Metformin associated with photodynamic therapy – A novel oncological direction

Journal of Photochemistry and Photobiology B: Biology 138 (2014) 80–91

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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

Metformin associated with photodynamic therapy – A novel oncological direction Iuliana Nenu a,1, Tiberiu Popescu a,1, Mihaela D. Aldea a, Lucian Craciun a, Diana Olteanu a, Corina Tatomir b, Pompei Bolfa c,d, Rodica M. Ion e, Adriana Muresan a, Adriana G. Filip a,⇑ a

Department of Physiology, ‘‘Iuliu Hatieganu’’ University of Medicine and Pharmacy, 1 Clinicilor Street, 400006 Cluj-Napoca, Romania Departments of Radiobiology and Tumor Biology, Oncology Institute ‘‘Prof. I. Chiricuta’’, 34-36 Republicii Street, 400015 Cluj-Napoca, Romania Department of Pathology, Cluj-Napoca, University of Agricultural Sciences and Veterinary Medicine, 3-5 Calea Manastur, 400372 Cluj-Napoca, Romania d Department of Biomedical Sciences, Ross University School of Veterinary Medicine Basseterre, PO Box 334, Saint Kitts and Nevis e National Research & Development Institute for Chemistry and Petrochemistry ICECHIM 202 Splaiul Independentei, 060021 Bucharest, Romania b c

a r t i c l e

i n f o

Article history: Received 6 January 2014 Received in revised form 15 April 2014 Accepted 28 April 2014 Available online 21 May 2014 Keywords: Metformin Photodynamic therapy TSPP Cancer

a b s t r a c t The aim of our study was to assess the effect of the combined treatment of Metformin (Metf) and 5, 10, 15, 20-tetra-sulfophenyl-porphyrin (TSPP)–mediated photodynamic therapy (PDT) on an in vivo tumour model. Wistar male rats were divided in 6 groups: group 1, treated with TSPP; groups 2 and 4 treated with TSPP and Metf, respectively, and irradiated 24 h thereafter; group 3 was treated with Metf and the last two groups received the combined treatment, Metf administered prior (group 5) or after (group 6) irradiation. 72 h from the start of the treatment, tumour tissue was sampled for the investigation of oxidative and nitrosative stress. The apoptotic rate, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions and matrix metalloproteinases activities were also quantified. Malondialdehyde and glutathione levels were significantly elevated in the groups treated with combined therapy (p < 0.05). Metf associated with TSPP–PDT reduced iNOS and COX-2 expressions and enhanced nitrotyrosine levels in both therapeutic regimens. Peroxynitrate formation and its cytotoxic effect on tumour cells were related to an elevated index of apoptosis and necrosis. Moreover, MMP-2 activity reached a minimum in the groups which received combined therapy. Our results confirmed that the association of Metf with PDT might prove a new and promising oncological approach. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Along with cardiovascular diseases, obesity and HIV infection, cancer has become nowadays a worldwide concern. Because of its increased frequency and high mortality rate, scientists are struggling to find more effective prevention methods and newer therapeutic means. This is the reason why recent research aim at developing various pharmacological schemes, discovering and improving new therapies and also at studying possible immunological and genetic approaches. Several studies have outlined lately a close connection between type two diabetes mellitus and the incidence of different cancers (e.g. colorectal, breast, pancreatic, hepatic, endometrial and renal cancer) and have linked the incriminated mitogenic effect to obes-

⇑ Corresponding author. Address: Department of Physiology, 1-3 Clinicilor Street, Cluj-Napoca, Romania. Tel.: +40 745 268704; fax: +40 264 597257. E-mail addresses: gabriela.filip@umfluj.ro, adrianafi[email protected] (A.G. Filip). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jphotobiol.2014.04.027 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

ity, dyslipidemia and hypertension, all hallmarks of the metabolic syndrome [1]. Treating diabetes mellitus seems to have preceded understanding the disease itself, as ancient Egyptians used Galega officinalis (also known as the ‘‘French lilac’’) as the source of an active substance called guanidine for ameliorating frequent urination (polyuria) and halitosis (sweet odour on breath) in type two diabetes mellitus (T2DM) [2]. However, it was not until the 1920s that scientists succeeded in linking two guanidine rings and developing the drug Metformin (Metf), which was later approved as an antihyperglycemic drug [3]. Nowadays, Metformin has become the most prescribed oral antidiabetic drug due to its ability to sensitize tissues to insulin and its consequent glucose lowering effect. Moreover, it is one of the most studied drugs in oncological research due to retrospective studies that revealed a decreased cancer incidence and cancer-related mortality in obese and diabetic patients treated with it. Several studies have demonstrated that Metf activates the adenosine monophosphate kinase (AMPK) pathway and induces phosphorylation of different intracellular proteins, fact that further

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leads to an increased apoptotic response and a decreased proliferation rate in tumoural cells [4]. In addition, in vitro studies revealed a reduced proliferation rate of several tumoural cell-lines after administration of Metf, either alone or as an adjuvant to chemotherapy [5]. The effect is remarkable not only against non-stem tumoural cells but most importantly against tumoural stem cells [6]. As an attempt to improve the outcome of anticancer therapy, our study proposes for the first time the association of Metf with a non-invasive oncological approach – photodynamic therapy. Greeks were among the first to acknowledge the curative effects of light and named this treatment heliotherapy. However, scientists refined and redefined this concept as photodynamic therapy (PDT). Modern PDT is based on three complementary components: visible light of a specific wavelength, an inert photosensitiser (PS) and oxygen, which leads to the formation of reactive oxygen species (ROS) with consequent cytotoxic effects on tumoural cells [7]. Several current clinical trials propose as possible PSs different compounds such as 5-aminolevulinic acid (5-ALA), its methylester or meso-tetra-hydroxylphenyl-chlorin (mTHPC). Although the action of activated porphyrins is important in clinical therapies there are some inconveniences that justify the permanent struggle for searching for an ideal PS. This has to be a PS that must accumulate in the tumour sufficiently enough so that it would generate large quantities of singlet oxygen that will radically cure it and 5, 10, 15, 20-tetra-sulfophenyl-porphyrin (TSPP) might be such a PS. Owing to its aggregation properties, which facilitate the distribution in tissues and raise the quantity of generated ROS [8], this synthetic porphyrin has demonstrated its efficiency on both in vitro and in vivo tumour models. In order to enhance the antitumoural response and to target different mechanisms involved in cellular death, our study proposes the association of the antidiabetic drug Metf with TSPP mediated-PDT on an in vivo experimental model. The effects of this combined therapeutic regimen were evaluated by determination of oxidative and nitrosative stress parameters, matrix metalloproteinases activities (MMPs), inducible nitric oxide synthase and cyclooxigenase-2 expressions and immunohistochemically quantification of apoptosis in correlation with the histopathological findings in the tumour. 2. Materials and methods 2.1. Reagents TSPP was obtained from Mrs. Rodica-Mariana Ion from the National Institute of R&D for Chemistry and Petrochemistry – ICECHIM, Bucharest, Romania (Fig. 1a). Firstly, the meso-tetra(4)-phenyl

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porphyrin (TPP) was synthesized from pyrrole and benzaldehyde in a reaction medium of propionic acid which was then purified with 2,3-dichloro-5,6-dicyanoquinone. To synthesize the tetrasulpho-phenyl-porphyrin (TSPP), TPP were dissolved in fuming H2SO4 (30% free SO3, heated at 85 °C and stirred). The resulting dark green precipitate was filtered and washed with HCl, then redissolved in NaOH and re-filtered from the water insoluble impurities. The filtrate was then neutralized with HCl, analysed by HPLC and stored at 0 °C till purification with chromatography [9]. 2-Thiobarbituric acid, 2,4-dinitrophenyl-hydrazine and guanidine hydrochloride were obtained from Merck KgA Darmstadt (Germany). Metformin (Fig. 1b), sodium dodecyl sulphate (SDS), Tris, Triton X-100, Coomassie brilliant blue R-250, NADPHdependent nitrate reductase, b-NADPH, N-(1-naphthyl) ethylenediamine hydrochloride, sulfanil-amide, trichloroacetic acid, Bradford reagent and o-phthalaldehyde were purchased from Sigma–Aldrich Chemicals GmbH (Germany), while absolute ethanol and n-butanol were provided by Chimopar (Bucharest). The TUNEL kit used to quantify apoptosis was supplied by Roche Applied Science, Mannheim (Germany), whereas the ELISA test for evaluation of nitrotyrosine levels was obtained from Blue Gene (China). Goat polyclonal IgG antibody for COX-2 and secondary antibody mouse anti-goat and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and rabbit polyclonal iNOS antibody from Thermo-Scientific (Germany).

2.2. Experimental design The experimental design included 6 groups (n = 48) of Wistar male rats (180 ± 20 g, 3 months old) bearing Walker 256 carcinosarcoma: group 1 received TSPP 10 mg/kg b.w. (TSPP), groups 2 and 4 received TSPP in the same dose and Metf, respectively, and were irradiated 24 h thereafter (TSPP + IR, Metf + IR), while group 3 received only Metf (20 mg/kg b.w.). The last two groups received the combined treatment with Metf prior to irradiation (TSPP + Metf + IR) and following irradiation (TSPP + IR + Metf). TSPP was administered intraperitoneally (i.p.) in 0.75 ml of phosphate bovine serum (PBS) and Metf was administered orally dissolved in distilled water. This pattern of drug (i.e. Metf) administration was chosen because of its similarity to human administration. Our experiment was designed at the Physiology Department and the Wistar albino rats were obtained from the Animal Department of ‘‘Iuliu Hatieganu’’ University of Medicine and Pharmacy, Cluj-Napoca. In order to acclimatize the animals, they were kept

Fig. 1. The chemical structures of photosensitiser (a) and metformin (b).

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for ten days in a 12 h dark and 12 h light regimen. They were fed a standard pellet diet and received water ad libitum. The rats were subcutaneously grafted small tumoural fragments of Walker 256 carcinosarcoma and when the tumour reached 1 cm3, PDT was administered. Irradiation was applied 24 h after drug administration and was performed with red light (k = 685 nm, 50 J/cm2, mean power 25 W/cm2, 10 kHz, spot diameter 10 mm, duration 15 min) provided by a Therapeutic LASER Model D-68. For this treatment the animals were shaved on the right thigh and were anaesthetized with an i.p. injection of ketamine + xylazine cocktail (90 mg kg 1 ketamine, 1090 mg kg 1 xylazine). 24 h after the irradiation, under anaesthesia, tumours from six animals were removed, fragmented and frozen at 80 °C until the biochemical investigation was performed. The remaining two tumours from each group were used for histopathological and immunohistochemical examination. All the experiments were performed according to the approved animal-care protocols of the Ethical Committee on Animal Welfare of the ‘‘Iuliu Hatieganu’’ University of Medicine and Pharmacy in accordance with the Romanian legislation, as part of Grant No. 22714/33/ 06.10.2011, and complying with the Guiding Principles in the Use of Animals in Toxicology. 2.3. Isolation of tumour samples For the biochemical analysis the fragments from the tumoural tissue were homogenized with a Polytron homogenizer (Brinkman Kinematica, Switzerland) following a standard protocol [10]. The Bradford method was used to measure the proteins content in the tumour homogenates [11]. For histopathological examination and immunohistochemistry, the tumoural samples were fixed in 10% buffered formalin and labelled. 2.4. Oxidative and nitrosative stress parameters 2.4.1. Malondialdehyde (MDA) MDA levels were determined using the fluorimetric method with 2-thiobarbituric acid [12]. The homogenate samples were mixed with a solution of 10 mM 2-thiobarbituric acid in 75 mM K2HPO4, pH 3, and afterwards heated in a boiling water bath for 1 h. Finally, after cooling, the reaction products were extracted in n-butanol. MDA was quantified spectrofluorimetrically in the organic phase using a synchronous technique with excitation at 534 nm and emission at 548 nm. MDA levels were expressed as nmoles/mg protein. 2.4.2. Protein carbonyls (PC) The reaction with 2,4-dinitrophenyl-hydrazine was used to obtain protein carbonyls [13]. The homogenates were treated with a solution of 2,4-dinitrophenyl-hydrazine 10 mM in 2.5 N HCl for 1 h, in the dark, at room temperature. Protein precipitation was obtained with 20% trichloroacetic acid followed by protein pellet centrifugation and washing (dissolved in guanidine hydrochloride 6 M). The absorbance was read at 355 nm and the carbonyl content was determined using the absorption coefficient for aliphatic hydrazones (22,000 M 1 cm 1). The final concentration of proteins in each sample was determined using a standard curve obtained with bovine serum albumin standards and was read at 280 nm. PC values were expressed as nmoles carbonyl/mg protein. 2.4.3. Reduced glutathione (GSH) Reduced glutathione (GSH) was determined using the fluorimetrical method based on o-phthalaldehyde reactive [14]. Samples were treated with trichloroacetic acid (10%) and centrifuged afterwards. A solution of o-phthalaldehyde (1 mg/ml in methanol) was added to supernatants diluted with sodium

phosphate buffer 0.1 M/EDTA 5 mM, pH 8.0. The fluorescence was recorded after 15 min (350 nm excitation and 420 nm emission) and GSH concentration was determined using a standard curve and expressed as nmoles/mg protein. 2.4.4. Nitric oxide (NO) Nitric oxide (NO) was measured by the Griess reaction model for nitrite plus nitrate by a two-step procedure [15]. In the first step the nitrate from the sample was reduced to nitrite using a conversion buffer containing glucose-6-phosphate (2.5 mM), glucose-phosphate dehydrogenase (final concentration 400U/l), and NADPH-dependent nitrate reductase (final concentration 200U/l) in 14 mM sodium phosphate buffer, pH 7.4, and a solution of NADPH (0.02 mmol). Incubation was followed by precipitation of proteins with ZnSO4 (3.5 M). After centrifugation the supernatant was treated with the Griess reagent (equal parts of 0.1 g N-1naphthyl) ethylene diamine hydrochloride in 100 ml water and 1 g sulfanil-amide in 100 ml 5% orthophosphoric acid). The absorbance of samples was read at 540 nm. The concentration of nitrite was calculated in comparison with a nitrite-standard curve and was expressed in nmoles/mg protein. 2.4.5. Nitrotyrosine Nitrotyrosine contents of tumour tissues were measured using the ELISA system, a quantitative sandwich immunoassay technique (Blue Gene, China) following the producer’s instructions. In order to measure the nitrotyrosine levels the results were compared to the standard curve of Optical Density. The results were expressed as pg/mg protein. 2.5. Evaluation of inflammation and angiogenesis markers In order to evaluate the inflammation, COX-2 and iNOS expressions were assessed. In addition, angiogenic markers (MMP-2 and MMP-9) were evaluated by zymography [16]. The paraffin embedded tumoural fragments were serially sectioned at 4 lm were used for immunohistochemical analyses. The following primary antibodies were used: rabbit polyclonal iNOS antibody (PA1-37925, Thermo-Scientific, dilution 1:100) and goat polyclonal COX-2 Antibody (N-20; sc-1746, Santa Cruz Biotechnology, dilution 1:200). Following deparaffinisation and rehydration, tissue sections were treated for 30 min with Peroxidase Block (RE7101, Novocastra) to block endogenous peroxidase. The next step was to boil the samples in 10 mM citrate buffer, pH 6.0 for 10 min for antigen retrieval, and then they were cooled at room temperature for 20 min. Primary antibody incubation was performed overnight at 4 °C for both antibodies. As negative controls, tumoural samples incubated only with antibody diluent (S0809, Dako) were used. After the sections were washed thoroughly with PBS, we used Novostain Universal Detection Kit reagents (Novocastra Laboratories Ltd., Benton Lane, Newcastle upon Tyne, UK). Thus, we incubated with biotinylated universal secondary antibody for 10 min, followed by incubation with ready-to-use streptavidin–peroxidase conjugate for 10 min. The immunostaining reaction product was developed using diaminobenzidine tetrahydrochloride (DAB) for 10 min and counterstained lightly with Gill’s Hematoxylin 2, followed by dehydration in ethanol, and mounting in an anhydrous mounting medium (Neo-Mount, Merck, Darmstadt, Germany). Two pathologists blinded for the treatment conditions evaluated the samples microscopically. Regarding COX-2 quantification, for every section, ten fields of 0.78 mm2 were randomly sampled and had their positive stained cells counted. The results were expressed as the mean percentage of COX-2 positive cells per field ± standard deviation. In case of iNOS expression, we evaluated the entire section, avoiding the areas with necrosis. The intensity of staining was scored as previously described by Aaltomaa et al. [17]

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as negative (0, no staining), weak (1, faint but clearly visible staining in the cell cytoplasm) and strong (2, clear strong staining). Tumour samples intended for zymography were homogenized with an ultraturrax homogenizer in a medium with glycerine and SDS in Tris–HCl buffer. The mixture was centrifuged at 12,000g and the content of protein was determined in supernatant. Five-microgram (5 lg) protein samples were subjected to electrophoresis on 10% polyacrylamide gels copolymerized with 1 g/ml gelatine at 100 V for 2 h. After electrophoresis, the gels were washed with 2.5% Triton X-100 for 1 h, then incubated for 18 h, at 37 °C, in 50 mM Tris–HCl buffer, pH 7.4, containing 5 mM CaCl2, 200 mM NaCl and 0.02% BRIJ-35. The gels were then stained with 1% Coomassie brilliant blue R-250 and discoloured in 10% acetic acid solution. The activity was evaluated with an automated Vilber-Lourmet (France) image analyser using the Bio 1-D program. Proteolytic activity was determined against a weight marker and was confirmed with an enzymatic marker. MMP-2 and MMP-9 levels were expressed as arbitrary units/lg protein.

2.6. Assessment of necrosis and apoptosis For the detection of necrosis, the sections, embedded in paraffin, were stained with haematoxylin–eosin. The Weibel scale was used to dimension the microscopic field. Each field had a surface of 0.156 mm2. The percentage of necrotic cells per mm2 at 400 magnification was determined. For the detection of apoptosis the formalin-fixed paraffin-embedded tumour tissue sections (4 lm thick) were deparaffinised by xylene and ethanol and then permeabilised with Pepsin 0.4% in HCl, pH 2 for 45 min at 37 °C. The TUNEL (TdT mediated dUTP nick end labelling) method was performed according to the manufacturer’s protocol (In Situ cell death detection kit, Roche Applied Science, Mannheim, Germany). Following incubation with TUNEL reaction mixture for 60 min at 37 °C, the slides were washed three times in PBS. For the negative controls the reaction mixture was not added to the slides. As a next step, the nuclei were stained with Blue pseudocolour (Draq5, Cell Signalling Technology, Massachusetts, USA) for 5 min in the dark, washed two times in PBS and then coverslips were mounted with aqueous antifade reagent (Mowiol 4-88-Carl Roth, Germany) and examined immediately. The tumoural tissue slides were viewed and images were collected using a Zeiss LSM 710 Confocal Laser Scanning unit (Carl Zeiss AG, Oberkochen, Germany) equipped with Argon and HeNe laser mounted on an Axio Observer Z1 Inverted Microscope. Specific visualization of TUNEL positive cells and of nuclei was achieved using the 488 and 633 nm excitation laser lines, and detected at 522 nm and 670 nm, respectively. Confocal images were recorded using a 10/0.45 Dry Plan-Apochromat Zeiss objective. The image combination, processing, and analysis were performed using the standard ZEN software package (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany). Quantification of TUNEL-positive cells was assessed by evaluating at least 3 random 10X fields/slide (approximately 1.92 mm2) and expressed as a percentage of positive cells/section, as previously described in Bolfa et al. [18]. At least 1000 nuclei were counted on each slide.

2.7. Statistical analysis The data are expressed as the means ± standard deviations in 8 animals. Comparisons were made by ANOVA one-way analysis of variance, with a Tukey’s Multiple Comparison Test. All statistical analyses were done by using ANOVA GraphPad Prism software, version 5.0. A p value less than 0.05 is considered to represent a statistically significant difference.

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3. Results 3.1. Effects of Metf and TSPP–PDT on oxidative and nitrosative stress parameters 3.1.1. Mda In order to quantify the action of reactive oxygen species on lipids, the malondialdehyde levels were evaluated in tumour homogenates. MDA levels were elevated in the groups subjected to the combined therapy (TSPP + Metf + IR: 0.44 ± 0.12 nmoles/ mg protein, p < 0.05 and TSPP + IR + Metf: 0.41 ± 0.1 nmoles/mg protein, p < 0.05) as compared with the group treated only with TSPP–PDT (0.18 ± 0.05 nmoles/mg protein). The greatest amount of lipid peroxides was recorded in the group which received the oral antidiabetic after TSPP and prior to irradiation (Fig. 2a). The administration of TSPP (0.22 ± 0.04 nmoles/mg protein) and Metf (0.12 ± 0.03 nmoles/mg protein) without irradiation maintained low levels of lipid peroxides in the tumour tissue. 3.1.2. Pc The reaction of proteins with reactive dicarbonyls will induce the inactivation of essential cellular proteins with important functional consequences. In our experimental groups, the treatments applied did not influence the protein carbonyls formation (p > 0.05) (Fig. 2b). The administration of Metf decreased PC levels but this decrease was statistically insignificant when compared to control, i.e. the TSPP–PDT group (6.01 ± 0.1 vs. 9.22 ± 3.00 nmoles/ mg protein, p > 0.05). 3.1.3. Gsh Glutathione is an important non-enzymatic cellular compound that prevents the destruction caused by reactive oxygen species. In the group that received TSPP and Metf prior to irradiation, GSH levels increased as an adaptive response to oxidative stress (5.31 ± 1.81 nmoles/mg proteins) as compared with the groups that received only the anti-diabetic drug (Metf: 1.22 ± 0.17 nmoles/mg proteins, p < 0.05, and Metf + IR: 1.56 ± 0.21 nmoles/mg proteins, p < 0.05, respectively) (Fig. 2c). The last group, where Metf was administered after the PDT, shows no significant results when compared to the other groups (2.77 ± 2.94 nmoles/mg proteins, p > 0.05). 3.1.4. NO and nitrotyrosine To evaluate the nitrosative stress induced by PDT and Metf we examined nitric oxide and nitrotyrosine levels. We did not find a statistically significant difference between TSPP + IR group and the group that received only TSPP (p > 0.05) which suggests that TSPP–PDT does not induce NO formation (Fig. 2d). The administration of Metf decreased the amount of NO found in tumour homogenates when compared to the group treated only with TSPP + IR (3.11 ± 0.71 vs. 8.09 ± 0.97 nmoles/mg protein, p < 0.05). The irradiation of tumours after Metf treatment significantly decreased NO formation when compared to the control group (2.45 ± 1.09 vs. 8.19 ± 4.3 nomles/mg protein; p < 0.05). Furthermore, the combined therapies, especially the association of Metf prior to irradiation, significantly reduced NO tumour levels when compared to TSPP–PDT group (3.75 ± 1.15 vs. 8.19 ± 4.3 nmoles/mg protein; p < 0.05). Metf administered after TSPP–PDT maintained the same low values in tumour, close to group 5, but the results were not statistically significant (3.75 ± 1.15 nmoles/mg protein, p > 0.05). The decreased levels of NO in the last two groups are due to the consumption of NO in the reaction with superoxide anion and nitrotyrosine production. As a result, nitrotyrosine levels in the groups treated with TSPP + Metf + IR (73.50 ± 12.03 pg/mg protein) and TSPP + IR +

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Fig. 2. The effects of TSPP-mediated PDT and Metf administration on MDA (a), PC (b), GSH (c), NO (d) and nitrotyrosine (e) levels in tumour tissue in Wistar W256-grafted rats. The rats were subjected to different treatments as described in the experimental design. 72 h After initiating the treatment oxidative stress markers were evaluated. The groups subjected to the combined therapeutic regimen showed an increase in lipid peroxidation (a) and nitrotyrosine levels (e) (p < 0.001), while NO levels were reduced (d). As an adaptive response to oxidative stress, GSH levels elevated in the group treated with TSPP and Metf prior to irradiation (c). The statistical analysis was done by one-way ANOVA followed by Tukey’s multiple comparison posttest (p < 0.01, p < 0.001). Data were expressed as mean ± standard deviation (SD).

Metf (85.84 ± 15.49 pg/mg protein) are higher than in the group treated with Metf + IR (p < 0.01) (Fig. 2e). This fact may lead to a prooxidant role of Metf in association with TSSP-based PDT. We have also obtained a statistical difference between Metf administered as single treatment and Metf administered prior to irradiation in the combined treatment (27.79 ± 5.61 vs. 73.50 ± 12.03 pg/mg protein; p < 0.001). 3.2. Evaluation of inflammation and angiogenesis after combined treatment 3.2.1. COX-2 expression COX-2 quantified by western blot technique revealed a reduction of its expression after the treatment with TSPP and Metf (Fig. 3b and c). Thus, both of the combined regimens significantly decreased COX-2 expression (TSPP + Metf + IR: 1.01 ± 0.03; TSPP + IR + Metf: 0.73 ± 0.10) as compared with TSPP–PDT group (1.58 ± 0.05; p < 0.001). The comparison of the results obtained in the groups treated with combined therapy revealed that the greatest reduction was noticed in the group which received Metf after irradiation (p < 0.01). Immunohistochemically, we noticed a significant decrease in COX-2 expression in the groups that were subjected to the combined therapeutic regimens when compared to the control group or the group treated with Metf (Fig. 3a). 3.2.2. iNOS expression Overall, iNOS expression was mainly observed in the cytoplasm of tumoural cells, but also in macrophages from stroma and some endothelial cells (Fig. 4). TSPP and TSPP + IR groups had similar

patterns of iNOS expression, with the staining pattern being either weak (25% for TSPP and 37.5% for TSPP + IR) or strong (75% for TSPP and 62.5% for TSPP + IR). The animals that received only Metf presented a reduction in staining intensity in the majority of the tumours (62.5%) showing a weak iNOS expression (both in Metf and Metf + IR). In one case (in the Metf group) one tumour specimen was totally negative regarding iNOS expression. A strong positive iNOS expression was observed in less than 50% of the tumours from animals with different combinations of Metf treatment (25% in the Metf and TSPP + Metf + IR groups, 37.5% in the Metf + IR group and 50% in the TSPP + IR + Metf group). Combined therapies resulted in a reduction of iNOS expression as compared to the TSPP groups (with or without irradiation), with most of the animals showing a weak expression (75% for TSPP + Metf + IR group and 50% of the tumours from the TSPP + IR + Metf group). 3.2.3. MMP-2 and MMP-9 activities MMPs’ activity was used as a surrogate marker to assess angiogenesis and vascular remodelling. Analysis of zymographic bands revealed only the presence of MMP-2 in all the groups we studied (Fig. 5a, b and d). The activity of MMP-9 could not be quantified. MMP-2 total activity decreased in all the groups treated with Metf (Fig. 5c). Besides, there was a strong statistically significant difference between the groups that received Metf (Metf: 550.7 ± 17.09 pg/mg protein, Metf + IR: 640.6 ± 46.01 pg/mg protein and TSPP + IR + Metf: 604.2 ± 47.12 pg/mg protein) when compared to control: 1068 ± 209.8 pg/mg protein (p < 0.001). Nonetheless, the association of Metf prior to irradiation (TSPP + Metf + IR, 740.6 ± 80.65 pg/mg protein) was not as efficient

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Fig. 3. The effects of TSPP-mediated PDT and Metf administration on tumour tissue COX-2 expression in Wistar rats. W256 carcinosarcoma-grafted Wistar rats were subjected to treatment as shown in the experimental design. 72 h After the initiation of the treatment COX-2 expression in the tumour was evaluated by immunohistochemistry and western blot technique. (a) Photomicrographs of tumour tissue COX-2 from each experimental group (TSPP, TSPP + IR, Metf, Metf + IR, TSPP + Metf + IR, TSPP + Ir + Metf) were shown in upper panels. (b and c) Representative images of immunoblotting for COX-2 and GAPDH in tumour tissue and the results of statistical analysis are shown in the lower panels. The statistical analysis was done by one-way ANOVA followed by Tukey’s multiple comparison posttest (p < 0.05).

as the combined regimen that associated the adjuvant drug following PDT (p < 0.01, when compared to TSPP + IR). 3.3. Assessment of necrosis and apoptosis after treatment with Metf and TSPP–PDT The tumour specimens stained with haematoxylin–eosin (HE) were examined in order to detect the onset of intra-tumour necrosis (Fig. 6). In the groups treated with Metf and TSPP–PDT amorphous cells with pyknotic or absent nuclei, without clear

intercellular limits, interspersed with viable cell areas were observed. In some areas the necrosis was largely extended and the architecture was altered. The necrotic percentage was increased in the group treated with Metf before TSPP–PDT (23.1 ± 1.6 necrotic cells) compared to group treated only with TSPP + IR (15.02 ± 3.0% positive cells; p < 0.05) or Metf + IR (15.02 ± 3.0% necrotic cells; p < 0.01). The necrosis rate was also increased in group treated with Metf after TSPP–PDT (30.05 ± 3.6 necrotic cells; p < 0.001) as compared to TSPP + IR. The magnification of the histological images was 40 (Fig. 6).

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Fig. 4. Immunohistochemical assessment of tumour iNOS expression in Wistar W256-grafted rats. The experimental groups were subjected to different treatments as described in the study design. Tumour samples taken at 72 h after the treatment started were used to quantify iNOS expression. iNOS expression was mainly observed in the cytoplasm of tumoural cells but also in macrophages from stroma and some endothelial cells. The intensity of staining was scored as negative (0, no staining), weak (1, faint but clearly visible staining in the cell cytoplasm) and strong (2, clear strong staining). The combined therapies decreased iNOS expression when compared to the TSPP groups (with or without irradiation), with most of the animals showing a weak expression (75% for TSPP + Metf + IR group and 50% of the tumours from the TSPP + IR + Metf group).

The apoptotic rate was determined as mean percentage ± standard deviation of TUNEL-positive cells/field. Interestingly, the majority of the TUNEL positive cells were located in the mesenchymal component of the tumour (Fig. 7a). Groups subjected to the combined therapeutic regimen expressed a significantly higher apoptotic rate (Fig. 7b). Therefore, an increased index was observed in the group that received Metf prior to irradiation (TSPP + Metf + IR: 96 ± 0.5 positive cells) when compared to TSPP + IR (60 ± 2.5 positive cells; p < 0.001). The index was also raised in the group in which Metf administration followed PDT (TSPP + IR + Metf, 107.5 ± 13.5 positive cells) compared to the control (p < 0.001), suggesting a better therapeutic outcome.

4. Discussions Experimental research, clinical and epidemiological data state that Metformin is a potential anticarcinogenic agent, particularly for tumours associated with an increase amount of insulin such as breast, colon, ovarian and prostate cancer [19]. Based on these findings, the purpose of our study was to strengthen the beneficial role of Metf in a Walker-256 carcinosarcoma experimental model and also to associate it with a promising and attested oncological approach, the photodynamic therapy. For this reason, we have evaluated the effects of 5, 10, 15, 20-tetra-sulfophenyl-porphyrin

(TSPP)-mediated PDT by assessing oxidative and nitrosative stress parameters and matrix metallo-proteinases activities in correlation with COX-2 and iNOS expressions as markers of inflammation and angiogenesis. Also, we have immunohistochemically assessed the tumour cellular death rate. A plethora of both fundamental and clinical data is a consisting proof of biguanide’s efficiency as a new direction in cancer research [20]. Consequently, Metf induced apoptosis in breast, colon, ovarian, glioma and prostate tumoural cell cultures and was involved in preventing the epithelial to mesenchymal transition (EMT) [21]. Also, Metf administered to mice bearing breast, colon and lung tumours unveiled a decreased tumour progression rate when associated with Trastuzumab and also revealed reduced Her-2 positive breast cells in transgenic animals [22]. In rats bearing the Walker-256 tumour, biguanides induced an increased area of tumoural necrosis and also reduced the adiposity of the obese animals [23]. Metf had contradictory effects though when it was administered to cancer patients. According to a non-placebo controlled study conducted on non-diabetic women with operable breast cancer, the biguanide reduced the tumour cell proliferation [24]. In other clinical trials, Metf administered in a dose of 250 mg/ day decreased the proliferation of both the aberrant crypt foci and the proliferative epithelium in colon cancer [25]. On the other side, there were some studies that revealed Metformin’s lack of importance in improving patient’s oncological outcome [26].

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Fig. 5. The effects of TSPP-mediated PDT and Metf administration on MMP-2 activities in tumour tissue. W256 carcinosarcoma-grafted Wistar rats were subjected to TSPP and Metf alone (TSPP, Metf), TSPP and Metf-mediated PDT (TSPP + IR, Metf + IR) and to a combined therapeutic regimen, TSPP + Metf + IR and TSPP + IR + Metf. At 72 h after the treatment started the inactive (a), active (b) and total (c) activities of MMP-2 in the tumour was assessed. (d) On zymography proMMP-2 and MMP-2 activities significantly decreased in all the groups that received Metf when compared to control (p < 0.001), with a smaller decrease in the group that associated Metf prior to irradiation (p < 0.01). The statistical analysis was done by one-way ANOVA followed by Tukey’s multiple comparison posttest (p < 0.01, p < 0.001). Data were expressed as mean ± SD.

In order to increase Metformin’s efficiency in reduced insulin level cancers, we propose for the first time the association of this antidiabetic drug with PDT. Walker 256 carcinosarcoma was chosen as tumoural model because, on the one hand, it can be easily transplanted to rats and, on the other hand, it has been demonstrated to be associated with altered levels of insulin. Zepp A. stated that rats bearing Walker 256 carcinosarcoma have reduced circulating insulin levels when compared to non-tumour-bearing rats and suggested that the tumoural cells would contain serine proteases that degrade the hormone [27]. PDT has been used as treatment because it is an attested anticancer therapy and it has already been used in a wide variety of tumours such as breast, lung, bladder, gastric and colorectal cancer and also cholangiocarcinoma with good results [28]. TSPP is a synthetic PS which has extensively been studied in PDT, both in in vitro and in in vivo studies [29]. It has been demonstrated that TSPP is a good candidate as a PS due to its solubility and aggregability properties, which allow it to be easily incorporated by the malignant cells. Furthermore, several studies have demonstrated its antineoplastic role by the generation of singlet oxygen, the strongest cytotoxic agent of all ROS for tumoural cells [30]. Our study confirms the promising outcome following the combined regimens, and states that Metf associated with PDT has a prooxidant role, induces a higher index of apoptosis when compared to the administration of Metf or PDT alone. In addition, the association of Metf with PDT diminished the expressions of iNOS and COX-2 in tumour tissue. The citophototoxic effect of PDT mediated by ROS generation and consequently by the increase of lipid

peroxidation was amplified when Metf was associated with PDT. These results showed a prooxidant effect when Metf was combined with PDT, both prior irradiation and after PDT. The complex cell response to oxidative stress involves the presence of ROS sensors which react to changes in the cellular redox potential and to the accumulation of ROS. GSH is an essential factor for GPx peroxidases, which detoxify peroxides (hydrogen peroxide, lipid peroxides) by reacting them with GSH. In order to prevent oxidative damage, the GSSG is reduced to GSH by GSSG reductase at the expense of NADPH, forming a redox cycle. In extreme conditions of oxidative stress, as was observed in the groups treated with Metf and PDT, the ability of the cell to reduce GSSG to GSH may be higher as adaptive response. The cytotoxic role of the combined treatment was also confirmed by the large tumour apoptotic areas. This is even more important as Metf was shown to be an antioxidant agent in different biological systems, preventing DNA mutations and ROS-induced caspase cellular death [31] without interacting with protein oxidation [32]. It is known that tumours which secrete high levels of NO would decrease patient outcome because of vasorelaxation and consequently because of promotion of tumour development [33]. In our experiment, in the groups treated with Metf alone and in those treated with Metf in combination with PDT, NO levels decreased, consequently increasing nitrotyrosine formation. The low levels of NO were also correlated with the decreased activity of iNOS expression in the same two groups. The high nitrotyrosine levels state that the superoxide anion produced by PDT in combination

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Fig. 6. Assessment of necrotic cells in tumours stained with HE. For the detection of necrosis, the sections, embedded in paraffin, were stained with haematoxylin–eosin. The Weibel scale was used to dimension the microscopic field. Each field had a surface of 0.156 mm2. The percentage of necrotic cells per mm2 at 400 magnification was determined (scale bar = 50 respectively 20 lm). The necrotic percentage was increased in the group treated with Metf before TSPP–PDT compared to group treated only with TSPP + IR (p < 0.05) or Metf + IR (p < 0.01). The necrosis rate was also increased in group treated with Metf after TSPP–PDT (p < 0.001) as compared to TSPP + IR. The statistical analysis was done by one-way ANOVA followed by Tukey’s multiple comparison posttest (p < 0.01, p < 0.001). Data were expressed as mean ± standard deviation (SD).

with the oral anti-diabetic drug generated peroxynitrite anions that further reacted with tyrosine and finally led to the formation of nitrotyrosine. As a result, Metf inhibited the tissue growth by lowering NO levels and decreasing iNOS activity and generated highly cytotoxic compounds, thus revealing and strengthening its antitumoural effect. MMP-2 and 9 activities and COX-2 expression were evaluated because of their role in angiogenesis by modulating VEGF bioavailability and finally their involvement in tumour metastasis [34]. Metf was known to inhibit cancer invasion through the AMPK enzyme. Experimental data demonstrated that Metf limited the invasion of melanoma by suppressing the matrix metalloproteinases 2 and 9 through a not fully elucidated AMPK dependent manner [35]. These properties could explain the results obtained in our experiment. Thus, Metf in association with PDT reduced MMP-2 activity and COX-2 expression as compared to the group treated only with PDT. Also, another study revealed that Metf blocked a component of a signal transduction pathway that was transmitted to NF-jB and a subsequent inflammatory response. Thus, the inhibition of the inflammatory process may be one explanation for the antitumoural effect of Metf in human cancers [36]. It seems that the intricate molecular pathway that acts through the AMPK enzyme may also be involved in a high apoptotic response in different tumours [37]. Several studies showed that a poor prognosis of a cancer patient was associated with a dysfunction of the AMPK enzyme. Zheng et al. revealed that the

antidiabetic did not only activate AMPK by inhibiting hepatocellular carcinoma cells growth in vitro and in vivo, but also augmented cisplatin tumouricidal effects [38]. The mechanisms involved are not fully elucidated yet. Thus, some authors demonstrated that the biguanides were involved in cellular cycle arrest and did not induce apoptosis [39]. Other authors suggested that cellular death-PDT induced was triggered by ROS which damaged DNA and induced activation of caspases [40]. In addition, Metf inhibited the Bcl-2 anti-apoptotic proteins and increased the apoptotic rate when administered in combination with other cytotoxic drugs, as shown in epithelial ovarian cancer [41]. In our study, in the groups treated with Metf in a single administration, the apoptosis was comparable with that obtained after TSPP treatment, probably due to the low dose or to the type of tumour used. Instead, the apoptotic index was strongly increased at the combined regimen in correlation with ROS formation and reduced angiogenesis. Last but not least, our results concerning an increased apoptotic index and limited invasion are associated with the raised levels of nitrotyrosine and high levels of glutathione, indirect hallmarks of reactive oxygen species and reactive nitrogen species production. It is known that Metf partially inhibits the complex one from the respiratory chain and generates superoxide anion which will react with nitric oxide released from mitochondrial or cellular membrane to form reactive nitrogen species such as ONOO . ONOO will further activate AMPK enzyme via c-Src-mediated, PI3 Kdependent pathway and will lead to the hypoglycemia [42]. Our

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Fig. 7. Assessment of apoptotic cells by the TUNEL assay in the experimental groups. W256 carcinosarcoma-grafted Wistar rats were subjected to TSPP and Metf alone (TSPP, Metf), TSPP and Metf-mediated PDT (TSPP + IR, Metf + IR) and to a combined therapeutic regimen, TSPP + Metf + IR and TSPP + IR + Metf. Tumour samples sampled at 72 h after the treatment started were fixed in 4% buffered formaldehyde and paraffin-embedded. (a) Apoptotic nuclei (bright green) and viable nuclei (Draq5 labelled) were observed using a confocal microscope. (b) Quantification of the TUNEL-positive cells in the tumour. Results are expressed as mean percentage of positive cells ± SD. The apoptotic index was higher in the groups treated with combined therapies (p < 0.001) with more apoptotic cells revealed in the group that received Metformin after TSPPmediated PDT. Statistical analysis was done by a one-way ANOVA with Tukey’s multiple comparisons posttest (p < 0.05; p < 0.01; p < 0.001).

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combined regimen, Metf and TSPP–PDT, increased nitrotyrosine formation, a stable marker for reactive nitrogen species generation and thus confirmed the beneficial role of them in activating AMPK and consequently in inhibition of tumour cell growth. However, the antitumoural mechanisms involved are not fully clarified. Metf can act by both dependent and independent insulin pathway, by activating the AMPK enzyme, thus blocking the mitogenic effect of insulin and also protein synthesis through the inhibition of the mTOR complex, an important relay centre for signals concerning tumour cell proliferation and angiogenesis [43,44].

5. Conclusions Due to the fact that both Metf and PDT are intensely studied by scientists and are bursting into oncological clinical trials, a future research challenge might be to associate them. Our study reveals for the first time the effects of the combined treatment that associates PDT with the antidiabetic drug Metf, defining a novel oncological approach. This combined therapeutic regimen induced oxidative stress, increased the apoptotic rate and reduced angiogenesis and tumour inflammation. The effects are independent of the chosen therapeutic associations. These first results are promising and could open new perspectives in oncology treatments. Nevertheless, further experimental data are needed for determining the exact doses of the biguanide and therapeutic scheme in order to obtain the best antitumoural response on future clinical trials. Finally, it is important to evaluate the exact molecular pathways involved in the combined action of the anti-diabetic and PS in order to exploit the additive effect of the two therapies.

Conflict of interests The authors declare that they have no competing interests. Acknowledgements The authors are grateful to ‘‘Iuliu Hatßieganu’’ University of Medicine and Pharmacy, Cluj-Napoca, Romania, for supporting their work through an Internal Grants Program as stated in contract no. 22714/33/06.10.2011. In addition, they wish to thank Rodica Boros, Doina Daicoviciu and Nicoleta Decea for their technical contribution and Remus Moldovan for animal handling. They are also thankful to Felix Mic for his kind antibody supply. References [1] S.D. Hursting, N.A. Berger, Energy balance, host-related factors, and cancer progression, J. Clin. Oncol. 28 (2010) 4058–4065, http://dx.doi.org/10.1200/ JCO.2010.27.9935. [2] L.A. Witters, The blooming of the French lilac, J. Clin. Invest. 108 (2001) 1105– 1107, http://dx.doi.org/10.1172/JCI200114178. [3] C.J. Bailey, R.C. Turner, Metformin, N. Engl. J. Med. 334 (1996) 574–579, http:// dx.doi.org/10.1056/NEJM199602293340906. [4] M. Zakikhani, R. Dowling, I.G. Fantus, N. Sonenberg, M. Pollak, Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells, Cancer Res. 66 (2006) 10269–10273, http://dx.doi.org/10.1158/0008-5472.CAN-06-1500. [5] G. Chen, S. Xu, K. Renko, M. Derwahl, Metformin inhibits growth of thyroid carcinoma cells, suppresses self-renewal of derived cancer stem cells, and potentiates the effect of chemotherapeutic agents, J. Clin. Endocrinol. Metab. 97 (2012), http://dx.doi.org/10.1210/jc.2011-1754. E510–20. [6] H.A. Hirsch, D. Iliopoulos, P.N. Tsichlis, K. Struhl, Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumour growth and prolong remission, Cancer Res. 69 (2009) 7507–7511, http:// dx.doi.org/10.1158/0008-5472.CAN-09-2994. [7] P. Agostinis, K. Berg, K.A. Cengel, T.H. Foster, A.W. Girotti, S.O. Gollnick, S.M. Hahn, M.R. Hamblin, A. Juzeniene, D. Kessel, M. Korbelik, J. Moan, P. Mroz, D. Nowis, J. Piette, B.C. Wilson, J. Golab, Photodynamic therapy of cancer: an update, CA Cancer J. Clin. 61 (2011) 250–281, http://dx.doi.org/10.3322/ caac.20114.

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