Hoechst 33258 solution (H33258, 1 mg/mL) and all other chemicals (formaldehyde, Triton X-100, BSA, NaCl, NaOH, Tris base, Na2EDTA·2H2O, PBS 10x, cisplatin, staurosporine, camptothecin, TiO2 P25) were purchased from Sigma Aldrich, USA.
Cell culture and treatment
All materials for cell culture were purchased from Sigma-Aldrich (USA) if not otherwise specified. HepG2 cells (ATCC® HB-8065™), a human hepatocellular carcinoma cell line, were purchased from ATCC (Manassas, VA, USA). HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium with high glucose content (4500 g/L, w/wo phenol red) supplemented with 10% (v/v) fetal bovine serum, 50 µg/mL penicillin, 50 µg/mL streptomycin, 10 mM HEPES and 2 mM glutamine, maintained at 37°C in a sterile, humidified atmosphere of 5% CO2. All the experiments were conducted using HepG2 cells in passages 4–15.
Human kidney, HK-2 cells (ATCC® CRL-2190™), a proximal tubular epithelial cell line derived from normal adult human kidney cells immortalized by transduction with human papillomavirus (HPV 16) DNA fragment 64, were purchased from the ATCC (Manassas, VA, USA). The cells were cultured in supplemented Dulbecco’s modified Eagle’s medium (DMEM/F12 = 1:1) with 5% (v/v) fetal bovine serum, 1 mM pyruvate, 10 µg/mL insulin, 5.5 µg/mL transferrin, 5 ng/mL sodium selenite, 50 µg/mL penicillin, 50 µg/mL streptomycin, and 5 ng/mL epidermal growth factor according to a published protocol 65,66. All the experiments were conducted using the HK-2 cells in passages 5–15.
HepG2 and HK-2 cells were tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). All cells used in the experiments were mycoplasma free. Short tandem repeat (STR) analysis (i.e., DNA fingerprinting) was used for HepG2 and HK-2 cell line authentication using a commercial kit in Generi Biotech (Czech Republic). The STR analysis proved 100% conformity of both HepG2 and HK-2 cells with the reference standards.
The HepG2 and HK-2 cells were seeded in 100 µL of appropriate cell culture medium in 96-well plates at density of 1.5x104 and 2x104 cells per well for 24 h (if not stated otherwise). To induce cell impairment, we used: cisplatin (CisPt, 0-100 µM), camptothecin (CAM; 0–5 µM), staurosporine (STA; 0-100 nM) and TiO2 P25 nanoparticles (NPs; 0–10 µg/mL). All compounds were diluted in appropriate cell culture mediums to obtain final concentrations. After seeding, the culture medium was replaced by 100 µL of medium containing a tested compound and the cells were treated for 6, 24 and 48 h. To characterize the extent of cell impairment, we used the newly developed spectrofluorometric assay using H33258 together with caspase activity and proteins expression measurements, TUNEL and DNA ladder assays.
Hoechst 33258 spectrofluorometric assay
To develop a spectrofluorometric method for detection of changes in nuclear condensation and fragmentation in intact cells, we used a fluorescence dye H33258. To optimize the assay, we used HepG2 and HK-2 cells at confluency 50–70%. After treatment with tested compounds (CisPt, CAM, STA, NPs) for 6, 24 and 48 h, the cells grown in a 96well plate were centrifuged (5 min, 8000g) at RT. Then, 70 µL of a supernatant was replaced with 70 µL of warmed phosphate-buffered saline (PBS 1x, 37°C) and 10 µL of H33258 solution (in PBS 1x) was added to a well. The final concentrations of H33258 in a well were 0.1-5 µg/mL. Then, the cells were incubated with H33258 for 60 minutes during optimization of the assay, or for 5 min at optimal conditions and the spectrofluorometric measurement was performed at EX/EM = 352/461 nm (EX/EM slit widths 25/25 nm) using a Tecan Spark fluorescence microplate reader (Tecan, Switzerland) while incubated at 37°C. The samples were measured at least in triplicates. After background subtraction, the fluorescence signal was presented in Relative Fluorescence Units (RFU) as mean ± SEM. All spectrofluorometric measurements presented here were repeated at least in three independent experiments.
Dehydrogenase activity measurement
Dehydrogenase activity was evaluated by WST-1 test (Roche, Germany). The WST-1 test measures the activity of intra- and extramitochondrial dehydrogenases. After cell treatment, 10 µL of WST-1 reagent was added to the treated cells. The absorbance change (0–1 h) was measured spectrophotometrically at wavelength of 440 nm using a Tecan Spark fluorescence microplate reader (Tecan, Switzerland). The cell viability was expressed as the percentage of intra- and extramitochondrial dehydrogenases activity relative to that in control cells (= 100%). The results were expressed as mean ± SD.
Glutathione levels were measured using the monochlorobimane spectrofluorometric assay 67. After cell treatment, 20 µL of the bimane solution was added to cells to obtain the final concentration 40 µM and spectrofluorometric measurement was started immediately. The fluorescence increase (EX/EM = 394/490 nm) was measured for 10 min using a Tecan Spark fluorescence microplate reader (Tecan, Switzerland). The fluorescence was expressed as the slope of change in fluorescence over time. The glutathione levels were expressed as the percentage relative to the glutathione levels in control cells (= 100%). The results were expressed as mean ± SD.
Caspase 3/7 activity measurement
Caspase 3/7 activity in HepG2 and HK-2 cells was detected using ApoONE® Homogeneous Caspase-3/7 Assay (Promega, USA) according to the manufacturer´s instructions. Briefly, 100 µL of the caspase 3/7 working solution was added to treated cells. After mixing, the cells were incubated for 30 min. Then, the fluorescence (EX/EM = 485/535 nm) was measured in duplicates using a Tecan Spark fluorescence microplate reader (Tecan, Switzerland) while incubated at 37°C. The caspase 3/7 activity levels were expressed as the percentage relative to the caspase 3/7 activity levels in control cells (= 100%). The results were expressed as mean ± SEM.
Capillary Western Immunoassay
Capillary Western Immunoassay was performed in protein lysates from HepG2 and HK-2 cells cultured in 6-well plates at density of 5x105 and 1.3x106 cells per well, respectively. After seeding, the culture medium was replaced by 2 mL of CisPt solution and the cells were treated for 6, 24 and 48 h. After treatment, the cells were washed twice with PBS 1x and protein lysates were prepared by lysing cells with 400 µL of RIPA buffer (Sigma-Aldrich, USA) with MS-safe protease and phosphatase inhibitor (Sigma-Aldrich, USA) on ice. Capillary Western Immunoassay was performed according to manufacturer´s instructions (Protein Simple, USA). Briefly, protein lysates were analyzed on a Wes system (ProteinSimple, USA) using a 12–230 kDa Separation Module (Biotechne, UK). Levels of phosphorylated JNK (pJNK, primary antibody 1:50, Promega, USA), poly-(ADP-ribose) polymerase-1 (PARP-1; primary antibody 1:100; Cell Signalling, USA) were normalized using the reference protein β-actin (primary antibody 1:500, Sigma-Aldrich, USA). The peaks were analyzed using Compass software (Protein Simple, USA). Two criteria were used for the discrimination of signals from the background: 1) the peak high must be higher or equal to 1000 and 2) the peak´s signal-to-noise ratio given by the software must be higher or equal to 10. The results were counted as:
TUNEL assay followed by H33258 staining of nuclei was performed in HepG2 and HK-2 cells cultured in 200 µL of appropriate cell culture medium on cell culture chamber slides at density of 1.5x105 and 2x105 cells per well, respectively. After seeding, the culture medium was replaced by 200 µL of CisPt solutions and the cells were treated for 6, 24 and 48 h. TUNEL assay was performed using ClickiT™ TUNEL Alexa Fluor™ 488 Imaging Assay kit (ThermoFisher Scientific, USA) according to manufacturer´s instructions. The cells were fixed with 12% formaldehyde for 15 min at 37°C. Then, the cells were permeabilized with 0.2% Triton X-100 for 15 min at 37°C, washed with PBS 1x and incubated with terminal deoxynucleotidyl transferase (TdT) buffer for 10 min at 37°C. After incubation, cells were mixed with a TdT reaction mixture (TdT buffer, 5-Ethynyl-2'-deoxyuridine 5'-triphosphate, TdT) and incubated for 1 h at 37°C. Then, the cells were washed with 3% bovine serum albumin (BSA) and Click IT reagent for fluorescent staining was added for 30 min at 37°C. After PBS 1x washing, H33258 at a final concentration of 2 µg/mL was used to visualize the cell nuclei. DNA strand breaks (FITC filter, 480/30 nm) and cell nuclei (DAPI filter, 375/28 nm) were visualized with an Eclipse 80i fluorescence microscope (Nikon, Japan).
DNA ladder was performed in HepG2 and HK-2 cells cultured in 6-well plates at density of 5x105 and 1x106 cells per well, respectively. After seeding, the culture medium was replaced by 2 mL of CisPt and the cells were treated for 6, 24 and 48 h. DNA was isolated from treated cells using The ApoTarget™ Quick Apoptotic DNA Ladder Detection Kit (Invitrogen, USA). Isolated DNA samples were loaded onto a 1.5% agarose gel with 0.5 mg/mL ethidium bromide (Top-Bio, Czech Republic) followed by electrophoresis (5 V/cm). Finally, DNA was visualized by an ultraviolet gel documentation system (Vilber Lourmat, Germany) at wavelength 254 nm. GeneRuler 100 bp DNA ladder (ThermoFisher Scientific, USA) was used as a DNA size standard.
Statistical analysis was performed using OriginPro 9.0.0 (OriginLab, USA). Statistical significance was analyzed after normality testing using one-way analysis of variance (ANOVA) followed by Tukey´s test at significance level p = 0.05 (*, p < 0.05; **, p < 0.01; ***, p < 0.001).