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Chemopreventive Effect of Quercetin in MNU andTestosterone Induced Prostate Cancer of Sprague-Dawley RatsGovindaraj Sharmila a , Thavadurainathan Athirai a , Balakrishnan Kiruthiga a , KalimuthuSenthilkumar a , Perumal Elumalai a , Ramachandran Arunkumar a & Jagadeesan Arunakarana

a Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences ,University of Madras , Chennai , IndiaPublished online: 09 Dec 2013.

To cite this article: Govindaraj Sharmila , Thavadurainathan Athirai , Balakrishnan Kiruthiga , Kalimuthu Senthilkumar ,Perumal Elumalai , Ramachandran Arunkumar & Jagadeesan Arunakaran (2014) Chemopreventive Effect of Quercetinin MNU and Testosterone Induced Prostate Cancer of Sprague-Dawley Rats, Nutrition and Cancer, 66:1, 38-46, DOI:10.1080/01635581.2014.847967

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Nutrition and Cancer, 66(1), 38–46Copyright C© 2014, Taylor & Francis Group, LLCISSN: 0163-5581 print / 1532-7914 onlineDOI: 10.1080/01635581.2014.847967

Chemopreventive Effect of Quercetin in MNUand Testosterone Induced Prostate Cancerof Sprague-Dawley Rats

Govindaraj Sharmila, Thavadurainathan Athirai, Balakrishnan Kiruthiga,Kalimuthu Senthilkumar, Perumal Elumalai, Ramachandran Arunkumar,and Jagadeesan ArunakaranDepartment of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences,University of Madras, Chennai, India

Prostate cancer becomes an ideal target for chemopreventionbecause of its high incidence and extended natural history. Theconsumption of quercetin (plant flavonoid) in diet is associatedwith decreased risk of disease and many cancers but then this wasnot elucidated in prostate malignancy. Hence, a study in which themale Sprague-Dawley rats were induced prostate cancer by hor-mone (testosterone) and carcinogen (MNU) and simultaneouslysupplemented with quercetin (200 mg/Kg body weight) thrice aweek, was conducted. After the treatment period, rats were killed;ventral and dorsolateral lobes of the prostate were dissected. His-tology and oxidative stress markers LPO, H2O2, and antioxidantGSH level were measured in both lobes. The lipid peroxidation,H2O2, in (MNU+T) treated rats were increased and GSH level wasdecreased, whereas simultaneous quercetin-treated rats revertedback to normal level in both ventral and dorsolateral regions. Thedifferent patterns of PIN were observed with associated hyperpla-sia and dysplasia; changes in these regions and the occurrence ofthis lesion were reduced in simultaneous quercetin-treated rats.The study concluded that dietary quercetin prevented MNU + T-induced prostate carcinogenesis on both ventral and dorsolaterallobes of Sprague-Dawley rats.

INTRODUCTION

Prostate cancer accounts for the second highest cancer-related deaths in men. The etiology of prostate cancer is un-known but it is usually associated with age, race, family history,hormonal, dietary factors, etc. (1). Prostate cancer progressesfrom premalignant lesion or prostatic intraepithelial neoplasia

Submitted 14 December 2012; accepted in final form 12 August2013.

Address correspondence to Jagadeesan Arunakaran, Depart-ment of Endocrinology, Dr. ALM Post Graduate Institute of Ba-sic Medical Sciences, University of Madras, Taramani, Chennai600113, India. Tel.: 91-44-24547043. Fax: 91-44-24540709. E-mail:j arunakaran@hotmail.com

(PIN) to several stages of metastasis and hormone refractory dis-ease (2). The lack of appropriate animal model systems leavesa major obstacle in the development of strategies for prostatecancer chemoprevention (4) though the progress of prostaticcarcinoma in to invasive phase can be studied in a few trans-plantable systems (3).

However, the combination of carcinogen, N-methyl-N-nitrosourea (MNU), and testosterone (T) has the advantage ofinducing higher incidence of prostate carcinogenesis in Wistar-Unilever rat that mimics the human prostate cancer model (5).Earlier reports from our laboratory had shown that the combinedhormone and carcinogen (MNU) treatment consistently gener-ated prostatic dysplasia, a putative preneoplastic lesion that isexclusively within prostates (6–8). The evaluation of anticancereffects of various plant-derived agents has gained good attentionin recent years due to the development of resistance against theavailable chemotherapeutic agents (9).

Among chemopreventers, flavonoids are the most studiedantioxidant compounds that are largely present in fruits, vegeta-bles, tea, red wine, and aromatic plants, (10–12). Quercetin isa principal flavanoid compound of onion (3, 3′, 4′, 5, 7-Pentahydroxyl flavanone), which possesses a wide spectrum of phar-macological properties (13, 14).

Various animal and in vitro studies showed that quercetin wasfound to have antiproliferative effects by inhibiting cell growthand induces apoptosis in numerious cancer cell like breast,leukemia, colon, ovary, endometrial, gastric, and lung (14–19).Quercetin also enhances TRAIL-induced apoptosis in prostatecancer cells through increased protein stability of death receptor5 (20). Recent findings from our laboratory have confirmed thatquercetin downregulates insulin like growth factor signallingand upregulates intrinsic as well as extrinsic pathway-mediatedapoptosis in androgen-independent prostate cancer cells (PC-3)(21). Quercetin also inhibits invasion, migration, and signallingmolecules involved in cell survival and proliferation of prostatecancer cell line (PC-3) (22).

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CHEMOPREVENTION OF PROSTATE CANCER BY QUERCETIN 39

However, there is no report available on the role of quercetinon prostate malignant in vivo model. Therefore, this studywas conducted to characterize the chemopreventive activity ofquercetin in the prostate carcinogenesis using an animal model(Sprague-Dawley rats).

MATERIALS AND METHODS

ChemicalsQuercetin and MNU were purchased from Sigma-Aldrich

Chemicals Private Ltd. (St. Louis, MO). Testosterone unde-canoate (T) was purchased from M/S Schering-Plough, Apel-doorn, The Netherlands. Other chemicals were obtained fromSISCO Research Laboratories Pvt. Ltd, Mumbai, India. All thechemicals were extra pure and of analytical grade.

AnimalsHealthy adult male Sprague-Dawley rats weighing approx-

imately 200–250 g were used. The animals were housed inclean polypropylene cages and maintained in an air-conditionedanimal house with constant 12-h light/dark cycle. Rats werepermitted free access to drinking water throughout the exper-imental period. The animals were fed with standard rat pelletdiet (Lipton India Ltd., Mumbai, India). Experiment was ap-proved by the Institute Animal Ethical Committee (IAEC No.01/01/12).

Prostate Tumor InductionRats were induced prostate cancer using carcinogen (MNU)

and hormone (testosterone undecanoate) by a modified protocol(5, 23). First, each rat received daily intraperitoneal (IP) injec-tions of testosterone undecanoate (50 mg/kg body weight) for21 consecutive days. At Day 23, rats received daily IP injectionsof 100 mg testosterone undecanoate /kg body weight in 0.3 mlpropylene glycol for 3 days. At Day 27, all the rats received asingle intravenous (IV) dose (50 mg/kg body weight) of MNU(dissolved in saline at 10 mg/ml) through the tail vein. After1 wk of MNU administration, rats received an IP injection of4 mg testosterone undecanoate /kg body weight alternatively for16 wk.

Experimental DesignA total of 40 rats were divided into 4 groups and each group

consisted of 10 rats. In Group I, rats that received vehicle (propy-lene glycol) alone by IP injection, considered as controls. InGroup II, rats were induced prostate cancer by using carcinogenplus hormone. In Group III, rats were induced prostate cancerwith simultaneous supplementation of quercetin (200 mg/kgbody weight) thrice a week for 16 wk through oral gavage.Quercetin supplementation was begun a week before the ad-ministration of the initial dose of testosterone undecanoate ad-ministration and throughout the studies. The dose was selectedbased on the earlier studies (24). In Group IV, rats receivedquercetin (200 mg/kg body weight) alone. After the treatment

period, rats were sacrificed by cervical decapitation. Prostaticfluid was removed, both ventral and dorsolateral prostate lobeswere dissected from the adhering connective tissue and washedseveral times with physiological saline, weighed accurately, andseparated. The ventral prostate and dorsolateral lobes were thenfixed with Bouin’s fluid for histopathological examination.

Biochemical Assays

Preparation of Tissue HomogenateVentral prostate and dorsolateral lobes (100 mg) were ho-

mogenized in 0.1 M/L of Tris–HCl buffer, pH 7.4, and cen-trifuged at 3000 × g for 15 min at 4◦C. The remaining super-natant was used for the biochemical analysis and the proteinconcentration was estimated by Lowry’s method (25).

Lipid Peroxidation and Hydrogen Peroxide (H2O2) LevelsLipid peroxidion (LPO) was measured according to the

method described by Devasagayam and Tarachand (26). Theconcentration of thiobarbituric acid reactive substance (TBARS)of the samples was expressed in nanomoles of TBARS formedper milligram protein. The H2O2 generation was assessed by thespectrophotometric method of Pick and Keisari (27). The H2O2

level was expressed in micromoles per minute per milligramprotein.

Nonenzymatic Antioxidant Level: Reduced GlutathioneGlutathione (GSH) level was measured according to the

method described by Moron et al. (28), and it was expressedin microgram per milligram protein.

HistologyThe fixed ventral prostate and dorsolateral lobes of 10 an-

imals in each group were further dissected sequentially (29).All samples were embedded in paraffin, and paraffin-embeddedtissue blocks were sectioned at 3-μm thickness using rotarymicrotome and fixed on microscopic slides. The sections werecovered with glass coverslip. After staining and destaining withhematoxylin and eosin, they were permanently mounted withdibutyl phthalate xylene (DPX) mount and viewed under aNikon eclipse 80i fluorescence microscope (10×, 20×, and 40×magnifications).

Tumor Incidence (%)The percentage of tumor incidence in each group [control,

cancer induced, cancer induced + quercetin) was obtained byobserving the histological evidence of each animal in all fourgroups and calculated by (number of animals with hyperplasia(or) dysplastic changes/total number of animal per group) ×100 and expressed as a percentage. The observation was doneonly once.

Statistical AnalysisThe data were subjected to statistical analysis using one way

analysis of variance followed by Student’s–Newman–Keul’s

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40 G. SHARMILA ET AL.

(SNK) tests to assess the significance of variation betweenthe means of control and treatment groups respectively, usingcomputer-based software (SPSS Inc., Chicago, IL). The valueswere considered significant if the P values were less than 0.05.

RESULTS

Body WeightBody weight was significantly decreased in MNU + T-treated

rats compared with controls, whereas simultaneous treatmentwith quercetin increased the boby weight.quercetin alone treatedrats did not show any change in body weight (Table 1).

Organ weight of Ventral and Dorsolateral ProstateThe absolute ventral prostate and dorsolateral prostate

weights were significantly increased in MNU + T-treated ratscompared to controls, whereas simultaneous quercetin treatmentshowed decreased ventral and dorsolateral prostate weight com-pared to MNU + T-induced rats. However quercetin alone treat-edgroup did not show any significant change in organ weight(Table 2).

Tumor Incidence (%)The percentage of tumor incidence in both ventral and dor-

solateral prostate was summarized in Fig. 1. MNU + T-treatedrats showed hyperplasia and dysplasia of approximately 70% (7)and 60% (6), respectively, in the ventral lobe were about 60%(6) and 50% (5) in the dorsolateral lobe of the prostate. PINor prostatic intraepithelial neoplasia is a preneoplastic lesion inwhich the epithelial cell number is increased and protruding intothe lumen. The tubules was composed of more than 1 layer ofepithelial cells with varying sizes. The tufting and micopapillaryprojections were present in the MNU + T-treated rats. PIN wasfound in 4 (40%) of 10 rats in the ventral prostate lobe and 20%(2) adenocarcinoma was observed in dorsolateral lobes of rats.

Approximately 10% of rats developed PIN in both ventral anddorsolateral prostate of quercetin supplemented rats. Hyperpla-sia (30%) and dysplasia (20%) were seen in the dorsolateralprostate of quercetin supplemented rats, respectively. No tumorincidence was observed in rats treated with quercetin alone andcontrol rats.

TABLE 2Effect of Quercetin (Q) on organ weight of ventral and

dorsolateral prostate of Sprague Dawley rats treated withN-methyl-N-nitrosourea (MNU) +testosterone (T), Q, and

vehicle

Absolute organ weight (mg)

Group VP DLP

Control 381.8 ± 4.26 267.1 ± 7.97Cancer induced

(MNU & T)900.4 ± 9.50a 410.0 ± 12.17a

Cancer induced +quercetin(200 mg/kg b.wt)

685.4 ± 13.16a,b 344.6 ± 14.26a,b

Quercetin (200 mg/kgb.wt)

404.6 ± 4.65b,c 303 ± 7, 26a,b,c

Each value represents mean ± SEM of 10 animals. The statisti-cal significance was considered at the level of P < 0.05 followingStudent’s–Newman–Keul’s test. VP = ventral prostate; DLP = dorso-lateral prostate; b.wt = body weight.

aControl vs. others. bCancer induced vs. cancer induced + Q-treatedrats. cCI + Q vs. quercetin alone.

Effect of Quercetin on Lipid peroxidation and H2O2

LevelsThe levels of lipid peroxidation and H2O2 were significantly

increased in MNU +T-treated rats compared with that of controlrats. However, simultaneous quercetin treatment prevented thelipid peroxidation and maintained H2O2 level (Table 3).

Effect of Quercetin on GSH LevelMNU + T-treated group showed a significant decrease in

the GSH level compared with that of the control group. How-ever, quercetin treatment maintains the level of GSH similar tothat in control. Quercetin alone treated group showed a signifi-cant increase in GSH level when compared with that of control(Table 3).

TABLE 1Body weight of Sprague Dawley rats treated with N-methyl-N-nitrosourea (MNU) + testosterone (T), quercetin (Q), and vehicle

Body weight (g)

Group 1st wk 5th wk 10th wk 15th wk 20th wk

Control 208 ± 2 222 ± 5 244 ± 4 262 ± 6 286 ± 7Cancer induced (MNU & T) 230 ± 3a 239 ± 4a 218 ± 6a 212 ± 4a 188 ± 4a

Cancer induced + quercetin (200 mg/kg b.wt) 214 ± 2a,b 229 ± 7a 229 ± 3a,b 242 ± 7a,b 255 ± 5a,b

Quercetin (200 mg/kg b.wt) 220 ± 3a,b,c 243 ± 5b,c 271 ± 4a,b,c 288 ± 3a,b,c 318 ± 4a,b,c

Each value represents mean ± SEM of 10 animals. The statistical significance was considered at the level of P < 0.05 followingStudent’s–Newman–Keul’s test. b.wt = body weight.

aControl vs. others. bCancer induced vs. cancer induced + Q-treated rats. cCI + Q vs. quercetin alone.

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CHEMOPREVENTION OF PROSTATE CANCER BY QUERCETIN 41

FIG. 1. Effect of quercetin on the% tumor incidence in ventral and dorsolateral prostate of Sprague Dawley rats.

Histopathology

Ventral Prostate

Tissue section of Group I: Control rats displayed a normalventral prostate architecture. The connective tissue between theacini and the epithelial tubules were thin, condensed around theacne and tubules of the gland. The epithelial cells were linedas single layer with columnar shape infolding into the lumen(Fig. 3a). Tissue section of Group II: MNU + T-induced an-imal showed the hyperplastic sites within the same glandularepithelium. Architectural pattern of high-grade PIN with tuft-

ing, micropapillary, and compression of prostatic epithelium(Fig. 3b1) was seen. The cell number in PIN was markedlyincreased, with normal cell lining below (arrow headed) wasobserved (Fig. 3b2 and 3b3). Tissue section of Group III:MNU+T+quercetin-simultaneous quercetin treatment illus-trated the absence of dysplastic and hyperplastic nodules, withorganized epithelia cell as normal (Fig. 2C). Tissue section ofGroup IV: Quercetin alone treated group showed the normalappearance of epithelial tubules lining the lumen secretionsand the tissue were tightly packed as seen in the control group(Fig. 4D).

TABLE 3Effect of quercetin (Q) on lipid peroxidation and H202 levels in ventral and dorsolateral prostate of Sprague Dawley rats treated

with N-methyl-N-nitrosourea (MNU) + testosterone (T), Q, and vehicle

Lipid peroxidation (nmol ofTBARS formed/mg protein) H2O2 (μmol of H2O2/mg protein) GSH (nmol of GSH/mg protein)

Group VP DLP VP DLP VP DLP

Control 32.76 ± .95 36.54 ± 1.62 6.24 ± 0.48 5.17 ± 0.33 3.98 ± 0.166 6.57 ± 0.51Cancer induced

(MNU & T)72.57 ± 1.4a 76.72 ± 2.66a 11.47 ± 0.43a 10.25 ± 0.21a 1.45 ± 0.07a 2.38 ± 0.09a

Cancer induced +Quercetin(200 mg/kg b.wt)

43.84 ± 1.76a,b 42.73 ± 2.32b 7.24 ± 0.25b 6.20 ± 0.30a,b 4.87 ± 0.29b 4.38 ± 0.14a,b

Quercetin(200 mg/kg b.wt)

30.44 ± 3.19b,c 25.48 ± 1.06b,c 5.12 ± 0.2b,c 4.37 ± 0.18b,c 4.32 ± 0.39b 8.27 ± 0.74a,b,c

Each value represents mean ± SEM of 10 animals. The statistical significance was considered at the level of P < 0.05 followingStudent’s–Newman–Keul’s test. TBARS = thiobarbituric acid reactive substance; GSH = glutathione; VP = ventral prostate; DLP = dor-solateral prostate; b.wt = body weight.

aControl vs. others. bCancer induced vs. cancer induced + Q-treated rats. cCI + Q vs. quercetin alone.

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42 G. SHARMILA ET AL.

FIG. 2. Histology of ventral prostate 10× magnification-treated with N-methyl-N-nitrosourea (MNU) + testosterone (T) + quercetin (200 mg/Kg body weight).A: Normal ventral prostate epithelium (VP). B1-B3: MNU + T treated rats. The epithelium of VP shows increased cell number when compared to control.Different architectural pattern of PIN was observed. C: MNU + T + quercetin treated groups shows the less common hyper plastic and dysplastic changes.D: Quercetin control group showing the normal appearance of epithelial cells as seen in control group. E = epithelium; L = lumen; S = stroma (color figureavailable online).

Dorsolateral ProstateThe dorsolateral prostate tissue section from rats in Group

I: Control animals displayed normal dorsolateral prostate ar-chitecture; the tubules were lined by a single layer of cuboidalcells lining with secretion in the lumen (Fig. 5A, 5E, and 5I).The dorsolateral prostate tissue section from rats in Group II:MNU + T-induced animal showed the hyperplastic and dysplas-tic changes along with proliferating adenocarcinoma within thesame glandular epithelium (Fig. 5C, 5G, and 5K). The differ-ent pattern of PIN like tufting, micropapillary, cribriform wasnot observed in those rats. The dorsolateral prostate tissue sec-tion from rats in Group III: MNU + T + quercetin showedremarkable decrease in the epithelial cell layer proliferation, in-dicating significant growth inhibition compared with (MNU/Talone) cancer induced (Fig. 5D, 5H, and 5L). The dorsolateralprostate tissue section from rats in Group IV: Quercetin alonetreated group showed the appearance of epithelial tubules liningthe lumen secretions as seen in the control group (Fig. 5B, 5F,and 5J).

DISCUSSIONCancer chemoprevention by antioxidant is a key strategy for

inhibiting, delaying, or even reversing the process of carcino-genesis (30). Quercetin (3,3′, 4′, 5,7-pentahydroxyflavone) is 1of several naturally occurring dietary polyphenolic flavonoidsthat is present in fruits and vegetables like apples, cranberries,blueberries, and onions, respectively.

A significant reduction in the body weight and increasedventral and dorsolateral prostatic weight were observed in car-cinogen and hormone-induced prostate carcinogenesis. The in-crease in prostate weight may be due to MNU that after a singledose along with continuous stimulation of testosterone inducesfibromuscular tissue and multiplication of squamous epitheliumof the prostate (31). The exact mechanism of decrease in bodyweight is unclear but it may be due to the formation of hydrox-ylated bases of DNA, an important event in chemical carcino-genesis (32). Whereas, simultaneous quercetin treatment of ratscaused a decreased tumor incidence with an associated reduc-tion in ventral and dorsal prostatic weight and increased body

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CHEMOPREVENTION OF PROSTATE CANCER BY QUERCETIN 43

FIG. 3. Histology of ventral prostate 20× magnification-treated with N-methyl-N-nitrosourea (MNU) + testosterone (T) + quercetin (200 mg/Kg body weight).a: Normal ventral prostate epithelium (VP). The epithelum was tall columinar with regular in size in tubules of the Gland. The connective tissue between the aciniand epithelium were thin and condensed. b1-b3: MNU + T treated rats. Hyperplastic and dysplastic changes are seen within the glandular epithelium.The PIN withdifferent pattern like tufting (b1), micropapillary (b2), and epithelial cell showed increased in number (b3) when compared to control groups. c: MNU+T+quercetintreated groups -shows the absence of hyper plastic and dysplastic changes. d: Quercetin control group showing the normal appearance of epithelial cells as seen incontrol group. E = epithelium; L = lumen; S = stroma (color figure available online).

weight, respectively suggests the potency of anticancer activityof quercetin in the initiation process.

Imbalance ROS production and antioxidant defense in liv-ing organism results in oxidative stress. Severe oxidative stresscauses mutation of tumor suppressor genes through DNA dam-age that results in the initiation of carcinogenesis (33), but thisalso promotes the multistep carcinogenesis (34). The free rad-icals may spontaneously react with nucleophilic centers of thecell and thereby covalently bound to DNA, RNA and proteinwhich may lead to cytotoxicity and carcinogenicity (35). Lipids,especially polyunsaturated fatty acids, are very susceptible tofree radical attack, which can initiate lipid peroxidation (36)that acts to control of cell division (37). The end product of lipidperoxidation, malondialdehyde is highly cytotoxic and inhibitedto protective enzymes and suggested to act as a tumor promoterand a cocarcinogenic agent (38).

A corresponding increase in lipid peroxidation, reactive oxy-gen level, and H2O2 were observed after carcinogen and hor-mone treatment in both ventral and dorsolateral prostates while

quercetin administration prevents prostate cancer initiation andprogression. Quercetin is considered to be a strong antioxi-dant that scavenges free radicals and bind transition metal ions.These properties help to inhibit lipid peroxidation (39). Thus,this report supports that upon quercetin treatment, the level oflipid peroxides and H2O2 got decreased due to its scavengingproperties of free radicals and thereby inhibits prostate cancerinitiation.

The increase in lipid peroxidation, H2O2 correlate with lowerlevel of antioxidants upon carcinogen and hormone administra-tion. The reduced GSH level was found to be significantly re-duced in MNU+T treated groups whereas, the quercetin-treatedgroup retrieved the GSH level and prevents prostate carcino-genesis initiation and hence decreased malondialdehyde in di-ethylnitrosamine induced hepatocarcinogen was found (40). Thesame results were found in DMBA induced breast cancer in ratsin which quercetin was supplemented in the diet (41).

The chemopreventive effect of quercetin was confirmedby histological examination in both ventral and dorsolateral

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44 G. SHARMILA ET AL.

FIG. 4. Histology of ventral prostate 40× magnification-treated with N-methyl-N-nitrosourea (MNU) + testosterone (T) + quercetin (200 mg/Kg body weight).E: Normal ventral prostate epithelium (VP) cells of glandular epithelium was tall columinar in shape and regular in size.the secretion of gland within the lumenwas seen. F1-F3: MNU + T treated rats. epithelium. The PIN with different pattern like tufting (F1), micropapillary (F2), and epithelial showed increased in cellnumber (F3) was observed. The epithelial cells are crowded and stratification with irregular spacing with hyperchromatin was seen. G: MNU + T + quercetintreated groups shows the absence of hyper plastic and dysplastic changes upon quercetin intake. H: Quercetin control group showing the normal appearance ofepithelial cells as seen in control group. E = epithelium; L = lumen; S = stroma (color figure available online).

prostate tissue. The pathological changes closely share the histo-logical features found in human prostatic dysplasia, also termedPIN. PIN lesion is widely accepted as a premalignant conditionof prostate cancer with four architectural patterns such as tuft,micropapillary, flat, and cribriform. The tufting pattern is mostcommonly associated with 97% of positive cases although theyhave multiple pattern associated (42). The MNU + T-treatedanimals, showed lesions such as micropapillary and tufting pat-terns in ventral prostate that shows the presence of hyperplasia,dysplasia, and PIN architectural patterns as seen in human HG-PIN (42). In this study, HGPIN exhibited disruption of the basallayer of cells due to changes in cell polarity. The number of cellswas higher in HGPIN, but the basement membrane remained in-tact. These data were supported by our earlier studies from ourlaboratory (7) that on chemoprevention of zinc in MNU + T-induced model, which states that the increase in epithelial cellproliferation may be due to MNU + T administration. The study

also showed a different pattern of PIN that was not observed inthe dorsolateral prostate of MNU + T treated rats. Hyperplasia(60%) and dysplasia (40%) were observed whereas in simul-taneous quercetin-treated group, the occurrence of hyperplasiaand dysplasia lesion was found to be significantly decreased.This could be due to the presence of quercetin that inhibitedlipid peroxidation and increased GSH level that exerted pro-tectitive effects against oxidative damage evidenced in hepaticcancer (24). Oral administration of quercetin causes its completemetabolization and metabolites still retain antioxidant proper-ties (43), thereby quercetin can donate electrons or hydrogenand scavenges H2O2 and superoxide anion (44).

To conclude, this study is first of its kind that provides suitablehistological evidence for chemoprevention of prostate carcino-genesis by a flavonoid quercetin in both ventral and dorsolateralprostate of male Sprague-Dawley rats. Its chemopreventive ef-fect is through scavenging free radicals (H2O2), inhibiting lipid

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CHEMOPREVENTION OF PROSTATE CANCER BY QUERCETIN 45

FIG. 5. Histology of dorsolateral prostate captured at 10× (A–D), 20× (E–H), and 40× (I–L) magnification under microscope-treated with N-methyl-N-nitrosourea (MNU) + testosterone (T) + quercetin (200 mg/Kg body weight). A,E,I: Normal dorsolateral prostate epithelium (DLP): The cells are tall columinaraligned in single layer with secretion in the lumen and surrounded by stroma. B,F,J: Quercetin control group showing the normal appearance of epithelial cells asseen in control group with single layer of epithelial cell lining the lumen secretion. C,G,K: MNU + T treated rats: The epithelial cell of DLP shows hyperplasticand dysplastic changes with increased number when compared to control groups. Two (20%) of 10 animals shown adenocarcinoma (ad) in which the glands isfully occupied by epithelial cell. It shows a glandur growth pattern within adundant amount of stromal tissue. D,H,L: MNU + T + quercetin treated groups showthe absence of hyper plastic and dysplastic changes. E = epithelium; L = lumen; S = stroma (color figure available online).

peroxidation and increasing GSH level. The molecular mecha-nism underlying the chemoprevention of quercetin and its levelin serum are the future prospects of the study. Quercetin may beused to target the signaling molecules involved in cell survival,proliferation, and apoptosis.

FUNDINGThis work was supported by the Council of Scientific and

Industrial Research (CSIR), India, in the form of CSIR-SRF(Grant no: 9/115 (0737)/2011-EMR-I date 28.03.2011) to Ms.G. Sharmila and DBT, Govt., of India to Dr. J. Arunakaran.

REFERENCES1. Jemal A, Siegel R, Xu J, and Ward E: Cancer statistics. CA Cancer J Clin

60, 277–300, 2010.2. Syed DN, Khan N, Afaq F, and Mukhtar H: Chemoprevention of prostate

cancer through dietary agents: progress and promise. Cancer EpidemiolBiomarkers Prev 16, 2193–2203, 2007.

3. Pollard M and Luckert PH: Transplantable metastasizing prostate adeno-carcinoma in rats. J Natl Cancer Inst 54, 643–649, 1975.

4. Chiarodo A: National Cancer Institute roundtable on prostate cancer: futureresearch directions. Cancer Res 5, 2498–2505, 1991.

5. McCormick DL and Rao KV: Chemoprevention of hormone-dependentprostate cancer in the Wistar-Unilever rat. Euro Urol 35, 464–467, 1999.

6. Arunkumar A, Vijayababu MR, Venkataraman P, Senthilkumar K, andArunakaran J: Chemoprevention of rat prostate carcinogenesis by dial-lyl disulfide, an organosulfur compound of garlic. Biol Pharm Bull 29,375–379, 2006.

7. Banudevi S, Elumalai P, Arunkumar R, Senthilkumar K, Sharmila G, et al.:Chemopreventive effects of zinc on MNU and testosterone induced prostatecancer in male SpragueDawley rats. J Clin Cancer Oncol Res 137, 677–686,2010.

8. Arunakaran J, Banudevi S, and Arunkumar A: Chemopreventive Targetfor Prostate Cancer: Prostatic Intraepithelial Neoplasia, IntraepithelialNeoplasia, Srivastava S. (ed.). InTech, 2012. http://www.intechopen.com/books/intraepithelial-neoplasia/chemopreventive-target-for-prostate-cancer-prostatic-intraepithelial-neoplasia (Accessed November 18, 2013).

9. Kelloff GJ, Higley HR, Brawer MK, Lucia MS, Sigman CC, et al.: Chemo-prevention strategies in the prostate: an overview. Rev Urol 4, 69–77, 2002.

10. Ramos S: Effects of dietary flavonoids on apoptotic pathways related tocancer chemoprevention. J Nut. Biochem 18, 427–442, 2007.

11. Bravo L: Polyphenols: chemistry, dietary sources, metabolism and nutri-tional significance. Nutrition Reviews 56, 317–333, 1998.

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46 G. SHARMILA ET AL.

12. Rice-Evans C: Flavonoid antioxidants. Current Medicinal Chemistry 8,797–807, 2001.

13. Aviram M and Fuhrman B: Wine flavonoids protect against LDL oxidationand atherosclerosis. Ann NY Acad Sci 957, 146–161, 2002.

14. Shaik YB, Castellani ML, Perrella A, Conti F, Salini V, et al.: Role ofquercetin (a natural herbal compound) in allergy and inflammation. J BiolRegul Homeost Agents 20, 47–52, 2006.

15. Choi JA, Kim JY, Lee JY, Kang CM, Kwon HJ, et al.: Induction of cellcycle arrest and apoptosis in human breast cancer cells by quercetin. Int JOncol 19, 837–844, 2001.

16. Kuo PC, Liu HF, and Chao JI: Survivin and p53 modulate quercetin-inducedcell growth inhibition and apoptosis in human lung carcinoma cells. J BiolChem 279, 55875–55885, 2004.

17. Yang CS, Tran E, Nguyen TT, Ong CK, Lee SK, et al.: Quercetin-inducedgrowth inhibition and cell death in nasopharyngeal carcinoma cells areassociated with increase in Bad and hypophosphorylated retinoblastomaexpressions. Oncol Rep 11, 727–733, 2004.

18. Vijayababu MR, Kanagaraj P, Arunkumar A, Ilangovan R, DharmarajanA, et al.: Quercetin induces p53-independent apoptosis in human prostatecancer cells by modulating Bcl-2-related proteins: a possible mediation byIGFBP-3. Oncol Res 16, 67–74, 2006.

19. Choi EJ, Bae SM, and Ahn WS: Antiproliferative effects of Quercetinthrough cell cycle arrest and apoptosis in human breast cancerMDA-MB-453 cells. Arch Pharm Res 31, 1281–1285, 2009.

20. Jung YH, Heo J, Lee JY, Kwon KT, and Kim HK: Quercetin enhancesTRAIL-induced apoptosis in prostate cancer cells via increased proteinstability of death receptor 5. Life Sci 86, 351–357, 2010.

21. Senthilkumar K, Elumalai P, Arunkumar R, Banudevi S, Gunadharini ND,et al.: Quercetin regulates insulin like growth factor signaling and inducesintrinsic and extrinsic pathway mediated apoptosis in androgen independentprostate cancer cells (PC-3). Mol Cell Biochem 344, 173–184, 2010.

22. Senthilkumar K, Arunkumar R, Elumalai P, Sharmila G, Gunadharini ND,et al.: Quercetin inhibits invasion, migration and signalling molecules in-volved in cell survival and proliferation of prostate cancer cell line (PC-3).Cell Biochem Funct 29, 87–95, 2011.

23. Liao Z, Boileau TWM, Erdman JW, and Clinton SK: Interrelationshipsamong angiogenesis, proliferation, and apoptosis in the tumor microen-vironment during N-methyl-N-nitrosourea androgen-induced prostate car-cinogenesis in rats. Carcinogenesis 23, 1701–1712, 2002.

24. Seufi AM, Ibrahim SS, Elmaghraby TK, and Hafez EE: Preventive effectof the flavonoid, quercetin, on hepatic cancer in rats via oxidant/antioxidantactivity: molecular and histological evidences. J Exp Clin Cancer Res 28,80, 2009.

25. Lowry OH, Rosenbrough NJ, Farr AL, and Randall RJ: Protein measure-ment with the Folin phenol reagent. J Biol Chem 193:256–75, 1951.

26. Devasagayam TP and Tarachand U: Decreased lipid peroxidation in therat kidneys during gestation. Biochem Biophys Res Commun 145,134–138,1987.

27. Pick E and Keisari Y: Superoxide anion and H2O2 production by chemi-cally elicited peritoneal macrophages-induced by multiple nonphagocyticstimuli. Cell Immunol 59, 301–318, 1981.

28. Moron MS, Depierre JW, and Mannervik B: Levels of glutathione, glu-tathione reductase and glutathione-S-transferase activities in rat lung andliver. Biochim Biophys Acta 582, 67–78, 1979.

29. Jesik CJ, Holland JM, and Lee C: An anatomic and histologic study of therat prostate. Prostate 3, 81–97, 1982.

30. Shureiqi I, Reddy P, and Brenner DE: Chemoprevention: general perspec-tive. Crit Rev Oncol/Hematol 33, 157–167, 2000.

31. Senthilkumar K, Arunkumar A, Sridevi N, Vijayababu MR, Kanagaraj P,et al.: Chemoprevention of MNU and testosterone induced prostate car-cinogenesis by calcitriol (vitamin D3) in adult male albino Wistar rats. AnnCancer Res Therap 14, 12–18, 2006.

32. Bartsch H and Nair J: Potential role of lipid peroxidation derived DNAdamage in human colon carcinogenesis: studies on exocyclic base adductas stable oxidative stress markers. Cancer Detect Prev 26, 308–312,2002.

33. Kang DH: Oxidative stress, DNA damage and breast cancer. AACN Clin13, 540–549, 2002.

34. Ahmed MI, Fayed ST, Hossein H, and Tash FM: Lipid peroxidation andantioxidant status in human cervical carcinoma. Dis Markers 15, 283–291,1999.

35. Tampa Y and Tsukamoto M: The antioxidant action of 2-methyl- 6-(pmethoxyphenyl3, 7- dihydroimidazo [1,2-alpha] pyrazin-3-one (MCLA),a chemiluminescence probe to detect superoxide anions. FEBS Lett 430,348–352, 1998.

36. Halliwell B and Gutteridge MC: Free radicals in biology and medicine (3rded.). Oxford, London, UK, 1999.

37. Diplock AT, Rice-Evans AC, and Burton RH: Is there a significantrole for lipid peroxidation in the of free causation of malignancy andfor antioxidants in cancer prevention? Cancer Res 54, 19525–19526,1994.

38. Otamiri T and Sjodahl R: Increased lipidperoxidation in malig-nant tissues of patients with colorectal cancer. Cancer 61, 122–125,1989.

39. Sakanashi Y, Oyama K, Matsui H, Oyama TB, Oyama TM, et al.: Possibleuse of quercetin, an antioxidant, for the protection of cells suffering froman overload of intracellular Ca2+: a model experiment. Life Sciences 83,164–169, 2008.

40. Gupta C, Vikram A, Tripathi DN, Ramarao P, and Jena GB: Antioxidantand antimutagenic effect of quercetin against DEN induced hepatotoxicityin rat. Phytother Res 24, 119–128, 2009.

41. Reddy NS, Nirmala P, Chidambaram N, and Kumar PA: Quercetin indimethyl benzanthracene induced breast cancer in rats. Am J PharmacolToxicol 7, 68–72, 2012.

42. Bostwick DG: High-grade prostatic intraepithelial neoplasia: the most likelyprecursor of prostate cancer. Cancer 75, 1823–1836, 1995.

43. Manach C, Morand C, Crespy V, Demigne C, Texier O, et al.: Quercetin isrecovered in human plasma as conjugated derivatives which retainantioxi-dant properties. FEBS Lett 426, 331–336, 1998.

44. Heijnen CG, Haenen GR, Vekemans JA and Bast A: Peroxynitrite scaveng-ing of flavonoids: structure activity relationship. Environ Toxicol Pharmacol10, 199–206, 2001.

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