p21 up-regulation and suppresses metastasis in 7,12-dimethylbenz(α)anthracene-induced rat mammary carcinoma

Available online at www.sciencedirect.com

ScienceDirect Journal of Nutritional Biochemistry xx (2014) xxx – xxx

Tangeretin, a citrus pentamethoxyflavone, exerts cytostatic effect via p53/p21 up-regulation and suppresses metastasis in 7,12-dimethylbenz(α)anthracene-induced rat mammary carcinoma☆ Lakshmi Arivazhagan, Subramanian Sorimuthu Pillai⁎ Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600 025, India

Received 14 February 2014; received in revised form 2 June 2014; accepted 12 June 2014

Abstract Breast cancer is the most commonly diagnosed cancer among women worldwide, which is characterized by unregulated cell growth and metastasis. Many bioactive compounds of plant origin such as tangeretin have been shown to possess potent antioxidant and anticancerous properties. In the present study we have investigated the chemotherapeutic effect of tangeretin against 7,12-dimethylbenz(α)anthracene (DMBA)-induced rat mammary carcinogenesis and studied its underlying mechanism of action. Breast cancer was induced by “air pouch technique” with a single dose of 25mg/kg of DMBA. Tangeretin (50 mg/kg) was administered orally for four weeks. Remarkably, tangeretin treatment controlled the growth of cancer cells which was clearly evidenced by morphological and histological analysis. Also, serum levels of estradiol, progesterone and prolactin; lipid bound sialic acid and total sialic acid and the tissue levels of nitric oxide and protein carbonyls of cancer induced animals were decreased upon tangeretin treatment. Staining of breast tissues for nucleolar organizer regions, mast cells, glycoproteins, lipids and collagen showed that tangeretin treatment to breast cancer induced rats significantly reduced tumorigenesis. Oral tangeretin treatment also effectively reduced the tumor cell proliferation markers such as PCNA, COX-2 and Ki-67. Further, tangeretin treatment arrested the cancer cell division at the G1/S phase via p53/p21 up-regulation and inhibited metastasis by suppressing matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor. Taken together, the data provides new evidence on the mechanism of action of tangeretin in breast cancer and hence extends the hypothesis supporting its potential use in chemotherapy. © 2014 Elsevier Inc. All rights reserved. Keywords: Breast cancer; DMBA; Tangeretin; G1/S arrest; Metastasis; Chemotherapeutic agent

1. Introduction Breast cancer is one of the most common malignancies in women with its incidence increasing alarmingly in both industrialized and developing countries. The highest rates of breast cancer are reported in North America, Australia and northwest Europe, and the lowest rates in Africa, Asia and the Middle East [1]. In India, breast cancer is the second most common cancer next to cervical cancer and accounts for 7% of global burden [2]. The general risk factors include nuliparity, early menarche, late age at first birth, late menopause, hormone replacement therapy, postmenopausal obesity, extended use of oral contraceptives, family history and previous benign breast disease [3]. Biochemical and molecular genetic studies have suggested the fact that breast cancer starts by subtle molecular changes within a cell, called the induction phase followed by the oncogenesis process comprising three stages: initiation, promotion and malignant progression [4]. These stages bring about numerous genetic modifica☆ Conflict of interest: The authors declare no conflict of interest. ⁎ Corresponding author at: Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600 025, India. Tel.: +91 9443026668. E-mail address: [email protected] (S. Sorimuthu Pillai).

http://dx.doi.org/10.1016/j.jnutbio.2014.06.007 0955-2863/© 2014 Elsevier Inc. All rights reserved.

tions that progressively accumulate in the neoplasic cells that cause malignant neoplasm, most often leading to disseminated disease and eventually cause mortality. However, most patients die of distant metastases that are frequently unresponsive to cancer therapy. In order to metastasize, cells need to be able to migrate and invade into the surrounding tissue, intravasate in to the blood vessel or lymphatic system, survive in circulation, extravasate and finally proliferate at a distant site [5]. Current treatment for breast cancer includes surgery, radiotherapy, chemotherapy, hormone therapy and radiotherapy coupled with chemotherapy. However, chemotherapy causes deleterious side effects for which, treatment with plant based bioactive compounds as chemotherapeutic drugs with minimal side effects is desired. Polycyclic aromatic hydrocarbons are one of the major environmental contaminants from oil furnaces and diesel engines and are well recognized for their capacity to produce free radicals that may induce various diseases including cancer. 7,12-dimethylbenz(α) anthracene (DMBA) is an important carcinogen that induces mammary tumors in rodents which mimics human breast cancer morphologically, clinically and histopathologically [6]. The DMBA induced tumors are generally hormone dependent adenocarcinomas [7]. DMBA carcinogen interacts with rapidly proliferating cells in the

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Table 1 Effect of Tangeretin on body weight gain and tumor weight of control and experimental animals. Groups

Initial body weight (g)

Final body weight (g)

Body weight gain (%)

Tumor weight (g)

Control Control + Tangeretin DMBA DMBA + Tangeretin

106.86±3.32 104.64±3.35 105.83±3.94* 103.61±3.07#

206.74±5.12 217.52±5.62 170.43±4.82* 189.51±3.12#

35.51±3.88 40.38±4.21 22.45±2.71* 27.42±3.72#

10.33±0.51* 2.94±0.16#

Values are given as mean±S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the group as follows: *Control rats; #DMBA induced rats. Values are statistically significant at Pb.05.

terminal end buds, forming DNA adducts, transforming the normal terminal end bud cells to malignant cells which is classically known as “the window of susceptibility” [8]. Flavonoids are a large family of polyphenolic compounds synthesized by plants as secondary metabolites [9]. Flavonoids exhibit a multitude of biological activities such as antioxidant, anti-inflammatory, antiallergic, vasodilatory and anticarcinogenic properties [10]. Flavonoids are indicated for cancer therapy due to their ability to suppress cancer cell proliferation, induce cell-cycle arrest and promote apoptosis [11]. Many of the biological effects of flavonoids appear to be related to their ability to modulate cell-signaling pathways, rather than their antioxidant activity. Due to their abundance in dietary products and their potential pharmacological and nutritional effects, the flavonoids are of considerable interest for drug discovery as well as health food supplements. Citrus fruit juices are a rich source of flavonoids and have been known to possess a number of medicinal properties [12]. Tangeretin (5, 6, 7, 8, 4′-pentamethoxyflavone), a polymethoxylated flavonoid concentrated in the peel of citrus fruits possesses several biological activities including the ability to enhance gap junctional intercellular communication [13], cytostatic [14], anti-metastatic [15], apoptotic [16] and neuroprotective [17] properties. Tangeretin has also been shown to be a potent inhibitor of xenobiotic-induced genotoxicity in vitro [18]. More recently, we have reported the chemotherapeutic

effect of tangeretin in the breast tissue of DMBA induced rat mammary carcinoma [19]. In the present study, an attempt has been made to investigate the mechanism of action of tangeretin and its anti-metastatic property in DMBA-induced rat mammary carcinogenesis. 2. Materials and methods 2.1. Chemicals DMBA was purchased from Sigma chemical company (St. Louis MO, USA) and tangeretin from Indofine Chemical, USA. All other chemicals used were of analytical grade procured from local commercial sources. 2.2. Experimental animals Virgin female Wistar rats, 7 weeks of age were purchased from Tamilnadu Veterinary and Animal Sciences University, Madhavaram, Chennai, India, and were used in the present study. Rats were housed spaciously in individual cages and maintained under standard experimental conditions: temperature 25±1°C, relative humidity 60±5% and 12±1 h (light/dark cycle) and fed with commercially available balanced pellet diet (Amrut laboratory Animal Feed, Bangalore, India) and water ad libitum. The animals were acclimatized for one week prior to the initiation of experiments. The experimental design was performed in accordance with the current ethical norms approved by the Ministry of Social Justice and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines (IAEC No: 01/059/09). 2.3. Experimental design The rats were divided into four groups with a minimum of six rats each as follows:

• Group I: normal control rats administered with 1 ml of phosphate-buffered saline (PBS) containing 0.1% dimethyl sulfoxide (DMSO) • Group II: control rats orally administered with tangeretin (50 mg/kg dissolved in 1 ml of PBS containing 0.1% DMSO per rat) for 4 weeks • Group III: DMBA induced breast cancerous rats • Group IV: DMBA induced breast cancerous rats orally administered with tangeretin (50 mg/kg dissolved in 1 ml of PBS containing 0.1% DMSO/rat) for 4 weeks

Fig. 1. Representative images showing control rat (A); DMBA induced mammary tumor bearing rat (B); tangeretin treated DMBA induced rat (C); structure of tangeretin (D); gross pathology of tumor from DMBA induced rat (E); gross pathology of tumor from tangeretin treated DMBA induced rat (F).

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2.4. Carcinogen treatment Mammary tumors were induced by DMBA using the “airpouch technique” as described by Arun et al. [20] with slight modifications. Sterile air (1–2 ml) was carefully injected subcutaneously just beneath the mammary fat pad so as to produce a pouch containing sterile air. The air inside was allowed to remain for a day to form a pouch. A single dose of DMBA (25 mg/kg per rat) in 0.5 ml of corn oil was carefully injected into the air pouch. 2.5. Drug treatment All rats were palpated every week to monitor the onset of tumorigenesis. Tumor mass and size was stabilized 90 days after the initiation with DMBA. Tangeretin (50 mg/kg dissolved in 1 ml of PBS containing 0.1% DMSO/rat) was administered orally for 4 weeks. After the experimental period, all animals were fasted overnight and sacrificed by sodium pentothal anesthesia followed by cervical decapitation. Blood was collected with and without anticoagulant and the serum was centrifuged at 5000 rpm for 15 min to obtain a clear supernatant and stored at −80°C until its use for further biochemical analysis. Mammary tissues from control and experimental groups of rats were immediately excised, washed in ice-cold PBS to remove the blood stains, blotted, weighed and homogenized in Tris-HCL buffer (0.1 M, pH 7.4) using a Teflon homogenizer to prepare 10% (w/v) tissue homogenate. This homogenate was centrifuged at 12,000×g for 30 min at 4°C to obtain a clear supernatant. 2.6. Body weight and tumor volume The total body weight gain of the control and experimental animals was recorded periodically throughout the experimental period. The tumor volume was estimated according to the method of Geran et al. (1972) [21]. Briefly, the resultant solid tumor was considered to be prelate ellipsoid with one long axis and two short axes. The two

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short axes were measured with vernier calipers. The tumor volume was calculated using the formula:   Tumor volume ¼ length ðcm Þ  width 2 ðcm Þ =2

2.7. Biochemical analysis 2.7.1. Evaluation of endocrine derangement hormonal levels Serum levels of estradiol, progesterone and prolactin were measured using enzyme linked immunosorbent assays (ELISA) kits procured from UBI MAGIWELL (USA). 2.7.2. Evaluation of markers of tumorigenicity The serum levels of total sialic acid (TSA) and lipid bound sialic acid (LSA) were evaluated according to the method of Plucinsky et al. [22]. 2.7.3. Assay for protein carbonylation and nitric oxide (NO) measurement Protein carbonylation was measured by 2,4-dinitrophenylhydrazine coupling method described by Levine et al. (1990) [23]. NO levels in the breast tissues were measured by the evaluation of total nitrate and nitrites using a method described by van Bezooijen et al. (1998) [24]. 2.8. Histopathology The breast tissue was immediately fixed in 10% neutral buffered formalin, embedded in paraffin, 5-μm section was cut using a microtome and then rehydrated with xylene and graded series of ethanol. The specimens were then stained with haematoxylin and eosin (H&E). The H&E stained breast specimens were examined by a pathologist to histopathologically classify the tumors as described by Russo et al. (1990) [25].

Fig. 2. Panel A shows the effect of tangeretin on the levels of Prolactin, Estradiol and Progesterone in serum of control and experimental animals. Prolactin is represented as ng/ml, estradiol as pg/ml and progesterone as ng/ml. Panel B shows the effect of tangeretin on the levels of TSA and LSA in serum of control and experimental animals. TSA and LSA are represented as ng/dl. Figure 2C shows the effect of tangeretin on the levels of protein carbonyl and NO in the breast tissue of control and experimental animals. Protein carbonyl is represented as nmol/ml and NO is represented as nmol/mg protein. Values are given as mean±S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the group as follows: *Control rats; #DMBA induced rats. Values are statistically significant at Pb.05.

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2.9. Pathological studies

2.11. Western blotting analysis

Five-micrometer sections of the paraffin-embedded tissues were cut using a microtome and then rehydrated with xylene and graded series of ethanol. The specimens were then stained accordingly as mentioned below. Argyrophilic nucleolar organizer regions (AgNORs) staining was performed according to the method of Ploton et al. [26]. Histochemical analysis of mast cells by toluidine blue staining was carried out by the method of Migliaccio et al. [27]. Glycoprotein content in the breast tissues was stained with Periodic Acid Schiff (PAS) base according to the method of Yamabayashi (1987) [28]. Fat accumulation was studied by Oil Red O staining performed according to the method of Young et al. [29]. Collagen deposition in the tissue was analysed by staining with Masson’s tri-chrome and Picrosirius red.

The breast tissues were removed from animals, resuspended in Tris/sucrose buffer with protease inhibitors and homogenized immediately. The protein was quantified by Bradford method and 20 μg of protein was separated by electrophoresis on 10% SDS-PAGE gels. It was then transferred to nitrocellulose membrane and blocked with 5% BSA in 0.1% TBST (Tris-buffered saline with 0.1% Tween-20) for 3 h. The membranes were incubated overnight with the respective primary antibodies procured from Santa Cruz Biotech, USA. The membranes were washed with 0.1% TBST for 5 min each and probed with secondary antibody (horse radish peroxidase conjugated secondary antibody) for 1 h at room temperature. The bands in the membranes were detected using the ECL Plus Western Blotting Detection System (Amersham Biosciences, UK). 2.12. Semiquantitative and quantitative PCR analysis

2.10. Transmission electron microscopy (TEM) A portion of the breast tissue from control and experimental groups of rats was fixed in 3% gluteraldehyde in sodium phosphate buffer (200 mM, pH 7.4) for 3 h at 4°C for ultrastructural studies. Tissue samples were washed with the same buffer, post-fixed in 1% osmium tetroxide and sodium phosphate buffer for 1 h at 4°C. Then, the samples were washed with the same buffer at 4°C, dehydrated with graded series of ethanol and embedded in Araldite. Thin sections were cut with ultramicrotome using a diamond knife, mounted on a copper grid and stained with 2% uranyl acetate and Reynolds lead citrate [30]. The sections were screened in JEOL JEM 1400 Transmission Electron Microscope at 80 kV. The micrographs were taken using Olympus Keenview CCD camera attached to the microscope.

The breast tissues were snap frozen and stored at −80°C prior to RNA isolation. Samples were homogenized with TRIzol (Invitrogen, USA) and mRNA was extracted according to the manufacturer's protocol. First-strand cDNA was synthesized using SuperScript First-Strand Synthesis System (Invitrogen, USA) and semiquantitative and quantitative PCR was done with GAPDH as the internal control. 2.13. Immunofluorescence microscopic analysis Paraffin embedded tissue sections were processed as described earlier and were then immunostained with the primary antibodies for PCNA, COX-2 and vascular endothelial growth factor (VEGF) from Santa Cruz Biotech, USA at a concentration of 1

Fig. 3. Representative images showing the pathological change in the mammary tissues of control and experimental group of animals is represented in Panels A–L. Photomicrographs of histopathological changes (A–D) in the mammary tissues of control (A) and tangeretin control (B) rats showing normal ductal epithelial architecture of the terminal epithelial buds; Mammary tissues of DMBA induced rat (C) showing invasive ductal carcinoma with abnormal cellular proliferation and infiltrated ducts; mammary tissues of tumor bearing rats treated with tangeretin (D) showing improved ductal architecture. AgNORs (E-H) and toluidine blue (I–L) staining showing normal levels in control (E and I) and tangeretin control (F and J) animals; the DMBA administered rats (G and K) showed increased number of the AgNORs dots/nuclei and increased mast cell accumulation when compared with control rats. However, oral tangeretin treatment to cancer induced rats showed decreased number of AgNORs dots/nuclei levels and decreased mast cell accumulation (H and L) in the breast tissues (magnification at 20x).

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μg/ml with 3% BSA in TBS and incubated overnight at 4°C. After washing the slides thrice with TBS the sections were then incubated with the respective FITC conjugated secondary antibodies (Bangalore Genei, India), diluted 1:40 with 3% BSA in TBS and incubated for 2 h at room temperature. The sections were then visualized by confocal laser scanning microscopy (Leica TCS-SP2 XL, Germany) using appropriate wavelength (DAPI: excitation 350 nm; emission 470 nm; FITC excitation 488 nm; emission 520).

2.14. Immunohistochemical analysis Paraffin embedded tissue sections were processed as described earlier and were then immunostained with the primary antibodies for p53, p21 and Ki-67 from Santa Cruz Biotech, USA at a concentration of 1 μg/ml with 3% BSA in TBS and incubated overnight at 4°C. After washing the slides thrice with TBS the sections were then incubated with the respective HRP conjugated secondary antibodies (Bangalore Genei, India), diluted 1:200 with 3% BSA in TBS and incubated for 2 h at room temperature. Sections were then washed with TBS and incubated for 5–10 min in a solution of 0.02% diaminobenzidine containing 0.01% hydrogen peroxide. Counter staining was performed using hematoxylin, and the slides were visualized under a light microscope (Nikon XDS-1B).

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3. Results 3.1. General observations The body weight gain of control and experimental groups of rats is summarized in Table 1. The average initial body weight of animals in each group was similar. DMBA administration significantly decreased the whole body weight, however tangeretin treatment to DMBA induced animals prevented the weight loss in animals. The whole body weight of control and tangeretin control animals increased significantly and remained nearly similar throughout the experimental period. Fig. 1 shows the structure of tangeretin and mammary gland whole mounts with lesions in rats with mammary tumors induced by DMBA. The DMBA induced rats had large tumors which were adenocarcinomas, whereas tangeretin treatment to DMBA induced rats showed significantly smaller tumors, proving the chemotherapeutic effect of tangeretin.

2.15. Gelatin zymographic analysis of matrix metalloproteinases (MMPs)

3.2. Effect of tangeretin on the markers of endocrine derangement Gelatin zymography for the detection of MMPs was performed as described by Stetler-Stevenson et al. [31]. Briefly, 40 μg of protein from homogenate was loaded into each well on 8% native polyacrylamide gel with 0.1% gelatin as substrate. Separation was carried out at 4°C with a constant current of 100 V.

2.16. Statistical analysis Statistical analysis was performed using SPSS 16.0 (SPSS, Inc., Chicago) statistical package. The results were expressed as mean±S.E.M. Analysis of variance (ANOVA) followed by post hoc test LSD was used to correlate the difference between the variables. Values were considered statistically significant if Pb.05.

Alterations in the level of endocrine derangement markers are shown in Fig. 2A. As compared to the control group, there was a significant increase in the prolactin and estradiol levels and significant decrease in the progesterone levels in DMBA induced group. Alternatively, there was a significant decrease in the prolactin and estradiol and significant increase in the progesterone levels in the DMBA induced animals treated with tangeretin. However, the levels of these hormones remained normal in the control and tangeretin control animals.

Fig. 4. Representative images showing the PAS staining (A-D), Oil Red O staining (E–H) and ultrastructural changes (I–N) in the breast tissues of control and experimental animals. PAS and Oil Red O staining showing normal levels in control (A and E) and tangeretin control (B and F) animals; the DMBA administered rats (C and G) showing increased glycoprotein expression and lipid accumulation when compared with control rats. However, oral tangeretin treatment to cancer-induced rats showing decreased glycoprotein and lipid accumulation (D and H) in the breast tissues (original magnification ×20). Ultrastructural studies showing lesser number of lipid droplets in control (I) and tangeretin control (J) animals; increased number of lipid droplets in DMBA induced (K and M) animals; decreased number of lipid droplets in tangeretin treated DMBA induced animals (L and N).

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3.3. Effect of tangeretin on the markers of tumorigenicity Fig. 2B shows the effect of tangeretin on the levels of serum tumor markers in the control and experimental groups of animals. The markers of tumorigenicity examined in the DMBA group showed a significant increase in the TSA and LSA levels. Tangeretin treatment to DMBA induced animals significantly decreased both the TSA and LSA levels. However, the sialic acid content remained normal in the control and tangeretin control animals. 3.4. Effect of tangeretin on DMBA induced free radicals Fig. 2C shows that DMBA induced animals showed a significant increase in the levels of free radicals such as protein carbonyl and NO and that tangeretin treatment reduced the free radical levels significantly. However, the activities of these oxidative stress markers were not altered in the control and tangeretin control animals. 3.5. Pathological observations in the mammary gland The pathological changes in the mammary tissues of control and experimental group of animals are represented in Fig. 3A–L. The hematoxylin and eosin stained (Fig. 3A–D) control and tangeretin alone

treated animals showed normal ductal epithelial architecture. DMBA-induced tumor bearing animals showed altered ductal epithelial lining indicating invasive ductal carcinoma. Tumor bearing animals treated with tangeretin showed improved ductal architecture. Fig. 3E–H and I–L shows the effect of tangeretin on the histochemical analysis of AgNORs staining and Toluidine blue staining respectively. The DMBA administered animals showed significantly increased number of AgNORs dots/nuclei when compared with control rats. However, oral tangeretin treatment to cancer induced rats showed decreased area of AgNORs dots/nuclei levels (Fig. 3 E-H). Toluidine blue staining showed a significant increase in the mast cell population (Fig. 3I–L) in the mammary tissues of DMBA induced animals. However, tangeretin treatment reduced the mast cell accumulation in the mammary tissue. Control and tangeretin alone treated groups of rats exhibited no AgNORs and inflammatory mast cells.

3.6. Effect of tangeretin on glycoprotein and lipid accumulation The glycoprotein and lipid accumulation in the mammary tissues of control and experimental group of animals are represented in Fig. 4A–N. PAS staining (Fig. 4A–D) showed the excess accumulation of glycoproteins in the mammary tissue of DMBA induced animals. Tangeretin treatment significantly reduced the glycoprotein

Fig. 5. Effect of tangeretin on the protein expression of PCNA, COX-2, Ki-67, p53, p21, cyclin D1, cyclin E, CDK4 and CDK2 in breast tissue of rats is shown in Figures 5 A and B. GAPDH was used as an internal standard. Lane 1: breast tissue of control animals; lane II: breast tissue of tangeretin control rats; lane III: breast tissue of DMBA induced rats; lane IV: breast tissue of induced rats treated with tangeretin. Values are given as mean±S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the group as follows: *Control rats; #DMBA induced rats. Values are statistically significant at pb0.05.

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accumulation proving its anti-neoplastic property. Fig. 4E–H and I–N shows the effect of tangeretin on the accumulation of lipids by Oil Red O staining and ultrastructural observations (TEM) respectively. The DMBA administered animals showed significantly increased accumulation of lipids when compared with control rats. However, oral tangeretin treatment to cancer induced rats showed decreased lipid accumulation (Fig. 4E–H). Ultrastructural studies also showed a significant increase in the accumulation of lipid droplets (Fig. 4I–N) in the mammary tissues of DMBA induced animals. However, tangeretin treatment reduced the lipid accumulation in the mammary tissue further confirming its anti-invasive property. Control and tangeretin alone treated groups of rats exhibited very little glycogen and lipid accumulation. 3.7. Western blot analysis Fig. 5 shows the immunoblot analysis of the expression of cell proliferation markers (Fig. 5A) such as PCNA, COX-2 and Ki-67 and cell cycle markers (Fig. 5B) such as p53, p21, Cyclin D1, Cyclin E, cyclin-dependent protein kinase (CDK) 4 and CDK2 in mammary tissues of control and experimental groups of rats. There was a significant increase in the levels of PCNA, COX-2, Ki-67, Cyclin D1,

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Cyclin E, CDK4 and CDK2 and significant decrease in the levels of p53 and p21 in DMBA induced animals. However, the levels of these genes were altered significantly in the mammary tissues of DMBA induced animals upon tangeretin treatment. Control and tangeretin alone treated animals showed normal expression of these genes. 3.8. Semi-quantitative and quantitative PCR analysis Fig. 6 shows the semi-quantitative mRNA expression pattern (Fig. 6A) and quantitative mRNA expression pattern (Fig. 6B) of cell cycle markers such as p53, p21, Cyclin D1 and Cyclin E in mammary tissues of control and experimental groups of rats. The semi-quantitative and quantitative PCR analysis of p53, p21, Cyclin D1 and Cyclin E revealed that the mRNA expression was consistent with the protein expression pattern observed for these genes. 3.9. Immunofluorescence analysis Fig. 7A and B show the immunofluorescence expression pattern of PCNA and COX-2 respectively in control and experimental groups of rats. There was a significant increase in the levels of PCNA and COX-2

Fig. 6. Effect of Tangeretin on the mRNA expression of p53, p21, cyclin D1 and cyclin E in breast tissue of rats is shown in Figure 6. GAPDH was used as an internal standard. The semi-quantitative and quantitative PCR analysis revealed that the mRNA expression pattern was consistent with the protein expression pattern. Values are given as mean±S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the group as follows: *Control rats; #DMBA induced rats. Values are statistically significant at Pb.05.

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in DMBA induced animals. However, the levels of these markers were decreased significantly in DMBA induced animals treated with tangeretin. Control and tangeretin alone treated animals showed very low expression of these markers.

3.10. Immunohistochemical analysis Fig. 8A–C shows the immunohistochemical expression pattern of Ki-67, p53 and p21 respectively in control and experimental groups of

Fig. 7. Immunofluorescence analysis of expression pattern of PCNA (7A) and COX-2 (7B) in the breast tissue of control and experimental animals. PCNA and COX-2 proteins in breast tissue of control and tangeretin treated control animals showing normal levels of expression; PCNA and COX-2 proteins in breast tissue of DMBA induced rats showing increased expression; PCNA and COX-2 proteins in breast tissue of tumor induced rats treated with tangeretin showing comparatively decreased expression. Values are given as mean±S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the group as follows: *Control rats; #DMBA induced rats. Values are statistically significant at Pb.05.

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rats. There was significant increase in the levels of Ki-67 and significant decrease in the levels of p53 and p21 in DMBA induced animals. However, the levels of these markers were altered significantly upon tangeretin treatment. Control and tangeretin alone treated animals showed normal expression of these markers. 3.11. Metastasis analysis Fig. 9A–C show the expression pattern of metastasis markers such as MMP-2, TIMP-2, MMP-9 and VEGF by Western blotting, qPCR and gelatin zymography respectively in control and experimental groups of rats. There was significant increase in the levels of MMP-2, MMP-9 and VEGF expression and significant decrease in the levels of TIMP-2 in DMBA induced animals. However, the levels of these genes were altered significantly in DMBA induced animals treated with tangeretin. Control and tangeretin alone treated animals showed normal expression of these metastasis markers. Fig. 10A–C show the immunofluorescence analysis of VEGF and staining for collagen with Masson’s tri-chrome and Picrosirius red respectively in control and experimental groups of rats. There was significant increase in the expression of VEGF and irregular collagen distribution in DMBA induced animals. However, the levels of VEGF and collagen distribution pattern were altered significantly in DMBA induced animals treated with tangeretin. Control and tangeretin alone treated animals showed very low expression of VEGF and collagen. The possible mechanism of action of tangeretin during DMBA induced mammary carcinogenesis is schematically depicted in Fig. 11.

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4. Discussion Dysregulated proliferation is one of the main mechanisms for breast tumorigenesis which involves different stages viz. initiation, promotion and progression of tumor growth, thereby increasing the tumor burden, and initiating metastasis. This dysregulation mainly occurs due to disrupted G1/S phase cell cycle transition during the development of malignant neoplasm [32]. Current treatment methods against breast cancer are limited by the number of drugs and their side effects which warrants the need for new chemotherapeutic agents against breast cancer. Even though tangeretin has been shown to possess anticancer property in many cancer models there is no data available in the literature regarding its chemotherapeutic effect and mechanism of action against breast cancer. Therefore, the present study was aimed to evaluate the cytostatic activity of tangeretin during DMBA induced rat mammary carcinogenesis. To accomplish the goal, an animal model comprising of control, tangeretin control, DMBA-induced and DMBA induced group treated with tangeretin. Overall, the results of the current study have demonstrated that tangeretin suppressed breast cancer proliferation by up-regulating p53/p21 proteins and inducing G1/S phase cell cycle arrest. Chemotherapy for cancer requires a long-term administration of the drug which requires that the compound possesses little or no toxicity. Tangeretin was well tolerated by rats in the mammary carcinogenesis model as there was no observable change in the gross behavior and no significant weight loss as shown by previous studies [33]. Tangeretin has improved bioavailability due to the presence of five methoxylated

Fig. 8. Immunohistochemical analysis of expression pattern of Ki-67 (8A), p53 (8B) and p21 (8C) in the breast tissue of control and experimental animals. Ki-67, p53 and p21 in the breast tissue of control and tangeretin treated control animals showing normal levels of expression; breast tissue of DMBA induced rats showing increased expression of Ki-67 and decreased expression of p53 and p21 proteins; breast tissue of tumor induced rats treated with tangeretin showing comparatively decreased expression of Ki-67 and increased expression of p53 and p21 proteins. Values are given as mean±S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the group as follows: *Control rats; #DMBA induced rats. Values are statistically significant at Pb.05.

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Fig. 9. Effect of tangeretin on the protein and mRNA expression pattern of metastatic oncogenes such as MMP2, TIMP2, MMP9 and VEGF in breast tissue of rats is shown in Figures 9 A and B respectively. Figure 9C shows the gelatin zymography for matrix metalloproteinase 2 and 9 in control and experimental groups of rats. MMP2, TIMP2, MMP9 and VEGF in the breast tissue of control and tangeretin treated control animals showing normal levels of expression; breast tissue of DMBA induced rats showing increased expression of MMP2, MMP9, VEGF and decreased expression of TIMP2; breast tissue of tumor induced rats treated with tangeretin showing comparatively decreased expression of MMP2, MMP9, VEGF and increased expression of TIMP2. Values are given as mean±S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the group as follows: *Control rats; #DMBA induced rats. Values are statistically significant at Pb.05.

groups that contribute to its increased metabolic stability and membrane transport in the intestine/liver. Walle has also shown that methoxylated flavones possess superior anti-cancer properties compared to hydroxylated flavones [34]. Methoxylated compounds inhibit the bioactivation of polycyclic aromatic hydrocarbons by directly binding to DNA and proteins; diminishing the CYP1A1/1B1 transcription and through aromatase inhibition at the initiation stage. At the promotion stage, the proliferation of cancer cells, but not normal cells, is inhibited [35]. This shows that the chemotherapeutic property of tangeretin may be rendered in part due to the presence of polymethoxylated groups. The administration of DMBA was associated with the development of mammary carcinomas, elevated levels of markers of tumorigenicity, endocrine derangement and oxidative stress. Prolactin plays a major role in mammary cancer pathogenesis and progression: intact prolactin molecule is responsible for lactogenesis, whereas its cleavage product (16 kDa fragment) stimulates uncontrolled cell division in the epithelial tissues of the mammary gland [36]. The underlying carcinogenic effects of estrogen include stimulation of DNA synthesis, promotion of cell division and induction of synthesis of various peptide growth factors. Progesterone exhibits cytoprotective effects against mammary dysplasia [37]. Our findings show increased levels of prolactin and estradiol and decreased levels of progesterone in the serum of DMBA induced animals which are supported by previous findings [38]. However, tangeretin treatment to DMBA induced animals significantly altered the serum levels of these hormones. Sialic acid, a family of acetylated derivatives of neuraminic acid, is found either as TSA or LSA in glycoproteins and glycolipids. Its level rapidly increases in breast cancer [39,40]. The increase in the serum

levels of TSA and LSA after the injection of DMBA could be due to an increase in cell number, henceforth, the levels are indicative of metastasis. However, tangeretin treatment significantly altered the sialic acid content. Breast cancer cells have increased ROS generation which promotes tumor growth and metastasis by enhancing invasive, angiogenic and migratory capacities of tumor cells [41]. ROS may damage all types of biological molecules including proteins, lipids, or DNA. However, proteins are possibly the most immediate targets because they are the most often used biomolecules. Protein carbonyl content is widely measured as a marker of protein oxidation and accumulation of protein carbonyls has been observed in several human diseases including cancer [42]. The high levels of NO and protein carbonylation in the DMBA group agree with that of previous reports [43]. Tangeretin treatment to DMBA induced animals significantly altered the levels of these oxidative stress markers in the breast tissue which may be due to its antioxidant property. Histopathological examination of mammary tissues in DMBAinduced animals showed altered ductal epithelial lining indicating invasive ductal adenocarcinoma with increased mitotic activity. Tumor bearing animals treated with tangeretin showed regression in tumor with improved ductal architecture indicating the chemotherapeutic effect of tangeretin. The control and tangeretin alone treated animals showed normal ductal epithelial architecture suggesting the non-toxic as well as beneficial role of tangeretin. Nucleolar organizer regions (NORs) that bind to silver ions through a set of acidic, non-histone proteins are chromosomal loops of DNA involved in the ribosomal synthesis [44]. Malignant cells exhibit larger number of AgNOR protein patterns indicating increased cell proliferation [45]. Tangeretin treatment significantly reduced the amount of AgNORs and, hence, served as an inhibitor of cell proliferation. In several types

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Fig. 10. Figure 10 shows the immunofluorescence analysis of VEGF (A), Masson’s tri-chrome (B) and Picrosirius red (C) staining for collagen in the breast tissue of control and experimental animals. Breast tissue of control and tangeretin treated control animals showing normal levels of expression of VEGF and regular collagen distribution; breast tissue of DMBA induced rats showing increased expression of VEGF expression and irregular collagen distribution; VEGF expression and collagen distribution were altered significantly upon tangeretin treatment to DMBA induced animals. Values are given as mean±S.E.M. for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the group as follows: *Control rats; #DMBA induced rats. Values are statistically significant at Pb.05.

of cancers, mast cells have been shown to have important proangiogenic effects and induce tumor growth [46]. Increase in the number of inflammatory mast cells creates a tumor microenvironment that reflects a persistent inflammatory state, thereby fostering tumor progression, angiogenesis and metastasis [47]. Our data shows the presence of increased mast cell number in the breast tissues of

DMBA-induced animals, inducing inflammation and tangeretin treatment significantly reduced the mast cell mediated inflammation by down-regulating MMPs and COX-2. The PAS reaction is a non-specific indicator for glycogen, which is present in the basement membranes, including those of blood vessels and tumor cells involved in the neoplastic process and distant

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Fig. 11. Mechanism of action of tangeretin. Schematic representation of the possible mechanism of action of tangeretin during DMBA-induced mammary carcinogenesis. DMBA induction leads to mammary tumorigenesis notably via increased expression of ROS, leading to breast cancer and metastasis. Treatment with tangeretin increases the expression of cell cycle arrest markers via p53/p21 up-regulation, thereby suppressing mammary carcinogenesis.

metastasis [48]. Ramos and Taylor [49] have shown by electron microscopy that lipids originate from a secretory product of the neoplastic cells. Llaverias et al. (2011) have shown that increased intracellular levels of lipids promote metastatic potential by driving invasion [50]. In our study, the PAS, Oil Red O staining and ultrastuctural observations showed the presence of glycoproteins and neutral lipid droplets in DMBA-induced animals, which were reduced upon tangeretin treatment. This is consistent with published high-resolution NMR studies that found malignant tissues contained greater amounts of lipids than normal breast tissues [51]. PCNA is a 36-kDa protein and functions as an important cell proliferation marker in several cancers including breast cancer [52]. Cyclo-oxygenase (COX), an inflammatory enzyme induced by cytokines, catalyzes the conversion of arachidonic acid to prostaglandins [53]. Recent reports on COX-2 expression in cancers show that this enzyme can stimulate angiogenesis (via VEGF induction) and is associated with tumor growth, invasion and metastasis (via induction of MMPs) [54]. Cell proliferation markers, such as Ki-67, which are expressed only in cycling cells and not in resting cells, are often used to assess proliferative activity of tumor cells. In a number of cancers, elevated Ki-67 expression has been found associated with higher aggressiveness and invasiveness [55]. Elevated expression of PCNA, COX-2 and Ki-67 in the mammary tissues of DMBA-induced animals indicates the massive proliferation of tumor cells. Tangeretin treatment resulted in decreased expression of these proliferative markers, which clearly revealed its anti-proliferative efficacy. One of the most common incidents required for human cancer development is dysregulation of the cell cycle mechanism. Cell cycle regulatory proteins are the potential molecular targets for cancer therapy/prevention because their functions are well regulated in normal cells, but they are differently regulated in tumor cells. The tumor suppressor p53 is inert in the absence of stress, but upon activation, it can lead the cells to apoptosis or to cell cycle arrest [56]. Among the transcriptional targets of p53, the CDK inhibitor p21WAF/CIP1 inhibits cell cycle progression and mediates the G1 phase arrest. The mammalian cell cycle is divided into four distinct phases namely the G1,

S, G2, and M phases. The CDKs are important regulators of the eukaryotic cell cycle progression, and the activities of these kinases require the binding of a positive regulatory subunit, known as cyclin. The cyclins (e.g., cyclins D, E, and A) activate the CDK2 and CDK4/6 and these cyclin/CDK complexes phosphorylate members of the retinoblastoma protein (Rb) family. This event, in turn, promotes cellular proliferation and S-phase progression through the release of a family of transcriptional regulators, collectively named the E2Fs, which are normally bound to hypophosphorylated Rb. Released E2F elicits the expression of various genes associated with S-phase progression [57]. In the present study, we report that p53-mediated G1/S arrest of DMBA-induced mammary carcinoma by tangeretin is elicited via p21 up-regulation. This is a phenomenon that has been observed previously in colorectal carcinoma cells after tangeretin treatment [14]. The increase in the levels of p53 and p21 and the decrease in the levels of cyclin D1, cyclin E, CDK2 and CDK4 upon tangeretin treatment to DMBA-induced animals indicates that the CDK inhibitor p21WAF/CIP1 acts via p53 up-regulation. p21WAF/CIP1 binds and inhibits the cyclin D1 and E dependent kinases and regulates the G1 to S phase transition of the cell cycle and hence cell proliferation. In summary, we have shown that growth-inhibitory response to tangeretin, including the inhibition of CDK2 and CDK4 activities, and the increase in the level of p21 is dependent on p53 activity. MMPs are zinc and calcium-dependent proteases that digest most of the extracellular matrix components and are produced by cancer cells during invasion. They are secreted as latent proenzymes and are converted to the active form by proteolytic cleavage of an aminoterminal domain [58]. MMPs have been implicated in processes leading to cancer invasion and metastasis, and may also play a major role in tumor angiogenesis [59]. VEGF is regarded as the major angiogenic factor during epithelial carcinogenesis in many malignant human cancers and in tumor metastases [60]. In the present study, DMBA-induced animals showed increased expression of MMP-2, MMP-9 and VEGF which was altered significantly upon tangeretin treatment which confirms the anti-metastatic and anti-angiogenic potential of tangeretin.

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In summary, the results of the present study clearly establish the chemotherapeutic activity of tangeretin against DMBA-induced mammary carcinoma in rats. Tangeretin inhibited tumor cell proliferation by down-regulating PCNA, COX-2 and Ki-67 levels. It arrested the division of breast cancer cells at the G1/S phase via p53/p21 up-regulation; by inhibiting CDK kinase activities and hence inhibiting cyclin D1 and cyclin E expressions. Tangeretin also exhibited anti-metastatic and antiangiogenic activities by down-regulating MMPs and VEGF expressions. To conclude, the potent anti-proliferative, anti-cancer and anti-metastatic effects of tangeretin are more pronounced when it is used as a successful chemotherapeutic agent in the treatment of breast cancer. Acknowledgement The Research Fellowship of the University Grant Commission, New Delhi, India, in the form of UGC-BSR-RF to the first author Ms. Lakshmi Arivazhagan is gratefully acknowledged. The authors are thankful to Dr. R. Arivazhagan, Head of Clinical Biochemistry Department, Cancer Institute (WIA), Chennai for his immense support and Dr. V. Pushpa, Professor, Cancer Institute (WIA), Chennai, India for analyzing samples under Transmission electron microscope. References [1] Jemal A, Bray F, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69–90. [2] Datta K, Choudhuri M, Guha S, Biswas J. Breast cancer scenario in a regional cancer centre in Eastern India over eight years — still a major public health problem. Asian Pac J Cancer Prev 2012;13(3):809–13. [3] McPherson K, Steel CM, Dixon JM. ABC of breast diseases. Breast cancerepidemiology, risk factors, and genetics. BMJ 2000;321(7261):624–8. [4] Michor F, Iwasa Y, Nowak MA. Dynamics of cancer progression. Nat Rev Cancer 2004;4(3):197–205. [5] Koumoutsakos P, Pivkin I, Milde F. The fluid mechanics of cancer and its therapy. Annu Rev Fluid Mech 2013;45:325–55. [6] Costa I, Solanas M, Escrich E. Histopathologic characterization of mammary neoplastic lesions induced with 7, 12 dimethylbenz(α)anthracene in the rat: a comparative analysis with human breast tumors. Arch Pathol Lab Med 2002;126(8):915–27. [7] Anderson WA, Perotti ME, McManaway M, Lindsey S, Eckberg WR. Similarities and differences in the ultrastructure of two hormone-dependent and one independent human breast carcinoma grown in athymic nude mice: comparison with the rat DMBA-induced tumor and normal secretory mammocytes. J Submicrosc Cytol 1984;16(4):673–90. [8] Russo J, Tait I, Russo IH. Susceptibility of the mammary gland to carcinogenesis. III. The cell of origin of rat mammary carcinoma. Am J Pathol 1983;113(1):50–66. [9] Beecher GR. Overview of dietary flavonoids: nomenclature, occurrence and intake. J Nutr 2003;133(10):3248S–54S. [10] Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 1996;20(7):933–56. [11] Zhu Y, Mao Y, Chen H, Lin Y, Hu Z, Wu J, et al. Apigenin promotes apoptosis, inhibits invasion and induces cell cycle arrest of T24 human bladder cancer cells. Cancer Cell Int 2013;13(1):54. [12] Kawaii S, Tomono Y, Katase E, Ogawa K, Yano M. Quantitation of flavonoid constituents in citrus fruits. J Agric Food Chem 1999;47(9):3565–71. [13] Chaumontet C, Bex V, Gaillard-Sanchez I, Seillan-Heberden C, Suschetet M, Martel P. Apigenin and tangeretin enhance gap junctional intercellular communication in rat liver epithelial cells. Carcinogenesis 1994;15(10):2325–30. [14] Pan MH, Chen WJ, Lin-Shiau SY, Ho CT, Lin JK. Tangeretin induces cell-cycle G1 arrest through inhibiting cyclin-dependent kinases 2 and 4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal carcinoma cells. Carcinogenesis 2002;23(10):1677–84. [15] Seo J, Lee HS, Ryoo S, Seo JH, Min BS, Lee JH. Tangeretin, a citrus flavonoid, inhibits PGDF-BB-induced proliferation and migration of aortic smooth muscle cells by blocking AKT activation. Eur J Pharmacol 2011;673(1–3):56–64. [16] Hirano T, Abe K, Gotoh M, Oka K. Citrus flavone tangeretin inhibits leukaemic HL-60 cell growth partially through induction of apoptosis with less cytotoxicity on normal lymphocytes. Br J Cancer 1995;72(6):1380–8. [17] Datla KP, Christidou M, Widmer WW, Rooprai HK, Dexter DT. Tissue distribution and neuroprotective effects of citrus flavonoid tangeretin in a rat model of Parkinson's disease. Neuroreport 2001;12(17):3871–5. [18] Lake BG, Beamand JA, Tredger JM, Barton PT, Renwick AB, Price RJ. Inhibition of xenobiotic-induced genotoxicity in cultured precision-cut human and rat liver slices. Mutat Res 1999;440(1):91–100. [19] Lakshmi A, Subramanian S. Chemotherapeutic effect of tangeretin, a polymethoxylated flavone studied in 7, 12-dimethylbenz(a)anthracene induced mammary carcinoma in experimental rats. Biochimie 2014;99:96–109.

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