Inhibition of Carcinogen-Activating Enzymes by 16␣-Fluoro-5-Androsten-17-One

[CANCER RESEARCH 62, 3685–3690, July 1, 2002] Inhibition of Carcinogen-activating Enzymes by 16␣-Fluoro-5-androsten-17-one Henry P. Ciolino,1 Christopher J. MacDonald, and Grace Chao Yeh Cellular Defense and Carcinogenesis Section, Basic Research Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, NIH, Frederick, Maryland 21702- 1201 ABSTRACT decrease in the incidence of a number of different types of cancer in humans (5, 6). In animal models, DHEA has been shown to inhibit In the present study, we examined the effect of a synthetic analogue of both spontaneous and chemically induced carcinogenesis in rodents the chemopreventive hormone dehydroepiandrosterone, 16␣-fluoro-5- (7–9). Specifically, DHEA inhibits both skin and mammary tumori- androsten-17-one, also known as fluasterone, on the activity and expression of carcinogen-activating enzymes in MCF-7 cells. The increase in genesis caused by the PAH DMBA (10–13). DHEA has been shown cytochrome P450 (CYP) 1A1 and 1B1 activity, as measured by to inhibit DMBA activation in vitro (14) and DMBA-DNA binding in ethoxyresorufin-O-deethylase activity, in cells treated with the carcino- vivo (15–18). The dramatic decline of DHEA levels in humans with gens dimethylbenzanthracene (DMBA) or 2,3,5,7-tetrachlorodibenzo-p- advancing age is, therefore, of substantial concern. However, DHEA dioxin (TCDD), was inhibited by cotreatment with fluasterone. However, supplementation is problematic, because it can be converted to both treatment of the cells with fluasterone after induction with DMBA or testosterone and estrone (19), and it exhibits considerable liver tox- TCDD failed to decrease enzyme activity, indicating that inhibition was icity (20) and hepatocarcinogenicity (21). Thus, DHEA’s clinical not the result of direct enzyme inhibition. Therefore, we examined the usefulness is limited. Recently, a synthetic analogue of DHEA, 16␣- effect of fluasterone on gene expression at the mRNA level. Both DMBA fluoro-5-androsten-17-one (fluasterone), was developed that lacks the and TCDD caused a dramatic increase in the amount of CYP1A1 and liver toxicity and hormone-related side effects of DHEA (22). Fluas- CYP1B1 mRNA, the two major isoforms involved in carcinogen activation in these cells. In cells cotreated with fluasterone, however, there was a terone has been shown to be chemopreventive against DMBA- dose-dependent decrease in CYP1A1 and CYP1B1 mRNA. Fluasterone induced carcinogenesis (23), and, like DHEA, it prevents DMBA also inhibited the basal level of CYP1A1 mRNA but not CYP1B1. Fluas- activation and DMBA-DNA adduct formation (10, 22). However, the terone inhibited the rate of CYP1A1 promoter-controlled transcription, mechanism of this activity is unknown. Therefore, we have tested the indicating that it affects the transcriptional regulation of the gene. Acti- capacity of fluasterone to modulate the effects of DMBA and TCDD nomycin D chase experiments showed that fluasterone also caused an on carcinogen-activating enzyme activity and expression in vitro. increase in the degradation of CYP1A1 mRNA, while leaving CYP1B1 DMBA is a classic model aryl hydrocarbon. TCDD is a widespread mRNA unaffected. These results indicate that fluasterone inhibits the environmental contaminant produced during trash incineration, increase in the expression of CYP1A1 normally caused by exposure to bleaching of paper pulp, synthesis of pesticides, and as a by-product carcinogens by both transcriptional and post-transcriptional mechanisms of combustion during various industrial processes (24, 25). and that CYP1B1 expression is not susceptible to the same post-transcriptional mechanism. MATERIALS AND METHODS INTRODUCTION Materials. Human breast cancer MCF-7 and human liver carcinoma HepG2 cells were from the American Type Culture Collection (Rockville, Many environmental compounds are carcinogenic only after met- 2 MD). RPMI 1640, glutamine, fetal bovine serum, trypsin/EDTA, and PBS abolic activation. Exposure to carcinogens, such as PAH, causes an were from BioFluids (Rockville, MD). Actinomycin D, ␣-NF, DMBA, EDTA, increase in the expression of the enzymes responsible for this activa- ethoxyresorufin, resorufin, Tris-HCl, and DMSO were from Sigma (St. Louis, tion. These enzymes consist of members of the CYP 1A and 1B MO). [32P]dATP was from DuPont NEN (Boston, MA). TCDD was from the subfamilies. They generate genotoxic epoxide metabolites of the Midwest Research Institute (Kansas City, MO). RT-PCR was performed with parent aryl hydrocarbon, which can bind DNA, forming adducts (1). an Omniscript kit from Qiagen (Valencia, CA). Tris/borate/EDTA gels, run- These adducts, if not repaired, can cause specific mutations leading to ning buffer, and high-density sample buffer were from Novex (San Diego, cellular transformation. Therefore, the activity and expression of CA). Primers for GPDH PCR and the ␤-galactosidase-containing reporter carcinogen-activating enzymes are key components in chemically vector were from Clontech (Palo Alto, CA). TRIzol reagent and Lipo- induced carcinogenesis, and the inhibition of their activity, either by fectAmine were from Life Technologies, Inc. (Gaithersburg, MD). CAT ELISA assay kit was from Boehringer Mannheim (Indianapolis, IN). Fluas- direct enzyme inhibition or through modulation of their expression, is terone was a gift from Dr. Thomas Wang (Phytonutrients Laboratory, USDA, thought to be an important mechanism in the prevention of carcino- Beltsville, MD). genesis (2). Cell Culture. MCF-7 and HepG2 were grown in RPMI 1640 with 2 mM DHEA is the most abundant steroid hormone in humans (3). Al- glutamine and 10% fetal bovine serum and subcultured weekly using 0.25% though its physiological function remains unclear, it is associated with trypsin/0.05% EDTA. All experiments were carried out on confluent cultures a number of beneficial health effects in humans (4). A considerable in growth medium, unless otherwise noted. body of evidence indicates that DHEA is also associated with a Assay of EROD Activity. Confluent MCF-7 or HepG2 cells in 24-well plates were treated with 1 ml of growth medium containing 1 ␮M DMBA or 1nM TCDD in the presence of DMSO (vehicle control) or fluasterone for 24 h. Received 1/11/02; accepted 5/2/02. The costs of publication of this article were defrayed in part by the payment of page The final DMSO concentration in both control and treated cultures for this and charges. This article must therefore be hereby marked advertisement in accordance with all other experiments was 0.1%. The medium was removed, and the wells were 18 U.S.C. Section 1734 solely to indicate this fact. washed two times with fresh growth medium. EROD activity was determined 1 To whom requests for reprints should be addressed, at Basic Research Laboratory, in intact cells using 1 ␮M ethoxyresorufin in growth medium as a substrate in Building 560/Room 12-05, National Cancer Institute at Frederick, NIH, Frederick, MD 21702-1201. Phone: (301) 846-5160; Fax: (301) 846-6709; E-mail: [email protected]. the presence of 1.5 mM salicylamide to inhibit conjugating enzymes. The assay 2 The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; CAT, chloram- was carried out at 37°C. The fluorescence of resorufin generated from the phenicol acetyltransferase; CYP, cytochrome P450; DHEA, dehydroepiandrosterone; conversion of ethoxyresorufin by CYP1A1/CYP1B1 was measured every 10 DMBA, dimethylbenzanthracene; EROD, ethoxyresorufin-O-deethylase; G6PDH, min for 60 min with a CytoFluor II multiwell fluorescence plate reader glucose-6-phosphate dehydrogenase; GPDH, glyceraldehyde-3-phosphate dehydrogenase; ␣-NF, ␣-naphthoflavone; RT-PCR, reverse transcription-PCR; TCDD, 2,3,5,7-tetra- (PerSeptive Biosystems, Framingham, MA), with an excitation wavelength of chlorodibenzo-p-dioxin; XRE, xenobiotic-responsive element. 530 nm and emission at 590 nm. 3685 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2002 American Association for Cancer Research. FLUASTERONE INHIBITS CYP 1A1 AND 1B1 EXPRESSION Statistical Analysis. Statistical analyses were performed using StatView Statistical Analysis software (SAS Institute). Differences between group mean values were determined by a one-factor ANOVA, followed by Fisher’s protected least-significant difference post hoc analysis for pairwise comparison of means. RESULTS Fluasterone Inhibits CYP1A1/1B1 Enzyme Activity in Intact Cells. Carcinogen-activating enzyme activity was measured in intact MCF-7 cells by the EROD assay, which is specific for CYP1A1/1B1. The treatment of MCF-7 cells with 1 ␮M DMBA for 24 h resulted in an increase in activity from undetectable levels to 1.38 Ϯ 0.13 pmol/min/105 cells. In cells coincubated with DMBA and fluasterone, there was a dose-dependent decrease in EROD activity, with a IC50 of ϳ0.5 ␮M (Fig. 1A). In cells treated with 1 nM TCDD, the most potent inducer of these enzymes, there was an increase in EROD activity to 16.72 Ϯ 0.43 pmol/min/105 cells. This was also decreased in a dose-dependent fashion by coincubation with fluasterone, with a IC50 of ϳ2.5 ␮M (Fig. 1B). Fluasterone and DHEA also inhibited TCDD- induced EROD activity in human liver HepG2 cells (Fig. 1C). MCF-7 cells were preincubated with DMBA or TCDD and postin- cubated for 3 h with fluasterone. As shown in Table 1, ␣-NF, a known inhibitor of EROD activity, caused a significant decrease in activity, but fluasterone had no effect. In microsomes isolated from cells that had been incubated with TCDD to induce activity, there was likewise no effect of fluasterone on EROD activity, whereas ␣-NF completely abolished activity. Fluasterone Inhibits CYP1A1 and CYP1B1 mRNA Levels. MCF-7 cells were treated with DMBA or TCDD with or without fluasterone for 6 h, and the amount of CYP1A1 mRNA was determined by RT-PCR. As shown in Fig. 2A, there was a 4-fold increase in CYP1A1 mRNA in DMBA-treated cells compared with DMSO- treated cells. In cells coincubated with fluasterone, this increase was Fig. 1. Effect of fluasterone on cellular EROD activity induced by DMBA (A), TCDD significantly inhibited at all doses tested.

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