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Phytomedicine 13 (2006) 732–734 www.elsevier.de/phymed

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Anti-proliferative properties of prenylated flavonoids from hops (Humulus lupulus L.) in human prostate cancer cell lines L. Delmullea, A. Bellahce`neb, W. Dhoogec, F. Comhairec, F. Roelensa, K. Huvaerea, A. Heyericka, V. Castronovob, D. De Keukeleirea, a

Ghent University-UGent, Faculty of Pharmaceutical Sciences, Laboratory of Pharmacognosy and Phytochemistry, B-9000 Ghent, Belgium b Metastasis Research Laboratory, University of Lie`ge, B-4000 Lie`ge, Belgium c Ghent University Hospital, Department of Endocrinology, B-9000 Ghent, Belgium

Abstract Chalcones xanthohumol (X) and desmethylxanthohumol (DMX), present in hops (Humulus lupulus L.), and the corresponding flavanones isoxanthohumol (IX, from X), 8-prenylnaringenin (8-PN, from DMX), and 6prenylnaringenin (6-PN, from DMX), have been examined in vitro for their anti-proliferative activity on human prostate cancer cells PC-3 and DU145. X proved to be the most active compound in inhibiting the growth of the cell lines with IC50 values of 12.371.1 mM for DU145 and 13.271.1 mM for PC-3. 6-PN was the second most active growth inhibitor, particularly in PC-3 cells (IC50 of 18.471.2 mM). 8-PN, a highly potent phytoestrogen, exhibited pronounced anti-proliferative effects on PC-3 and DU145 (IC50 of 33.571.0 and 43.171.2 mM, respectively), and IX gave comparable activities (IC50 of 45.271.1 mM for PC-3 and 47.471.1 mM for DU145). DMX was the least active compound. It was evidenced for the first time that this family of prenylated flavonoids from hops effectively inhibits proliferation of prostate cancer cells in vitro. r 2006 Elsevier GmbH. All rights reserved. Keywords: Prenylflavonoids; Humulus lupulus L.; Prostate cancer cells; Antiproliferative activity

Introduction Hops (Humulus lupulus L.), an essential raw material for beer brewing, is a rich source of prenylated flavonoids. Xanthohumol (X; 1) is the most abundant prenylated chalcone present in hops (concentrations up to 1%, w/w). It is accompanied by its homologue, desmethylxanthohumol (DMX; 2), albeit in lower concentrations (Stevens et al., 1997). Both chalcones are readily isomerized (Michael-type intramolecular

Corresponding author. Tel.: +32 9 264 8055; fax: +32 9 264 8192.

E-mail address: [email protected] (D. De Keukeleire). 0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.01.001

reaction) to the corresponding flavanones, X giving rise to isoxanthohumol (IX; 3) and DMX being converted to a mixture of 6-prenylnaringenin (6-PN; 4) and 8prenylnaringenin (8-PN; 5) (Stevens et al., 1999). These five prenylated flavonoids constitute a set of highly interesting bioactive compounds and the ability of X, DMX, and IX to inhibit proliferation of human breast cancer (MCF-7), colon cancer (HT-29), and ovarian cancer (A2780) cell lines in vitro has been demonstrated (Miranda et al., 1999). Cancer chemopreventive properties of X have been highlighted by cell- and enzymebased in vitro bioassays using markers relevant for the inhibition of carcinogenesis during the initiation, promotion, and progression phases (Gerhaeuser et al., 2002).

ARTICLE IN PRESS L. Delmulle et al. / Phytomedicine 13 (2006) 732–734

OH OH

HO

4' OH

R3 HO

8

O

Cell viability (% of control)

120 100 80 60 X

40

DMX

IX 20

8PN 6PN

0 1

10

(Α)

100

Concentration (µM) 160

Cell viability (% ofcontrol)

Despite intensive studies, particularly focused on X and 8-PN, no data are available about the effects of prenylated flavonoids from hops in prostate cancer cells. In this study, we aimed for the first time at evaluating their activity against proliferation of PC-3 and DU145 prostate cancer cell lines using the WST-1 colorimetric assay for quantification of cell proliferation rates (Figs. 1 and 2). X proved to be the most potent inhibitor with IC50 values of 12.371.1 mM for DU145 and 13.271.1 mM for PC-3. 6-PN appeared as the second most active compound with marked growth-inhibitory effects both on PC-3 (IC50 of 18.471.2 mM) and on DU145 (IC50 of 29.171.1 mM). Remarkably, the highly potent phytoestrogen 8-PN (Milligan et al., 1999; Zierau et al., 2002) showed pronounced anti-proliferative effects on PC-3 and DU145 prostate cancer cells (IC50 of 33.571.0 and 43.171.2 mM, respectively), while also IX gave activities on prostate cancer cells comparable to those of 8-PN (IC50 of 45.271.1 mM for PC-3 and 47.471.1 mM for DU145). DMX was the least active compound (IC50 of 49.971.0 mM for PC-3 and 53.871.1 mM for DU145). The observed differences in sensitivity between DU145 and PC-3 prostate cancer cells may be attributed to the varying expression profiles of the estrogen receptors ER-a and ER-b. It has been demonstrated that PC-3 prostate cancer cells express low levels of ERa and high levels of ER-b mRNAs (Maruyama et al., 2000; Lau et al., 2000). For the DU145 prostate cancer cell line, exclusive expression of high levels of ER-b has been shown. Besides estrogen receptor-related actions, phytoestrogens may exert their activities by interacting with some of the key enzymes in sex steroid production or by other non-hormonal functions. Previous studies on structure–activity relationships of chalcones indicated that their potency to inhibit proliferation of colon cancer cells (CCL 220.1) is dependent on various structural features (Yit and Das, 1994; De Vincenzo et al., 1995). Insights into the observed effects reveal that the methoxy group at the 60 -position in the A-ring of a chalcone, such as in X, increases the inhibitory capacity, when compared to DMX having a hydroxyl group. In the report by

733

140 120 100 80 60

X

40

IX

DMX 8PN 6PN

20 0 1

(Β)

10

100

Concentration (µM)

Fig. 2. Dose–response relationships for hop-derived prenylflavonoids [xanthohumol (X), desmethylxanthohumol (DMX), isoxanthohumol (IX), 8-prenylnaringenin (8PN) and 6-prenylnaringenin (6PN)] on the growth of human prostate cancer cells (A: PC-3, B: DU145) after 2 days of exposure. Data are means7SD (n ¼ 3).

Miranda et al. (1999), transformation of a chalcone to a flavanone (e.g., X to IX) resulted in a reduced antiproliferative activity with respect to breast, colon, and ovarian cancer cells. Our observations indicate that conversion of the chalcone DMX to flavanones 8-PN and 6-PN results in an increased activity in prostate cancer cells. In conclusion, potent anti-proliferative activities of xanthohumol (X) and 6-prenylnaringenin (6-PN) were found for the prostate cancer cells PC-3 and DU145. Further research on the non-genomic effects of these prenylated flavonoids is necessary to understand the mechanism of action and to confirm whether these compounds may be of use in the chemoprevention of prostate cancer.

6

R2 OR

O

1: R = Me 2: R = H

OR1 O 3: R1 = Me, R2 = H, R3 = prenyl 4: R1 = R3 = H, R2 = prenyl 5: R1 = R2 = H, R3 = prenyl

Fig. 1. Structures of xanthohumol (X; 1), desmethylxanthohumol (DMX; 2), isoxanthohumol (IX; 3), 8-prenylnaringenin (8PN; 4) and 6-prenylnaringenin (6PN; 5).

Materials and methods Prenylated flavonoids from hops were isolated as described previously (Milligan et al., 1999). Cell culture media, L-glutamine, non-essential amino acids (NEAA), penicillin (10,000 IU/ml) and streptomycin (10,000 mg/ml), foetal bovine serum (FBS), and sodium

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L. Delmulle et al. / Phytomedicine 13 (2006) 732–734

pyruvate were obtained from Invitrogen (Merelbeke, Belgium). HEPES was purchased from Sigma Chemical (Bornem, Belgium). Charcoal was provided by Merck (Darmstadt, Germany) and dextran by Amersham Pharmacia Biotech (Uppsala, Sweden). Established human epithelial cancer cell lines from prostatic (PC-3, DU145) origin were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA). Cells were maintained in cell specific media, at 37 1C in a humidified atmosphere of 5% carbon dioxide. The cells were passed at 70% confluency and used within 10 passages for all experiments. The cells were cultured in DMEM/F12 nutrient mixture (1:1), which was supplemented with 10% FBS and antibiotic agents (penicillin 10,000 IU/ml and streptomycin 10,000 mg/ml). Experiments with DU145 cells were performed in high glucose (4500 mg/l) DMEM without phenol red, experiments with PC-3 cells in phenol red-free RPMI 1640. Both media were supplemented with 10% steroid-free dextran-coated charcoaltreated FBS and 1% antibiotic agents (penicillin 10,000 IU/ml and streptomycin 10,000 mg/ml). In addition, 2 mM L-glutamine, 0.1 mM NEAA and 1 mM sodium pyruvate were added to the DMEM medium and 1 mM sodium pyruvate and 10 mM HEPES were added to the RPMI 1640 medium. The WST1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)2H-5-tetrazolio]-1,3-benzene) assay (Roche Diagnostics, Vilvoorde, Belgium) was carried out in accordance with the manufacturer’s instructions. Statistics were performed using SPSS 10.0 (SPSS Inc., Chicago, Illinois, USA).

Acknowledgments LD is grateful to the IWT-Vlaanderen (Institute for the Promotion of Innovation by Science and Technology in Flanders, Brussels, Belgium) and FR to FWOVlaanderen (Fund for Scientific Research – Flanders, Brussels, Belgium) for providing pre-doctoral grants. Part of this work was also funded by the Ministry of the Flemish Community through their Centre for Health and Environment.

References De Vincenzo, R., Scambia, G., Benedetti Panici, P., Ranelletti, F.O., Bonanno, G., Ercoli, A., Delle Monache, F., Ferrari, F., Piantelli, M., Mancuso, S., 1995. Effect of synthetic and naturally occurring chalcones on ovarian cancer cell growth: structural–activity relationships. Anticancer Drug Res. 10, 481–490. Gerhaeuser, C., Alt, A., Heiss, E., Gamal-Eldeen, A., Klimo, K., Knauft, J., Neumann, I., Scherf, H.R., Frank, N., Bartsch, H., Becker, H., 2002. Cancer chemopreventive activity of xanthohumol, a natural product derived from hop. Mol. Cancer Ther. 1, 959–969. Lau, K.-M., LaSpina, M., Long, J., Ho, S.-M., 2000. Expression of oestrogen receptor (ER)-a and ER-b in normal and malignant prostatic epithelial cells: regulation by methylation and involvement in growth regulation. Cancer Res. 60, 3175–3182. Maruyama, S., Fujimoto, N., Asano, K., Ito, A., Usui, T., 2000. Expression of oestrogen receptor alpha and beta mRNAs in prostate cancer treated with leuprorelin acetate. Eur. Urol. 38, 635–639. Milligan, S.R., Kalita, J.C., Heyerick, A., Rong, H., De Cooman, L., De Keukeleire, D., 1999. Identification of a potent phytooestrogen in hops (Humulus lupulus L.) and beer. J. Clin. Endocrinol. Metab. 83, 2249–2252. Miranda, C.L., Stevens, J.F., Helmrich, A., Henderson, M.C., Rodriguez, R.J., Yang, Y.H., Deinzer, M.L., Barnes, D.W., Buhler, D.R., 1999. Anti-proliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines. Food Chem. Toxicol. 37, 271–285. Stevens, J.F., Ivanc¸ic, M., Hsu, V.L., Deinzer, M.L., 1997. Prenylflavonoids from Humulus lupulus. Phytochemistry 44, 1575–1585. Stevens, J.F., Taylor, A.W., Clawson, J.E., Deinzer, M.L., 1999. Fate of xanthohumol and related prenylflavonoids from hops to beer. J. Agric. Food Chem. 47, 2421–2428. Yit, C.C., Das, N.P., 1994. Cytotoxic effect of butein on human colon adenocarcinoma cell proliferation. Cancer Lett. 82, 65–72. Zierau, O., Gester, S., Schwab, P., Metz, P., Kolba, S., Wulf, M., Vollmer, G., 2002. Estrogenic activity of the phytoestrogens naringenin, 6-(1,1-dimethylallyl)naringenin and 8prenylnaringenin. Planta Med. 68, 449–451.