The clonogenic assay was performed in triplicate

The clonogenic assay was performed in triplicate. Wound healing assay A wound healing assay was performed as previously described (22). catalyzes the reaction of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate to generate farnesyl pyrophosphate (FPP) (5). This results in an increase in geranylgeranyl pyrophosphate (GGPP), which plays an important role in the production of small GTPases, including Ras, Rac, Rho and cell division control protein 42 homolog (CDC42) (11), and can subsequently control cancer cell proliferation. BPs strongly inhibit FPPS, which reduces the levels of FPP and GGPP and expression of small GTPases (5). Furthermore, BPs cause an accumulation of IPP that is converted to the cytotoxic adenosine 5-triphoshpate analogue ApppI, which induces cancer cell death (12). It has been suggested that BPs stimulate cancer cell death and apoptosis by inhibiting the mevalonate pathway and reducing the number of small GTPases (13,14). This inhibits integrin-mediated cancer cell adhesion to the bone (15), increase Rap1 unprenylation, reduce the growth of mesothelioma cells (16) and deactivate Rho protein, which leads to inhibition of cancer cell migration (17). Small GTPases affect cancer cell cycle progression and/or growth by modulating the transcription of certain genes, including cyclin D, which stimulates the G1 to S transition and cancer cell proliferation (18,19). The transcription of cyclin D1 is controlled by a number of transcription factors, including activator protein-1 and nuclear factor-B, the activity of which are regulated by small GTPases (18,19). Accordingly, direct inhibition of small GTPase activity induces cell cycle arrest and apoptosis in cancer cells by leading to decreased cell function and eventually programmed cell death (20). Zoledronic acid exhibits pronounced antiproliferative and proapoptotic effects in breast cancer MDA-MB-231 cells by increasing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) production and enhancing the TRAIL/osteoprotegerin (OPG) ratio, which affects cell integrity and survival (21). The present study investigated the anticancer properties of three second-generation BPs, alendronate, risedronate and pamidronate, in MCF-7 human breast cancer cells using sulforhodamine B (SRB), colony formation and flow cytometry assays. The mechanism of BP-induced apoptosis was also explored by analyzing expression levels of apoptosis-associated proteins, reactive oxygen species (ROS) production, caspase-3 activity and mitochondrial function. Finally, effects of BPs on cancer cell migration were determined using a wound healing assay, gelatin zymography and by analyzing expression levels of genes associated with migration. The current study provides a valuable overview of the cytotoxic, apoptotic and antimigratory effects of different BPs on MCF-7 breast cancer cells. Materials and methods Chemicals and reagents Alendronate, risedronate, pamidronate, protease inhibitor cocktail, dihydroethidium (DHE), radioimmunoprecipitation assay (RIPA) lysis buffer, Caspase-3 Fluorometric assay kit, GGPP, doxorubicin and SRB were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Primary antibodies against cyclin D1 (cat. no. 2992), p21 (cat. no. 2947), cytochrome c (cat. no. 4272), caspase-3 (cat. no. 9662) and the internal control -actin (cat. no. 4967), and the anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody (cat. no. 7074) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). All cell culture reagents were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Cell lines, culture condition The human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained according to the manufacturer’s protocol. The MCF-7 cell line was cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) consisting of 10% fetal bovine serum, 100 U/ml penicillin G and 100 mg/ml streptomycin, and maintained under an atmosphere of 5% CO2 at 37C. The cells were subcultured every 3 days or after cells reached 70C80% confluence using 0.25% trypsin-EDTA. Cells were plated in new complete DMEM until required for future experiments. Cell viability assay A SRB assay was used to measure the effect of the BPs alendronate, risedronate and pamidronate on the viability of MCF-7 cells. The assay was Tulobuterol hydrochloride performed as previously described (22). In brief, MCF-7 cells were seeded into 96-well culture plates at 1104 cells/well and allowed to attach for 24 h. Subsequently, cultured cells were treated with various concentrations of BPs (0C1,000 M alendronate and risedronate, 0C250 M pamidronate) for 24 and 48 h at 37C. Cells were then washed, fixed with 10% ice-cold trichloroacetic acid for 30 min at 4C, stained with 0.4% SRB for 30 min at room temperature, washed with 1% acetic acid to remove unbound dye and the remaining protein-bound dye was then solubilized with 10 mM unbuffered Tris. The absorbance was measured at 540 nm using a microplate reader. Cell viability was expressed in terms.no. in the mevalonate pathway, including farnesyl pyrophosphate synthase (FPPS) (11). FPPS is a key enzyme that catalyzes the reaction of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate to generate farnesyl pyrophosphate (FPP) (5). This results in an increase in geranylgeranyl pyrophosphate (GGPP), which plays an important role in the production of small GTPases, including Ras, Rac, Rho and cell division Tulobuterol hydrochloride control protein 42 homolog (CDC42) (11), and can subsequently control cancer cell proliferation. BPs strongly inhibit FPPS, which reduces the levels of FPP and GGPP and expression of small GTPases (5). Furthermore, BPs cause an accumulation of IPP that is converted to the cytotoxic adenosine 5-triphoshpate analogue ApppI, which induces cancer cell death (12). It has been suggested that BPs stimulate cancer cell death and apoptosis by inhibiting the mevalonate pathway and reducing the number of small GTPases (13,14). This inhibits integrin-mediated cancer cell adhesion to the bone (15), increase Rap1 unprenylation, reduce the growth of mesothelioma cells (16) and deactivate Rho protein, which leads to inhibition of cancer cell migration (17). Small GTPases affect cancer cell cycle progression and/or growth by modulating the transcription of certain genes, including cyclin D, which stimulates the G1 to S transition and cancer cell proliferation (18,19). The transcription of cyclin D1 is controlled by a number of transcription factors, including activator protein-1 and nuclear factor-B, the activity of which are regulated by small GTPases (18,19). Accordingly, direct inhibition of small GTPase activity induces cell cycle arrest and apoptosis in malignancy cells by leading to decreased cell function and eventually programmed cell death (20). Zoledronic acid exhibits pronounced antiproliferative and proapoptotic effects in breast tumor MDA-MB-231 cells by increasing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) production and enhancing the TRAIL/osteoprotegerin (OPG) percentage, which affects Tulobuterol hydrochloride cell integrity and survival (21). The present study investigated the anticancer properties of three second-generation BPs, alendronate, risedronate and pamidronate, in MCF-7 human being breast tumor cells using sulforhodamine B (SRB), colony formation and circulation cytometry assays. The mechanism of BP-induced apoptosis was also explored by analyzing manifestation levels of apoptosis-associated proteins, reactive oxygen species (ROS) production, caspase-3 activity and mitochondrial function. Finally, effects of BPs on malignancy cell migration were determined using a wound healing assay, gelatin zymography and by analyzing manifestation levels of genes associated with migration. The current study provides a valuable overview of the cytotoxic, apoptotic and antimigratory effects of different BPs on MCF-7 breast cancer cells. Materials and methods Chemicals and reagents Alendronate, Tulobuterol hydrochloride risedronate, pamidronate, protease inhibitor cocktail, dihydroethidium (DHE), radioimmunoprecipitation assay (RIPA) lysis buffer, Caspase-3 Fluorometric assay kit, GGPP, doxorubicin and SRB were from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Main antibodies against cyclin D1 (cat. no. 2992), p21 (cat. no. 2947), cytochrome c (cat. no. 4272), caspase-3 (cat. no. 9662) and the internal control -actin (cat. no. 4967), and the anti-rabbit IgG horseradish Rabbit Polyclonal to GPR174 peroxidase (HRP)-conjugated antibody (cat. no. 7074) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). All cell tradition reagents were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Cell lines, tradition condition The human being breast cancer cell collection MCF-7 was from the American Type Tradition Collection (Manassas, VA, USA) and managed according to the manufacturer’s protocol. The MCF-7 cell collection was cultured in total Dulbecco’s revised Eagle’s medium (DMEM) consisting of 10% fetal bovine serum, 100 U/ml penicillin G and 100 mg/ml streptomycin, and managed under an atmosphere of 5% CO2 at 37C. The cells were subcultured every 3 days or after cells reached 70C80% confluence using 0.25% trypsin-EDTA. Cells were plated in fresh total DMEM until required for future experiments. Cell viability assay A SRB assay was used to measure the effect of the BPs alendronate, risedronate and pamidronate within the viability of MCF-7 cells. The assay was performed as previously explained (22). In brief, MCF-7 cells were seeded into 96-well tradition plates at 1104 cells/well and allowed to attach for 24 h. Subsequently, cultured cells were treated with numerous concentrations of BPs (0C1,000 M alendronate and risedronate, 0C250 M pamidronate) for 24 and 48 h Tulobuterol hydrochloride at 37C. Cells were then washed, fixed with 10% ice-cold trichloroacetic acid for 30 min at 4C, stained with 0.4% SRB for 30 min at space temperature, washed with 1%.