The four cancerous (Ishikawa/endometrium, MDA-MB-231/breast, Caco-2/colon, PC-3/prostate) and one normal (PNT-2/prostate) cell lines were obtained from the departmental cell bank at the University of Portsmouth. All cell lines were originally purchased from the European Collection of Authenticated Cell Cultures (Ishikawa, MDA-MB-231, Caco-2, PNT-2) or the American Type Culture Collection (PC-3). Cells were maintained in required media (suppl. Table 1) and harvested at 90% confluence for the downstream assays. All cell lines were authenticated using STR profiling and screened for mycoplasma contamination in our laboratories over the period of the investigation.
Stock solutions of cisplatin (CDDP) and dequalinium chloride hydrate (DQA) (Sigma, Dorset, UK) were prepared at 100 mM in DMSO and 2 mM in distilled water, respectively. Both drugs were added to the cells in various concentrations and incubated for 24 hours to determine their IC50s (IC50: the half maximal inhibitory concentration) which were used in all subsequent experiments. N-Acetyl-L-cysteine (NAC) (Sigma), a powerful antioxidant, was dissolved in distilled water at the concentration of 100 mM shortly before each experiment, and the pH was adjusted to 7.4 before diluting the solution to the working concentration (10 mM) with complete cell culture medium. Cells were pre-incubated with NAC (10 mM) for 1 hour prior to the CDDP and DQA treatments. All experiments related to NAC followed the same procedure as described in the sections below.
Cell viability assay
Cell viability was measured colorimetrically using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (CellTiter 96 Aqueous) (Promega, Southampton, UK). Briefly, 90 µl of cell suspension containing 10000 cells was added in each well of the 96-well plate and incubated for 24 hours. Cells were then treated with the 10 × drug solution (10 µl) for the desired amount of time. At the end of the experiment, the medium was replaced with 100 µl of fresh medium containing MTS (final concentration - 0.3 mg/ml) and incubated for 90-180 min according to the optimised protocol for each cell line. Absorbance was measured using the microplate reader (Multiskan® GO) at 490 nm. IC50 was calculated as the concentration of the drug that caused a 50% loss of metabolic activity.
Combination Index (CI) for synergism assessment
The synergistic effect of CDDP and DQA was assessed by the CI values calculated at different drug combinations according to the median-effect principle of the Chou and Talalay method, using the CompuSyn Software 1.4 . The CI values indicate how drug combinations influence the therapeutic efficacy, i.e. CI > 1 – Antagonistic; CI = 1 – Additive; CI < 1 – Synergistic.
Baseline intracellular ROS levels were measured in a 96-well plate format using the Cellular Reactive Oxygen Species Detection Assay Kit (Abcam, Cambridge, UK) based on a fluorogenic dye, H2DCFDA, according the manufacturer’s protocol. Cells (25000/well) were seeded in the 96-well black-wall plate (Corning, NY, USA) overnight prior to the experiments. In the following day, cells were washed with HBSS (150 ml; Gibco, ThermoFisher Scientific, Loughborough, UK), then staining buffer (100 ml, 20 µM of H2DCFDA in HBSS) was added to each well, and the plate was incubated for 40 minutes at 37 °C. The cells were then washed with HBSS, and HBSS (100 µl) was added to each well. Fluorescence was measured using the microplate reader (POLARstar Omega) at 485 nm (excitation) and 535 nm (emission). For determining intracellular ROS levels upon treatments, the staining buffer was added and then the plate was incubated with the treatment solution for the desired amount of time before fluorescence was measured.
To measure mitochondrial superoxide, the same procedure was conducted as described above using a mitochondrial ROS specific dye, MitoSOX™ (Life Technologies, ThermoFisher Scientific), instead of H2DCFDA. The cells were incubated with 5 µM of MitoSox in HBSS. Fluorescence was measured using the same microplate reader at 510 nm (excitation) and 580 nm (emission).
Mitochondrial membrane potential assay
MMP was measured by staining the cells with the JC-10 fluorescent dye (Enzo Life Sciences, Exeter, UK) according the manufacturer’s protocol. Briefly, 25000 cells were seeded in each well of the 96-well black-wall plate overnight prior to the experiments. The next day, cells were treated with required concentrations of the drugs for the desired amount of time. Following drug treatments, cells were washed with HBSS, then staining buffer (100 ml, 20 µM of JC-10 in HBSS) was added to each well and the plate was incubated for 40 min at 37 °C. Cells were then washed again with HBSS and HBSS (100 µl) was added to each well before reading the plate. Red and green fluorescence were measured using the microplate reader (POLARstar Omega) at 540 nm (excitation) / 590 nm (emission) and 490 nm (excitation) / 525 nm (emission), respectively.
DNA extraction and measurement of mtDNAcn by SYBR Green real-time PCR
Total DNA was isolated from untreated and treated (24-hour incubation) cells using the QIAmp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacture’s protocol. Relative quantification of mtDNAcn was measured using the QuantiTect SYBR Green PCR kit (QIAGEN) and run on a LightCycler® 96 System (Roche, Basel, Switzerland) according to previously published method . The relative quantity of mtDNA content in each sample was calculated by normalising the copy number of mtDNA against that of the housekeeping gene, β-actin. Three independent experiments were carried out and all samples were run in triplicates in each experiment.
RNA extraction, reverse transcription and TaqMan real-time PCR
Total RNA was isolated from untreated and treated (24-hour incubation) cells using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol. In the last step, RNA was eluted with RNA–free sterile water (40 μl). cDNA was synthesised using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, California, USA) with 1 µg of RNA template in a 20-µl reaction.
TaqMan real-time PCR was performed using the pre-designed assays (Integrated DNA Technologies, Belgium) to measure expression levels of antioxidant genes including SOD1, SOD2 and CAT (suppl. Table 2). The experiments were performed using the FastStart Essential DNA Probes Master (Roche) and run on a LightCycler® 96 System (Roche). The amplification procedure entailed 45 cycles of 95°C for 10 seconds followed by 60°C for 30 seconds. For each reaction, GAPDH was utilised as the endogenous control gene. The average mRNA fold change in drug-treated samples was normalised against untreated samples using the 2-∆∆CT method. Three independent experiments were carried out and all samples were run in triplicates in each experiment.
WB was performed to measure the BCL-XL and released cytochrome c proteins from mitochondria according to the established protocols in our laboratories. Briefly, protein extracts (20 µg) were resolved by SDS-PAGE electrophoresis, and the blots were visualised using a high-sensitivity CCD camera imaging platform (Chemidoc MP; Bio-Rad, Watford, UK). ImageJ software was used for the densitometric quantification of the western blot bands. The primary and secondary antibodies employed in the experiments are listed in suppl. Table 3.
Caspase activity assay
Caspase-3/7 activity was measured using the Caspase-Glo 3/7 reagent from the ApoTox-Glo™ Triplex Assay kit (Promega). According to the manufacture protocol, 90 µl of cell suspension containing 20000 cells was added in each well of the 96-well black-wall plate and incubated overnight. Cells were then treated with drugs for 24 hours. At the end of the experiment, fresh Caspase-Glo 3/7 reagent (100 µl) was added to each well, and the plate was incubated for 60 minutes at room temperature. Luminescence was then measured using the microplate reader (POLARstar Omega).
Data were analysed with the GraphPad Prism 8.0 program (Graphpad software, CA, USA) and presented as the mean ± SEM of at least three separate experiments. Specific statistical tests used to analyse various data sets are described in the associated figure legends. Differences between groups were considered statistically significant according the following criteria: *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.