Cell Viability Assay
The cytotoxicity of the TMZ alone and combined with VER was tested against the human neuroblastoma SH-SY5Y cell line (ATCC CRL-2266). The cells were cultured in a complete medium containing DMEM, 10% FBS, 1% L-glutamine, 100IU/ml penicillin, and 10 mg/ml streptomycin in 25cm2 polystyrene flasks and incubated in a humidified atmosphere with 5% CO2 at 37°C. When the cells reached the desired density (75%), they were passaged.
TMZ and VER stock solutions were prepared in DMSO. Each stock solution was sterilized by filtering with a 0.2 µm syringe tip filter (highest concentration DMSO ratio 0.1%). Then, serial dilutions were made from these stocks, and different concentrations of inhibitors to be applied to the cells were prepared (for TMZ; 100 µM-10 mM, and for VER; 0.25-10 µg/ml). IC50 values were determined by applying drugs in concentrations ranging from low to high.
The effect of drugs alone and in combinations on the viability of the SH-SY5Y cell line was investigated with the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxamide) test. The human neuroblastoma SH-SY5Y cells were seeded in 96-well plates at a density of 1 × 104, cells per well in 100 µl colorless DMEM medium, and the cells were treated with the TMZ alone (ranging from 100 µM to 10 mM or combined with Verapamil at 2.5 µg/ml (the concentration without the inhibitory effect on cell viability determined for VER in this study.) concentrations and incubated for 24 h and 48 h. The same procedure is applied at the end of 24 and 48 hours, and the XTT labeling mixture is added to each well and incubated at 37°C for 4 hours. The absorbance of occurred XTT-formazan was measured at 450 nm compared to the control via a microplate (ELISA) reader. The experiments were repeated three times independently of each other, and cell viability was evaluated through control (100% of viability).
Apoptosis Assay
Based on the IC50 values obtained from the cell viability test, we investigated the effect of TMZ alone and the combination of VER on the apoptosis of the SH-SY5Y cell line through the Muse® Annexin V & Dead Cell kit. The assay procedure briefly consists of SH-SY5Y cells seeded in 6 well plates, with 6 × 105 cells/well, incubated overnight to attach the cells. Apoptosis experimental groups were designed as control, TMZ (IC50 for 24 h), and 2.5 µg/ml VER + TMZ (IC50 for 24 h). The cells were incubated in a complete culture medium containing drugs for 24 h. Then, 100 µL of 1 × 106 cells in suspension were transferred to a new Eppendorf tube and incubated with 100 µL of Annexin V & Dead Cell Reagent (Luminex, Austin, USA) for 20 min at 20–22°C. Early and late apoptotic cells were determined by the Guava® Muse® Cell Analyzer (Luminex, Europe).
Wound healing assay and analysis
The wound healing (scratch) assay is a method to measure two-dimensional cell migration. An artificial gap is generated on a confluent cell monolayer, and movement is tracked via microscopy or other imaging. SH-SY5Y cells were seeded in 24-well plates at 2 × 105 cells/mL and incubated for 24–48 h. After cells reached 100% confluence, wounds were generated using a 1 mL micropipette tip. Media was removed, cells were washed with 500 µL PBS, and 500 µL of complete culture media containing compounds were added into each well. Images were acquired immediately following media replacement (T = 0) and every six h for 24 h via multi-mode plate reader at 10×. After exporting images, wound areas were measured using ImageJ. Briefly, a polygon selection tool was used to indicate wound area and quantified. Extents of closure at T0 and T24 were calculated by subtracting the area at T0; percentage closure was determined by normalizing the difference to the area at T0.
Statistical analysis
Statistical analysis was realized using GraphPad Prism 7 version, and all data are described as mean ± SEM. Groups were crosschecked statistically using general linear models of analysis of variance (ANOVA) followed by the Tukey test.