Acta Histochemica 116 (2014) 1454–1461

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Combination of Pitavastatin and melatonin shows partial antineoplastic effects in a rat breast carcinoma model Peter Kubatka a,∗ , Bianka Bojková b , Monika Kassayová b , Peter Orendáˇs b , Karol Kajo c,d , e ´ Desanka Vybohová , Peter Kruˇzliak f , Katarína Adamicová g , Martin Péˇc a,∗ , h Nadeˇzda Stollárová , Marián Adamkov i a

Department of Medical Biology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia Department of Animal Physiology, Institute of Biological and Ecological Sciences, Faculty of Science, P. J. Sˇ afárik University, Koˇsice, Slovakia c Department of Pathology, Slovak Medical University, Bratislava, Slovakia d St. Elisabeth Oncology Institute, Bratislava, Slovakia e Department of Anatomy, Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia f International Clinical Research Center, St. Anne’s University Hospital and Masaryk University, Brno, Czech Republic g Department of Pathological Anatomy, Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia h Department of Biology and Ecology, Faculty of Education, Catholic University, Ruˇzomberok, Slovakia i Department of Histology and Embryology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia b

a r t i c l e

i n f o

Article history: Received 17 August 2014 Received in revised form 22 September 2014 Accepted 28 September 2014 Keywords: Mammary carcinogenesis Pitavastatin Melatonin Apoptosis Angiogenesis Proliferation

a b s t r a c t Our previous results indicated significant tumor-suppressive effects of different statins in rat mammary carcinogenesis. The purpose of this experiment was to examine the chemopreventive effects of Pitavastatin alone and in combination with the pineal hormone melatonin in the model of Nmethyl-N-nitrosourea-induced mammary carcinogenesis in female Sprague-Dawley rats. Pitavastatin was administered dietary (10 mg/kg) and melatonin in an aqueous solution (20 ␮g/ml). Chemoprevention began 7 days prior to carcinogen administration and subsequently continued for 15 weeks until autopsy. At autopsy, mammary tumors were removed and prepared for histopathological and immunohistochemical analysis. Compared to controls, Pitavastatin alone reduced average tumor volume by 58% and lengthened latency by 8 days; on the other hand, the drug increased tumor frequency by 23%. Combined administration of Pitavastatin with melatonin decreased tumor frequency by 23%, tumor volume by 44% and lengthened tumor latency by 5.5 days compared to control animals. The analysis of carcinoma cells showed significant increase in caspase-3 expression in both treated groups and a tendency of increased caspase-7 expression after Pitavastatin treatment alone. Significant expression decrease of Ki67 was found in carcinoma cells from both treated groups. Compared to control carcinoma cells, Pitavastatin alone increased VEGF expression by 41%, however melatonin totally reversed its undesirable effect. Pitavastatin combined with melatonin significantly increased femur compact bone thickness in animals. Pitavastatin alone decreased plasma triglycerides and total cholesterol levels, however it significantly increased levels of glucose. In summary, our results show a partial antineoplastic effect of Pitavastatin combined with melatonin in the rat mammary gland carcinoma model. © 2014 Elsevier GmbH. All rights reserved.

Introduction The promising anti-cancer effects of statins in preclinical research have stimulated investigations into their possible clinical

∗ Corresponding authors at: Department of Medical Biology, Jessenius Faculty of Medicine, Comenius University, Malá Hora 4, SK-036 01 Martin, Slovakia. E-mail addresses: [email protected] (P. Kubatka), [email protected] (M. Péˇc). http://dx.doi.org/10.1016/j.acthis.2014.09.010 0065-1281/© 2014 Elsevier GmbH. All rights reserved.

implications as anticancer agents in specific cancer types. Several clinical trials have explored the potential benefits of statins in carcinogenesis. In some cases, promising results have been reported regarding their efficacy. Recently in large clinical studies, a decreased risk of esophageal malignancy (Alexandre et al., 2014), hepatocellular carcinoma (Singh et al., 2013), and colorectal cancer (Liu et al., 2014) was found. Some clinical studies showed that statins might be useful to prevent recurrence and improve survival in patients already suffering from some breast cancer types (Bonovas et al., 2014). Consequently, the possible role of statins

P. Kubatka et al. / Acta Histochemica 116 (2014) 1454–1461

in cancer disease and cancer chemoprevention is being seriously discussed among oncologists. As an essential step in biosynthesis of the mevalonate pathway, statins affect the levels of cholesterol and also other downstream products (isoprenoids), which are important in key physiological processes such as cell signalling, translation, post-translational modifications, proliferation, apoptosis, and differentiation (Kato et al., 2010; Wu et al., 2011; Liang et al., 2013). In our recent experimental study, significant chemopreventive effects of Simvastatin in rat mammary carcinogenesis was accompanied by a distinct decrease of proliferating cell nuclear antigen Ki67 in treated mammary carcinoma cells (Kubatka et al., 2012). In our previous experiments, we have shown a pro-apoptotic shift in Bax/Bcl-2 mRNA expression in rat mammary tumor cells after Atorvastatin treatment (Kubatka et al., 2011a). Our recent results suggest significant proapoptotic effects of Pravastatin by increasing caspase-3 and caspase-7 in rat mammary carcinomas (Orendáˇs et al., 2014, in press). Statins demonstrably decrease the levels of one of the most important molecules of angiogenesis, vascular endothelial growth factor (VEGF), and thereby inhibit capillary formation (Park et al., 2002). In rat mammary carcinomas treated with Fluvastatin, we have found a considerable decrease of VEGFR-2 expression (Kubatka et al., 2013). Several studies have raised the suggestion that the pineal hormone melatonin possesses anticancer properties (Sanchez-Barcelo et al., 2012). Melatonin was shown to be particularly effective in experimental colorectal and mammary carcinogenesis (Wang et al., 2012; Hong et al., 2014). The main aim of this study was to determine the preventive effects of Pitavastatin alone and in combination with melatonin after long-term administration in a model of N-methylN-nitrosourea (NMU)-induced mammary carcinogenesis in female rats. The combined administration of Pitavastatin and melatonin was evaluated with regard to find their additive antineoplastic effects. The histopathology of tumors and immunohistochemical analysis of mechanism of action were determined. Some side effects of the drugs after long-term administration in animals were evaluated. Material and methods The experimental protocols for the study were approved by the Ethical Commission of Jessenius Faculty of Medicine of Comenius University (Protocol No. EK 857/2011) and by the State Veterinary and Food Administration of the Slovak Republic (accreditation No. Ro-1762/12-221). Animals and induction of mammary carcinogenesis Female Sprague-Dawley strain rats (n = 60) (Charles River Laboratories, Sulzfeld, Germany) aged 32–36 days, were used in the study. The animals were adapted to standard vivarium conditions with a temperature 23 ± 2 ◦ C, relative humidity 40–60%, artificial regimen light:dark (12 h:12 h) (lights on from 6 a.m., light intensity 150 lx/cage). During the experiment the animals were fed the Ssniff diet (Ssniff Spezialdiäten GmbH, Soest, Germany) and allowed tap water ad libitum. Mammary carcinogenesis was induced by N-methyl-N-nitrosourea (NMU; Sigma, Deisenhofen, Germany) administered intraperitoneally in a single dose of 50 mg/kg body weight on average on postnatal day 42. Experimental design Chemoprevention with Pitavastatin (Livazo, Lilly, Switzerland) and melatonin (Sigma, Deisenhofen, Germany) began 1 week before carcinogen administration and lasted until the end of the experiment, 15 weeks after NMU administration. Pitavastatin was

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administered in the diet at a single concentration of 10 mg/kg (0.001%, i.e. 10 ppm). Based on our previous experience using statins in rat mammary carcinogenesis (rats demonstrate different pharmacokinetics and pharmacodynamics than humans), it was necessary to use doses of Pitavastatin 10 times higher than the maximal clinical dose to find its antineoplastic effect in this experiment. Relatively low dose of pitavastatin (10 mg/kg of the diet) was used in order to retain the possibility for analyzing an additive effect with melatonin in the combined administration. Melatonin was administered daily in drinking water (20 ␮g/ml). Based on average daily water intake in rats drinking melatonin (26.02 ml), the daily dose of melatonin per rat was 520.4 ␮g (2.10 mg/kg b.w./day). Melatonin solution was freshly prepared three times a week. Twenty mg of melatonin were diluted in 0.4 ml of 30% ethanol and mixed with tap water to the desired volume. The animals were divided into three groups: (1) control group without chemoprevention; (2) chemoprevention with Pitavastatin (PITAVA); (3) chemoprevention with Pitavastatin and melatonin (PITAVA + MEL). Each group consisted of 20 animals. The animals were weighed weekly and from week 6 post-NMU administration palpated in order to register the presence, number, location and size of each palpable tumor. Food and water intake were monitored during 24 h in week 6 and 12 of the experiment. In the final week of the experiment (week 15), the animals were quickly decapitated, the blood from each animal was collected, mammary tumors and femora were excised and tumor size was recorded. Macroscopic changes in selected organs (liver, spleen, stomach, and ovaries) were evaluated at autopsy. Selected parameters of the lipid and carbohydrate metabolism were analyzed. Triglycerides and glucose were determined using commercial sets (Pliva-Lachema, Brno, Czech Republic), and total cholesterol according to Zlatkis et al. (1953). Histopathological evaluation Tissue samples of each mammary tumor and decalcified femora (taken from the mid-diaphysis of the femur) were routinely formalin-fixed and embedded in paraffin wax. The tumors were classified according to the criteria for the classification of rat mammary tumors (Russo and Russo, 2000). An additional parameter, the grade of invasive carcinomas, was used. Tumor samples were divided into low-grade (LG) and high-grade (HG) carcinomas. The criteria for categorization: solidization, cell atypia, mitotic activity index, and necrosis, were chosen according to the standard diagnostic method of classification. As HG carcinomas were considered tumors with ≥2 positive criteria, LG carcinomas were tumors with ≤1 positive criterion. The solidization was considered if >30% of tumor sample displays solid growth, high mitotic activity index if ≥10 mitosis is observed in 10 high power fields and necrosis if the occurrence of comedo (not infarct) was determined. Histopathological examination and measurements (anterior, posterior, medialis, and lateralis) of thickness of the compact bone of the femur were performed by light microscopy. Immunohistochemical analysis of carcinoma cells The most relevant part of the mammary tumor in paraffin blocks (which includes the typing characteristics and having the largest representation of vital tumor epithelial component, i.e. without regressive changes such as extensive necrosis) was chosen for immunohistochemical analysis. The detection of selected proteins was carried out using an indirect immunohistochemical method on whole paraffin sections, using commercially available rat specific antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Dako, Glostrup, Denmark; Abcam, Cambridge, UK). After deparaffinization, endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide in methanol for 30 min. Sections

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Table 1 Effects of Pitavastatin alone and in combination with melatonin in N-methyl-N-nitrosourea-induced mammary carcinogenesis in female rats at the end of experiment. Group

Controls

Pitavastatin

Pitavastatin and melatonin

All animals/tumor bearing animals Tumor incidence (%) Tumor frequency per group* Tumor latency* (days) Average tumor volume* (cm3 ) Cumulative tumor volume** (cm3 )

20/12 60.0 1.10 ± 0.28 88.36 ± 3.82 0.90 ± 0.24 19.80

20/11 55.0 (−8%) 1.35 ± 0.45 (+23%) 96.52 ± 3.22 (+8 days) 0.38 ± 0.17 (−58%) 10.15 (−49%)

20/11 55.0 (−8%) 0.85 ± 0.26 (−23%) 93.72 ± 3.81 (+5.5 days) 0.50 ± 0.29 (−44%) 9.02 (−54%)

Values in brackets are calculated as %-ual deviation from the 100% of non-influenced control group (with exception of latency). * Data are expressed as means ± SEM. ** Data are expressed as a sum of volumes per group.

were pre-treated in a microwave oven for 15 min in 10 mM citrate buffer (pH 6.0) and incubated with primary antibody in PBS containing 1% BSA for 60 min at room temperature. The primary antibodies were visualized by a secondary staining system (EnVision, Dual Link System-HRP, cat. No. K4061, Dako North America, Carpinteria, CA, USA) using diaminobenzidine tetrahydrochloride (DAB) as chromagen. The sections were counterstained with hematoxylin, dehydrated and mounted in Canadian balsam. Negative controls included sections where primary antibody was omitted in the staining procedure. Immunohistochemically detected antigen expression was evaluated by precise morphometric method. Sections were examined and digital images of microscopic views acquired at magnifications of ×200 with an Olympus Evolt E420 digital camera installed on an Olympus BX41N microscope. Expression of VEGF, caspase-3, and caspase-7 was analyzed in the cytoplasm of tumor cells. Ki67 was detected within the nucleus. Receptors for VEGF were observed in the cell membrane. Expression of proteins was quantified as the average percentage of antigen positive area in standard fields (0.5655 mm2 ) of tumor “hot spot” areas. Morphometric analysis of the digital images was done using QuickPhoto Micro version 2.3 software (Promicra, Prague, Czech Republic). Antigen positive area was evaluated by phase analysis with standard thresholds for weak, mild and strong intensity of immunoreactivity. The values of protein expression were compared between treated (PITAVA, PITAVA + MEL) and non-treated (control) tumor cells of female rats; at least 60 images for each protein were analyzed (a total of 310 images for 5 proteins). Statistical analysis Statistical analyses were conducted with GraphPadPrism software, version 5.01 (GraphPad Software, La Jolla, CA, USA). Mann–Whitney and Kruskal–Wallis tests and one-way analysis of variance were used in data evaluation. P values of 0.05 or less were considered statistically significant. Tumor volume was calculated according to the formula: V = . (S1 )2 . S2 /12 (S1 , S2 are tumor diameters; S1 < S2 ). Results Parameters of mammary carcinogenesis and histopathology of tumors The effects of Pitavastatin and a combination of Pitavastatin with melatonin in the chemoprevention of rat mammary gland cancer are summarized in Table 1. Compared to the control group, only slight (non-significant) oncostatic effects were found after administration of Pitavastatin alone: tumor latency was lengthened by 8 days, average tumor volume was decreased by 58% and incidence by 8%, however tumor frequency was increased by 23%. Pitavastatin combined with melatonin decreased all determined parameters: tumor frequency by 23%, tumor volume by 44%, incidence by 8%, and lengthened tumor latency by 5.5 days

Table 2 Histopathological classification and number of mammary tumors. Mammary tumors Malignant lesions P, C C, P C C, CO P P,C,CO Total number

Control

Pitavastatin

Pitavastatin and melatonin

9 8 4 1 – –

14 10 2 – 1 –

8 2 4 – 2 1

22

27

17

Type: Invasive carcinoma (C, cribriform; P, papillary; CO, comedo). Dominant type in mixed tumors is the first in order.

compared to control animals. The histopathological classification of all mammary tumors is summarized in Table 2. In all experimental groups, the most frequently occurring lesions were mixed cribriform/papillary, papillary/cribriform, and cribriform carcinomas. After the histopathological analysis, any apparent shift in the rate of poorly differentiated (high grade, HG) and well differentiated (low grade, LG) mammary carcinomas after the treatment with Pitavastatin and Pitavastatin + melatonin was found [control group: 5/17 (HG/LG); PITAVA: 5/22 (P = 0.73 vs CONT); PITAVA+MEL: 8/9 (P = 0.12 vs CONT)]. Immunohistochemical evaluation As seen in Fig. 1, immunohistochemical analysis of carcinoma cells showed a significant increase in the expression of caspase3 after Pitavastatin (by 114%) and combined (by 109%) treatment in comparison with control (untreated) cells (also Fig. 2). A clear tendency in the expression increase of caspase-7 by 37% after Pitavastatin alone treatment was found. Compared to control cells, there was a significant decrease in the expression of Ki67 by 31% after Pitavastatin treatment and by 33% after combined treatment. In tumors treated with Pitavastatin, the expression of VEGF was elevated by 41% when compared to non-treated tumors; however, this parameter was significantly decreased by 42% after the combination of Pitavastatin + melatonin when compared with cells in Pitavastatin treated alone group. Concerning the expression of VEGFR-2, there was a trend of increased expression in tumor cells in both treated groups (increase by 10% and 8%, respectively) in comparison with the control group (Fig. 2). Side effects At autopsy, no macroscopic changes due to long-term administration of the drugs in the body organs (e.g. liver steatosis, hepato/splenomegaly, ovarian cysts, apparent hematopoietic disorders, and gastritis) were observed. Pitavastatin combined with melatonin significantly increased femur compact bone thickness compared to controls (Table 3, Fig. 3). Pitavastatin alone and in combination with melatonin significantly increased concentrations of

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Fig. 1. Immunohistochemical evaluation of caspase-3, caspase-7, Ki67, VEGF, and VEGFR-2 expression in rat mammary carcinoma cells after treatment with Pitavastatin alone and in combination with melatonin. Data are expressed as means. Figure represents the expression of proteins quantified as the average percentage of antigen positive area in standard fields (0.5655 mm2 ) of tumor “hotspot” areas. The values of protein expression were compared between treated (Pitavastatin, Pitavastatin and melatonin) and non-treated (control) carcinoma cells of female rats; at least 60 images for one protein were analyzed. Significantly different, ** P < 0.01 vs Control, + P < 0.05 vs Pitavastatin. CONT – control, PITAVA – group with administered Pitavastatin, PITAVA + MEL – group with administered Pitavastatin + melatonin.

serum glucose. On the other hand, a tendency of decreased triglycerides and total cholesterol levels in the Pitavastatin alone group compared to control animals were found. When compared to controls, melatonin potentiated the effect of Pitavastatin on serum triglycerides (Table 3). The evaluation of final body weight gain in rats did not reveal changes in both treated groups (Table 3). Chemoprevention in both groups significantly increased daily food intake in animals (by approx. 2 g) when compared to controls. Discussion In our previous experiments, we observed significant antitumor effects of dietary administered lipophilic statins (Atorvastatin, Simvastatin, and Fluvastatin) in the chemoprevention of rat mammary tumors (Kubatka et al., 2011a, 2012, 2013). Our experiments with hydrophilic statins: Rosuvastatin (Kubatka et al., 2011b) and Pravastatin (Orendáˇs et al., 2014, in press) have shown lower (non-significant antineoplastic activity) than lipophilic statins in this model of experimental breast cancer. Apparent antineoplastic effects of statins in our experiments Table 3 Effects of Pitavastatin alone and in combination with melatonin on plasma glucose, triglycerides, and cholesterol levels, and on body weight and femur compact bone thickness. Group Glucose (mmol/l) Triglycerides (mmol/l) Total cholesterol (mmol/l) Body weight (g) Compact bone thickness (␮m)

Control 3.71 ± 0.08 0.83 ± 0.06 4.93 ± 0.20 252.84 ± 3.29 0.51 ± 0.01

Data are expressed as means ± SEM. Significantly different: ** P < 0.01, *** P < 0.001 vs Control, + P < 0.05 vs Pitavastatin.

Pitavastatin 4.78 ± 0.15*** 0.68 ± 0.03 4.38 ± 0.18 256.52 ± 2.88 0.54 ± 0.01

Pitavastatin and melatonin 4.46 ± 0.18** 0.60 ± 0.06** 4.77 ± 0.20 248.01 ± 4.58 0.59 ± 0.02** , +

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confirmed also results from histopathological analyzes. Pravastatin significantly reduced the high/low grade carcinomas ratio and this clear trend was also observed in carcinomas after Atorvastatin, Simvastatin and Rosuvastatin treatment. Other authors also focused on the evaluation of tumor-preventive effects of statins in rodents. Simvastatin and Pravastatin (in similar doses to those used in our experiments) significantly reduced tumor frequency in colon carcinogenesis in ICR mice (Narisawa et al., 1994). In other experiments in rats, Pravastatin reduced the incidence and volume of hepatic neoplastic nodules (Tatsuta et al., 1998) and inhibited colon carcinogenesis (Narisawa et al., 1996). Despite the above described strong evidence on the anti-neoplastic effects of statins in several animal models, there are also in vivo experimental data suggesting minimal tumor-suppressive activity (Lubet et al., 2009) or even potential carcinogenicity of these drugs, however, in several times higher doses (Robison et al., 1994). This study is the first report to evaluate the effects of Pitavastatin in experimental breast cancer in female rats. Only a slight chemopreventive effect of lipophilic Pitavastatin administered alone was recorded (characterized by decreased tumor volume and lengthened tumor latency). However, the decrease in tumor volume by 58% after Pitavastatin treatment was accompanied by a significant decrease in proliferation and induction of apoptosis in carcinoma cells. Relatively few in vitro and in vivo studies have evaluated anti-neoplastic effects of Pitavastatin in different cancer models. In the study of Kamigaki et al. (2011), Pitavastatin dramatically suppressed cell proliferation and induced apoptosis in human cholangiocarcinoma cell lines. Feeding with 10 ppm Pitavastatin (the same dose as in our study) significantly inhibited the development of hepatic premalignant lesions in male db/db mice by inducing apoptosis and inhibiting cell proliferation (Shimizu et al., 2011). Similarly, the treatment with Pitavastatin at doses of 20 and 40 ppm significantly decreased the total number of polyps in Min mice (Teraoka et al., 2011). All these results indicated that Pitavastatin has potential benefits in the suppression of carcinogenesis in different organ sites. Data on the effects of statins on cancer risk reduction and prognosis are still lacking, with the exception of consistent evidence that statins are associated with reduced risk of advanced prostate cancer (Bansal et al., 2012). However, statins affect several important cellular functions, and therefore their interactions with other anticancer drugs are of particular interest to oncologists as they may influence the success of cancer treatment. Thus statins could also be combined with specific anticancer drugs and potentiate their effects, ameliorate their side-effects or prevent the development of resistance (Bonovas et al., 2014). Another aim of this study was to use a lower Pitavastatin dose in combination with the pineal hormone melatonin to enhance the effects of the statin. Oncostatic effects of several substances (Bexarotene, Resveratrol, Celecoxib, Pravastatin) co-administered with melatonin using the same breast cancer model in our recent experiments were observed (Orendas et al., 2009, 2012; Kiskova et al., 2012; Orendáˇs et al., 2014, in press). Based on the evaluation of the most sensitive parameter in this model (tumor frequency), this experiment confirmed our previous results and demonstrated an additive tumor-suppressive effect of a combination of Pitavastatin and melatonin in rat mammary carcinogenesis. Recently, in vitro and in vivo studies have demonstrated pleiotropic effects of statins in cells, which include influence on regulatory mechanisms such as the apoptosis, cell cycle, differentiation, or vascularization. Sequential activation of caspases plays a central role in the execution-phase of apoptosis. Caspase-3 cleaves and activates caspases 6 and 7; and the protein itself is processed and activated by caspases 8, 9, and 10. Caspase-7 and has also been shown to be an executioner protein of apoptosis (Porter and Jänicke, 1999). Statins seem to be suitable for activation of caspases. Cafforio et al. (2005) observed that Cerivastatin induced apoptosis in human

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Fig. 2. Representative images of caspase-3 (a), caspase-7 (b), Ki67 (c), VEGF (d), and VEGFR-2 (e) expression in rat mammary carcinoma cells after treatment with Pitavastatin alone and in combination with melatonin using immunohistochemical analysis. Images were selected according to results summarized in Fig. 1. For detection polyclonal caspase-3 antibody (Abcam, Cambridge, UK), polyclonal caspase-7 antibody (Santa Cruz Biotechnology, CA, USA), monoclonal Ki67 antibody (Dako, Glostrup, Denmark), monoclonal VEGF, and VEGFR-2 antibodies (Santa Cruz Biotechnology Inc., CA, USA) were used.

myeloma cells by activating caspase-3, caspase-8, and caspase-9. Tuerdi et al. (2013) observed that intrinsic apoptotic marker and caspase-3 activation was induced only in the presence of Atorvastatin or Simvastatin in human malignant mesothelioma. Lovastatin caused programmed cell death through activation of caspase-7 in a prostate cancer cell line (Marcelli et al., 1998) and by activation of caspase-3 in leukemic cells (Wang et al., 2000). In our recent study using the same model as in this study, mild anti-neoplastic effects of Pravastatin were accompanied by a significant increase of caspase-3 and caspase-7 in rat mammary tumor cells; moreover, using a combination of Pravastatin + melatonin, this effect on both caspases was noticeably enhanced (Orendáˇs et al., 2014, in press). On the other hand, highly effective treatment of rat mammary carcinomas by Fluvastatin caused only a slight increase in

caspase-3 expression (Kubatka et al., 2013). In this study, a significant increase of caspase-3 cell expression pointed to pro-apoptotic effects of Pitavastatin alone and in combination with melatonin in rat mammary carcinoma cells. The expression of Ki67 protein is strictly associated with cell proliferation. The fact that the Ki67 protein is present during all active phases of the cell cycle, but is absent from resting cells, makes Ki67 a good tumor marker (Zorc et al., 2003). It is considered that Ki67 protein expression is an absolute requirement for progression through the cell-division cycle. In this in vivo study, we found a significant decrease of Ki67 expression in rat mammary carcinoma cells after treatment with Pitavastatin alone and in combination with melatonin. Similar in vitro results with statins were found by other authors. Pitavastatin inhibited the proliferation

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Fig. 3. Representative images of femur compact bone thickness in rats after the treatment with Pitavastatin and combined treatment. Specimens of femora (3-mm long) taken from the mid-diaphysis were decalcified. All samples were embedded in paraffin using conventional automated systems. The blocks were cut to obtain 4 to 5 ␮m thick sections and were stained with hematoxylin-eosin. Histopathological examination was performed by light microscopy.

and suppressed the nuclear expression of NF-kappa B in human breast cancer cell lines (Wang and Kitajima, 2007). In a recent study, Fluvastatin inhibited proliferation of the C6 glioma cells (Slawinska-Brych et al., 2014). In our previous study, we found a decrease of Ki67 protein expression by 27% in Simvastatin treated rat mammary tumor cells (Kubatka et al., 2012). However, the tumor cell expression of Ki67 was not changed after Fluvastatin administration in the same model (Kubatka et al., 2013). In contrast with the above cited results, Pravastatin in our previous experiment significantly increased the Ki67 carcinoma cell expression by 40% compared to controls (this result is probably related to hydrophilic nature of Pravastatin); however, melatonin completely reversed this adverse effect of Pravastatin on proliferation of tumor cells. The most prominent target of anti-angiogenic agents is vascular endothelial growth factor (VEGF) and its receptors. VEGF has been shown to stimulate vascular endothelial cell mitogenesis and cell migration (Petrovic, 2010). VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF (Holmes et al., 2007). Recent studies have shown that statins may reduce VEGF (Dworacka et al., 2014) or VEGFR-2 (Kubatka et al., 2013) expression in different cells. An anti-angiogenic effect of melatonin, characterized by VEGFR-2 expression decrease was found in an xenograft model of breast cancer (Jardim-Perassi et al., 2014). In our recent study, an apparent tendency in the decrease in VEGFR-2 levels in carcinoma cells after Pravastatin and combined treatment with melatonin was observed (Orendáˇs et al., 2014, in press). Contrary to this result, Pitavastatin in this study increased VEGF expression by 41% in comparison with untreated cells; however melatonin completely reversed this adverse effect. Future research will focus on determining the tumor types and stages that will benefit most from antiangiogenic therapy with statins. Preclinical and clinical research has shown that pleiotropic effects of statins also include an influence on bone metabolism. In our model, significant negative effects of Fluvastatin (Kubatka et al., 2013), neutral effects of Pravastatin (Orendáˇs et al., 2014, in press), or mild positive effects of Pitavastatin in this study on femur compact bone thickness in rats were found. Regarding statins, due to differences in their polarity and bone availability, their individual bone effects might differ (Hanayam et al., 2009). A recent systematic review of clinical results (involving 34.877 patients) on the relationship between use of statins and bone mineral density was reported (Liu et al., 2013). The results suggested that statins may help improve and maintain bone mineral density at the lumbar spine, hip and femoral neck, especially in Caucasians and Asians. Melatonin is considered as the agent with bone-protective effects in model studies (Uslu et al., 2007). This assumption was confirmed

in this study, where significant positive effects of the combined treatment with Pitavastatin and melatonin on femur compact bone thickness in rats were observed. Statins and melatonin may have beneficial effects on the skeleton, but more longitudinal studies on humans (e.g. of different ethnicities) are warranted. Pitavastatin administered alone demonstrated only slight antitumor effects in breast cancer model in rats. On the other hand, combined treatment of Pitavastatin and melatonin was more effective. Obtained results pointed to pro-apototic and antiproliferative effects of Pitavastatin alone and in combination with melatonin in rat mammary carcinoma cells. The results with statins used as a single agent in patients for cancer treatment or risk reduction are controversial. It seems likely that statins will be utilized as a combination with other anti-cancer drug, which could potentiate their anti-cancer effects, ameliorate their side-effects or prevent the development of resistance. Based on these and our previous results, statins co-administered with other suitable drugs (e.g. melatonin) should be further evaluated for tumor-preventive properties.

Acknowledgements This work was supported by the Scientific Grant Agency, Ministry of Education, Slovak Republic (contract no. VEGA1/0043/12 and VEGA 1/0071/13). This work was supported by the project “Martin Biomedical Centre”, ITMS: 26220220187, co-funded from EU sources and European Regional Development Fund. This study was elaborated within the framework of the grant of European Regional Development Fund-Project FNUSA-ICRC (No. CZ.1.05/1.1.00/02.0123), both projects co-funded from EU sources and European Regional Development Fund. We would like to thank Andrea Kapinová and Martina Piterová for technical support.

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