Inhibition of precancerous lesions development in kidneys by chrysin via regulating hyperproliferation, inflammation and apoptosis at pre clinical stage

Archives of Biochemistry and Biophysics 606 (2016) 1e9

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Inhibition of precancerous lesions development in kidneys by chrysin via regulating hyperproliferation, inflammation and apoptosis at pre clinical stage Summya Rashid, Sana Nafees, Abul Vafa, Shekh Muhammad Afzal, Nemat Ali, Muneeb U. Rehman, Syed Kazim Hasan, Aisha Siddiqi, Preeti Barnwal, Ferial Majed, Sarwat Sultana* Section of Molecular Carcinogenesis and Chemoprevention, Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 April 2016 Received in revised form 3 July 2016 Accepted 4 July 2016 Available online 9 July 2016

Chrysin (CH) is natural, biologically active compound, belongs to flavoniod family and possesses diverse pharmacological activities as anti-inflammatory, anti-oxidant and anti-cancer. It is found in many plants, honey and propolis. In the present study, we investigated the chemopreventive efficacy of CH against N-nitrosodiethylamine (DEN) initiated and Fe-NTA induced precancerous lesions and its role in regulating oxidative injury, hyperproliferation, tumor incidences, histopathological alterations, inflammation, and apoptosis in the kidneys of Wistar rats. Renal cancer was initiated by single intraperitoneal (i.p.) injection of DEN (200 mg/kg bw) and promoted by twice weekly injection of ferric nitrilotriacetate (Fe-NTA) 9 mg Fe/kg bw for 16 weeks. CH attenuated Fe-NTA enhanced renal lipid peroxidation, serum toxicity markers and restored renal anti oxidant armory significantly. CH supplementation suppressed the development of precancerous lesions via down regulation of cell proliferation marker like PCNA; inflammatory mediators like TNF-a, IL-6, NFkB, COX-2, iNOS; tumor incidences. CH up regulated intrinsic apoptotic pathway proteins like bax, caspase-9 and caspase-3 along with down regulation of Bcl-2 triggering apoptosis. Histopathological and ultra structural alterations further confirmed biochemical and immunohistochemical results. These results provide powerful evidence for the chemopreventive efficacy of CH against chemically induced renal carcinogenesis possibly by modulation of multiple molecular pathways. © 2016 Elsevier Inc. All rights reserved.

Keywords: Renal cell carcinoma Chrysin ROS Inflammation Apoptosis Hyper proliferation

1. Introduction Renal cell carcinoma (RCC) is the most common malignancy of adult kidney [1]. It has been reported to be the most therapy

Abbreviations: CAT, Catalase; CH, Chrysin; DEN, Di ethyl nitrososamine; Fe-NTA, Ferric Nitriloacetic acid; BSA, bovine serum albumin; CDNB, 1-chloro 2, 4dinitrobenzene; DTNB, 5, 50 -dithio bis-[2-nitrobenzoic acid]; EDTA, ethylene diamine tetra acetic acid; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; NADPH, reduced nicotinamide adenine dinucleotide phosphate; ROS, reactive oxygen species; TBA, thiobarbituric acid; LDH, lactate dehydrogenase; BUN, Blood urea nitrogen; MDA, malondialdehyde. * Corresponding author. E-mail address: [email protected] (S. Sultana). http://dx.doi.org/10.1016/j.abb.2016.07.004 0003-9861/© 2016 Elsevier Inc. All rights reserved.

resistant cancer and responds either very less or not at all to the conventional therapies [2]. Many novel chemotherapeutic agents have been developed over the past decade, besides an increase in deciphering molecular mechanisms implicated in the development of RCC, yet it is not curable and a fatal disease [3]. Oxidative stress is one of the risk factors for human RCC and plays an essential role in Fe-NTA induced carcinogenesis as well [4]. Diethylnitrosamine (DEN) is an effective carcinogenic N-nitroso compound broadly recognized for the induction of preneoplastic lesions in experimental animals [5]. Nitrilotriacetate (NTA) is chemically amino tricarboxylic acid and a potent nephrotoxic agent. Fe-NTA, ferric nitrilotriacetate is an iron chelate formed by combination of Fe and NTA, which on repeated administration produces acute and sub acute renal proximal tubular necrosis that

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ultimately results in high prevalence of renal adenocarcinoma in rats and mice. DEN and Fe-NTA stands as an outstanding model for inducing renal carcinogenesis in vivo via free radical induced damage coupled with widespread peroxidation of membrane lipids which is further illustrated by high frequency of tumor associated mortality, prevalence of pulmonary metastasis and peritoneal invasion [6,7]. Presently much research is being carried on evaluation of safe and efficient plant based products with diversified pharmacological properties chiefly against cancer because of their positive results in animal models, clinical trials as well as poor alternatives the modern system of medicine recommends for the treatment. Flavonoids are plant polyphenolic compounds with variable phenolic structures and are extensively distributed in medicinal plants, fruits, health beverages and teas. Epidemiological studies further prove importance of flavoniods via mentioning high fruit and vegetable consumption is associated with a decreased risk of cardiovascular disease and several types of cancers including breast, colon, lung, pancreas, oral and prostate both in vitro and in vivo. This has increased the public’s interest in the use of flavonoids for their potential health benefits and a growing attention in the revelation of the biological roles of flavonoids, the major components of some traditional medicinal plants by both consumers and food manufacturers. Chrysin (5,7-dihydroxyflavone) is a natural flavonoid present in many plant extracts, honey and propolis. It possesses numerous biological and pharmacological properties including antioxidant, apoptotic, anticancer, antiinflammatory etc. Earlier studies have confirmed the importance of distribution and quantity of the hydroxl groups are related to antioxidant property of flavonoids particularly depending on their hydroxylation of ring B [8]. Recent studies have revealed that chrysin regulates key molecules involved in inflammation, cancer and aging [9]. It has also been reported that chrysin inhibits proliferation and induces apoptosis in cancer cells, making it a possible candidate as anticancer agent [10]. In vitro studies reveal that chrysin inhibits the growth of Hela cells by down regulating the expression of proliferating cell nuclear antigen (PCNA), induces apoptosis via caspase activation and Akt inactivation in various cancer cell lines. In vivo findings showed that dietary administration of chrysin significantly inhibited the development of AOM-induced colonic ACF in rats [11]. It inhibited tumor angiogenesis in vivo, which is a key step in cancer cell metastasis. It also significantly sensitizes TNF-a induced apoptosis in a number of human cancer cells as well as inhibited COX-2 expression and IL-6 signaling which suggests its antiinflammatory property [12]. Therefore the current study was planned to investigate its potential against Fe-NTA induced nephrotoxicity and its preclinical chemo preventive efficacy against two stage renal carcinogenesis induced by DEN initiation and Fe-NTA promotion for 16 weeks in Wistar rats by studying its possible potential molecular targets. To this effect, we studied oxidative stress markers, anti oxidant armory profile, histopathological alterations, ultra structural changes, expressions of inflammatory marker proteins, instrinsic apoptotic pathway proteins and hyper proliferation marker PCNA known to be deregulated in cancer cells and hence might be one of the novel targets of the chemopreventive activity of CH.

All other chemicals and reagents were of the highest purity grade and commercially available. 2.2. Ethical statement All procedures for using experimental animals were checked and permitted by the ‘‘Institutional Animal Ethical Committee (IAEC)’’that is fully accredited by the Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The animals were provided by the Central Animal House Facility, Jamia Hamdard, whose registration number and date of renewal are (IAEC No: 173/Go/Re/S/2000/CPSCEA) and 30th march, 2015. Approval ID/project number for performing this study by permission of IAEC is 1059. 2.3. Animals Male Wistar rats (150e200 g), 6e8 weeks old, were obtained from the Central Animal House of Hamdard University, New Delhi, India. Rats were housed in polypropylene cages in groups of four rats per cage and were kept in a room maintained at 25 ± 2  C with a 12 h light/dark cycle. They were allowed to acclimatize for one week before the experiments and were given free access to standard laboratory animal diet and water ad libitum. 2.4. Preparation of Fe-NTA The Fe-NTA solution was prepared by the method of Awai et al. briefly, ferric nitrate (0.16 mM/5.0 ml) solution was mixed with a fourfold molar excess or disodium salt of NTA (0.64 mM/5.0 ml) and the pH was adjusted to 7.4 with sodium bicarbonate. The solution was freshly prepared immediately before use and was injected on the basis of 10 ml/kg b wt [13]. 2.5. Experimental design The treatment regimen for CH and the proposal of verifying its chemopreventive efficacy against renal carcinogenesis was based on the preliminary dose dependent pilot study which was carried out in our laboratory. To study the protective effects of CH on biochemical and serological changes induced by toxicity of Fe-NTA in rats, 24 male Wistar rats were randomly divided into four equal groups.

Groups (n ¼ 6)

Treatment from 1st to 13th day

Treatment on 13th day

Group I (control) Group II (only FeNTA) Group III (FeNTA þ CHD1) Group IV (FeNTA þ CHD2)

Distilled water Distilled water

Normal saline only (0.9% i.p.) Fe-NTA 9 mg/kg b.wt.i.p (13th day) Fe-NTA 9 mg/kg b.wt.i.p (13th day) Fe-NTA 9 mg/kg b.wt.i.p (13th day)

CH 50 mg/kg b.wt. CH 100 mg/kg b.wt.

2. Materials and methods 2.1. Chemicals DEN, Fe-NTA, BSA, BUN, CDNB, DTNB, EDTA, GPx, GR, GSH, GSSG, LDH, NADPH, TBA, CH etc were obtained from Sigma-Aldrich, USA.

All animals were sacrificed within 1 h exactly 12 h after Fe-NTA administration. Kidney tissues were processed for biochemical estimations. Blood was collected and serum separated out and

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processed for serological studies. These doses were further used in the long term cancer study.

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enzymes. 2.7. Biochemical estimations

✓ To study the effect of treatment of CH on DEN initiated and FeNTA promoted renal carcinogenesis. The complete treatment regimen followed in tumor study was as follows:

Lipid peroxidation (LPO) Lipid peroxidation was measured by the method of Wright et al. [15]. Catalase activity (CAT) Catalase activity was measured by the method of Claiborne [16]. Reduced Glutathione (GSH) Reduced glutathione was estimated by the method of Jollow et al. [17]. Glutathione reductase (GR) activity GR activity was measured by the method of Carlberg et al. [18]. Glutathione Peroxidase Activity (GPx) GPx activity was measured by the method of Mohandas et al. [19]. Estimation of creatinine Serum creatinine levels were measured by the method of Hare [20]. Estimation of blood urea nitrogen (BUN) Serum blood urea nitrogen was measured by the method of Kanter [21]. Estimation of protein The protein concentration in all samples was determined by the method of Lowry et al. using BSA as standard [22]. Immunohistochemistry was done as described by Ahmad et al. [23]. 2.8. Histopathological examination and electron microscopy The kidneys were quickly removed after sacrifice and preserved in 10% neutral buffered formalin for histopathological processing. The kidneys tissue was embedded in paraffin wax and longitudinally sectioned with a microtome. Hematoxylin and eosin staining of the sections was observed under an Olympus microscope. For electron microscopy, tissue samples of 1 mm3 were fixed in glutaraldehyde (3%) and phosphate buffered saline (pH 7.2) at 4  C for 3 h and post-fixed with 1% osmium tetroxide for 1 h. Osmium tetroxide was washed away with the same buffer. Ethyl alcohol was used for dehydration and samples were embedded in Araldite CY212. Thin sections were double stained with saturated uranyl acetate (20 min) and lead citrate. Each sample was visualized under an electron microscope. 2.9. Statistical analysis

At the end of 24 weeks, all the animals were sacrificed by mild anesthesia. Kidneys were quickly removed and processed for various molecular, histopathological and immunohistochemical studies. 2.6. Tissue processing Post mitochondrial supernatant of kidney samples was prepared by the method of Rashid et al. [14]. In brief, the kidneys were removed quickly, cleaned of extraneous material and immediately perfused with ice-cold saline (0$85% NaCl). The kidneys were homogenized in chilled phosphate buffer (0$1 M, pH 7$4) containing KCl (1$17%) using a PottereElvehjen homogenizer. The homogenate was filtered through muslin cloth and centrifuged at 800 g for 5 min at 4  C by a REMI cooling centrifuge to separate the nuclear debris. The aliquot obtained was centrifuged at 12000 rpm for 20 min at 4  C to obtain the PMS, which was used as a source of

Differences between groups were analyzed using analysis of variance (ANOVA) followed by Tukey Kramer’s test. All data points are presented as the treatment groups mean ± standard error of the mean (SEM). 3. Results 3.1. Effect of CH on Fe-NTA induced oxidative stress and anti oxidant armory The protective effects of CH on Fe-NTA induced depletion of antioxidant armory, elevation in serum kidney toxicity markers are shown in Table 1. Fe-NTA administration elevated LPO substantially (p < 0.001) in group II as compared to group I and treatment by CH at both the doses mitigated LPO levels significantly (p < 0.01 and p < 0.001). CAT activity was markedly decreased (p < 0.001) due to Fe-NTA administration in group II as compared to group I. Its activity was increased significantly by both the doses of CH treatment (p < 0.05 and p < 0.001). GSH was depleted in group II (p < 0.001)

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due to Fe-NTA administration as compared to group I. However both the doses of CH replenished GSH significantly (p < 0.01 and p < 0.001). There was also concomitant and significant decrease in the activity of glutathione dependent antioxidant enzymes (glutathione peroxidase and glutathione reductase) as compared to control group (P < 0.001 and P < 0.01). However, prophylactic treatment of CH at both the doses replenished the activity of the above said enzymes considerably (p < 0.05, p < 0.01 and P < 0.001). Serological chemistry revealed that Fe-NTA induced acute nephropathy as evident by the significant increase in kidney serum toxicity markers like BUN and creatinine in group II as compared to group I (p < 0.001). On the other hand, prophylactic treatment of CH at both the doses attenuated Fe-NTA induced elevation in the serum levels of BUN (p < 0.001) and creatinine (P < 0.01 and P < 0.001) respectively (Table 1). 3.2. Effect of CH on pro inflammatory cytokines and inflammatory marker proteins in DEN initiated and Fe-NTA promoted renal carcinogenesis Levels of pro inflammatory cytokines TNF-a and IL-6 was found to be elevated significantly in DEN initiated and Fe-NTA promoted group for 16 weeks in comparison to untreated control (P < 0.001). Pre and post treatment with CH in groups III and IV (p < 0.05, p < 0.01 and p < 0.001) alleviated TNF-a and IL-6 levels significantly (Table 2). The effect of CH was investigated on NFkB, iNOS and COX-2 protein expressions in DEN initiated and Fe-NTA promoted groups. Immuno histochemical data revealed administration of DEN þ Fe-NTA in tumor group (group II) up regulated the expression of NFkB, COX-2 and iNOS in rat kidneys which is well evident by intense staining of NFkB, COX-2 and iNOS. (Fig. 2I, II, III). There was negligible expression of these three proteins i,e NFkB, COX-2 and iNOS in kidneys of control group rats. However Pre and post treatment with CH markedly attenuated DEN þ Fe-NTA induced expression of NFkB, COX-2 and iNOS at both the doses respectively. 3.3. Effect of CH on tumor incidences, histopathological alterations and ultra structural changes against DEN initiated and Fe-NTA promoted renal carcinogenesis The data given in Table 3 gives a summary of the percentage incidence of renal tumors (RTs) in different treatment groups. Group I did not show any incidences of kidney tumors. However DEN-initiation and Fe-NTA promotion in animals for 16 weeks enhanced the development of RTs to 78.57% in the animals studied. In comparison, tumor incidences in the group III i.e., group that was co-treated with CH (50 mg/kg b wt) along with DEN initiation and Fe-NTA promotion was 50.00% whereas in the group receiving the higher dose of CH (100 mg/kg b wt) along with DEN initiation and Fe-NTA promotion the tumor incidences were reduced to 28.57%. The histology of the rat kidney tissues showed normal histo architecture in the control group. Kidneys of rats in the group II that were DEN initiated and chronically promoted with Fe-NTA for 16 weeks resulted in disruption of the normal renal architecture which

was well evident by infiltration of inflammatory cells, increased eosinophilic staining, hyperchromatism, blood sinusoids, interstitial hemorrhages, glomerular, tubular congestion and atrophy (Fig. 1). Furthermore, CH treatment showed protective changes in the glomeruli and tubules. Group III treated with CH (50 mg/kg b wt) showed mild inflammatory cell invasion, glomerular and tubular congestion and very few hyperchromatic deposits, whereas higher dose of CH (100 mg/kg b wt) restored renal histological features nearly to normal. Ultra structural changes of kidneys of group II animals showed swollen mitochondria with rupture of the outer membrane and vacuolation of the inner compartment. Disruption, fragmentation and granulation of the cristae were also seen. All these changes were reversed by higher dose treatment of CH in group IV (Fig. 4). 3.4. Effect of CH on proliferative marker protein against DEN initiated and Fe-NTA promoted renal carcinogenesis Immuno histochemical expressions showed intense expression of PCNA positive cells in DEN initiated and Fe-NTA promoted rats for 16 weeks compared to untreated controls (Fig. 3I). Moderate expression of PCNA was found in the low dose modulator (50 mg/kg b.wt) treated group as compared to group II (tumor group). However, higher dose of CH (100 mg/kg b.wt) treated rats showed considerably lesser expression of PCNA positive cells which means that CH has attenuated renal cell proliferation in kidneys in group IV as compared to group II. Therefore deciphering anti proliferative potential of CH. 3.5. Effect of CH on induction of apoptotic marker proteins in DEN initiated and Fe-NTA promoted renal carcinogenesis Intrinsic apoptotic pathway proteins like Bax, Bcl-2, caspase-3 and caspase-9 were evaluated. There was no significant immunopositive staining of bax in rats subjected to DEN initiated and FeNTA promotion for 16 weeks as per immune histochemical results in group II (Fig. 3III). However, pre and post treatment of CH in group III and group IV activated and increased the expression of bax at both the doses respectively. There was an up regulation in the expression of Bcl-2 in rat kidneys in DEN þ Fe-NTA promoted group i,e tumor group (group II) as compared to group I (Fig. 3II). However Pre and post treatment with CH markedly attenuated DEN þ FeNTA induced expression of bcl-2 at both the doses respectively. Hence triggering apoptosis by down regulation of Bcl2, which is an anti apoptotic protein. There was also down regulation of caspase-9 and caspase-3 found by colorimetric analysis in DEN þ Fe-NTA promoted group (p < 0.001) inhibiting apoptosis as compared to control. However, treatment with CH at both the doses up regulated the levels significantly (p < 0.05, p < 0.01 and P < 0.001) (Table 2) thereby resulting in the induction of apoptosis. 3.6. Effect of CH on CAT, LPO, GSH, GR, GPx, BUN and creatinine against Fe-NTA administration

Table 1 Levels of CAT, LPO, GSH, GR, GPx, BUN and Creatinine in rat kidney. Groups

CAT (nmol H2O2 consumed/ LPO (nmol MDA formed/ GSH (m mol min/mg protein) h per g tissue) GSH/g tissue)

Group Group Group Group

57.5 18.1 32.7 40.7

I II III IV

± ± ± ±

4.1 2.3*** 3.5# 3.3###

6.6 ± 0.9 25.6 ± 2.6*** 15.1 ± 0.5## 11.6 ± 1.1###

0.76 0.24 0.58 0.69

±0 .07 ±0 .02*** ± 0.03## ± .05###

GR (m mol NADPH oxidized/min/mg protein) 226.1 121.1 188.6 214.6

± ± ± ±

16.8 8.9*** 12.8## 14.1###

GPX (m mol NADPH oxidized/min/mg)

BUN (mg/dl) Creatinine (mg/dl)

197.4 ± 13.3 94.3 ± 19.2** 179.3 ± 13.3# 191.1 ± 20.3#

17.01 ± 1.8 49.8 ± 0.9*** 33.5 ± 1.3### 27.9 ± 1.2###

0.91 ±0 .1 3.7 ±0 .4*** 1.9 ±0 .04## 1.7 ±0 .07###

Each value is represented as mean ± SE (n ¼ 6animals/group). Significant differences were indicated by***p < 0.001 when compared with control, #p < 0.05,##p < 0.01 and###p < 0.001 were used to show significance when compared with Fe-NTA administered animals.Group I (control), Group II (only Fe-NTA administered), Group III (FeNTA þ CHD1) and Group IV (FeNTA þ CHD2) where CH ¼ Chrysin; CHD1 ¼ 50 mg/kg/b wt; CHD2 ¼ 100 mg/kg/b wt.

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3.7. Effect of chrysin (CH) on IL-6, TNF-a, Caspase-9 and Caspase-3 against DEN initiated and Fe-NTA promoted renal carcinogenesis

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3.9. Effect of chrysin (CH) on ultra structural changes against DEN initiated and Fe-NTA promoted renal carcinogenesis

Table 2 Levels of IL-6, TNF-alpha, caspase-9 and caspase-3 in rat kidney. Groups

IL-6 (pg/ml)

TNF-a ((pg/ml))

Group Group Group Group

806.9 ± 31.02 2535.9 ± 77.58*** 2197.13 ± 94.5# 1848.05 ± 68.5###

310.8 587.5 469.9 434.5

I II III IV

± ± ± ±

7.3 13.6*** 22.4## 21.1###

Caspase-9 0.782 0.281 0.492 0.596

± ± ± ±

0.07 0.02*** 0.05## 0.07###

Caspase-3 0.612 0.210 0.375 0.443

± ± ± ±

0.06 0.04*** 0.07## 0.05###

Each value is represented as mean ± SE (n ¼ 6animals/group). Significant differences were indicated by***p < 0.001 when compared with control, #p < 0.05,##p < 0.01 and###p < 0.001 were used to show significance when compared with Fe-NTA administered animals. Group I (control), Group II (only Fe-NTA administered), Group III (FeNTA þ CHD1) and Group IV (FeNTA þ CHD2) where CH ¼ Chrysin; CHD1 ¼ 50 mg/kg/b wt; CHD2 ¼ 100 mg/kg/b wt.

Fig. 4. Ultra structural examination of rat kidneys (A) Normal ultra structure of kidneys (B) Disruption of the normal ultra structure by DEN þ Fe-NTA administration was observed (C and D) treatment with CHD1 and CHD2 showed protective changes were restored on higher dose of CH (50 & 100 mg/kg b.wt.).

3.8. Effect of chrysin on histopathology against DEN initiated and Fe-NTA promoted renal carcinogenesis

Fig. 1. Histopathological examination of rat kidney (A) Normal histology of kidney (B) Disruption of the normal renal architecture by DEN þ Fe-NTA administration was observed (C and D) treatment with CHD1 and CHD2 showed protective changes in the glomeruli and tubules and the morphology was restored on higher dose of CH (50 & 100 mg/kg b.wt.).

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3.10. Effect of chrysin (CH) on the expression of NFkB, COX2 and iNOS against DEN initiated and Fe-NTA promoted renal carcinogenesis

Fig. 2. (I, II, III): Representative photomicrographs of NFkB(I), Cox-2 (II)& iNOS(III) determined by immunohistochemistry. (A) There is negligible expression of NFkB, Cox-2 & iNOS in the renal sections of control group. (B) DEN þ Fe-NTA administration increased strongly NFkB, Cox-2 & iNOS expressions in renal tissues. (C) There was partial inhibition of NFkB, Cox-2 & iNOS expression as evidenced by weak immunostaining in the rat kidneys treated with lower dose of CHD1 (50 mg/kg BW). (D) In contrast, there was almost complete suppression of NFkB, Cox-2 & iNOS in rats treated with higher dose of CHD2 (100 mg/kg BW) as evident from the figure, as the tubular structures within the inner cortical regions do not show any substantial immunostaining.

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3.11. Effect of chrysin (CH) on the expression of PCNA, Bcl2 and Bax against DEN initiated and Fe-NTA promoted renal carcinogenesis

Fig. 3. (I, II, III): Representative photomicrographs of PCNA, Bcl2, Bax determined by IHC (A) Negligible expression of Bax, Bcl2, PCNA observed in control group. (B) DEN þ Fe-NTA administration increased the number of PCNA positive cells and staining of Bcl2 in glomerular and tubular region of renal sections of animals and vice versa for Bax (C) DEN þ FeNTA þ CHD1 treated animals showed slightly less number of PCNA positive cells and less staining intensity of Bcl2 and vice versa for Bax(D) DEN þ Fe-NTA þ CHD2 animals showed lesser number of PCNA positive cells and staining of Bcl2 and vice versa for Bax.

4. Discussion Fe being an indispensable element in the human body contributes much of the toxicity of ROS in living organisms via generation of .OH and other powerful oxidants through fenton reaction. Thereby, playing an imperative role in oxidative injury [24,25]. ROS has been reported to induce DNA damage leading to activation of proto oncogenes, inactivation of tumor suppressor genes demonstrated by various in vitro experiments, forming a

critical step in the process of changing a normal cell to malignancy. Henceforth free radical generation resulting in lipid peroxidation, cell membrane damage triggers inflammation, tumor promotion, anti apoptosis may be postulated as one possible mechanism behind DEN initiated and Fe-NTA promoted renal carcinogenesis. Oxidative stress plays an essential role in the pathogenesis of nephrotoxicity caused by Fe-NTA playing fundamental role not only in nephrotoxicity but also in tumorigenesis as reported previously [23,26]. As a result of Fe NTA, iron gets deposited in kidneys

Table 3 Modulatory effect of Chrysin (CH) on tumor data in DEN-initiated and Fe-NTA promoted renal carcinogenesis. Treatment regimen per group Number of animals treated Number of animals survived Number of animals with renal cell tumors Incidence of renal cell tumors (%) Group Group Group Group

I II III IV

15 20 15 15

15 14 12 14

0 11 6 4

0.0 78.57 50.00 28.57

Group I (untreated control); group II (tumor group) e DEN þ Fe-NTA; group III e DEN þ Fe-NTA þ CH (60 mg/kg b wt); group IV e DEN þ Fe-NTA þ CH (120 mg/kg b wt). Tumor group showed highest percentage of tumor incidences which was abrogated by the administration of CH in groups III and IV.

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generating ROS resulting in oxidative damage and lipid per oxidation. In the present study also, LPO was increased in Fe-NTA administered group as compared to control group as reported previously which was attenuated by CH treatment [27]. Renal anti oxidant armory including GSH, GR, GPx and CAT were depleted in Fe-NTA administered group as compared to control which was in concurrence with earlier reports. Nevertheless, it is apparent from the present results that prophylactic treatment of CH restored and augmented antioxidant armory which was formerly used in scavenging the ROS produced by Fe-NTA. Serum toxicity markers like BUN and Creatinine simultaneously were found to be elevated in Fe-NTA administered group as compared to control. On the other hand, CH alleviated BUN and creatinine as compared to group II. Therefore CH deciphers its nephro protective efficacy possibly by alleviation in lipid per oxidation, serum toxicity markers and replenishment of anti oxidant armory. Histopathological examination of the kidneys of animals administered with DEN and FeNTA for 16 weeks revealed tubular necrosis, glomerular congestion, massive inflammatory cell infiltration and hyperchromatism. Treatment with CH abrogated pathological alterations at both the doses as compared to group II. Ultra structural changes revealed that there was massive destruction at ultra structural level in group II which was regulated by treatment of CH at both the doses respectively. Our data was further supported by immune histochemical results of hyper proliferation, inflammation, apoptosis marker proteins and tumor incidences which were highest in tumor group. CH treatment at both the doses reduced tumor incidences significantly. It has been reported that oxidative stress plays an essential role in tumor promotion. Inflammation has been recognized as seventh hallmark of cancer and evading apoptosis is another hallmark of cancer cells [29]. CH is expected to suppress cell proliferation, modulation of oncogenic pathways, inhibition of inflammation and induction of apoptosis as reported earlier [5,30]. Sustained proliferative signaling has also been recognized as one of the hallmark of cancer. In the present study, there was an up regulation of PCNA positive nuclei, a proliferative marker after 16 weeks of Fe-NTA administration in DEN initiated and Fe-NTA promoted group as compared to control group. CH interfered in cellular proliferation resulting in inhibition of growth and proliferation of tumor cells as well as induction of apoptosis. Similarly PCNA was down regulated by prophylactic treatment of CH at both the doses suggesting its anti tumor and anti proliferative potential. TNF-a and IL-6 are pro inflammatory marker cytokines of inflammation which play an essential role in various diseases including cancer because of their mutagenic and proliferative capacity. Therefore, TNF-a plays an essential role in alteration and later survival of altered cells which result into tumor development by promoting the accumulation of mutations. It also stimulates the activation of other inflammatory mediators such as ROS via oxidative stress-responsive genes which augments and extends inflammation [31]. Chronic exposure of Fe-NTA for 16 weeks in tumor group elevated the expression of TNF-a and IL-6 as compared to control group. Prophylactic treatment of CH down regulated the expressions dose dependently further supports its anti-inflammatory role. NFkB is a pro-tumorigenic and has proliferative potential besides found constitutively active in various types of cancers resulting in up-regulation of anti apoptotic genes. It is being implicated in RCC as well and henceforth emerges to be an attractive target for the development of novel therapeutics against RCC [32]. COX-2, a key enzyme in prostaglandin synthesis is said to be over expressed in various cancers including RCC [33] and is associated with metastases hence a positive prognostic factor in metastatic RCC. Thus, inhibition of the inflammatory pathway proteins is now accepted as an important tool to prevent

carcinogenic development. Fe-NTA exposure was found to activate inflammatory pathway proteins in renal tissues in our study which is consistent with earlier reports [26]. We found in the present study that NFkB, COX-2 and iNOS were up regulated in tumor group which depicts renal inflammation and tumor promotion. However, administration of CH at both the doses attenuated pro inflammatory cytokines and inflammatory marker proteins respectively. Apoptosis is commonly inactivated in tumor cells where it permits continued survival, progression and resistance to therapy. Among the best-studied regulators of the apoptotic pathway is the Bcl-2 family of proteins. Increased expression of Bcl-2 has been reported in virtually all cancer types including RCC [34]. It has been hypothesized that the principal behind the mechanism of chemoprevention of cancer by antioxidants is via attenuation of DNA damaging free radicals and activation of apoptosis in tumor cells directly [28]. Intrinsic apoptotic pathway proteins were studied in the present study because activation of apoptosis in cancer cells is one of the best achieved treatment strategy against cancer. We found that there was down regulation of bax, caspase-9 and caspase-3 in DEN initiated and Fe-NTA promoted group and up regulation of Bcl-2. However CH treatment up regulated the expression of bax, caspase-9, capase-3 proteins and simultaneously down regulated Bcl-2 at both the doses resulting in activation of apoptosis. Therefore, in the present study CH treatment scavenged the ROS, inhibited proliferation, inflammation, decreased tumor incidences and triggered apoptosis. 5. Conclusion In conclusion, the present study suggests that CH suppresses pre neoplastic lesion formation as a result of DEN exposure and chronic Fe-NTA administration in rats, probably by multiple mechanisms such as altered the redox balance to prevent oxidative stress, reduced cell proliferation through control of expression of key regulatory proteins, inflammatory mediators and induced apoptosis by altering the balance between pro and anti apoptotic proteins. Chrysin may therefore be considered a potential compound for both cancer prevention and treatment. Our results provide important insights into the molecular mechanisms involved in the anti cancer activity of chrysin. But further studies are needed to fully elucidate the mechanism involved in cell death. Funding SS is thankful to University Grants Commission, New Delhi India under SAP (Special Assistance Programme) for Departmental Research Support II with grant number 3-76/2009 (SAP-II) for the award of project to carry out the study. SR is thankful to ICMR for the award of senior research fellowship. Conflict of interest The authors declare that they have no conflict of interest. References [1] K. Sahin, B. Cross, N. Sahin, K. Ciccone, S. Suleiman, A.O. Osunkoya, et al., Lycopene in the prevention of renal cell cancer in the TSC2 mutant Eker rat model, Arch. Biochem. Biophys. 15 (2015) 36e39. [2] T. Shi, L.S. Liou, P. Sadhukhan, Z.H. Duan, A.C. Novick, J.G. Hissong, et al., Effects of resveratrol on gene expression in renal cell carcinoma, Cancer Biol. Ther. 3 (2004) 882e888. [3] A. Bullock, D.F. McDermott, M.B. Atkins, Management of metastatic renal cell carcinoma in patients with poor prognosis, Cancer Manag. Res. 2 (2010) 123e132. [4] F.A. Aguilar-Alonso, J.D. Solano, C.Y. Vargas-Olvera, I. Pacheco-Bernal, rez, M.E. Ibarra-Rubio, MAPKs’ status at early stages of renal T.O. Pariente-Pe

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