Cell Culture
A selection of 5 human SCLC cell lines (H69, DMS79, H446, H841, and SW1271) were purchased from American Type Culture Collection (Manassas, VA, USA). All these cell lines were cultured in RPMI-1640 medium (Gibco®, Life Technologies, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco®). They were maintained in a CO2 incubator which was set as a humidified chamber with 5% CO2 at 37°C.
Obtained as a suspension cell line, H69 was cultured over time and an adherent subpopulation was acquired. This study employed this adherent subpopulation, and is referred to as H69-Adherent henceforth. Comparably, H841 was obtained as a mixed cell line comprising both suspension and adherent subpopulations. Culturing over time, the adherent subpopulation dominated and was then used throughout the study and is referred to as H841-Adherent henceforth. The remaining 3 cell lines – DMS79, H446 and SW1271 – remained in their original forms (fully suspension, mixed, and fully adherent, respectively) when cultured over time.
Cell Viability Assay
The effect of thymoquinone (TQ, Merck, 274666) on the cell viability of SCLC cells was determined through two methods: 3-(4,5-dimethyl-thiazoyl-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method (22) and the crystal violet staining method (23). Cell lines containing suspended cells, DMS79 and H446, were exposed to the MTT method and cell lines comprising adherent cells, H69-Adherent, H841-Adherent and SW1271 were subjected to the crystal violet staining protocol. Cells were treated to a serial dilution range of TQ for 24, 48, and 72 hours. Optical density was measured at 570 nm on the microplate reader (FLUO star OPTIMA, Bmg Labtec GmbH, Ortenberg, Germany). A minimum of 3 independent experiments were carried out, with each dose performed in sextuplicate in each experiment.
Cell viability assay with N-acetylcysteine (NAC)
H841-Adherent cells (5000 cells/well) were exposed to various concentrations of NAC for 24 hours. Cell viability was then determined.
Flow Cytometry Assays
Various cellular events including mitochondrial membrane depolarization (MMD), apoptosis, cell cycle arrest, and reactive oxygen species (ROS) levels were researched through flow cytometry. CytoFLEX S (Beckman Coulter, CA, USA) was employed for all samples in all assays studied through flow. All processes were computed and deciphered through FlowJo software v10 (BD Biosciences).
Annexin V-PE/7-aminoactinomycin D (AAD) staining
Apoptosis was investigated through phycoerythrin (PE)-conjugated annexin-V/7-aminoactinomycin D (AAD) kit (BD Biosciences). Briefly, 100,000 cells/well were seeded in 6-well plates and exposed to TQ for 24 hours, then harvested, washed, and re-suspended in binding buffer. Cells were stained for an hour at room temperature with FITC-annexin V (Ex/Em = 494 nm/518 nm)/7-AAD (Ex/Em = 546 nm/647 nm), and signals read by CytoFLEX S with FL1/FL12 (FITC and PC5.5, respectively) channels (Beckman Coulter, CA, USA). The cell populations within the FITC+/7-AAD + and FITC+/7-AAD- quadrants were relevant to result analysis (22).
Cell Cycle Arrest
Cell cycle arrest was examined on cells treated with TQ for 48 hours and stained with propidium iodide (PI). Briefly, cells were harvested, washed with phosphate buffered saline (PBS), and fixed with 70% ethanol at -20°C overnight. Fixed cells were centrifuged, washed once with PBS, and incubated at 4°C in darkness for 30–40 minutes, with RNase A (50 µg/ml) and PI (25 µg/ml) in serum-free medium. The intensity of fluorescence was measured by FL10 channel in CytoFLEX S (22).
Measurement of mitochondrial membrane depolarization (MMD)
Briefly, 100,000 cells/well were seeded in 6-well plates and TQ-treated for 24 hours. They were then harvested, washed, and stained at 37°C in the dark for 25 mins with 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, 2 µM). Cells were read through the FL1/FL10 (FITC and PE, respectively) channels of CytoFLEX S (22).
Detection of ROS
ROS was examined through 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, Ex/Em = 500/520 nm, Thermofisher Scientific) staining. Briefly, 100,000 cells/well were seeded in 6-well plates and TQ-treated for 24 hours. They were then harvested, washed, and incubated with 1 µM of H2DCFDA in serum-free medium for 15 minutes at 37°C in darkness, and read out from FL1 (FITC) channel on CytoFLEX S (22).
Protein extraction, gel electrophoresis, and immunoblotting
Cells treated with respective concentrations of TQ for 72 hours were lysed with RIPA buffer and total protein lysate acquired. Blocking was done according to the manufacturer’s directions (with either 5% BSA or 5% milk), after which membranes were incubated at 4°C overnight with primary antibody: cyclin A2, (Cell Signaling Technology, 4656P), cyclin E1 (Cell Signaling Technology, 4129T), Cyclin B1 (Cell Signaling Technology, 4138P), cyclin D3 (Cell Signaling Technology, 2936P), cdc2 (Cell Signaling Technology, 9116T), Bcl-2 (Cell Signaling Technology, 15071S), PARP-1 (Santa Cruz Biotechnology, sc-7150) and β-actin (Sigma Aldrich, A1978), followed by incubation with the corresponding secondary antibody at room temperature for at least 2 hours. An enhanced chemiluminescence (ECL) kit (GE Healthcare) was used to detect protein expression. Beta-actin was selected as a house-keeping protein (22).
Tumor growth inhibition in H446 xenograft model
TQ was dissolved in 20% Kolliphor EL (Sigma Aldrich) in PBS and sonicated for 20 minutes. Tumor xenograft model was established with nude mice (female, 4 weeks old, BALB/cAnN-nu, Charles River Laboratories, Wilmington, USA). H446 cells (5×106) were subcutaneously inoculated into the upper back of each mouse, causing a palpable tumor of approximately 50–100 mm3 post-inoculation 3–4 weeks. Mice were then split into 3 treatment groups, with 5 mice in each group: control (20% Kolliphor EL in PBS) and TQ (2.5 and 5 mg/kg). Treatments were intraperitoneally injected on alternate days. Tumor size (with a standard caliper) and body weight were measured. Tumor volumes (V) were calculated using the formula V= (length x width x depth)/2. Mice were sacrificed when humane endpoints (tumor volume > 600 mm3 or 20% drop in body weight) were reached. Post-treatment weight was then determined (22). The study protocol was approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong (CULATR 5631-21) and reported in accordance with ARRIVE guidelines.
Statistical analysis
Experiments were replicated at least three times and data analyzed. Student’s two-tailed t-test was used for comparison of pairs. The difference between more than two groups was evaluated using variance analysis (ANOVA) by Prism (GraphPad Software, La Jolla, Southern California, United States). A p-value < 0.05 was considered statistically significant (*: p < 0.05, **: p < 0.01, ***: p < 0.001).