4-HPR seen RA (38). These values indicate that combinations of 4-HPR and in this study. RA that we found to be synergistic are obtainable in patients. It On a cellular basis our results could be explained if 4-HPR may also be noteworthy that we saw synergistic effects with acted by a mechanism that increased the availability of RA for combinations of 4-HPR and RA at concentrations of RA that are either transcriptional regulation of RA responsive genes or for close to the physiological range of about 10 nM (39, 40). Thus, retinoylation of target proteins. In cells exposed to both RA and our results raise the possibility that biological effects of 4-HPR 4-HPR, 4-HPR may displace RA from sites where it is seques- in vivo may in part derive from influences on the effective tered. Another possibility is that 4-HPR alters the efficiency of biological availability of endogenous RA. This further raises the the cytochrome P450-mediated catabolism of RA to 4-hydroxy- prospect that the spectrum of cancers responding to 4-HPR may retinoic acid. This hydroxylation occurs in HL-60 cells (34). be greater if RA were combined with 4-HPR. In particular, However, we are not aware of any report that 4-HPR affects this 4-HPR may be useful in the treatment of acute promyelocytic reaction. Experiments to distinguish these possibilities are in leukemia or squamous neoplasms. Although cytodifferentiation progress. therapy by RA for acute promyelocytic leukemia patients is

encouraging, there are several limitations preventing better din- 9. Rotmensz, N., De Palo, G., Formelli, F., Costa, A., Marubini, E., ical outcomes. A high percentage of patients in complete remis- Campa, T., Crippa, A., Danesini, G. M., Delle Grottaglie, M., Di Mauro, M. G., Filiberti, A., Gallazzi, M., Guzzon, A., Magni, A., Malone, W., sion induced by RA alone relapse within a few months (41-43). Mariani, L., Palvarini, M., Perloff, M., Pizzichetta, M., and Veronesi, U. Most relapsed patients are resistant to further treatment with Long-term tolerability of fenretinide (4-HPR) in breast cancer patients. RA, although their leukemia cells may respond to RA in vitro Eur. J. Cancer, 27: 1127-1131, 1991. (43-45). 10. Decensi, A., Bruno, S., Costantini, M., Torrisi, R., Curotto, A., One explanation for the resistance of relapsing patients to Gatteschi, B., Nicolo, G., Polizzi, A., Perloff, M., Malone, W. F., and RA treatment is an increased systemic cytochrome P450-medi- Bruzzi, P. 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Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 1995 American Association for Cancer Research. N-(4-hydroxyphenyl)retinamide (Fenretinide) in combination with retinoic acid enhances differentiation and retinoylation of proteins.

N Takahashi, E A Sausville and T R Breitman

Clin Cancer Res 1995;1:637-642.

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