ANTICANCER RESEARCH 33: 4453-4462 (2013) Repression of Cell Proliferation and Androgen Receptor Activity in Prostate Cancer Cells by 2’-Hydroxyflavanone MITSUO OFUDE1, ATSUSHI MIZOKAMI1, MISAKO KUMAKI1, KOUJI IZUMI1, HIROYUKI KONAKA1, YOSHIFUMI KADONO1, YASUHIDE KITAGAWA1, MINKYOUNG SHIN1, JIAN ZHANG2, EVAN T. KELLER3 and MIKIO NAMIKI1 1Department of Integrative Cancer Therapy and Urology, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Ishikawa, Japan; 2Center for Translational Medical Research, Guangxi Medical University, Guangxi, P.R. China; 3Department of Urology, School of Medicine, University of Michigan, Ann Arbor, MI, U.S.A. Abstract. Background: Prevention of the development of line hormonal therapy using medical castration, such as castration-resistant from hormone-naïve prostate cancer is luteinizing hormone releasing hormone (LH-RH) agonists, an important issue in maintaining the quality of life of the LH-RH antagonist, and antiandrogen for advanced PCa. patients. We explored the effect of 2’-hydroxyflavanone on After an initial response to ADT, however, PCa eventually proliferation and androgen responsiveness using prostate loses responsiveness to ADT and progresses into what is cancer cell lines. Materials and Methods: To investigate the termed castration-resistant PCa (CRPC). effect of 2’-hydroxyflavanone on proliferation, prostate The AR axis also plays an important part in the process cancer cells were treated with 2’-hydroxyflavanone. when hormone- sensitive PCa become CRPC. Although Androgen-responsiveness in LNCaP cells was confirmed by serum testosterone decreases to less than 5% before starting luciferase assay after transfection of luciferase reporter ADT, PCa adapts to low serum testosterone level by several driven by prostate specific antigen promoter. To detect mechanisms. One other important factor is the adrenal androgen receptor (AR) expression, reverse transcriptase androgen, dehydroepiandrostenedione (DHEA). DHEA is polymerase chain reaction and western blot analysis were metabolized into testosterone, and then converted to conducted. Results: 2’-Hydroxyflavanone inhibited the dihydrotestosterone (DHT) by 5α-reductase in PCa tissue, proliferation of PC-3 and DU145 cells by induction of which then activates the AR. In fact, the concentration of apoptosis. 2’-Hydroxyflavanone inhibited the proliferation of DHT in PCa tissue remains 20 to 40% of pretreatment LNCaP cells stimulated by androgens and attenuated values (1-3). Interaction of epithelial and stromal cells androgen-responsiveness through down-regulation of AR plays an important role in the production of DHT in PCa protein. Conclusion: 2’-Hydroxyflavanone not only inhibited tissue. After castration, adrenal androgen DHEA is proliferation of prostate cancer cells, but also repressed metabolized into DHT in stromal cells and epithelial cells androgen-responsiveness, suggesting that it might be a useful coordinately (4). Moreover, CYP17A inhibitors, abiraterone agent in preventing recurrence of prostate cancer. acetate and TAK-700, which inhibit conversion from pregnenolone into DHEA, is effective for more than 70% Since the androgen receptor (AR) axis is the main route for of CRPC after docetaxel-treatment. These results indicate development and progression of prostate cancer (PCa), that the AR axis affects the recurrence of PCs even in androgen-deprivation therapy (ADT) is conducted as a first- patients resistant to docetaxel. Important enzyme in intratumoral androgen synthesis mediating through interaction of epithelial and stromal cells are type 3 and type 5 17β-hydroxysteroid dehydrogenase Correspondence to: Atsushi Mizokami, Department of Integrative (HSD17B3 and HSD17B5) and 3β-hydroxysteroid Cancer Therapy and Urology, Kanazawa University Graduate dehydrogenase (3β-HSD). In particular, HSD17B3 catalyzes School of Medical Sciences, 13-1 Takaramachi, Kanazawa, the formation of testosterone from 4-androstenedione (adione) Ishikawa, 920-8640, Japan. Tel: +81 762652393, Fax: +81 762226726, e-mail: [email protected] in the testis and peripheral tissues (5). However, the function of the testes is lost in PCa after ADT, the main androgen Key Words: Prostate cancer, androgen receptor, 2’-hydroxyflavanone, synthesis enzyme in CRPC is HSD17B5. It is known that androgen. AKR1C3 aldo-keto reductase acts as a HSD17B5 (6, 7). 0250-7005/2013 $2.00+.40 4453 ANTICANCER RESEARCH 33: 4453-4462 (2013) AKR1C3 was found to be up-regulated in patients with PCa, Recombinant plasmid constructs. Recombinant plasmid pEGFP-fAR especially, in those with metastatic PCa and CRPC (8-11). that expresses full-length wild-type AR fused with green fluorescent Moreover, overexpression of AKR1C3 promoted PCa protein (GFP) was constructed by inserting the full-length AR cDNA of pSGAR2, which is driven by SV40 promoter (19) (at -24 to 3110 proliferation (12). These findings suggest that hyperactivation bp of start codon), into pEGFP-C1 (Invtrogen, CA, USA). The insert of AKR1C3 might affect the recurrence of PCa. configurations of fAR cDNAs were confirmed by sequence analysis. Flavonoids are a large group of polyphenolic compounds present in foods and beverages of plant origin, and are Luciferase assay. To evaluate AR transcriptional activity, 24 h after subdivided into six subclasses: flavonols, flavones, plating 5×104 cells on 12-well plates in DMEM-5% CCS, LNCaP flavanones, flavan-3-ols, anthocyanidins, and isoflavones (13). and PC-3 cells were transfected using Lipofectamine transfection Flavonoids display a broad range of pharmacological activity, reaction (Invitrogen) using 0.5 μg of luciferase reporter plasmid, pGL3PSAp-5.8, driven by a 5.8 kb PSA promoter (20). Twenty-four such as antioxidative, anti-inflammatory, and antiproliferative hours after transfection, cells were treated by the addition of DHT activities (13-15). Flavonoids have been shown to inhibit with and without 2’HF for 24 h. After treated cells were harvested, AKR1C3 activity in vitro (16, 17). 2’-Hydroxyflavanone cells were lysed in luciferase lysis buffer (Promega, Madison, WI, (2’HF), which is a flavanones in particular had a strong USA) and the luciferase activity was quantitated by a luminometer. inhibitory effect on AKR1C3 in vitro (16). For overexpression of EGFP-fAR in LNCaP cells, 5×104 LNCaP In the present study, we investigated the effect of 2’HF on cells were co-transfected with 0.1 μg of pEGFP-fAR and 0.4 μg of proliferation and androgen responsiveness using PCa cell pGL3PSAp-5.8, and then cells were further treated with adione, DHT and/or 2’HF for 24 h. lines. Apoptosis assay. To investigate whether 2’HF causes PCa cells to Materials and Methods undergo apoptosis, the Annexin-V-FLUOS Staining kit (Roche, Mannheim, Germany) was used according to the manufacture’s 5 4 Cell lines and cell proliferation assay. LNCaP and DU145 cells protocol. In brief, 1×10 LNCaP, and DU145 cells, and 5×10 PC-3 (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified cells were seeded in 6-well plates with DMEM-5% CCS. They were Eagle Medium (DMEM) supplemented with 1% treated with 10 μM 2’HF for 72 h. After removing the media and penicillin/streptomycin (P/S; Invitrogen, Carlsbad, CA, USA) and washing by PBS, cells were incubated with 100 μl Annexin-V- 5% fetal bovine serum (FBS; Sigma–Aldrich, St. Louis, MO, USA), FLUOS labeling solution added with propidium iodide for 15 min at respectively. PC-3 cells (ATCC) were cultured in RPMI -1640 room temperature. The stained cells were analyzed by fluorescence supplemented with 1% P/S (Invitrogen) and 5% FBS. Twenty-four microscope, FSX100 (Olympus, Tokyo, Japan). hours after plating at a density of 5×104 cells onto 12-well plates Liquid chromatography mass spectometry/mass spectrometry (LC- with DMEM-5% charcoal-stripped fetal calf serum (CCS; Thermo MS/MS). 4 Scientific HyClone, UK), cells were treated with ethanol, adione, After plating 5×10 cells on 12-well plates in DMEM-5% testosterone, DHT and/or 2’HF in DMEM-5% CCS and the media CCS, PC-3 cells were treated with 10 nM adione or 10 nM were changed every two days. In each experiment, cells were testosterone in the absence and presence of 10 μM 2’HF. Then the harvested and the numbers of the cells were counted in triplicate media were collected 24 hours later. The concentration of adione, using a hemocytometer. The data shown represent the means±SD of testosterone, and DHT in media was measured by LC-MS/MS three replicates. (Division of Pharmacological Research, Aska Pharma Medical Co. Ltd., Kawasaki, Japan). Reverse transcriptase polymerase chain reaction (RT-PCR) and Visualization of AR localization. Twenty-four hours after western blot analysis. For RT-PCR, 24 h after plating at a density transfection of pEGFP-fAR into PC-3 cells, cells were treated with of 1×105 cells onto 6-well plates with DMEM-5% CCS, cells were or without 10 μM 2’HF for 24 h. Consequently cells were cultured treated with or without Adione, DHT and/or 2’HF for 24 h and total in the absence or presence of 10 nM DHT for 8 h, and fAR fused to RNA was extracted. Total RNA extraction from cells and RT-PCR green fluorescent protein (GFP) was visualized by FSX100. for AR, prostate specific antigen (PSA), and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was performed as described Statistical analysis. Statistical significance was determined by using previously (2, 4). Prism 6.0 software, the χ2 test was utilized to assess the significance For western blot analysis, total protein was extracted from cells between different proportions. Analysis of continuous variables as described previously (18). Protein was quantified according to between different groups was assessed by one-way analysis of the method of Bradford, and equal amounts of protein were variance followed by Fisher’s protected least significant difference electrophoresed on a 10% or 12.5% Ready Gel J (Bio-Rad, test. *, **, and *** in Figure represent significant difference p<0.05, Hercules, CA, USA). Membranes were incubated with mouse p<0.01, and p<0.001, respectively. monoclonal antibody against AR (NH27) (19) and GAPDH (Novus Biologicals, Littleton, CO, USA).