Pharmacological Reports 66 (2014) 121–129
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Fluvastatin inhibits growth and alters the malignant phenotype of the C6 glioma cell line Adrianna Sławin´ska-Brych a,*, Barbara Zdzisin´ska b, Martyna Kandefer-Szerszen´ b a b
Department of Cell Biology, Maria Curie-Sklodowska University, Lublin, Poland Department of Virology and Immunology, Maria Curie-Sklodowska University, Lublin, Poland
A R T I C L E I N F O
Article history: Received 15 November 2012 Received in revised form 7 July 2013 Accepted 2 August 2013 Available online 1 February 2014 Keywords: Glioma cells Fluvastatin VEGF MMP-9 MAPKs Actin cytoskeleton
A B S T R A C T
Background: Fluvastatin is a member of the family of HMG-CoA reductase inhibitors (statins) extensively used in medical practice. Increasing evidence suggests that fluvastatin may be implicated in suppression of cancer growth and development. The aim of the present study was to investigate the anti-cancer potential of fluvastatin in C6 rat malignant glioma cells. Methods: First, the effects of fluvastatin on cell viability (MTT assay), proliferation (BrdU assay), cell morphology, and cytoskeleton were examined. Subsequently, its effect on extracellular signal regulated kinase 1 and 2 (ERK1/2) and c-Jun N-terminal kinase 1 and 2 (JNK 1/2) expression was estimated by Western blot. Finally, the influence of fluvastatin on cell migration and production of MMP-9 and VEGF was determined using a wound-healing assay and ELISA test, respectively. Results: The results obtained showed that fluvastatin had a remarkable inhibitory and cytotoxic effect on tumor C6 cells (IC50 = 8.6 mM, 48 h), but did not inhibit the growth of normal neuronal cells. The concentrations from 1 to 10 mM induced marked morphologic alterations typical for apoptosis including shrinkage of cytoplasm, chromatin condensation, and nucleus breakdown. Conclusion: The inhibitory effects of fluvastatin on cell proliferation seemed to be associated with decreased p-ERK1/2 expression, upregulation of p-JNK1/2, and reduction in the MMP-9 and VEGF concentrations in culture media. The high anticancer (antiproliferative, proapoptotic, antiinvasive) activity of fluvastatin and lack of its toxicity against normal cells indicate a potential use of this statin in the treatment of malignant glioma. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Gliomas are the most common and most serious type of brain cancer in adults and account for nearly 70% of all intracranial neoplasms [29]. The incidence rate for glioblastoma is approximately 3–4 per 100,000 people per year. Men are more frequently affected than women [2]. Glial tumors are characterized by highly proliferative growth, aggressive behavior, and drug-resistance. In spite of the current therapeutic options, including surgery, chemotherapy, radiotherapy, or other novel modalities, the median survival of patients still remains short. Therefore, novel pharmaco-
Abbreviations: HMG-CoA, 3-hydroxy 3-methyl-glutaryl coenzyme A; MAPKs, mitogen-activated protein kinases; MMPs, matrix metalloproteinases; GPP, geranylpyrophosphate; GGPP, geranylgeranylpyrophosphate; FPP, farnesylpyrophosphate; BrdU, bromodeoxyuridine; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide. * Corresponding author. E-mail address: [email protected] (A. Sławin´ska-Brych).
logical substances are constantly sought after to improve the results of treatment of patients with malignant gliomas [7,15,28]. There is some evidence that lipid lowering drugs called statins may exhibit potential activity against brain tumors [28]. Statins are now widely used in the treatment of hypercholesterolemia and cardiovascular diseases [45,46]. These drugs have also been demonstrated to be beneficial for many other human diseases, in particular, macular degeneration, ischemic stroke, Alzheimer’s disease, and multiple sclerosis [6,26]. As the structural competitive analogs of 3-hydroxy-3-methylglutaryl-CoA, statins not only inhibit cholesterol biosynthesis but also decrease the production of nonsterol isoprenoids. These include geranylpyrophosphate (GPP), geranylgeranylpyrophosphate (GGPP), and farnesylpyrophosphate (FPP). They participate in the post-translational modification of many intracellular molecules, especially Ras and Rho proteins which are involved in intracellular signaling. Hence, reduced levels of these intermediates may have important biological consequences [14,25]. Some experiments in vitro and in vivo indicate putative association between non-lipid-related effects of statins and their
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2014.01.002
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anticancer activity [8]. Statins have recently been reported to possess cytotoxic, antiproliferative, proapoptotic, and antiinvasive potential in a broad range of human malignant tumor cell lines like neuroblastoma, medulloblastoma, melanoma, breast and colon carcinoma, head and neck squamous cell carcinoma, lung cancer, leukemia, and multiple myeloma [11,14,16,25,46]. Such a wide spectrum of the anticancer action of statins may explain the increasing interest of many oncologists in application of statins as antitumor drugs. However, up till now, there are only few papers concerning the anticancer effects of statins in glioma cells. Therefore, in the present study, we investigated the antiproliferative and antiinvasive activity of fluvastatin in the rat C6 cell line in order to evaluate its efficacy in treatment of gliomas.
ranging from 0.5 to 100 mM. After 48-h incubation at 37 8C in a humidified atmosphere of 5% CO2, the cytotoxic effect of fluvastatin was estimated by the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Briefly, the cells were incubated for 3 h with the MTT solution (5 mg/ml, Sigma–Aldrich). MTT was metabolized by viable cells to purple formazan crystals, which were solubilized overnight in SDS buffer (10% SDS in 0.01 N HCl) and the reaction product was quantified spectrophotometrically by measuring absorbance at 570 nm using an E-max Microplate Reader (Molecular Devices Corporation). The absorbance of the control wells was taken as 100% and the results were expressed as percentage of the control.
Materials and methods
DNA synthesis in proliferating cells was evaluated by measuring bromodeoxyuridine (BrdU) incorporation using a commercial Cell Proliferation ELISA System (Roche Molecular Biochemicals, Germany). The C6 cells were seeded into 96-well microplates at a density of 1 104 cells/ml in a proliferation medium containing 10% FBS. Next day, the medium was replaced with a fresh medium and the cells were exposed to 1–50 mM of fluvastatin for 48 h. After that, the cells were incubated for 2 h with a BrdU labeling solution containing 10 mM BrdU. The assay was performed according to the manufacturer’s instructions. The absorbance values were measured at 450 nm using an ELISA reader. The culture medium alone was used as a control for nonspecific binding.
Reagents Fluvastatin was purchased from Calbiochem (La Jolla, Ca, USA). Stock Solution (100 mM) was prepared in DMSO (Sigma–Aldrich, St. Louis, MO, USA). The final concentration of DMSO in all experiments was less than 0.01%, and all treatment conditions were compared with vehicle controls. The drug was diluted in a culture medium immediately before use. TRITC-phalloidin, the cell culture media, antibiotics, 0.25% trypsin–EDTA, fetal bovine serum (FBS), protease inhibitor cocktail, and primary antibodies (phospho-ERK1/2, anti-phospho-JNK1/2, anti-b-actin) were purchased from Sigma–Aldrich. The antibodies against total ERK1 and total JNK were from Santa Cruz Biotechnology (USA). The secondary antibody coupled to horseradish peroxidase was obtained from Pierce. Cell cultures Rat glioblastoma cell line C6 was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured in DMEM containing 10% FBS, 100 units/ml penicillin, and 50 mg/ml streptomycin. The medium was changed every two days. The cells were grown in 75 cm2 flasks (Nunc, Roskilde, Denmark) and kept in a humidified atmosphere with 5% CO2 at 37 8C. Neuronal cell culture was prepared from cortices of 18-day-old Wistar rat fetuses as described previously [34]. The tissue was pooled into ice cold glucose (33 mM) Hanks’ Balanced Salt Solution (HBSS, Sigma–Aldrich), cut into small pieces, and incubated for 30 min at 37 8C with a 0.25% trypsin–EDTA solution. A single cell suspension was obtained by gentle pipetting of the cortex fragments in the presence of 10% FBS and 0.01% DNase I (Sigma). The cells were then filtered through a nylon filter and centrifuged (at 800 rpm for 10 min). The cells were resuspended in Neurobasal Medium (Life Technologies, Karlsruhe, Germany) supplemented with L-glutamine (0.5 mM, Sigma–Aldrich), B-27 supplement (2%), and an antibiotic–antimycotic solution (1%, Life Technologies), and then distributed (1 104 cells/well) into poly-L-lysine (4 mg/ml, Sigma–Aldrich) coated 96-plastic plates (Nunc). The cells were cultured in a humidified atmosphere containing 5% CO2 at 37 8C. The culture medium was changed every three days, until the culture reached a monolayer (about 14 days) in vitro. Cell viability (MTT assay) The C6 and neuronal cells were plated on 96-well flat-bottomed microplates at a density of 1 104 cells/well in 100 ml of a complete growth medium. Before drug treatment, the growth medium was replaced with a fresh medium containing 2% FBS. The C6 and neuronal cells were exposed to fluvastatin at a concentrations
Bromodeoxyuridine (BrdU) cell proliferation assay
Assessment of morphological changes and cytoskeleton staining The C6 cells at a density of 5 104 cells/ml were grown in Leighton tubes containing rectangular glass coverslips for 24 h. Then, the medium was replaced with a fresh medium containing 2% FBS and different concentrations of fluvastatin (1, 5 or 10 mM), and the cells were incubated for 48 h at 37 8C. Control cells were cultured in a medium without fluvastatin. After the treatment, the cells were fixed and stained according to the standard hematoxylin–eosin method. Observation was performed under an Olympus BX51 System Microscope (Olympus Optical, Tokyo, Japan) and the micrographs were prepared using analySiS software (Soft Imaging System GmbH, Munster, Germany). A part of the cell cultures were fixed in a 3% formaldehyde solution in phosphate-buffered saline (PBS), pH 7.4, for 30 min at room temperature, incubated with 0.5% Triton X-100 in PBS for 15 min at room temperature, blocked in PBS containing 3% bovine serum albumin (BSA, Sigma–Aldrich) for 1 h at room temperature, and incubated with TRITC-labeled phalloidin (0.5 mg/ml) in PBS for 1 h at room temperature in the dark. Representative fluorescence micrographs of these cells were performed using an Olympus BX51 System Microscope. Western blot analysis The C6 cells were grown in 6-well plastic plates (3 105 cells/ ml with 3 ml/well) in a medium with 10% of FBS for 24 h at 37 8C. Next day, the medium was replaced with fresh 2% FBS-DMEM and the cells were treated with fluvastatin at the concentrations of 1, 5 or 10 mM. After 24 h of incubation, the cells were washed in cold PBS and lysed in RIPA buffer (containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 1 mM sodium orthovanadate, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail) at 4 8C for 30 min. Next, the lysates were centrifuged at 10,000 g for 15 min at 4 8C. The protein concentration was quantified using a BCA protein assay kit (Pierce1 BCA Protein Assay Kit, Thermo Scientific, Rockford,
A. Sławin´ska-Brych et al. / Pharmacological Reports 66 (2014) 121–129
USA). 50 mg of the samples was mixed with Laemmli buffer, boiled for 5 min, loaded on (9%) SDS polyacrylamide gel (SDS-PAGE), and separated electrophoretically. The proteins were transferred onto the Immobilon P membrane (Merck, Darmstadt, Germany). Following the transfer, the membrane was blocked with blocking buffer (5% nonfat dried milk in TBS/0.1% Tween 20) for 1 h at room temperature and then probed with appropriate dilutions of primary antibodies overnight at 4 8C. The membranes were washed 3 times for 10 min with PBS containing 0.05% Triton X100 and incubated for 1 h with the horseradish peroxidase-labeled anti-mouse antibody. The membranes were visualized with ECM Western Blot Chemiluminescence Reagent (Amersham Biosciences, Germany) using a Kodak Biomax film (Sigma–Aldrich). The blots were reprobed with antibodies against b-actin to ensure equal loading and transfer of proteins. Wound healing assay (migration of cells) The C6 cells (4 105 cells/ml) were seeded in culture dishes (4 cm in diameter, Nunc) in 2 ml of complete medium. After 24 h, the monolayer of cells was scratched with a pipette tip (P300) to create one linear wound. The cells were then rinsed with PBS and covered with a fresh medium with 2% FBS supplemented with 0.5, 1 or 2.5 mM of fluvastatin. The control cells were cultured in a culture medium without fluvastatin. The next day, the plates were
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stained according to the May–Gru¨nwald–Giemsa method and then observed under an Olympus BX51 System Microscope. Cells that migrated to the wound areas were counted on micrographs and the results were expressed as the mean number of cells that had migrated to 50 selected fields taken from four micrographs. Gelatin zymography The C6 cells were seeded at a density of 2 104/well into 24well tissue culture plates in DMEM–10% FBS. The cells were allowed to adhere for 24 h and then they were incubated with various concentrations of fluvastatin (0.5, 2.5 or 5 mM). The conditioned media were collected 48 h after the treatment with fluvastatin, mixed with nonreducing Laemmli sample buffer, and subjected to electrophoresis in 10% SDS–PAGE containing 0.1% (w/ v) gelatin. The gel was washed with 2.5% Triton X-100 (v/v) for 30 min at room temperature to remove SDS and allow the protein to renature; subsequently it was incubated in substrate buffer (50 mM Tris–HCl, pH 7.5, 1 mM ZnCl2, and 5 mM CaCl2) at 37 8C for 24 h. The gel was then stained with 0.5% (w/v) Coomassie blue in 10% acetic acid (v/v) and 30% methanol (v/v), and then destained with the same solution without Coomassie blue. Gelatinolytic activities were detected as unstained bands against the background of Coomassie blue-stained gelatin. It is important to note that in this SDS-containing gel, the latent form of MMP-9,
Fig. 1. The influence of fluvastatin on the viability of C6 glioma cells (A) and primary neuronal cells (B). The antiproliferative effect of fluvastatin (C). The cells were treated with fluvastatin at various concentrations for 48 h. Cell viability was determined by the MTT assay. Cell proliferation was determined by the BrdU incorporation assay. The results represent the mean SD of three independent experiments in triplicates. *Statistically significant at p < 0.01 in comparison to the control.
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pro-MMP-9, and the activated gelatinase develop gelatinolytic activity. Therefore, the word ‘‘activity’’ was used to indicate the total gelatinolytic activity measured in the conditioned media. Determination of MMP-9 and VEGF concentration by ELISA assay The total MMP-9 level in the conditioned media of the C6 cells was detected using a commercial human MMP-9 immunoassay kit (RayBiotec, Norcross, GA, USA), following the manufacturer’s protocol. The VEGF protein released into the conditioned medium was measured by a VEGF enzyme immunoassay kit (Strathmann Biotec, Hamburg, Germany) according to the manufacturer’s instructions. The C6 cells (3 105 cells/ml) were seeded in 24-well plates in 1 ml of DMEM with 10% of FBS and incubated for 24 h. Subsequently, the growth medium was changed by DMEM containing 2% FBS and the C6 cells were treated with fluvastatin (0.5, 2.5 or 5 mM). After 48-h exposition, the culture medium was collected, centrifuged, and frozen immediately at 80 8C until quantification of the MMP-9 or VEGF concentration.
reduced cell viability to approximately 14% in comparison to the control. In contrast to the C6 glioma cells, the rat normal neuronal cells were resistant to the fluvastatin treatment. When they were incubated with fluvastatin at the concentrations from 1 mM to 50 mM, the number of living cells in the cultures was markedly higher, compared to the untreated cells (Fig. 1B). These results clearly indicate selective, dose-dependent cytotoxicity of fluvastatin against tumor cells. To examine the antiproliferative potential of fluvastatin, the C6 glioma cells were treated with 1, 5, 10, 25, or 50 mM of fluvastatin for 48 h and the BrdU assay was performed. Fluvastatin effectively reduced BrdU incorporation during the DNA synthesis (Fig. 1C). A significant decrease in cell division was found even at 1 mM. However, the most pronounced effect was observed with 50 mM fluvastatin, which suppressed proliferation of the C6 cells in 70%. These data correspond with the MTT results and indicated a dosedependent antiproliferative activity of fluvastatin in the cells studied. Fluvastatin changes the morphology of C6 cells and cytoskeletal assembly
Statistical analysis The data were expressed as the means standard error. Statistical analyses were performed using GraphPAD Prism 4 (GraphPAD Software Inc., CA, USA). The data were analyzed by one-way analysis of variance, followed by Dunnett’s test; p values
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Fluvastatin inhibits growth and alters the malignant phenotype of the C6 glioma cell line Adrianna Sławin´ska-Brych a,*, Barbara Zdzisin´ska b, Martyna Kandefer-Szerszen´ b a b
Department of Cell Biology, Maria Curie-Sklodowska University, Lublin, Poland Department of Virology and Immunology, Maria Curie-Sklodowska University, Lublin, Poland
A R T I C L E I N F O
Article history: Received 15 November 2012 Received in revised form 7 July 2013 Accepted 2 August 2013 Available online 1 February 2014 Keywords: Glioma cells Fluvastatin VEGF MMP-9 MAPKs Actin cytoskeleton
A B S T R A C T
Background: Fluvastatin is a member of the family of HMG-CoA reductase inhibitors (statins) extensively used in medical practice. Increasing evidence suggests that fluvastatin may be implicated in suppression of cancer growth and development. The aim of the present study was to investigate the anti-cancer potential of fluvastatin in C6 rat malignant glioma cells. Methods: First, the effects of fluvastatin on cell viability (MTT assay), proliferation (BrdU assay), cell morphology, and cytoskeleton were examined. Subsequently, its effect on extracellular signal regulated kinase 1 and 2 (ERK1/2) and c-Jun N-terminal kinase 1 and 2 (JNK 1/2) expression was estimated by Western blot. Finally, the influence of fluvastatin on cell migration and production of MMP-9 and VEGF was determined using a wound-healing assay and ELISA test, respectively. Results: The results obtained showed that fluvastatin had a remarkable inhibitory and cytotoxic effect on tumor C6 cells (IC50 = 8.6 mM, 48 h), but did not inhibit the growth of normal neuronal cells. The concentrations from 1 to 10 mM induced marked morphologic alterations typical for apoptosis including shrinkage of cytoplasm, chromatin condensation, and nucleus breakdown. Conclusion: The inhibitory effects of fluvastatin on cell proliferation seemed to be associated with decreased p-ERK1/2 expression, upregulation of p-JNK1/2, and reduction in the MMP-9 and VEGF concentrations in culture media. The high anticancer (antiproliferative, proapoptotic, antiinvasive) activity of fluvastatin and lack of its toxicity against normal cells indicate a potential use of this statin in the treatment of malignant glioma. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Gliomas are the most common and most serious type of brain cancer in adults and account for nearly 70% of all intracranial neoplasms [29]. The incidence rate for glioblastoma is approximately 3–4 per 100,000 people per year. Men are more frequently affected than women [2]. Glial tumors are characterized by highly proliferative growth, aggressive behavior, and drug-resistance. In spite of the current therapeutic options, including surgery, chemotherapy, radiotherapy, or other novel modalities, the median survival of patients still remains short. Therefore, novel pharmaco-
Abbreviations: HMG-CoA, 3-hydroxy 3-methyl-glutaryl coenzyme A; MAPKs, mitogen-activated protein kinases; MMPs, matrix metalloproteinases; GPP, geranylpyrophosphate; GGPP, geranylgeranylpyrophosphate; FPP, farnesylpyrophosphate; BrdU, bromodeoxyuridine; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide. * Corresponding author. E-mail address: [email protected] (A. Sławin´ska-Brych).
logical substances are constantly sought after to improve the results of treatment of patients with malignant gliomas [7,15,28]. There is some evidence that lipid lowering drugs called statins may exhibit potential activity against brain tumors [28]. Statins are now widely used in the treatment of hypercholesterolemia and cardiovascular diseases [45,46]. These drugs have also been demonstrated to be beneficial for many other human diseases, in particular, macular degeneration, ischemic stroke, Alzheimer’s disease, and multiple sclerosis [6,26]. As the structural competitive analogs of 3-hydroxy-3-methylglutaryl-CoA, statins not only inhibit cholesterol biosynthesis but also decrease the production of nonsterol isoprenoids. These include geranylpyrophosphate (GPP), geranylgeranylpyrophosphate (GGPP), and farnesylpyrophosphate (FPP). They participate in the post-translational modification of many intracellular molecules, especially Ras and Rho proteins which are involved in intracellular signaling. Hence, reduced levels of these intermediates may have important biological consequences [14,25]. Some experiments in vitro and in vivo indicate putative association between non-lipid-related effects of statins and their
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2014.01.002
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anticancer activity [8]. Statins have recently been reported to possess cytotoxic, antiproliferative, proapoptotic, and antiinvasive potential in a broad range of human malignant tumor cell lines like neuroblastoma, medulloblastoma, melanoma, breast and colon carcinoma, head and neck squamous cell carcinoma, lung cancer, leukemia, and multiple myeloma [11,14,16,25,46]. Such a wide spectrum of the anticancer action of statins may explain the increasing interest of many oncologists in application of statins as antitumor drugs. However, up till now, there are only few papers concerning the anticancer effects of statins in glioma cells. Therefore, in the present study, we investigated the antiproliferative and antiinvasive activity of fluvastatin in the rat C6 cell line in order to evaluate its efficacy in treatment of gliomas.
ranging from 0.5 to 100 mM. After 48-h incubation at 37 8C in a humidified atmosphere of 5% CO2, the cytotoxic effect of fluvastatin was estimated by the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Briefly, the cells were incubated for 3 h with the MTT solution (5 mg/ml, Sigma–Aldrich). MTT was metabolized by viable cells to purple formazan crystals, which were solubilized overnight in SDS buffer (10% SDS in 0.01 N HCl) and the reaction product was quantified spectrophotometrically by measuring absorbance at 570 nm using an E-max Microplate Reader (Molecular Devices Corporation). The absorbance of the control wells was taken as 100% and the results were expressed as percentage of the control.
Materials and methods
DNA synthesis in proliferating cells was evaluated by measuring bromodeoxyuridine (BrdU) incorporation using a commercial Cell Proliferation ELISA System (Roche Molecular Biochemicals, Germany). The C6 cells were seeded into 96-well microplates at a density of 1 104 cells/ml in a proliferation medium containing 10% FBS. Next day, the medium was replaced with a fresh medium and the cells were exposed to 1–50 mM of fluvastatin for 48 h. After that, the cells were incubated for 2 h with a BrdU labeling solution containing 10 mM BrdU. The assay was performed according to the manufacturer’s instructions. The absorbance values were measured at 450 nm using an ELISA reader. The culture medium alone was used as a control for nonspecific binding.
Reagents Fluvastatin was purchased from Calbiochem (La Jolla, Ca, USA). Stock Solution (100 mM) was prepared in DMSO (Sigma–Aldrich, St. Louis, MO, USA). The final concentration of DMSO in all experiments was less than 0.01%, and all treatment conditions were compared with vehicle controls. The drug was diluted in a culture medium immediately before use. TRITC-phalloidin, the cell culture media, antibiotics, 0.25% trypsin–EDTA, fetal bovine serum (FBS), protease inhibitor cocktail, and primary antibodies (phospho-ERK1/2, anti-phospho-JNK1/2, anti-b-actin) were purchased from Sigma–Aldrich. The antibodies against total ERK1 and total JNK were from Santa Cruz Biotechnology (USA). The secondary antibody coupled to horseradish peroxidase was obtained from Pierce. Cell cultures Rat glioblastoma cell line C6 was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured in DMEM containing 10% FBS, 100 units/ml penicillin, and 50 mg/ml streptomycin. The medium was changed every two days. The cells were grown in 75 cm2 flasks (Nunc, Roskilde, Denmark) and kept in a humidified atmosphere with 5% CO2 at 37 8C. Neuronal cell culture was prepared from cortices of 18-day-old Wistar rat fetuses as described previously [34]. The tissue was pooled into ice cold glucose (33 mM) Hanks’ Balanced Salt Solution (HBSS, Sigma–Aldrich), cut into small pieces, and incubated for 30 min at 37 8C with a 0.25% trypsin–EDTA solution. A single cell suspension was obtained by gentle pipetting of the cortex fragments in the presence of 10% FBS and 0.01% DNase I (Sigma). The cells were then filtered through a nylon filter and centrifuged (at 800 rpm for 10 min). The cells were resuspended in Neurobasal Medium (Life Technologies, Karlsruhe, Germany) supplemented with L-glutamine (0.5 mM, Sigma–Aldrich), B-27 supplement (2%), and an antibiotic–antimycotic solution (1%, Life Technologies), and then distributed (1 104 cells/well) into poly-L-lysine (4 mg/ml, Sigma–Aldrich) coated 96-plastic plates (Nunc). The cells were cultured in a humidified atmosphere containing 5% CO2 at 37 8C. The culture medium was changed every three days, until the culture reached a monolayer (about 14 days) in vitro. Cell viability (MTT assay) The C6 and neuronal cells were plated on 96-well flat-bottomed microplates at a density of 1 104 cells/well in 100 ml of a complete growth medium. Before drug treatment, the growth medium was replaced with a fresh medium containing 2% FBS. The C6 and neuronal cells were exposed to fluvastatin at a concentrations
Bromodeoxyuridine (BrdU) cell proliferation assay
Assessment of morphological changes and cytoskeleton staining The C6 cells at a density of 5 104 cells/ml were grown in Leighton tubes containing rectangular glass coverslips for 24 h. Then, the medium was replaced with a fresh medium containing 2% FBS and different concentrations of fluvastatin (1, 5 or 10 mM), and the cells were incubated for 48 h at 37 8C. Control cells were cultured in a medium without fluvastatin. After the treatment, the cells were fixed and stained according to the standard hematoxylin–eosin method. Observation was performed under an Olympus BX51 System Microscope (Olympus Optical, Tokyo, Japan) and the micrographs were prepared using analySiS software (Soft Imaging System GmbH, Munster, Germany). A part of the cell cultures were fixed in a 3% formaldehyde solution in phosphate-buffered saline (PBS), pH 7.4, for 30 min at room temperature, incubated with 0.5% Triton X-100 in PBS for 15 min at room temperature, blocked in PBS containing 3% bovine serum albumin (BSA, Sigma–Aldrich) for 1 h at room temperature, and incubated with TRITC-labeled phalloidin (0.5 mg/ml) in PBS for 1 h at room temperature in the dark. Representative fluorescence micrographs of these cells were performed using an Olympus BX51 System Microscope. Western blot analysis The C6 cells were grown in 6-well plastic plates (3 105 cells/ ml with 3 ml/well) in a medium with 10% of FBS for 24 h at 37 8C. Next day, the medium was replaced with fresh 2% FBS-DMEM and the cells were treated with fluvastatin at the concentrations of 1, 5 or 10 mM. After 24 h of incubation, the cells were washed in cold PBS and lysed in RIPA buffer (containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 1 mM sodium orthovanadate, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail) at 4 8C for 30 min. Next, the lysates were centrifuged at 10,000 g for 15 min at 4 8C. The protein concentration was quantified using a BCA protein assay kit (Pierce1 BCA Protein Assay Kit, Thermo Scientific, Rockford,
A. Sławin´ska-Brych et al. / Pharmacological Reports 66 (2014) 121–129
USA). 50 mg of the samples was mixed with Laemmli buffer, boiled for 5 min, loaded on (9%) SDS polyacrylamide gel (SDS-PAGE), and separated electrophoretically. The proteins were transferred onto the Immobilon P membrane (Merck, Darmstadt, Germany). Following the transfer, the membrane was blocked with blocking buffer (5% nonfat dried milk in TBS/0.1% Tween 20) for 1 h at room temperature and then probed with appropriate dilutions of primary antibodies overnight at 4 8C. The membranes were washed 3 times for 10 min with PBS containing 0.05% Triton X100 and incubated for 1 h with the horseradish peroxidase-labeled anti-mouse antibody. The membranes were visualized with ECM Western Blot Chemiluminescence Reagent (Amersham Biosciences, Germany) using a Kodak Biomax film (Sigma–Aldrich). The blots were reprobed with antibodies against b-actin to ensure equal loading and transfer of proteins. Wound healing assay (migration of cells) The C6 cells (4 105 cells/ml) were seeded in culture dishes (4 cm in diameter, Nunc) in 2 ml of complete medium. After 24 h, the monolayer of cells was scratched with a pipette tip (P300) to create one linear wound. The cells were then rinsed with PBS and covered with a fresh medium with 2% FBS supplemented with 0.5, 1 or 2.5 mM of fluvastatin. The control cells were cultured in a culture medium without fluvastatin. The next day, the plates were
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stained according to the May–Gru¨nwald–Giemsa method and then observed under an Olympus BX51 System Microscope. Cells that migrated to the wound areas were counted on micrographs and the results were expressed as the mean number of cells that had migrated to 50 selected fields taken from four micrographs. Gelatin zymography The C6 cells were seeded at a density of 2 104/well into 24well tissue culture plates in DMEM–10% FBS. The cells were allowed to adhere for 24 h and then they were incubated with various concentrations of fluvastatin (0.5, 2.5 or 5 mM). The conditioned media were collected 48 h after the treatment with fluvastatin, mixed with nonreducing Laemmli sample buffer, and subjected to electrophoresis in 10% SDS–PAGE containing 0.1% (w/ v) gelatin. The gel was washed with 2.5% Triton X-100 (v/v) for 30 min at room temperature to remove SDS and allow the protein to renature; subsequently it was incubated in substrate buffer (50 mM Tris–HCl, pH 7.5, 1 mM ZnCl2, and 5 mM CaCl2) at 37 8C for 24 h. The gel was then stained with 0.5% (w/v) Coomassie blue in 10% acetic acid (v/v) and 30% methanol (v/v), and then destained with the same solution without Coomassie blue. Gelatinolytic activities were detected as unstained bands against the background of Coomassie blue-stained gelatin. It is important to note that in this SDS-containing gel, the latent form of MMP-9,
Fig. 1. The influence of fluvastatin on the viability of C6 glioma cells (A) and primary neuronal cells (B). The antiproliferative effect of fluvastatin (C). The cells were treated with fluvastatin at various concentrations for 48 h. Cell viability was determined by the MTT assay. Cell proliferation was determined by the BrdU incorporation assay. The results represent the mean SD of three independent experiments in triplicates. *Statistically significant at p < 0.01 in comparison to the control.
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pro-MMP-9, and the activated gelatinase develop gelatinolytic activity. Therefore, the word ‘‘activity’’ was used to indicate the total gelatinolytic activity measured in the conditioned media. Determination of MMP-9 and VEGF concentration by ELISA assay The total MMP-9 level in the conditioned media of the C6 cells was detected using a commercial human MMP-9 immunoassay kit (RayBiotec, Norcross, GA, USA), following the manufacturer’s protocol. The VEGF protein released into the conditioned medium was measured by a VEGF enzyme immunoassay kit (Strathmann Biotec, Hamburg, Germany) according to the manufacturer’s instructions. The C6 cells (3 105 cells/ml) were seeded in 24-well plates in 1 ml of DMEM with 10% of FBS and incubated for 24 h. Subsequently, the growth medium was changed by DMEM containing 2% FBS and the C6 cells were treated with fluvastatin (0.5, 2.5 or 5 mM). After 48-h exposition, the culture medium was collected, centrifuged, and frozen immediately at 80 8C until quantification of the MMP-9 or VEGF concentration.
reduced cell viability to approximately 14% in comparison to the control. In contrast to the C6 glioma cells, the rat normal neuronal cells were resistant to the fluvastatin treatment. When they were incubated with fluvastatin at the concentrations from 1 mM to 50 mM, the number of living cells in the cultures was markedly higher, compared to the untreated cells (Fig. 1B). These results clearly indicate selective, dose-dependent cytotoxicity of fluvastatin against tumor cells. To examine the antiproliferative potential of fluvastatin, the C6 glioma cells were treated with 1, 5, 10, 25, or 50 mM of fluvastatin for 48 h and the BrdU assay was performed. Fluvastatin effectively reduced BrdU incorporation during the DNA synthesis (Fig. 1C). A significant decrease in cell division was found even at 1 mM. However, the most pronounced effect was observed with 50 mM fluvastatin, which suppressed proliferation of the C6 cells in 70%. These data correspond with the MTT results and indicated a dosedependent antiproliferative activity of fluvastatin in the cells studied. Fluvastatin changes the morphology of C6 cells and cytoskeletal assembly
Statistical analysis The data were expressed as the means standard error. Statistical analyses were performed using GraphPAD Prism 4 (GraphPAD Software Inc., CA, USA). The data were analyzed by one-way analysis of variance, followed by Dunnett’s test; p values
