[CANCER RESEARCH 58. 2379-2384, June 1, 1998| Increased UDP-glucuronosyltransferase Activity and Decreased Prostate Specific Antigen Production by Biochanin A in Prostate Cancer Cells Xiao-Ya Sun, Cathie A. Plouzek, James P. Henry,1 Thomas T. Y. Wang, and James M. Phang2 Laboratory of Nutritional and Molecular Regulation, National Cancer Institute-Frederick Cancer Research and Development Center. NIH. Frederick. Maryland 21702-1201 ABSTRACT Because androgen levels in the prostate may be clinically impor tant, the modulation of UDPGT and androgen metabolism by dietary Our laboratory has characterized androgen metabolism in an andro- factors is of considerable interest. Various studies have shown that gen-responsive prostate cancer cell line (LNCaP) and showed that these consumption of soybean is associated with low incidence of prostate cells accumulated intracellular testosterone primarily as glucuronidated metabolites. Using a cell-free assay with testosterone as substrate, we cancer in Asian populations (6). Flavonoids, a family of compounds showed that LNCaP had UDP-glucuronosyltransferase (UDPGT) activity. plentiful in fruits and vegetables and especially in soy products, may Because dietary factors, such as flavonoids in soy products, may reduce reduce the risk for hormone-dependent cancers (7). We undertook the the risk for hormone-dependent cancers, we studied the effects of fla current studies to examine the possible effects of flavonoids on vonoids on testosterone-UDPGT activity. LNCaP cells were exposed to testosterone-UDPGT activity and on the metabolism of testosterone in selected flavonoids for up to 6 days. The increase in UDPGT-specific prostate cancer cells. We found that UDPGT was induced by fla activity was linear over this period. Of the compounds tested, biochanin A vonoids in LNCaP cells, and this increase in UDPGT not only affected was the most potent, with increased activity at concentration range 0.5-50 the conversion of testosterone to its conjugated glucuronide in intact JIM. Activities were linear for time and protein and were unaffected by LNCaP cells but also correlated with altered production of PSA, an flavonoids added directly to the assay. Kinetics studies showed no change androgen-responsive marker of prostate cancer (8). in A,,, for testosterone in the face of these large increases in specific activity. Cellular metabolism of testosterone reflected the increase in enzyme activity. Intact cells treated with biochanin A produced testoster- MATERIALS AND METHODS one-glucuronide from testosterone at twice the rate of controls. The steroid form of the UDPGT transcript was expressed in LNCaP cells and Chemicals. Radiolabeled ([^Hj-l^.o^Hestosterone was obtained from was enhanced in biochanin A-treated LNCaP cells. Additionally, bio New England Nuclear (Boston. MA). Unlabeled testosterone. UDP-glucuronic chanin A markedly decreased prostate specific antigen (PSA) level against acid, biochanin A. apigenin. kaempterol. quercetin. naringcnin, and HPLC the effect of testosterone on PSA production. Biochanin A significantly solvents were obtained from Sigma Chemical Co. (St. Louis, MO). Daid/ein. decreased the testosterone-stimulated release of PSA, presumably because genistein, galangin. tbrmononetin. and prunetin were purchased from Indorine biochanin A increased UDPGT and increased the intracellular glucu- Chemical Co. (Somerville. NJ). Fresh stock solutions of various flavonoid ronidation of testosterone. These studies suggest that the modulation of compounds were prepared in DMSO at a concentration of 50 mM before each hormone metabolism by dietary factors may be important in the preven experiment. tion and treatment of prostate cancer. LNCaP Cell Culture. The human prostatic carcinoma cell line LNCaP was obtained from the American Type Culture Collection (Rockville, MD). INTRODUCTION Cell culture components were purchased from Biofluids. Inc. (Rockville. MD). Cells were cultured (37°C.5% CO2) in RPMI 1640 supplemented with 10% It is well established that androgens (testosterone and its metabo FBS. penicillin ( 100 U/ml) and streptomycin 1100 fig/ml). Cells were passaged lites) play an important role in the normal growth and function of the every 6-7 days by trypsinization. and the culture medium was changed every prostate ( 1). Changes in androgen metabolism or responsiveness to 2-3 days. Cell Treatment for Enzyme Assay. LNCaP cells (1 X IO5)were plated in androgens have been implicated in the formation of benign prostatic 75-cm2 flasks in the presence of 25 ml of growth medium and allowed to attach hypertrophy and prostate cancer ( 1). The conjugation of steroid hormones with UDP-glucuronic acid is for 1 day. On the following day (day 1). each of the selected tlavonoid catalyzed by the enzyme UDPGT3 (EC 2.4.1.17; Ref. 2). This reac compounds at varying concentrations was added to the incubation medium. The final concentration of DMSO was 0.1 c/t in the medium. On day 3. medium tion, which converts steroids to more polar derivatives, not only was replaced with fresh medium containing same additives with or without inactivates but also expedites its elimination from tissues (3). Al flavonoids and maintained for an additional 3 days. though conjugation of steroid hormones occurs primarily in the liver, Preparation of Cell Homogenates for Enzyme Assay. Treated cells were it occurs also in peripheral tissues and may play a role in regulating harvested by scraping the cells into PBS. centrifuged. and resuspended in androgen levels in the prostate. Our laboratory (4) and others (5) have 1 ml of HBSS containing 25 mM HEPES buffer at pH 7.4 (9). The characterized testosterone metabolism in an androgen-responsive resuspended cells were disrupted by sonication. and the resultant homoge- nate was stored at -70°C. Protein concentrations for these homogenates prostate cancer cell line (LNCaP) and showed that these cells accu were determined by Lowry's method (10) using BCA Protein Assay Rea mulated intracellular testosterone primarily as glucuronidated deriv atives. Using a cell-free assay with testosterone as substrate, we gent (Pierce. Rockford, IL). UDPGT Assay. UDPGT activity was measured according to methods showed that LNCaP prostate cancer cells had testosterone-UDPGT described previously (4). The reaction mixture contained: 1 /iCi ['Hltestos- activity (4). terone. 4 /IM unlabeled testosterone. 5 mM UDP-glucuronic acid, and 100 /il of homogenate protein obtained from LNCaP cells in a final volume of 1 ml (HBSS containing 25 mM HEPES buffer). After l-h incubation at 37°C,the Received 11/25/97: accepted 4/2/98. The costs of publication of this article were defrayed in part by the payment of page mixture was acidified with 25 /xl of l N HCI for 15 min to release androgen charges. This article must therefore be hereby marked advertisement in accordance with steroids trapped by binding proteins (11). The samples were then neutralized 18 U.S.C. Section 1734 solely to indicate this fact. ' Present address: Gillette Research Institute. Gaithersburg, MD 20879. with 1 ml of PBS and 5 /il of 10 N NaOH. Metabolites first were separated on 2 To whom requests for reprints should be addressed, at National Cancer Institute. C,g columns (Waters. Milford, MA) and then were analyzed on the Waters Laboratory of Nutritional and Molecular Regulation. Building 560. Room 12-91. Fred HPLC system with a Supelcosil 5 mm LC-18 reverse phase column (Supelco. erick. MD 21702. 3 The abbreviations used are: UDPGT. UDP-glucuronosyltransferase; PSA. prostate- Inc., Bellefonte. PA) (12). An isocratic mobile phase consisting of mclhanol: specific antigen; HPLC. high-performance liquid chromatography: FBS. fetal bovine acetonitrile:water (15:45:40, v/v/v) for organic metabolites was used, whereas serum. 35% acetonitrile. 0.1% trifluroacetic acid in water at a flow rate of I ml/min 2379 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1998 American Association for Cancer Research. EFFECT OF BIOCHANIN A ON LNCaP CELLS was used to identify polar metabolites. The effluent was fed into a FLO-ONE/ RESULTS Beta online radioactive flow detector (Radiomatic. Meriden, CT). The testos terone and conjugated testosterone were separated and identified by comparing Stimulation of UDPGT Activity by Selected Flavonoids on the characteristic clution times with integrated standard peaks: testosterone and LNCaP Cells. Flavonoidsarea largeclass of naturalcompoundsthat testosterone glucuronide. Characterization of metabolites in the HPLC eluant are found in a wide variety of fruits and vegetables. The selected was also performed with a mass spectrometer as described by Henry et al. (4). flavonoids were apigenin, galangin, kaempferol, quercetin, narin- UDPGT activity was expressed as testosterone-glucuronide formation in pmol/ genin, and quercetin, whereas the tested isoflavones were biochanin h-/xg protein. A, daidzein, formononetin. genistein, and prunetin. Structures of the Cellular Metabolism of Testosterone. LNCaP cells (1 x IO5)were plated selected flavonoids were shown in Fig. 1. on day 0. and biochanin A (5 ¿tM)where indicated was added to the medium LNCaP cells were exposed to each of the selected flavonoids for 6 on day I. This medium was replaced with or without biochanin A on day 3. On days. All of the selected flavonoids stimulated the activity of testos- day 6 at the beginning of the experiment, growth medium was removed and terone-UDPGT. which conjugates testosterone to testosterone-glucu substituted with 2 ml of fresh medium without serum or biochanin A. After an ronide. Fig. 2 illustrates the relative effect on testosterone-UDPGT by hour of equilibration, cells were exposed to 2 ml of fresh medium containing [ !H[testosterone (92.1 Ci/mmol) and unlabcled testosterone so that the final selected flavonoids at a concentration of 5 JU.M.Themost effective concentration of testosterone was 31.2 nM. This concentration was chosen to compound was biochanin A (5 /AM),which stimulated UDPGT activ approximate the total plasma testosterone concentration of 20 nM (13). Cells ity about 10-fold. were incubated at 37°C.After incubation, aliquots of media were collected and Increase of UDPGT Activity by Biochanin A on LNCaP Cells.