Novel Antiproliferative Flavonoids Induce Cell Cycle Arrest in Human Prostate Cancer Cell Lines

Prostate Cancer and Prostatic Diseases (2006) 9, 68–76 & 2006 Nature Publishing Group All rights reserved 1365-7852/06 $30.00 www.nature.com/pcan ORIGINAL ARTICLE Novel antiproliferative flavonoids induce cell cycle arrest in human prostate cancer cell lines AQ Haddad1, V Venkateswaran1, L Viswanathan1, SJ Teahan1, NE Fleshner2 and LH Klotz1 1Division of Urology, Sunnybrook & Women’s College Health Sciences Centre, Toronto, ON, Canada and 2Division of Urology, Princess Margaret Hospital, Toronto, ON, Canada Epidemiologic studies have demonstrated an inverse association between flavonoid intake and prostate cancer (PCa) risk. The East Asian diet is very high in flavonoids and, correspondingly, men in China and Japan have the lowest incidence of PCa worldwide. There are thousands of different naturally occurring and synthetic flavonoids. However, only a few have been studied in PCa. Our aim was to identify novel flavonoids with antiproliferative effect in PCa cell lines, as well as determine their effects on cell cycle. We have screened a representative subgroup of 26 flavonoids for antiproliferative effect on the human PCa (LNCaP and PC3), breast cancer (MCF-7), and normal prostate stromal cell lines (PrSC). Using a fluorescence-based cell proliferation assay (Cyquant), we have identified five flavonoids, including the novel compounds 2,20-dihydroxychalcone and fisetin, with antiproliferative and cell cycle arresting properties in human PCa in vitro. Most of the flavonoids tested exerted antiproliferative effect at lower doses in the PCa cell lines compared to the non-PCa cells. Flow cytometry was used as a means to determine the effects on cell cycle. PC3 cells were arrested in G2/M phase by flavonoids. LNCaP cells demonstrated different cell cycle profiles. Further studies are warranted to determine the molecular mechanism of action of 2,20-DHC and fisetin in PCa, and to establish their effectiveness in vivo. Prostate Cancer and Prostatic Diseases (2006) 9, 68–76. doi:10.1038/sj.pcan.4500845; published online 29 November 2005 Keywords: cell cycle; diet; flow cytometry; flavonoids Introduction and dietary factors on PCa incidence. A number of case– control studies have correlated increased flavonoid Diets rich in flavonoids have been associated with a intake with a reduced incidence of a number of reduced incidence and mortality of prostate cancer malignancies including PCa.1,7–10 (PCa). The lowest incidence of PCa worldwide is seen Flavonoids comprise over 4000 structurally related in populations consuming the largest amount of flavo- polyphenols,11 which are ubiquitous in plants, and noids.1 In East Asian countries (China and Japan), diets ingested to varying degrees in the diet. The estimated are up to 100 times more abundant in flavonoids than in average daily intake of flavonoids is up to 1 g.12 This by the West, due in part to the consumption of soy and far exceeds the intake of other antioxidants such as green tea.2,3 Correspondingly, the incidence of PCa in vitamin E, and highlights the potential importance of China and Japan is 60- to 80-fold lower than in North flavonoids in the diet. Flavonoids have been shown to America.4 Studies on Japanese migrants to the United possess a wide range of biological activity , including States have shown that migrants born in Japan and living antioxidant (greater than vitamin C),13 anti-inflamma- in the United States have a higher incidence of PCa tory, antithrombogenic, and antiangiogenic activity.14 compared to men living in Japan.5,6 The incidence rates The anticancer properties of flavonoids have been for Japanese Americans born in the United States demonstrated in a variety of cell types in vitro and in increases further, approaching that of American white vivo.15 Despite the large number of flavonoids, studies men. Although these studies are not definitive, they have focused only on a select few. The flavonoids most emphasize the importance of environmental, lifestyle intensely studied in PCa to date are the soy isoflavones (genestein/daidzein),16,17 the green tea catechins (EGCG- epigallocatechin-3-gallate)18,19 and the milk thistle flavo- Correspondence: Dr LH Klotz, Division of Urology, Sunnybrook & nones (silibinin/silymarin).20,21 Little is known of the Women’s College Health Sciences Centre, MG-408, 2075 Bayview biological effect of most other flavonoids.22,23 Avenue, Toronto, Ontario, Canada M4N 3M5. E-mail: [email protected] In an attempt to identify novel flavonoids with Received 5 April 2005; revised 21 September 2005; accepted 22 growth-arresting properties in PCa cells, we have September 2005; published online 29 November 2005 screened a number of compounds from each of the Antiproliferative flavonoids and prostate cancer AQ Haddad et al 69 Table 1 Chemical composition of the flavonoids tested Subgroup Flavonoids Flavonoid side chain position* 20 30 40 50 23 45 6 7 8 Flavones Acacetin OMea OH OH b 6-Aminoflavone NH2 Baicalein OH OH OH Baicalin OH OH O-gluc 7,8-Benzoflavone benzd benzd Chrysin OH OH Diosmin OH OMe OH O-glu Karanjin OMe Furane Luteolin OH OH OH OH 5-Methoxyflavone OMe Primuletin OH Flavonols Fisetin OH OH OH OH Galangin OH OH OH Geraldol OH OMe OH OH Gossypin OH OH OH OH OH O-glu Kaempferol OH OH OH OH Morin OH OH OH OH OH Myricetin OH OH OH OH OH OH Quercetin OH OH OH OH OH Isoflavonoids Formononetin OMe OH Prunetin OH OH OMe Chalcones 2,20-DHC OH OH Isoliquiritigenin OH OH OH Flavonones Pinostrobin OH OMe Catechins Epigallocatechin OH OH OH OH OH OH Anthocyanidins Pelargonidine OH OH OH OH aMethoxyl group. bAmino group. cGlycoside. dBenzene ring. eFuranone. *This figure applies only to flavone and flavonol subgroups. Conventional numbering of the side chains in employed for the remaining flavonoids in the table (ref.11). major flavonoid subgroups (Table 1). We have examined 5-methoxyflavone, acacetin, morin, 6-aminoflavone, 7,8- their antiproliferative effect on the PCa cell lines PC3 benzoflavone, epigallocatechin and epicatechin gallate (androgen independent) and LNCaP (androgen depen- were purchased from Sigma Aldrich (St Louis, MO, dent), MCF-7 breast cancer cell line and a nonmalignant USA). All other flavonoids were procured from Indofine prostate stromal cell line (PrSC). We have identified a Chemical Co. (Hillsborough, NJ, USA). Green tea number of novel flavonoids with antiproliferative and catechin-epigallocatechin (EGC) was dissolved in water. cell cycle effects in human PCa cells in vitro. The All other flavonoids were dissolved in dimethylsulph- compound identified with the greatest antiproliferative oxide (DMSO) to a stock concentration of 100 mM. effect was the synthetic flavonoid 2,20-dihydroxychal- Working standards were made up in serum containing cone (2,20-DHC). media. The final concentration of DMSO in culture did not exceed 0.2%. Flavonoids that were poorly soluble in DMSO were not studied further. These included the Materials and methods flavonoids prunetin, acacetin, formononetin, diosmin and karanjin. Chemicals The flavonoids, which have been included in this study, Tissue culture are presented in Table 1. Quercetin, pinostrobin, kaemp- The human PCa cell lines, LNCaP (mutated androgen ferol, pelargonidin, galangin, formononetin, prunetin, receptor (AR), p53 wild-type), PC3 (AR null, p53 null), Prostate Cancer and Prostatic Diseases Antiproliferative flavonoids and prostate cancer AQ Haddad et al 70 and the estrogen receptor positive breast cancer cell line concentration of 10–150 mM. Control wells were treated MCF-7 were obtained from the American Type Tissue with vehicle alone (DMSO 0.2%). After 72 h of treatment Collection (ATCC), Rockville, MD, USA. The nonmalig- at 371C, the media was discarded, and the plates frozen nant prostate stromal cell line, PrSC, was obtained from at À801C until use. On the day of the analysis, the plates Cambrex, NJ, USA. LNCaP cells were cultured in RPMI with the adherent cells were thawed and incubated with 1640 medium (Gibco, New York) supplemented with the CyQuant dye for 5 min in the dark. Fluorescence was 10% foetal bovine serum (FBS) and 100 IU/ml penicillin measured on an FL600 fluorescence micro-plate reader and 100 mg/ml streptomycin. PC3 cells were cultured in (Bio-Tek, VT) with filters set at 480 nm excitation and DMEM/F12 medium with 10% FBS and antibiotics. 520 nm emission. The IC50 for each flavonoid was MCF-7 cells were cultured in IMEM media supplemen- determined in the four cell lines tested (Table 2). Each ted with 5% FBS, antibiotics, and insulin. PrSC medium experiment was performed independently at least three consisted of stromal cell basal media, and the manufac- times. turer’s growth factor supplements (Cambrex, NJ, USA). All cells were cultured at 371C with 5% CO . 2 Flow cytometry and cell cycle analysis Cell cycle arrest pattern and S phase enumeration were Cell proliferation assay determined by flow cytometry on cells labelled with anti- BrdU FITC and propidium iodide. Asynchronously Proliferation was assessed using the CyQuant cell 5 proliferation assay (Molecular Probes, OR, USA). In this growing cells (5 Â 10 cells/plate) were plated in 10 cm assay, the proprietary CyQuant dye binds to DNA, and dishes and treated for 72 h with the flavonoid at the IC50 the fluorescence emitted by the dye is linearly propor- concentration as determined earlier. Control plates were tional to the number of cells in the well. Cells were plated treated with vehicle alone (DMSO 0.2%). The cells were in 96-well black fluorescence micro-titre plates, at a pulse labelled with bromodeoxyuridine (BrdU) for 2 h density of 4000 cells/well. At 24 h after plating, triplicate prior to harvesting. As a negative control, a no-BrdU wells were treated with the appropriate flavonoid at a control was also included. Cells were trypsinized, fixed in ice cold 70% ethanol and stored at À201C until further analysis.

Load more