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Chemopreventive Effect of Quercetin in MNU and Testosterone Induced Prostate Cancer of SpragueDawley Rats a
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Govindaraj Sharmila , Thavadurainathan Athirai , Balakrishnan Kiruthiga , Kalimuthu a
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Senthilkumar , Perumal Elumalai , Ramachandran Arunkumar & Jagadeesan Arunakaran a a
Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences , University of Madras , Chennai , India Published 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 Quercetin in MNU and Testosterone Induced Prostate Cancer of Sprague-Dawley Rats, Nutrition and Cancer, 66:1, 38-46, DOI: 10.1080/01635581.2014.847967 To link to this article: http://dx.doi.org/10.1080/01635581.2014.847967
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Nutrition and Cancer, 66(1), 38–46 C 2014, Taylor & Francis Group, LLC Copyright ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2014.847967
Chemopreventive Effect of Quercetin in MNU and Testosterone Induced Prostate Cancer of Sprague-Dawley Rats Govindaraj Sharmila, Thavadurainathan Athirai, Balakrishnan Kiruthiga, Kalimuthu Senthilkumar, Perumal Elumalai, Ramachandran Arunkumar, and Jagadeesan Arunakaran
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Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, India
(PIN) to several stages of metastasis and hormone refractory disease (2). The lack of appropriate animal model systems leaves a major obstacle in the development of strategies for prostate cancer chemoprevention (4) though the progress of prostatic carcinoma in to invasive phase can be studied in a few transplantable systems (3). However, the combination of carcinogen, N-methyl-Nnitrosourea (MNU), and testosterone (T) has the advantage of inducing higher incidence of prostate carcinogenesis in WistarUnilever rat that mimics the human prostate cancer model (5). Earlier reports from our laboratory had shown that the combined hormone and carcinogen (MNU) treatment consistently generated prostatic dysplasia, a putative preneoplastic lesion that is exclusively within prostates (6–8). The evaluation of anticancer effects of various plant-derived agents has gained good attention in recent years due to the development of resistance against the available chemotherapeutic agents (9). Among chemopreventers, flavonoids are the most studied antioxidant compounds that are largely present in fruits, vegetables, tea, red wine, and aromatic plants, (10–12). Quercetin is a principal flavanoid compound of onion (3, 3 , 4 , 5, 7-Penta hydroxyl flavanone), which possesses a wide spectrum of pharmacological properties (13, 14). Various animal and in vitro studies showed that quercetin was found to have antiproliferative effects by inhibiting cell growth and induces apoptosis in numerious cancer cell like breast, leukemia, colon, ovary, endometrial, gastric, and lung (14–19). Quercetin also enhances TRAIL-induced apoptosis in prostate cancer cells through increased protein stability of death receptor 5 (20). Recent findings from our laboratory have confirmed that quercetin downregulates insulin like growth factor signalling and upregulates intrinsic as well as extrinsic pathway-mediated apoptosis in androgen-independent prostate cancer cells (PC-3) (21). Quercetin also inhibits invasion, migration, and signalling molecules involved in cell survival and proliferation of prostate cancer cell line (PC-3) (22).
Prostate cancer becomes an ideal target for chemoprevention because of its high incidence and extended natural history. The consumption of quercetin (plant flavonoid) in diet is associated with decreased risk of disease and many cancers but then this was not elucidated in prostate malignancy. Hence, a study in which the male Sprague-Dawley rats were induced prostate cancer by hormone (testosterone) and carcinogen (MNU) and simultaneously supplemented with quercetin (200 mg/Kg body weight) thrice a week, was conducted. After the treatment period, rats were killed; ventral and dorsolateral lobes of the prostate were dissected. Histology and oxidative stress markers LPO, H2 O2 , and antioxidant GSH level were measured in both lobes. The lipid peroxidation, H2 O2 , in (MNU+T) treated rats were increased and GSH level was decreased, whereas simultaneous quercetin-treated rats reverted back to normal level in both ventral and dorsolateral regions. The different patterns of PIN were observed with associated hyperplasia and dysplasia; changes in these regions and the occurrence of this lesion were reduced in simultaneous quercetin-treated rats. The study concluded that dietary quercetin prevented MNU + Tinduced prostate carcinogenesis on both ventral and dorsolateral lobes of Sprague-Dawley rats.
INTRODUCTION Prostate cancer accounts for the second highest cancerrelated deaths in men. The etiology of prostate cancer is unknown but it is usually associated with age, race, family history, hormonal, dietary factors, etc. (1). Prostate cancer progresses from premalignant lesion or prostatic intraepithelial neoplasia
Submitted 14 December 2012; accepted in final form 12 August 2013. Address correspondence to Jagadeesan Arunakaran, Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600113, India. Tel.: 91-44-24547043. Fax: 91-44-24540709. E-mail: j [email protected]
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However, there is no report available on the role of quercetin on prostate malignant in vivo model. Therefore, this study was conducted to characterize the chemopreventive activity of quercetin in the prostate carcinogenesis using an animal model (Sprague-Dawley rats).
period, rats were sacrificed by cervical decapitation. Prostatic fluid was removed, both ventral and dorsolateral prostate lobes were dissected from the adhering connective tissue and washed several times with physiological saline, weighed accurately, and separated. The ventral prostate and dorsolateral lobes were then fixed with Bouin’s fluid for histopathological examination.
MATERIALS AND METHODS
Biochemical Assays
Chemicals Quercetin and MNU were purchased from Sigma-Aldrich Chemicals Private Ltd. (St. Louis, MO). Testosterone undecanoate (T) was purchased from M/S Schering-Plough, Apeldoorn, The Netherlands. Other chemicals were obtained from SISCO Research Laboratories Pvt. Ltd, Mumbai, India. All the chemicals were extra pure and of analytical grade.
Preparation of Tissue Homogenate Ventral prostate and dorsolateral lobes (100 mg) were homogenized in 0.1 M/L of Tris–HCl buffer, pH 7.4, and centrifuged at 3000 × g for 15 min at 4◦ C. The remaining supernatant was used for the biochemical analysis and the protein concentration was estimated by Lowry’s method (25).
Animals Healthy adult male Sprague-Dawley rats weighing approximately 200–250 g were used. The animals were housed in clean polypropylene cages and maintained in an air-conditioned animal house with constant 12-h light/dark cycle. Rats were permitted free access to drinking water throughout the experimental period. The animals were fed with standard rat pellet diet (Lipton India Ltd., Mumbai, India). Experiment was approved by the Institute Animal Ethical Committee (IAEC No. 01/01/12). Prostate Tumor Induction Rats were induced prostate cancer using carcinogen (MNU) and hormone (testosterone undecanoate) by a modified protocol (5, 23). First, each rat received daily intraperitoneal (IP) injections of testosterone undecanoate (50 mg/kg body weight) for 21 consecutive days. At Day 23, rats received daily IP injections of 100 mg testosterone undecanoate /kg body weight in 0.3 ml propylene glycol for 3 days. At Day 27, all the rats received a single intravenous (IV) dose (50 mg/kg body weight) of MNU (dissolved in saline at 10 mg/ml) through the tail vein. After 1 wk of MNU administration, rats received an IP injection of 4 mg testosterone undecanoate /kg body weight alternatively for 16 wk. Experimental Design A total of 40 rats were divided into 4 groups and each group consisted of 10 rats. In Group I, rats that received vehicle (propylene glycol) alone by IP injection, considered as controls. In Group II, rats were induced prostate cancer by using carcinogen plus hormone. In Group III, rats were induced prostate cancer with simultaneous supplementation of quercetin (200 mg/kg body weight) thrice a week for 16 wk through oral gavage. Quercetin supplementation was begun a week before the administration of the initial dose of testosterone undecanoate administration and throughout the studies. The dose was selected based on the earlier studies (24). In Group IV, rats received quercetin (200 mg/kg body weight) alone. After the treatment
Lipid Peroxidation and Hydrogen Peroxide (H2 O2 ) Levels Lipid peroxidion (LPO) was measured according to the method described by Devasagayam and Tarachand (26). The concentration of thiobarbituric acid reactive substance (TBARS) of the samples was expressed in nanomoles of TBARS formed per milligram protein. The H2 O2 generation was assessed by the spectrophotometric method of Pick and Keisari (27). The H2 O2 level was expressed in micromoles per minute per milligram protein. Nonenzymatic Antioxidant Level: Reduced Glutathione Glutathione (GSH) level was measured according to the method described by Moron et al. (28), and it was expressed in microgram per milligram protein. Histology The fixed ventral prostate and dorsolateral lobes of 10 animals in each group were further dissected sequentially (29). All samples were embedded in paraffin, and paraffin-embedded tissue blocks were sectioned at 3-μm thickness using rotary microtome and fixed on microscopic slides. The sections were covered with glass coverslip. After staining and destaining with hematoxylin and eosin, they were permanently mounted with dibutyl phthalate xylene (DPX) mount and viewed under a Nikon 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 by observing the histological evidence of each animal in all four groups 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 done only once. Statistical Analysis The data were subjected to statistical analysis using one way analysis of variance followed by Student’s–Newman–Keul’s
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(SNK) tests to assess the significance of variation between the means of control and treatment groups respectively, using computer-based software (SPSS Inc., Chicago, IL). The values were considered significant if the P values were less than 0.05.
TABLE 2 Effect of Quercetin (Q) on organ weight of ventral and dorsolateral prostate of Sprague Dawley rats treated with N-methyl-N-nitrosourea (MNU) +testosterone (T), Q, and vehicle
RESULTS
Absolute organ weight (mg)
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Body Weight Body weight was significantly decreased in MNU + T-treated rats compared with controls, whereas simultaneous treatment with quercetin increased the boby weight.quercetin alone treated rats did not show any change in body weight (Table 1). Organ weight of Ventral and Dorsolateral Prostate The absolute ventral prostate and dorsolateral prostate weights were significantly increased in MNU + T-treated rats compared to controls, whereas simultaneous quercetin treatment showed decreased ventral and dorsolateral prostate weight compared to MNU + T-induced rats. However quercetin alone treatedgroup did not show any significant change in organ weight (Table 2). Tumor Incidence (%) The percentage of tumor incidence in both ventral and dorsolateral prostate was summarized in Fig. 1. MNU + T-treated rats 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. PIN or prostatic intraepithelial neoplasia is a preneoplastic lesion in which the epithelial cell number is increased and protruding into the lumen. The tubules was composed of more than 1 layer of epithelial cells with varying sizes. The tufting and micopapillary projections were present in the MNU + T-treated rats. PIN was found 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 and dorsolateral prostate of quercetin supplemented rats. Hyperplasia (30%) and dysplasia (20%) were seen in the dorsolateral prostate of quercetin supplemented rats, respectively. No tumor incidence was observed in rats treated with quercetin alone and control rats.
Group
VP
Control Cancer induced (MNU & T) Cancer induced + quercetin (200 mg/kg b.wt) Quercetin (200 mg/kg b.wt)
DLP
381.8 ± 4.26 900.4 ± 9.50a
267.1 ± 7.97 410.0 ± 12.17a
685.4 ± 13.16a,b
344.6 ± 14.26a,b
404.6 ± 4.65b,c
303 ± 7, 26a,b,c
Each value represents mean ± SEM of 10 animals. The statistical significance was considered at the level of P < 0.05 following Student’s–Newman–Keul’s test. VP = ventral prostate; DLP = dorsolateral prostate; b.wt = body weight. a Control vs. others. bCancer induced vs. cancer induced + Q-treated rats. cCI + Q vs. quercetin alone.
Effect of Quercetin on Lipid peroxidation and H2 O2 Levels The levels of lipid peroxidation and H2 O2 were significantly increased in MNU +T-treated rats compared with that of control rats. However, simultaneous quercetin treatment prevented the lipid peroxidation and maintained H2 O2 level (Table 3). Effect of Quercetin on GSH Level MNU + T-treated group showed a significant decrease in the GSH level compared with that of the control group. However, quercetin treatment maintains the level of GSH similar to that in control. Quercetin alone treated group showed a significant increase in GSH level when compared with that of control (Table 3).
TABLE 1 Body weight of Sprague Dawley rats treated with N-methyl-N-nitrosourea (MNU) + testosterone (T), quercetin (Q), and vehicle Body weight (g) Group Control Cancer induced (MNU & T) Cancer induced + quercetin (200 mg/kg b.wt) Quercetin (200 mg/kg b.wt)
1st wk
5th wk
10th wk
15th wk
20th wk
208 ± 2 230 ± 3a 214 ± 2a,b 220 ± 3a,b,c
222 ± 5 239 ± 4a 229 ± 7a 243 ± 5b,c
244 ± 4 218 ± 6a 229 ± 3a,b 271 ± 4a,b,c
262 ± 6 212 ± 4a 242 ± 7a,b 288 ± 3a,b,c
286 ± 7 188 ± 4a 255 ± 5a,b 318 ± 4a,b,c
Each value represents mean ± SEM of 10 animals. The statistical significance was considered at the level of P < 0.05 following Student’s–Newman–Keul’s test. b.wt = body weight. a Control vs. others. bCancer induced vs. cancer induced + Q-treated rats. cCI + Q vs. quercetin alone.
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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 normal ventral prostate architecture. The connective tissue between the acini and the epithelial tubules were thin, condensed around the acne and tubules of the gland. The epithelial cells were lined as single layer with columnar shape infolding into the lumen (Fig. 3a). Tissue section of Group II: MNU + T-induced animal showed the hyperplastic sites within the same glandular epithelium. 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 markedly increased, with normal cell lining below (arrow headed) was observed (Fig. 3b2 and 3b3 ). Tissue section of Group III: MNU+T+quercetin-simultaneous quercetin treatment illustrated the absence of dysplastic and hyperplastic nodules, with organized epithelia cell as normal (Fig. 2C). Tissue section of Group IV: Quercetin alone treated group showed the normal appearance of epithelial tubules lining the lumen secretions and the tissue were tightly packed as seen in the control group (Fig. 4D).
TABLE 3 Effect of quercetin (Q) on lipid peroxidation and H2 02 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 of TBARS formed/mg protein) Group Control Cancer induced (MNU & T) Cancer induced + Quercetin (200 mg/kg b.wt) Quercetin (200 mg/kg b.wt)
VP
H2 O2 (μmol of H2 O2 /mg protein) GSH (nmol of GSH/mg protein)
DLP
VP
DLP
VP
DLP
32.76 ± .95 72.57 ± 1.4a
36.54 ± 1.62 76.72 ± 2.66a
6.24 ± 0.48 11.47 ± 0.43a
5.17 ± 0.33 10.25 ± 0.21a
3.98 ± 0.166 1.45 ± 0.07a
6.57 ± 0.51 2.38 ± 0.09a
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
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 following Student’s–Newman–Keul’s test. TBARS = thiobarbituric acid reactive substance; GSH = glutathione; VP = ventral prostate; DLP = dorsolateral prostate; b.wt = body weight. a Control vs. others. bCancer induced vs. cancer induced + Q-treated rats. cCI + Q vs. quercetin alone.
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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 figure available online).
Dorsolateral Prostate The dorsolateral prostate tissue section from rats in Group I: Control animals displayed normal dorsolateral prostate architecture; the tubules were lined by a single layer of cuboidal cells 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 dysplastic changes along with proliferating adenocarcinoma within the same glandular epithelium (Fig. 5C, 5G, and 5K). The different pattern of PIN like tufting, micropapillary, cribriform was not observed in those rats. The dorsolateral prostate tissue section from rats in Group III: MNU + T + quercetin showed remarkable decrease in the epithelial cell layer proliferation, indicating significant growth inhibition compared with (MNU/T alone) cancer induced (Fig. 5D, 5H, and 5L). The dorsolateral prostate tissue section from rats in Group IV: Quercetin alone treated group showed the appearance of epithelial tubules lining the lumen secretions as seen in the control group (Fig. 5B, 5F, and 5J).
DISCUSSION Cancer chemoprevention by antioxidant is a key strategy for inhibiting, delaying, or even reversing the process of carcinogenesis (30). Quercetin (3,3 , 4 , 5,7-pentahydroxyflavone) is 1 of several naturally occurring dietary polyphenolic flavonoids that is present in fruits and vegetables like apples, cranberries, blueberries, and onions, respectively. A significant reduction in the body weight and increased ventral and dorsolateral prostatic weight were observed in carcinogen and hormone-induced prostate carcinogenesis. The increase in prostate weight may be due to MNU that after a single dose along with continuous stimulation of testosterone induces fibromuscular tissue and multiplication of squamous epithelium of the prostate (31). The exact mechanism of decrease in body weight is unclear but it may be due to the formation of hydroxylated bases of DNA, an important event in chemical carcinogenesis (32). Whereas, simultaneous quercetin treatment of rats caused a decreased tumor incidence with an associated reduction in ventral and dorsal prostatic weight and increased body
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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 acini and epithelium were thin and condensed. b1 -b3: MNU + T treated rats. Hyperplastic and dysplastic changes are seen within the glandular epithelium.The PIN with different pattern like tufting (b1 ), micropapillary (b2 ), and epithelial cell showed increased in number (b3 ) when compared to control groups. c: MNU+T+quercetin treated groups -shows the absence of 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 figure available online).
weight, respectively suggests the potency of anticancer activity of quercetin in the initiation process. Imbalance ROS production and antioxidant defense in living organism results in oxidative stress. Severe oxidative stress causes mutation of tumor suppressor genes through DNA damage that results in the initiation of carcinogenesis (33), but this also promotes the multistep carcinogenesis (34). The free radicals may spontaneously react with nucleophilic centers of the cell and thereby covalently bound to DNA, RNA and protein which may lead to cytotoxicity and carcinogenicity (35). Lipids, especially polyunsaturated fatty acids, are very susceptible to free radical attack, which can initiate lipid peroxidation (36) that acts to control of cell division (37). The end product of lipid peroxidation, malondialdehyde is highly cytotoxic and inhibited to protective enzymes and suggested to act as a tumor promoter and a cocarcinogenic agent (38). A corresponding increase in lipid peroxidation, reactive oxygen level, and H2 O2 were observed after carcinogen and hormone treatment in both ventral and dorsolateral prostates while
quercetin administration prevents prostate cancer initiation and progression. Quercetin is considered to be a strong antioxidant 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 of lipid peroxides and H2 O2 got decreased due to its scavenging properties of free radicals and thereby inhibits prostate cancer initiation. The increase in lipid peroxidation, H2 O2 correlate with lower level of antioxidants upon carcinogen and hormone administration. The reduced GSH level was found to be significantly reduced in MNU+T treated groups whereas, the quercetin-treated group retrieved the GSH level and prevents prostate carcinogenesis initiation and hence decreased malondialdehyde in diethylnitrosamine induced hepatocarcinogen was found (40). The same results were found in DMBA induced breast cancer in rats in which quercetin was supplemented in the diet (41). The chemopreventive effect of quercetin was confirmed by histological examination in both ventral and dorsolateral
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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 lumen was seen. F1 -F3 : MNU + T treated rats. epithelium. The PIN with different pattern like tufting (F1 ), micropapillary (F2 ), and epithelial showed increased in cell number (F3 ) was observed. The epithelial cells are crowded and stratification with irregular spacing with hyperchromatin was seen. G: MNU + T + quercetin treated groups shows the absence of hyper plastic and dysplastic changes upon quercetin intake. H: Quercetin control group showing the normal appearance of epithelial cells as seen in control group. E = epithelium; L = lumen; S = stroma (color figure available online).
prostate tissue. The pathological changes closely share the histological features found in human prostatic dysplasia, also termed PIN. PIN lesion is widely accepted as a premalignant condition of prostate cancer with four architectural patterns such as tuft, micropapillary, flat, and cribriform. The tufting pattern is most commonly associated with 97% of positive cases although they have multiple pattern associated (42). The MNU + T-treated animals, showed lesions such as micropapillary and tufting patterns in ventral prostate that shows the presence of hyperplasia, dysplasia, and PIN architectural patterns as seen in human HGPIN (42). In this study, HGPIN exhibited disruption of the basal layer of cells due to changes in cell polarity. The number of cells was higher in HGPIN, but the basement membrane remained intact. These data were supported by our earlier studies from our laboratory (7) that on chemoprevention of zinc in MNU + Tinduced model, which states that the increase in epithelial cell proliferation may be due to MNU + T administration. The study
also showed a different pattern of PIN that was not observed in the dorsolateral prostate of MNU + T treated rats. Hyperplasia (60%) and dysplasia (40%) were observed whereas in simultaneous quercetin-treated group, the occurrence of hyperplasia and dysplasia lesion was found to be significantly decreased. This could be due to the presence of quercetin that inhibited lipid peroxidation and increased GSH level that exerted protectitive effects against oxidative damage evidenced in hepatic cancer (24). Oral administration of quercetin causes its complete metabolization and metabolites still retain antioxidant properties (43), thereby quercetin can donate electrons or hydrogen and scavenges H2 O2 and superoxide anion (44). To conclude, this study is first of its kind that provides suitable histological evidence for chemoprevention of prostate carcinogenesis by a flavonoid quercetin in both ventral and dorsolateral prostate of male Sprague-Dawley rats. Its chemopreventive effect is through scavenging free radicals (H2 O2 ), inhibiting lipid
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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-Nnitrosourea (MNU) + testosterone (T) + quercetin (200 mg/Kg body weight). A,E,I: Normal dorsolateral prostate epithelium (DLP): The cells are tall columinar aligned 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 as seen 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 hyperplastic and dysplastic changes with increased number when compared to control groups. Two (20%) of 10 animals shown adenocarcinoma (ad) in which the glands is fully occupied by epithelial cell. It shows a glandur growth pattern within adundant amount of stromal tissue. D,H,L: MNU + T + quercetin treated groups show the absence of hyper plastic and dysplastic changes. E = epithelium; L = lumen; S = stroma (color figure available online).
peroxidation and increasing GSH level. The molecular mechanism underlying the chemoprevention of quercetin and its level in serum are the future prospects of the study. Quercetin may be used to target the signaling molecules involved in cell survival, proliferation, and apoptosis. FUNDING This 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. REFERENCES 1. 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 Epidemiol Biomarkers Prev 16, 2193–2203, 2007. 3. Pollard M and Luckert PH: Transplantable metastasizing prostate adenocarcinoma in rats. J Natl Cancer Inst 54, 643–649, 1975.
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12. Rice-Evans C: Flavonoid antioxidants. Current Medicinal Chemistry 8, 797–807, 2001. 13. Aviram M and Fuhrman B: Wine flavonoids protect against LDL oxidation and atherosclerosis. Ann NY Acad Sci 957, 146–161, 2002. 14. Shaik YB, Castellani ML, Perrella A, Conti F, Salini V, et al.: Role of quercetin (a natural herbal compound) in allergy and inflammation. J Biol Regul Homeost Agents 20, 47–52, 2006. 15. Choi JA, Kim JY, Lee JY, Kang CM, Kwon HJ, et al.: Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin. Int J Oncol 19, 837–844, 2001. 16. Kuo PC, Liu HF, and Chao JI: Survivin and p53 modulate quercetin-induced cell growth inhibition and apoptosis in human lung carcinoma cells. J Biol Chem 279, 55875–55885, 2004. 17. Yang CS, Tran E, Nguyen TT, Ong CK, Lee SK, et al.: Quercetin-induced growth inhibition and cell death in nasopharyngeal carcinoma cells are associated with increase in Bad and hypophosphorylated retinoblastoma expressions. Oncol Rep 11, 727–733, 2004. 18. Vijayababu MR, Kanagaraj P, Arunkumar A, Ilangovan R, Dharmarajan A, et al.: Quercetin induces p53-independent apoptosis in human prostate cancer cells by modulating Bcl-2-related proteins: a possible mediation by IGFBP-3. Oncol Res 16, 67–74, 2006. 19. Choi EJ, Bae SM, and Ahn WS: Antiproliferative effects of Quercetin through cell cycle arrest and apoptosis in human breast cancerMDA-MB453 cells. Arch Pharm Res 31, 1281–1285, 2009. 20. Jung YH, Heo J, Lee JY, Kwon KT, and Kim HK: Quercetin enhances TRAIL-induced apoptosis in prostate cancer cells via increased protein stability 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 induces intrinsic and extrinsic pathway mediated apoptosis in androgen independent prostate 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 involved 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: Interrelationships among angiogenesis, proliferation, and apoptosis in the tumor microenvironment during N-methyl-N-nitrosourea androgen-induced prostate carcinogenesis in rats. Carcinogenesis 23, 1701–1712, 2002. 24. Seufi AM, Ibrahim SS, Elmaghraby TK, and Hafez EE: Preventive effect of the flavonoid, quercetin, on hepatic cancer in rats via oxidant/antioxidant activity: molecular and histological evidences. J Exp Clin Cancer Res 28, 80, 2009. 25. Lowry OH, Rosenbrough NJ, Farr AL, and Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:256–75, 1951. 26. Devasagayam TP and Tarachand U: Decreased lipid peroxidation in the rat kidneys during gestation. Biochem Biophys Res Commun 145,134–138, 1987. 27. Pick E and Keisari Y: Superoxide anion and H2 O2 production by chemically elicited peritoneal macrophages-induced by multiple nonphagocytic stimuli. Cell Immunol 59, 301–318, 1981.
28. Moron MS, Depierre JW, and Mannervik B: Levels of glutathione, glutathione reductase and glutathione-S-transferase activities in rat lung and liver. Biochim Biophys Acta 582, 67–78, 1979. 29. Jesik CJ, Holland JM, and Lee C: An anatomic and histologic study of the rat prostate. Prostate 3, 81–97, 1982. 30. Shureiqi I, Reddy P, and Brenner DE: Chemoprevention: general perspective. 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 carcinogenesis by calcitriol (vitamin D3) in adult male albino Wistar rats. Ann Cancer Res Therap 14, 12–18, 2006. 32. Bartsch H and Nair J: Potential role of lipid peroxidation derived DNA damage in human colon carcinogenesis: studies on exocyclic base adduct as stable oxidative stress markers. Cancer Detect Prev 26, 308–312, 2002. 33. Kang DH: Oxidative stress, DNA damage and breast cancer. AACN Clin 13, 540–549, 2002. 34. Ahmed MI, Fayed ST, Hossein H, and Tash FM: Lipid peroxidation and antioxidant 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 (3rd ed.). Oxford, London, UK, 1999. 37. Diplock AT, Rice-Evans AC, and Burton RH: Is there a significant role for lipid peroxidation in the of free causation of malignancy and for antioxidants in cancer prevention? Cancer Res 54, 19525–19526, 1994. 38. Otamiri T and Sjodahl R: Increased lipidperoxidation in malignant tissues of patients with colorectal cancer. Cancer 61, 122–125, 1989. 39. Sakanashi Y, Oyama K, Matsui H, Oyama TB, Oyama TM, et al.: Possible use of quercetin, an antioxidant, for the protection of cells suffering from an 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: Antioxidant and antimutagenic effect of quercetin against DEN induced hepatotoxicity in rat. Phytother Res 24, 119–128, 2009. 41. Reddy NS, Nirmala P, Chidambaram N, and Kumar PA: Quercetin in dimethyl benzanthracene induced breast cancer in rats. Am J Pharmacol Toxicol 7, 68–72, 2012. 42. Bostwick DG: High-grade prostatic intraepithelial neoplasia: the most likely precursor of prostate cancer. Cancer 75, 1823–1836, 1995. 43. Manach C, Morand C, Crespy V, Demign´e C, Texier O, et al.: Quercetin is recovered in human plasma as conjugated derivatives which retainantioxidant properties. FEBS Lett 426, 331–336, 1998. 44. Heijnen CG, Haenen GR, Vekemans JA and Bast A: Peroxynitrite scavenging of flavonoids: structure activity relationship. Environ Toxicol Pharmacol 10, 199–206, 2001.
Nutrition and Cancer Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/hnuc20
Chemopreventive Effect of Quercetin in MNU and Testosterone Induced Prostate Cancer of SpragueDawley Rats a
a
a
Govindaraj Sharmila , Thavadurainathan Athirai , Balakrishnan Kiruthiga , Kalimuthu a
a
a
Senthilkumar , Perumal Elumalai , Ramachandran Arunkumar & Jagadeesan Arunakaran a a
Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences , University of Madras , Chennai , India Published 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 Quercetin in MNU and Testosterone Induced Prostate Cancer of Sprague-Dawley Rats, Nutrition and Cancer, 66:1, 38-46, DOI: 10.1080/01635581.2014.847967 To link to this article: http://dx.doi.org/10.1080/01635581.2014.847967
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Nutrition and Cancer, 66(1), 38–46 C 2014, Taylor & Francis Group, LLC Copyright ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2014.847967
Chemopreventive Effect of Quercetin in MNU and Testosterone Induced Prostate Cancer of Sprague-Dawley Rats Govindaraj Sharmila, Thavadurainathan Athirai, Balakrishnan Kiruthiga, Kalimuthu Senthilkumar, Perumal Elumalai, Ramachandran Arunkumar, and Jagadeesan Arunakaran
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Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, India
(PIN) to several stages of metastasis and hormone refractory disease (2). The lack of appropriate animal model systems leaves a major obstacle in the development of strategies for prostate cancer chemoprevention (4) though the progress of prostatic carcinoma in to invasive phase can be studied in a few transplantable systems (3). However, the combination of carcinogen, N-methyl-Nnitrosourea (MNU), and testosterone (T) has the advantage of inducing higher incidence of prostate carcinogenesis in WistarUnilever rat that mimics the human prostate cancer model (5). Earlier reports from our laboratory had shown that the combined hormone and carcinogen (MNU) treatment consistently generated prostatic dysplasia, a putative preneoplastic lesion that is exclusively within prostates (6–8). The evaluation of anticancer effects of various plant-derived agents has gained good attention in recent years due to the development of resistance against the available chemotherapeutic agents (9). Among chemopreventers, flavonoids are the most studied antioxidant compounds that are largely present in fruits, vegetables, tea, red wine, and aromatic plants, (10–12). Quercetin is a principal flavanoid compound of onion (3, 3 , 4 , 5, 7-Penta hydroxyl flavanone), which possesses a wide spectrum of pharmacological properties (13, 14). Various animal and in vitro studies showed that quercetin was found to have antiproliferative effects by inhibiting cell growth and induces apoptosis in numerious cancer cell like breast, leukemia, colon, ovary, endometrial, gastric, and lung (14–19). Quercetin also enhances TRAIL-induced apoptosis in prostate cancer cells through increased protein stability of death receptor 5 (20). Recent findings from our laboratory have confirmed that quercetin downregulates insulin like growth factor signalling and upregulates intrinsic as well as extrinsic pathway-mediated apoptosis in androgen-independent prostate cancer cells (PC-3) (21). Quercetin also inhibits invasion, migration, and signalling molecules involved in cell survival and proliferation of prostate cancer cell line (PC-3) (22).
Prostate cancer becomes an ideal target for chemoprevention because of its high incidence and extended natural history. The consumption of quercetin (plant flavonoid) in diet is associated with decreased risk of disease and many cancers but then this was not elucidated in prostate malignancy. Hence, a study in which the male Sprague-Dawley rats were induced prostate cancer by hormone (testosterone) and carcinogen (MNU) and simultaneously supplemented with quercetin (200 mg/Kg body weight) thrice a week, was conducted. After the treatment period, rats were killed; ventral and dorsolateral lobes of the prostate were dissected. Histology and oxidative stress markers LPO, H2 O2 , and antioxidant GSH level were measured in both lobes. The lipid peroxidation, H2 O2 , in (MNU+T) treated rats were increased and GSH level was decreased, whereas simultaneous quercetin-treated rats reverted back to normal level in both ventral and dorsolateral regions. The different patterns of PIN were observed with associated hyperplasia and dysplasia; changes in these regions and the occurrence of this lesion were reduced in simultaneous quercetin-treated rats. The study concluded that dietary quercetin prevented MNU + Tinduced prostate carcinogenesis on both ventral and dorsolateral lobes of Sprague-Dawley rats.
INTRODUCTION Prostate cancer accounts for the second highest cancerrelated deaths in men. The etiology of prostate cancer is unknown but it is usually associated with age, race, family history, hormonal, dietary factors, etc. (1). Prostate cancer progresses from premalignant lesion or prostatic intraepithelial neoplasia
Submitted 14 December 2012; accepted in final form 12 August 2013. Address correspondence to Jagadeesan Arunakaran, Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600113, India. Tel.: 91-44-24547043. Fax: 91-44-24540709. E-mail: j [email protected]
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However, there is no report available on the role of quercetin on prostate malignant in vivo model. Therefore, this study was conducted to characterize the chemopreventive activity of quercetin in the prostate carcinogenesis using an animal model (Sprague-Dawley rats).
period, rats were sacrificed by cervical decapitation. Prostatic fluid was removed, both ventral and dorsolateral prostate lobes were dissected from the adhering connective tissue and washed several times with physiological saline, weighed accurately, and separated. The ventral prostate and dorsolateral lobes were then fixed with Bouin’s fluid for histopathological examination.
MATERIALS AND METHODS
Biochemical Assays
Chemicals Quercetin and MNU were purchased from Sigma-Aldrich Chemicals Private Ltd. (St. Louis, MO). Testosterone undecanoate (T) was purchased from M/S Schering-Plough, Apeldoorn, The Netherlands. Other chemicals were obtained from SISCO Research Laboratories Pvt. Ltd, Mumbai, India. All the chemicals were extra pure and of analytical grade.
Preparation of Tissue Homogenate Ventral prostate and dorsolateral lobes (100 mg) were homogenized in 0.1 M/L of Tris–HCl buffer, pH 7.4, and centrifuged at 3000 × g for 15 min at 4◦ C. The remaining supernatant was used for the biochemical analysis and the protein concentration was estimated by Lowry’s method (25).
Animals Healthy adult male Sprague-Dawley rats weighing approximately 200–250 g were used. The animals were housed in clean polypropylene cages and maintained in an air-conditioned animal house with constant 12-h light/dark cycle. Rats were permitted free access to drinking water throughout the experimental period. The animals were fed with standard rat pellet diet (Lipton India Ltd., Mumbai, India). Experiment was approved by the Institute Animal Ethical Committee (IAEC No. 01/01/12). Prostate Tumor Induction Rats were induced prostate cancer using carcinogen (MNU) and hormone (testosterone undecanoate) by a modified protocol (5, 23). First, each rat received daily intraperitoneal (IP) injections of testosterone undecanoate (50 mg/kg body weight) for 21 consecutive days. At Day 23, rats received daily IP injections of 100 mg testosterone undecanoate /kg body weight in 0.3 ml propylene glycol for 3 days. At Day 27, all the rats received a single intravenous (IV) dose (50 mg/kg body weight) of MNU (dissolved in saline at 10 mg/ml) through the tail vein. After 1 wk of MNU administration, rats received an IP injection of 4 mg testosterone undecanoate /kg body weight alternatively for 16 wk. Experimental Design A total of 40 rats were divided into 4 groups and each group consisted of 10 rats. In Group I, rats that received vehicle (propylene glycol) alone by IP injection, considered as controls. In Group II, rats were induced prostate cancer by using carcinogen plus hormone. In Group III, rats were induced prostate cancer with simultaneous supplementation of quercetin (200 mg/kg body weight) thrice a week for 16 wk through oral gavage. Quercetin supplementation was begun a week before the administration of the initial dose of testosterone undecanoate administration and throughout the studies. The dose was selected based on the earlier studies (24). In Group IV, rats received quercetin (200 mg/kg body weight) alone. After the treatment
Lipid Peroxidation and Hydrogen Peroxide (H2 O2 ) Levels Lipid peroxidion (LPO) was measured according to the method described by Devasagayam and Tarachand (26). The concentration of thiobarbituric acid reactive substance (TBARS) of the samples was expressed in nanomoles of TBARS formed per milligram protein. The H2 O2 generation was assessed by the spectrophotometric method of Pick and Keisari (27). The H2 O2 level was expressed in micromoles per minute per milligram protein. Nonenzymatic Antioxidant Level: Reduced Glutathione Glutathione (GSH) level was measured according to the method described by Moron et al. (28), and it was expressed in microgram per milligram protein. Histology The fixed ventral prostate and dorsolateral lobes of 10 animals in each group were further dissected sequentially (29). All samples were embedded in paraffin, and paraffin-embedded tissue blocks were sectioned at 3-μm thickness using rotary microtome and fixed on microscopic slides. The sections were covered with glass coverslip. After staining and destaining with hematoxylin and eosin, they were permanently mounted with dibutyl phthalate xylene (DPX) mount and viewed under a Nikon 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 by observing the histological evidence of each animal in all four groups 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 done only once. Statistical Analysis The data were subjected to statistical analysis using one way analysis of variance followed by Student’s–Newman–Keul’s
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(SNK) tests to assess the significance of variation between the means of control and treatment groups respectively, using computer-based software (SPSS Inc., Chicago, IL). The values were considered significant if the P values were less than 0.05.
TABLE 2 Effect of Quercetin (Q) on organ weight of ventral and dorsolateral prostate of Sprague Dawley rats treated with N-methyl-N-nitrosourea (MNU) +testosterone (T), Q, and vehicle
RESULTS
Absolute organ weight (mg)
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Body Weight Body weight was significantly decreased in MNU + T-treated rats compared with controls, whereas simultaneous treatment with quercetin increased the boby weight.quercetin alone treated rats did not show any change in body weight (Table 1). Organ weight of Ventral and Dorsolateral Prostate The absolute ventral prostate and dorsolateral prostate weights were significantly increased in MNU + T-treated rats compared to controls, whereas simultaneous quercetin treatment showed decreased ventral and dorsolateral prostate weight compared to MNU + T-induced rats. However quercetin alone treatedgroup did not show any significant change in organ weight (Table 2). Tumor Incidence (%) The percentage of tumor incidence in both ventral and dorsolateral prostate was summarized in Fig. 1. MNU + T-treated rats 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. PIN or prostatic intraepithelial neoplasia is a preneoplastic lesion in which the epithelial cell number is increased and protruding into the lumen. The tubules was composed of more than 1 layer of epithelial cells with varying sizes. The tufting and micopapillary projections were present in the MNU + T-treated rats. PIN was found 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 and dorsolateral prostate of quercetin supplemented rats. Hyperplasia (30%) and dysplasia (20%) were seen in the dorsolateral prostate of quercetin supplemented rats, respectively. No tumor incidence was observed in rats treated with quercetin alone and control rats.
Group
VP
Control Cancer induced (MNU & T) Cancer induced + quercetin (200 mg/kg b.wt) Quercetin (200 mg/kg b.wt)
DLP
381.8 ± 4.26 900.4 ± 9.50a
267.1 ± 7.97 410.0 ± 12.17a
685.4 ± 13.16a,b
344.6 ± 14.26a,b
404.6 ± 4.65b,c
303 ± 7, 26a,b,c
Each value represents mean ± SEM of 10 animals. The statistical significance was considered at the level of P < 0.05 following Student’s–Newman–Keul’s test. VP = ventral prostate; DLP = dorsolateral prostate; b.wt = body weight. a Control vs. others. bCancer induced vs. cancer induced + Q-treated rats. cCI + Q vs. quercetin alone.
Effect of Quercetin on Lipid peroxidation and H2 O2 Levels The levels of lipid peroxidation and H2 O2 were significantly increased in MNU +T-treated rats compared with that of control rats. However, simultaneous quercetin treatment prevented the lipid peroxidation and maintained H2 O2 level (Table 3). Effect of Quercetin on GSH Level MNU + T-treated group showed a significant decrease in the GSH level compared with that of the control group. However, quercetin treatment maintains the level of GSH similar to that in control. Quercetin alone treated group showed a significant increase in GSH level when compared with that of control (Table 3).
TABLE 1 Body weight of Sprague Dawley rats treated with N-methyl-N-nitrosourea (MNU) + testosterone (T), quercetin (Q), and vehicle Body weight (g) Group Control Cancer induced (MNU & T) Cancer induced + quercetin (200 mg/kg b.wt) Quercetin (200 mg/kg b.wt)
1st wk
5th wk
10th wk
15th wk
20th wk
208 ± 2 230 ± 3a 214 ± 2a,b 220 ± 3a,b,c
222 ± 5 239 ± 4a 229 ± 7a 243 ± 5b,c
244 ± 4 218 ± 6a 229 ± 3a,b 271 ± 4a,b,c
262 ± 6 212 ± 4a 242 ± 7a,b 288 ± 3a,b,c
286 ± 7 188 ± 4a 255 ± 5a,b 318 ± 4a,b,c
Each value represents mean ± SEM of 10 animals. The statistical significance was considered at the level of P < 0.05 following Student’s–Newman–Keul’s test. b.wt = body weight. a Control vs. others. bCancer induced vs. cancer induced + Q-treated rats. cCI + Q vs. quercetin alone.
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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 normal ventral prostate architecture. The connective tissue between the acini and the epithelial tubules were thin, condensed around the acne and tubules of the gland. The epithelial cells were lined as single layer with columnar shape infolding into the lumen (Fig. 3a). Tissue section of Group II: MNU + T-induced animal showed the hyperplastic sites within the same glandular epithelium. 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 markedly increased, with normal cell lining below (arrow headed) was observed (Fig. 3b2 and 3b3 ). Tissue section of Group III: MNU+T+quercetin-simultaneous quercetin treatment illustrated the absence of dysplastic and hyperplastic nodules, with organized epithelia cell as normal (Fig. 2C). Tissue section of Group IV: Quercetin alone treated group showed the normal appearance of epithelial tubules lining the lumen secretions and the tissue were tightly packed as seen in the control group (Fig. 4D).
TABLE 3 Effect of quercetin (Q) on lipid peroxidation and H2 02 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 of TBARS formed/mg protein) Group Control Cancer induced (MNU & T) Cancer induced + Quercetin (200 mg/kg b.wt) Quercetin (200 mg/kg b.wt)
VP
H2 O2 (μmol of H2 O2 /mg protein) GSH (nmol of GSH/mg protein)
DLP
VP
DLP
VP
DLP
32.76 ± .95 72.57 ± 1.4a
36.54 ± 1.62 76.72 ± 2.66a
6.24 ± 0.48 11.47 ± 0.43a
5.17 ± 0.33 10.25 ± 0.21a
3.98 ± 0.166 1.45 ± 0.07a
6.57 ± 0.51 2.38 ± 0.09a
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
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 following Student’s–Newman–Keul’s test. TBARS = thiobarbituric acid reactive substance; GSH = glutathione; VP = ventral prostate; DLP = dorsolateral prostate; b.wt = body weight. a Control vs. others. bCancer induced vs. cancer induced + Q-treated rats. cCI + Q vs. quercetin alone.
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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 figure available online).
Dorsolateral Prostate The dorsolateral prostate tissue section from rats in Group I: Control animals displayed normal dorsolateral prostate architecture; the tubules were lined by a single layer of cuboidal cells 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 dysplastic changes along with proliferating adenocarcinoma within the same glandular epithelium (Fig. 5C, 5G, and 5K). The different pattern of PIN like tufting, micropapillary, cribriform was not observed in those rats. The dorsolateral prostate tissue section from rats in Group III: MNU + T + quercetin showed remarkable decrease in the epithelial cell layer proliferation, indicating significant growth inhibition compared with (MNU/T alone) cancer induced (Fig. 5D, 5H, and 5L). The dorsolateral prostate tissue section from rats in Group IV: Quercetin alone treated group showed the appearance of epithelial tubules lining the lumen secretions as seen in the control group (Fig. 5B, 5F, and 5J).
DISCUSSION Cancer chemoprevention by antioxidant is a key strategy for inhibiting, delaying, or even reversing the process of carcinogenesis (30). Quercetin (3,3 , 4 , 5,7-pentahydroxyflavone) is 1 of several naturally occurring dietary polyphenolic flavonoids that is present in fruits and vegetables like apples, cranberries, blueberries, and onions, respectively. A significant reduction in the body weight and increased ventral and dorsolateral prostatic weight were observed in carcinogen and hormone-induced prostate carcinogenesis. The increase in prostate weight may be due to MNU that after a single dose along with continuous stimulation of testosterone induces fibromuscular tissue and multiplication of squamous epithelium of the prostate (31). The exact mechanism of decrease in body weight is unclear but it may be due to the formation of hydroxylated bases of DNA, an important event in chemical carcinogenesis (32). Whereas, simultaneous quercetin treatment of rats caused a decreased tumor incidence with an associated reduction in ventral and dorsal prostatic weight and increased body
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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 acini and epithelium were thin and condensed. b1 -b3: MNU + T treated rats. Hyperplastic and dysplastic changes are seen within the glandular epithelium.The PIN with different pattern like tufting (b1 ), micropapillary (b2 ), and epithelial cell showed increased in number (b3 ) when compared to control groups. c: MNU+T+quercetin treated groups -shows the absence of 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 figure available online).
weight, respectively suggests the potency of anticancer activity of quercetin in the initiation process. Imbalance ROS production and antioxidant defense in living organism results in oxidative stress. Severe oxidative stress causes mutation of tumor suppressor genes through DNA damage that results in the initiation of carcinogenesis (33), but this also promotes the multistep carcinogenesis (34). The free radicals may spontaneously react with nucleophilic centers of the cell and thereby covalently bound to DNA, RNA and protein which may lead to cytotoxicity and carcinogenicity (35). Lipids, especially polyunsaturated fatty acids, are very susceptible to free radical attack, which can initiate lipid peroxidation (36) that acts to control of cell division (37). The end product of lipid peroxidation, malondialdehyde is highly cytotoxic and inhibited to protective enzymes and suggested to act as a tumor promoter and a cocarcinogenic agent (38). A corresponding increase in lipid peroxidation, reactive oxygen level, and H2 O2 were observed after carcinogen and hormone treatment in both ventral and dorsolateral prostates while
quercetin administration prevents prostate cancer initiation and progression. Quercetin is considered to be a strong antioxidant 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 of lipid peroxides and H2 O2 got decreased due to its scavenging properties of free radicals and thereby inhibits prostate cancer initiation. The increase in lipid peroxidation, H2 O2 correlate with lower level of antioxidants upon carcinogen and hormone administration. The reduced GSH level was found to be significantly reduced in MNU+T treated groups whereas, the quercetin-treated group retrieved the GSH level and prevents prostate carcinogenesis initiation and hence decreased malondialdehyde in diethylnitrosamine induced hepatocarcinogen was found (40). The same results were found in DMBA induced breast cancer in rats in which quercetin was supplemented in the diet (41). The chemopreventive effect of quercetin was confirmed by histological examination in both ventral and dorsolateral
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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 lumen was seen. F1 -F3 : MNU + T treated rats. epithelium. The PIN with different pattern like tufting (F1 ), micropapillary (F2 ), and epithelial showed increased in cell number (F3 ) was observed. The epithelial cells are crowded and stratification with irregular spacing with hyperchromatin was seen. G: MNU + T + quercetin treated groups shows the absence of hyper plastic and dysplastic changes upon quercetin intake. H: Quercetin control group showing the normal appearance of epithelial cells as seen in control group. E = epithelium; L = lumen; S = stroma (color figure available online).
prostate tissue. The pathological changes closely share the histological features found in human prostatic dysplasia, also termed PIN. PIN lesion is widely accepted as a premalignant condition of prostate cancer with four architectural patterns such as tuft, micropapillary, flat, and cribriform. The tufting pattern is most commonly associated with 97% of positive cases although they have multiple pattern associated (42). The MNU + T-treated animals, showed lesions such as micropapillary and tufting patterns in ventral prostate that shows the presence of hyperplasia, dysplasia, and PIN architectural patterns as seen in human HGPIN (42). In this study, HGPIN exhibited disruption of the basal layer of cells due to changes in cell polarity. The number of cells was higher in HGPIN, but the basement membrane remained intact. These data were supported by our earlier studies from our laboratory (7) that on chemoprevention of zinc in MNU + Tinduced model, which states that the increase in epithelial cell proliferation may be due to MNU + T administration. The study
also showed a different pattern of PIN that was not observed in the dorsolateral prostate of MNU + T treated rats. Hyperplasia (60%) and dysplasia (40%) were observed whereas in simultaneous quercetin-treated group, the occurrence of hyperplasia and dysplasia lesion was found to be significantly decreased. This could be due to the presence of quercetin that inhibited lipid peroxidation and increased GSH level that exerted protectitive effects against oxidative damage evidenced in hepatic cancer (24). Oral administration of quercetin causes its complete metabolization and metabolites still retain antioxidant properties (43), thereby quercetin can donate electrons or hydrogen and scavenges H2 O2 and superoxide anion (44). To conclude, this study is first of its kind that provides suitable histological evidence for chemoprevention of prostate carcinogenesis by a flavonoid quercetin in both ventral and dorsolateral prostate of male Sprague-Dawley rats. Its chemopreventive effect is through scavenging free radicals (H2 O2 ), inhibiting lipid
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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-Nnitrosourea (MNU) + testosterone (T) + quercetin (200 mg/Kg body weight). A,E,I: Normal dorsolateral prostate epithelium (DLP): The cells are tall columinar aligned 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 as seen 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 hyperplastic and dysplastic changes with increased number when compared to control groups. Two (20%) of 10 animals shown adenocarcinoma (ad) in which the glands is fully occupied by epithelial cell. It shows a glandur growth pattern within adundant amount of stromal tissue. D,H,L: MNU + T + quercetin treated groups show the absence of hyper plastic and dysplastic changes. E = epithelium; L = lumen; S = stroma (color figure available online).
peroxidation and increasing GSH level. The molecular mechanism underlying the chemoprevention of quercetin and its level in serum are the future prospects of the study. Quercetin may be used to target the signaling molecules involved in cell survival, proliferation, and apoptosis. FUNDING This 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. REFERENCES 1. 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 Epidemiol Biomarkers Prev 16, 2193–2203, 2007. 3. Pollard M and Luckert PH: Transplantable metastasizing prostate adenocarcinoma in rats. J Natl Cancer Inst 54, 643–649, 1975.
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