Reduction of rat prostate weight by combined quercetin-finasteride treatment is associated with cell cycle deregulation

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Reduction of rat prostate weight by combined quercetin-finasteride treatment is associated with cell cycle deregulation
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  Reduction of rat prostate weight by combined quercetin–finasteridetreatment is associated with cell cycle deregulation Zengshuan Ma, Thanh Hung Nguyen, Thanh Hoa Huynh,Phuc Tien Do  and  Hung Huynh Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, National Cancer Center of Singapore, Singapore 169610(Requests for offprints should be addressed to Hung Huynh; Email: cmrhth @ nccs.com.sg) Abstract Benign prostate hyperplasia and prostate cancer are major public health problems. We report herein that dailytreatment of male rats with 50, 100 or 150 mg quercetinper kg body weight resulted in serum concentrations of quercetin equivalent to 25·3 µM, 43·3 µM and 54·3 µMrespectively. Concomitantly, serum testosterone levelswere increased by 1·79-, 1·83- and 3·48-fold, while serumdihydrotestosterone (DHT) levels were 125%, 92% and73% of the control. A slight increase in prostate weightcoupled with dilated prostate lumens full of secretorymaterials were observed. Finasteride alone caused a sig-nificant decrease in serum DHT level and prostate weight.Co-administration of quercetin with finasteride preventedthe finasteride-induced decrease in serum DHT levels butsignificantly enhanced the reduction in wet prostateweight, which was reduced by 26·9% in finasteride-treated animals to 31·8%, 40·0% and 48·2% after fina-steride given together with the three doses of quercetin.The combined treatment altered cell cycle-regulated pro-teins in a wide spectrum. The expressions of cyclin D1,CDK-4, cdc-2 and phospho-cdc-2 at tyrosine 15,phospho-MEK1/2, phospho-MAP kinase, phospho-pRbat serine 780 and serine 807/811 were significantlyinhibited, while the levels of p15, p21 and p27 wereincreased. In conclusion, quercetin–finasteride treatmentscaused wide cell cycle deregulation in rat prostates, which,in turn, decreased the proliferation rate, changed thesecretion activities of epithelial cells and resulted in amarked reduction in wet prostate weight. The resultssuggest that quercetin synergizes with finasteride toreduce the wet prostate weight through a cell cycle-related pathway, which may be androgen independent.  Journal of Endocrinology   (2004)  181,  493–507 Introduction Benign prostate hyperplasia (BPH) and prostate cancer aremajor public health problems (Stoner 1994). The aim of palliative treatment of BPH or prostate cancer is todownregulate the levels of circulating androgen or to blockthe transcription, activation and function of the androgenreceptor, or both. Finasteride acts as a competitive andspecific inhibitor of 5  -reductase, resulting in suppressionof serum and intraprostatic dihydrotestosterone (DHT)concentrations to castrated levels, with subsequent reduc-tion in prostatic size (The Finasteride Study Group 1993,Rittmaster 1994). Finasteride is approved for treatment of symptomatic BPH (Feigl  et al.  1995). The only clinicallysignificant side e ff   ects of finasteride are related to sexualfunction such as decreased libido and impotence (Stoner 1994).Androgen plays a critical role in the growth, mainten-ance and function of the normal prostate gland (Cunha et al.  1987). Androgen-deprivation therapy causes markedand characteristic changes in normal prostate and inprostate cancer (Murphy  et al.  1991, Ferguson  et al.  1994).The biological function of androgen in the prostate ismediated by the androgen receptor (AR). Activation of the AR leads to complex proliferative, apoptotic andangiogenic events, which are mediated by interaction witha series of co-activators and a smaller subset of co-repressors (Lu  et al.  1999, Gregory  et al.  2001, Petre  et al. 2002). The mitotic signal of androgens is thought to targetultimately the cell cycle machinery (Yamamoto  et al. 2000). Androgen stimulates the expression of the cell cyclegenes CDK-1, CDK-2 and CDK-4 and increases thelevels of cyclin A and cyclin B1 mRNAs (Lu  et al.  1997,Gregory  et al.  2001). The cyclin-dependent kinase (CDK)kinase activities can be further increased by repressing theexpression of CDK inhibitor p16 gene (Lu  et al.  1997),overexpressing phosphatase cdc25B (Ngan  et al.  2003) or binding to cyclin E (Akita  et al.  2001). Cyclin E has beenreported to associate with AR to potentiate its activity inprostate cancer (Yamamoto  et al.  2000). Although studieshave also established the functional link of AR with cyclinD1 (Petre  et al.  2002) and p21 (Lu  et al.  1999), themultiple roles they play in cell proliferation, di ff   eren-tiation, and apoptosis are still intriguing. Cyclin D1 may 493  Journal of Endocrinology   (2004)  181,  493–5070022–0795/04/0181–493   2004 Society for Endocrinology  Printed in Great Britain Online version via http://www.endocrinology.org  play a mitogenic role (CDK-4-dependent) and an anti-mitogenic role (dependent on regulation of the AF-1domain) that can collectively control the rate of androgen-dependent cellular proliferation (Petre  et al.  2002).On the other hand, epidemiological studies have shownthat the consumption of vegetables, fruit and tea is associ-ated with a low risk of cancer (Steinmetz & Potter 1991, Yang & Wang 1993). Quercetin, one of the most commonflavonoid glycones, has a wide range of biological activitiesincluding inhibition of protein kinase C (Agullo  et al. 1997), tyrosine kinase (Akiyama  et al.  1987), phospha-tidylinositide 3-kinase (PI-3 kinase) (Agullo  et al.  1997)and DNA topoisomerase II (Constantinou & Huberman1995). Importantly, quercetin has antiproliferative activity in vitro  against several cancer cells by inhibiting the expres-sion of cyclin A (Yoshida  et al.  1992), cyclin B1 (Choi  et al. 2001), cyclin D1 (Kaneuchi  et al.  2003), cdc2 (Choi  et al. 2001, Yoshida  et al.  1992) and CDK-4 (Bhatia  et al.  2001).Treatment with quercetin arrests cell cycle progressioneither at the G1/S phase (Bhatia  et al.  2001) or at theG2/M transitional boundary (Choi  et al.  2001).  In vivo synergy of quercetin with cisplatin against a lung cancer xenograft has been reported (Hofmann  et al.  1988).Quercetin also inhibits cell invasion and induces apoptosisthrough a pathway involving heat shock proteins (Wei et al.  1994). Although the mechanisms of the antiprolifer-ation e ff   ects of quercetin remain to be illustrated, there isevidence suggesting that the action of quercetin is probablymediated by interaction with the type II estrogen bindingsites (Ranelletti  et al.  1992) or the aryl hydrocarbon recep-tor (Ashida  et al.  2000). These activities of quercetin makeit a promising candidate for the treatment and preventionof various cancers including prostate cancer. The cell cycle-regulated proteins are considered to be thecommon downstream e ff  ectors mediating the e ff  ects of finasteride and quercetin in the prostate gland. In this study,we report that quercetin when co-administered with finas-teride prevented finasteride-induced changes in serumDHT levels, but caused reduction in wet prostate weight.The combined e ff  ects of quercetin and finasteride on wetprostate weight were associated with their ability to modu-late the expression of cell cycle-regulated proteins. Theexpressions of cyclin D1, CDK-4, cdc-2 and phospho-cdc-2 at tyrosine 15, phospho-mitogen extracellular kinase1/2 (MEK1/2), phospho-MAP kinase (MAPK), phospho-pRb at serine 780 and serine 807/811 were significantlydown-regulated while the levels of p15, p21 and p27 wereincreased. The combined treatment also significantlyreduced the ratio of hyperphosphorylated pRb to total pRb. Materials and Methods Reagents Rabbit anti-phospho MEK1/2 (Ser217/221), mouse anti-phospho p44/42 MAP kinase (Thr202/Tyr204), rabbitanti-cdc-2, rabbit anti-phosphorylated cdc-2 (Tyr15),mouse anti-retinoblastoma (pRb), rabbit anti-phosphorylated pRb (Ser780), rabbit anti-phosphorylatedpRb (Ser795), rabbit anti-phosphorylated pRb (Ser807/811) and rabbit anti-phospho-Akt-1 (Ser473) antibodieswere purchased from New England BioLabs (Beverly,MA, USA). Rabbit anti-ERK-1, rabbit anti-p85 subunitof PI-3 kinase, mouse anti-  -tubulin, rabbit anti-cyclin A,rabbit anti-epidermal growth factor receptor (EGFR),rabbit anti-Raf, rabbit anti-pRaf, rabbit anti-cyclin B1,mouse anti-cyclin E, mouse anti-cyclin D1, mouse anti-CDK-2, rabbit anti-CDK-4, mouse anti-CDK-6, mouseanti-p21, mouse anti-p27 and mouse anti-p15 antibodieswere from Santa Cruz Biotechnology (Santa Cruz, CA,USA). Mouse anti-Ki-67 was from NeoMarkers Inc.(Fremont, CA, USA). Anti-  -tubulin antibody was usedat a final concentration of 0·5 µg/ml. Other anti-bodies were diluted into Tris–bu ff   ered saline Tween 20(TBST) solution at a final concentration of 1 µg/ml, asrecommended by the manufacturers.  Animals Animals were maintained and treated according tothe guidelines of the Local Animal Care Committee.Ten-week-old male Sprague-Dawley rats, provided bythe Animal Holding Unit, National University of Singapore, were divided into 8 groups ( n =8). To testthe e ff   ect of quercetin on rat prostate, rats were dailygavaged with quercetin (Sigma, St Louis, MO, USA),dissolved in 5% dimethyl sulfoxide (DMSO) aqueoussolution, at a dose of 50, 100 or 150 mg per kg bodyweight (BW). To block the conversion of testosteroneto DHT, one group of rats was treated daily withfinasteride 1 mg/kg BW (5 mg/tablet, Merck Frost,Quebec, Canada) by gavage. We have previouslyreported that at this dose the weight of the prostate wasreduced to 80% of the control, while the DHT levelwas decreased to 39·5% and the testosterone level wasincreased 2·4-fold as compared with the control (Huynh et al.  1998). To investigate the combined e ff   ects of quercetin and finasteride on prostate weight and serumandrogen levels, rats were treated daily with 1 mgfinasteride/kg BW plus 50, 100 or 150 mg quercetin/kgBW. Control rats received the same dose of vehicles. Todetermine the concentration of quercetin in the serum, therats were immobilized in a restraint device at 4 h post drugadministration and the blood was collected with a 23-gauge needle after the tail vein was slightly enlarged bymopping with 70% alcohol. After 10 days of treatment,animals were weighed and killed, and wet prostate,pancreas, liver, kidney, testis and serum were collected.After weighing, a portion of the prostate, together withother tissues, was fixed in 10% bu ff   ered formalin for para ffi n embedding; the remaining tissue was immediatelyfrozen in liquid nitrogen for further analysis. Z MA  and others ·  Cell cycle in rat prostate 494 www.endocrinology.org  Journal of Endocrinology   (2004)  181,  493–507  Immunohistochemistry and histology Fixed prostate, pancreas, liver, kidney and testis wereroutinely processed in a tissue processor and embedded inpara ffi n. Sections of 5 µm were cut and subjected toimmunohistochemical study using the ImmunoCruzStaining System (Lab Vision Corporation, Fremont, CA,USA). Briefly, the slides were depara ffi nized, rehydratedgradually through graded alcohols, and incubated in 3%H 2 O 2  for 20 min to block endogenous peroxidase activity.The antigens were retrieved by boiling the slides in10 mM citrate bu ff   er (pH 6·0) for 15 min. After blockingunspecific background with 5% skim milk for 20 min atroom temperature, the slides were incubated with mouseanti-Ki-67 antibody overnight at 4  C. The slides werethen incubated with the appropriate biotinylated second-ary antibody, followed by peroxidase-conjugated strepta-vidin complex and diaminobenzidene. The sections werefinally counterstained with hematoxylin. The slides wereexamined under an Olympus Provis microscope (BX60,Olympus Optical Corp., Toyota, Japan) and images werecaptured digitally with the Olympus DP11 camera(Olympus Optical Corp.). The Ki-67 labeling index wasexpressed as the percentage of clearly labeled Ki-67reactive nuclei in total epithelial cells in randomly selectedfields at a magnification of   200 ( n =6). Quantitation of apoptosis Sections of 5 µm were used to quantitate apoptosisin prostate tissues. Fragmented DNA was labeled usingthe ApoAlert DNA fragmentation assay (ClontechLaboratories, Palo Alto, CA, USA) which is based on theterminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL) as described (Nickerson  et al. 1998). Labeling indices were obtained by counting thenumber of labeled cells among at least 100 epithelial cellsper region and were expressed as percentage values. Serum androgen measurement  Serum testosterone and DHT levels were determinedusing their respective enzyme-linked immunosorbentassay (ELISA) kits (IBL Immuno-Biological Laboratories,Hamburg, Germany) as described by the manufacturer.Briefly, an unknown amount of antigen present in thesample and a fixed amount of enzyme-labeled antigencompeted for the binding sites of the antibodies coatedonto the wells. After incubation, the wells were washedto stop the competition reaction and the tetramethyl-benzidine (TMB) substrate solution was added. Themeasured optical density of the standards was used toconstruct a calibration curve against which the concen-trations of the unknown samples were calculated. Theconcentration of antigen present in the samples wasinversely proportional to the optical density measured at awavelength of 450 nm. Determination of the concentration of total quercetin metabolitesin serum After ingestion of quercetin, the major circulating metabo-lites in rat blood are glucurono-sulfo conjugates of isorhamnetin and of quercetin (Morand  et al.  1998).Quercetin, quercetin glycosides, quercetin glucuronidesand quercetin sulfates were hydrolyzed to their aglyconesby incubating 180 µl rat serum, acidified to pH 4·9 with20 µl 0·58 M acetic acid solution, for 30 min at 37  C inthe presence of 10 µl enzyme mixture (5  10 6 U/l  -glucuronidase and 2·5  10 5 U/l sulfatase). The agly-cones were then extracted with 500 µl methanol/HCl(200 mM) followed by centrifugation (14 000  g  for 10 min) (Morand  et al.  2000). The concentrations of aglycones were determined by a reversed-phase highperformance liquid chromatography (RP-HPLC) methodin a chromatograph (Waters 2695 Separation Module,Milford, MA, USA) equipped with a Photodiode Array Table 1  Effects of quercetin (Q; 50, 100, 150 mg/kg), finasteride (F; 1 mg/kg) and finastrideplus quercetin (FQ) on the body weight, wet prostate weight, serum testosterone anddihydrotestosterone (DHT) levels of rats (means  S.E.M. ,  n = 8) Body weight (g) Wet prostate weight (mg) Serum testosterone (ng/ml) Serum DHT (pg/ml) Treatment Control 343·7  7·6 455·0  15·5 a 1·79  0·29 a 227·9  19·9 a,b Q50 334·1  6·7 495·7  25·1 a 3·21  0·67 b 284·4  25·3 a Q100 353·9  5·5 509·8  22·3 a 3·28  0·87 b 210·5  27·4 a,b Q150 338·7  7·2 474·6  21·4 a 6·23  1·14 c 166·5  35·1 b F1 343·2  3·7 332·4  19·5 b 5·65  0·92 c 118·4  24·3 c F1Q50 343·4  3·2 310·3  18·7 b,c 3·43  0·72 b 275·2  39·4 a F1Q100 348·7  5·6 273·2  17·2 c 5·16  0·49 c 205·9  34·2 a,b F1Q150 336·0  5·8 235·5  14·8 c 4·85  0·98 c 172·1  23·5 b Numbers within columns with different superscript letters are significantly different from one another at P  < 0·05 (ANOVA). Cell cycle in rat prostate  ·  Z MA  and others 495 www.endocrinology.org  Journal of Endocrinology   (2004)  181,  493–507  detector (Waters 2996) and a Waters Symmetry column(C 18 , 5 µm, 3·9  150 mm). The mobile phase consistedof 60% methanol and 40% phosphoric acid solution (0·5%).The injection volume, the flow rate and the detectionwavelength were 20 µl, 0·7 ml/min and 370 nm respect-ively. The total quercetin metabolites in serum weredetermined by summing the concentrations of the agly-cones, i.e. isorhamnetin, quercetin and tamarixetin, whichwere determined against their respective standard curves. Western blot analysis To determine the changes in the expression levels of cellcycle-regulated proteins, the frozen rat prostate was hom-ogenized in lysis bu ff   er (1 mM CaCl 2 , 1 mM MgCl 2 , 1%NP-40, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µM phe-nylmethylsulfonyl fluoride, and 100 µM NaVO 4 ). Onehundred micrograms proteins were subjected to Westernblot analysis as described (Huynh  et al.  1995). Blots wereincubated with the indicated primary antibodies followedby horseradish peroxidase-conjugated donkey antimouseor antirabbit secondary antibody (1:7500). Blots werevisualized with a chemiluminescent detection system(Amersham Pharmacia Biotech UK Ltd, UK) as describedby the manufacturer. For quantitative analysis, the densityof bands corresponding to the protein blotting with theantibodies under study was calculated with the QualityOne software (Bio-Rad Laboratories, Hercules, CA,USA) and was normalized to that of    -tubulin. Statistical analysis Comparison between the di ff   erent groups was performedby one-way ANOVA (SPSS 10, SPSS Inc. Chicago, IL,USA) with the Student’s unpaired  t  -test applied for pairedcomparisons of means.  P   values of 0·05 or less wereconsidered significant, while values of 0·01 or less wereconsidered very significant. Results E   ff   ects of finasteride and quercetin on the rat body weight and wet prostate weight  Treatment of rats with 50, 100 or 150 mg quercetin/kg for 10 days resulted in an insignificant increase in wet prostateweight regardless of the doses used (Table 1). Finasteride Figure 1  Effects of quercetin, finasteride and finasteride plus quercetin on prostate gland morphology. Rats were treated for 10 days with(A) vehicle, (B) finasteride (1 mg/kg body weight daily), (C) quercetin (150 mg/kg body weight daily), or (D) finasteride plus quercetin.Hematoxylin-eosin stain was used; scale bar = 10   m. Z MA  and others ·  Cell cycle in rat prostate 496 www.endocrinology.org  Journal of Endocrinology   (2004)  181,  493–507  at a dose of 1 mg/kg body weight caused a significant26·9% reduction in wet prostate weight as compared withcontrols ( P  , 0·001) (Table 1). Co-administration of finasteride with 50, 100 and 150 mg quercetin/kg BWdecreased the wet prostate weight by 31·8%, 40·0% and48·2% respectively. There was a linear relationshipbetween the wet prostate weight and the quercetin dose inthe co-treated groups (R 2 =0·991), with the most signifi-cant e ff   ect being observed when finasteride was adminis-tered together with 150 mg quercetin ( P  , 0·001). Therewas, however, no statistically significant change in thebody weight of rats. E   ff   ects of finasteride and quercetin on the histology of rat  prostate, pancreas, liver, kidney and testis As shown in Fig. 1A, the alveoli of control prostates werelined with a layer of tall columnar epithelial cells with ahigh cytoplasm/nuclear ratio. The luminal epithelial cellsshowed a marked reduction in cytoplasm and their secre-tory activity became diminished after finasteride treatmentfor 10 days (Fig. 1B). Compared with the control prostategland, the lumens of prostate glands derived fromfinasteride-treated rats were markedly reduced in size(Fig. 1B compared with Fig. 1A). Quercetin treatment, onthe other hand, caused a dramatic dilation of the prostatelumen. The lumens were filled with secretory materials(Fig. 1C), indicating that quercetin enhanced the secretoryactivity of epithelial cells. The increase in luminal volumewas accounted for by a significant ( P  , 0·01) decrease inthe average cell number per unit area, reaching 63% of control rats (Table 2). Co-administration of finasteride andquercetin, however, led to additive e ff   ects on the thicknessof the prostate epithelium. The cytoplasm was greatlyreduced and the epithelial layer became very thin(Fig. 1D). No marked change in the cell morphology or histology was observed in the slides of pancreas, liver,kidney and testis (data not shown). E   ff   ects of finasteride and quercetin on serum androgen levels Quercetin and finasteride, when administered as a singleagent for 10 days, significantly increased serum testoster-one levels ( P  , 0·001) after 10 days of treatment (Table 1).As expected, finasteride significantly decreased serumDHT levels to half that of the control rats (118 vs228 pg/ml,  P  , 0·01) (Table 1). Quercetin treatment,however, resulted in a biphasic change in serum DHTconcentration, which was slightly increased at a dose of 50 mg/kg BW, but decreased at a dose of 150 mg/kg BW.Similar e ff   ects were seen when quercetin was giventogether with finasteride. It was noteworthy that theintake of quercetin dampened the e ff   ects of finasteride onthe levels of both serum testosterone and DHT. Compared  T   a     b     l   e     2     E     f     f   e   c    t   s   o     f   q   u   e   r   c   e    t     i   n     (     Q   ;    5    0 ,    1    0    0 ,    1    5    0   m   g    /     k   g     ) ,     fi   n   a   s    t   e   r     i     d   e     (    F   ;    1   m   g    /     k   g     )   a   n     d     fi   n   a   s    t   r     i     d   e   p     l   u   s   q   u   e   r   c   e    t     i   n     (    F     Q     )   o   n    t     h   e   p   r   o   s    t   a    t   e   e   p     i    t     h   e     l     i   a     l    t     h     i   c     k   n   e   s   s ,     l   u   m   e   n     d     i   a   m   e    t   e   r ,   c   e     l     l   n   u   m     b   e   r   s   p   e   r   s   e     l   e   c    t   e     d     fi   e     l     d   a   n     d    K     i  -    6    7     i   n     d     i   c   e   s     (   m   e   a   n   s             S .    E .     M .  ,      n           8     )      C   o   n    t   r   o     l     Q     5     0     Q     1     0     0     Q     1     5     0     F     1     F     1     Q     5     0     F     1     Q     1     0     0     F     1     Q     1     5     0     E   p     i    t     h   e     l     i   a     l    t     h     i   c     k   n   e   s   s     (          m     )    6   ·    1            1   ·    5     a     5   ·    1            1   ·    2        b     4   ·    5            1   ·    3        b     4   ·    7            1   ·    1        b     4   ·    7            1   ·    4        b     5   ·    0            1   ·    3        b     4   ·    1            1   ·    1        b     4   ·    0            1   ·    2        b     L   u   m   e   n     d     i   a   m   e    t   e   r     (          m     )    6    6   ·    8            1    1   ·    3     a     8    5   ·    5            1    3   ·    1     a ,       b     9    5   ·    7            2    1   ·    0        b     1    1    4   ·    4            2    7   ·    6        b     3    5   ·    9            1    3   ·    4     c     7    2   ·    3            1    5   ·    8     a     7    9   ·    8            1    6   ·    6     a ,       b     9    7   ·    6            1    9   ·    4     a ,       b      C   e     l     l   n   u   m     b   e   r    1    3    3    0            2    3    0     a     1    3    1    9            1    9    4     a     1    0    7    7            2    6    1     a ,       b     8    4    5            1    7    0        b     1    5    3    9            3    4    5     a     1    1    3    3            2    0    6     a ,       b     1    0    1    1            1    9    6     a ,       b     1    0    6    8            1    3    4     a ,       b     K     i  -    6    7     i   n     d   e   x     (    %     )    1   ·    5    0            0   ·    3    1     a     1   ·    6    7            0   ·    2    8     a     2   ·    5    9            0   ·    4    7        b     2   ·    6    6            0   ·    6    8        b     0   ·    1    9            0   ·    1    3     c     0   ·    1    2            0   ·    0    9     c     0        d     0   ·    0    2            0   ·    0    1        d      N   u   m     b   e   r   s   w     i    t     h     i   n   c   o     l   u   m   n   s   w     i    t     h     d     i     f     f   e   r   e   n    t   s   u   p   e   r   s   c   r     i   p    t     l   e    t    t   e   r   s   a   r   e   s     i   g   n     i     fi   c   a   n    t     l   y     d     i     f     f   e   r   e   n    t     f   r   o   m   o   n   e   a   n   o    t     h   e   r   a    t        P       <     0   ·    0    5   a   s     d   e    t   e   r   m     i   n   e     d     b   y   o   n   e  -   w   a   y     A     N     O    V     A . Cell cycle in rat prostate  ·  Z MA  and others 497 www.endocrinology.org  Journal of Endocrinology   (2004)  181,  493–507
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