Roswell Park Memorial Institute Medium 1640 (RPMI 1640), fetal bovine serum, and antibiotics (penicillin and streptomycin) were obtained from GIBCO BRL Co. (Grand Island, NY, USA). Shikonin, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), propidium iodide (PI), and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against caspase-3, poly(ADP-ribose) polymerase (PARP), AMPK, mTOR, Beclin, ATG5, LC3B, p62/SQSTM1, and GFP were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against RIP1 were purchased from Abcam, Inc. (Cambridge, UK). 3-Methyl adenine (3-MA) and bafilomycin A were purchased from Sigma-Aldrich. Caspase inhibitor Z-VAD-FMK, Z-DEVD-FMK, Z-IETD-FMK, and Z-LEHD-FMK were purchased from R&D Systems (Minneapolis, MN, USA). Necrostatin-1 (Nec-1) was purchased from Tocris Bioscience (Ellisville, MO, USA). Anti-rabbit IgG-conjugated horseradish peroxidase (HRP) antibodies and enhanced chemiluminescence (ECL) kits were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK).
A549 human lung cancer cells were obtained from the Korean Cell Line Bank (Seoul, Korea) and grown in RPMI 1640 containing 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 10% fetal bovine serum. The cells were incubated in a humidified atmosphere of 5% CO2 in air at 37 °C and maintained in log-phase growth.
Multiplexed viability and cytotoxicity assays
The CellTox Green Cytotoxicity Assay (Promega, Madison, WI, USA) was used to measure the DNA in dead cells and the CellTiter-Glo 2.0 Assay (Promega) was then used to measure ATP as a marker of viable cells. A549 cells were seeded in 96-well plates at 20,000 cells/well in 50 µL and incubated at 37 °C for 24 h in a 5% CO2 incubator, followed by the addition of 50 μL/well CellTox Green Cytotoxicity Assay Reagent (Promega). After 15 min of incubation at room temperature, the resulting fluorescence was measured in the 485Ex 530Em channels using a SpectraMax M3 Plate Reader. After the readings were obtained, 100 µL of CellTiter-Glo 2.0 assay reagent was added to all wells and the resulting luminescence was measured using a SpectraMax M3 Plate Reader after 10 min of incubation at 37 °C.
Caspase 8 assay
A549 cells were dispensed in culture medium at 2 × 104 cells/well in white-walled 96-well luminometer plates and incubated for 24 h at 37 °C in the presence or absence of test materials, followed by the addition of 100 μL/well Caspase-Glo 8 Reagent (Promega). After 30 min of incubation at room temperature, the luminescence intensity was measured using a SpectraMax M3 Plate Reader.
After cells were treated with the specified drugs, MTT was added to the cell suspension and incubated for 4 h. The cells were then washed three times with phosphate-buffered saline (PBS; pH 7.4), and the insoluble formazan product was dissolved in dimethyl sulfoxide. The optical density (OD) at 595 nm in each well was measured using a microplate reader (Titertek Multiskan; Flow Laboratories, North Ryde, New South Wales, Australia). The OD resulting from formazan production in control cells was defined as 100% cell viability, and all other measurements were expressed as a percentage of the control cell value.
Annexin V/PI assay
Annexin V-FITC and PI staining were used to analyze whether A549 cells were undergoing early/late apoptosis or necroptosis. Cells in the exponential growth phase (2.5 × 105 cells) were seeded in 35-mm2 dishes and were incubated at 37 °C for the indicated times in the presence or absence of specified test drugs. The cells, both adherent and floating, were then harvested and analyzed by the annexin V assay according to the manufacturer’s instructions. Pelleted cells were briefly washed with PBS and resuspended in annexin-binding buffer. They were then incubated with annexin V-FITC and PI for 15 min at room temperature. After incubation, the stained cells were analyzed using a FACSCalibur system and Cell Quest software (Becton–Dickinson, San Jose, CA, USA).
Tumor xenograft studies in nude mice
Five- to six-week-old BALB/c athymic nude mice (Charles River, Tokyo, Japan) were housed in cages with HEPA-filtered air (12-h light/dark cycle). Food and autoclaved water were provided ad libitum. A549 cells were injected subcutaneously (s.c.) into both hind legs of each mouse. When the implanted tumors reached a volume of 90–130 mm3, the mice were randomly assigned to one of two groups (n = 5 per group). For the experimental group, shikonin (2.0 mg/kg, diluted PBS) was injected intraperitoneally once per da. The control group received PBS alone. Animals were monitored for 14 days or until the tumors reached a volume of 1300 mm3.
Tumor orthotopic studies in nude mice
Nude mice were intraperitoneally injected with thiopental sodium (0.08 mL/kg of body weight) to induce anesthesia; subsequently, the mice were placed in the right lateral decubitus position. Then, 50 μL of the A549 single cell suspension (1.5 × 106) prepared using a 1-mL injector was rapidly inoculated percutaneously into the upper margin of the sixth intercostal rib on the left anterior axillary line to a depth of about 5 mm. The needle was then promptly removed. The mice were maintained in the left lateral decubitus position after injection and were observed until complete recovery.
Micro-CT imaging analysis
Mice with lung cancer underwent micro-CT scanning weekly after cell inoculation. Whole lungs were scanned for the detection of tumors at 20× magnification. For each tumor, micro-CT images were used to reconstruct three-dimensional images (axial, coronal, and 3D). Tumor size was evaluated using imaging software (Xelis; Infinitt, Seoul, Korea). The diameter of the tumor was defined as the maximum diameter of the tumor in a 2D plane. The tumor volume was evaluated using volume analysis software (VGStudio MAX, Heidelberg, Germany). Small tumors were not included in the data analysis owing to the inability to measure the size of these tumors.
At the end of the study, the whole tumor was harvested from each mouse and fixed in 100 mL of buffered formalin for 24 h. Formalin-fixed tissue was paraffin-embedded, sectioned (3–5 μm), and stained with H&E. Sections were evaluated for necrotic degree of tumors and counted under a microscope.
Acridine orange staining
Autophagy is characterized by the formation and promotion of acidic vesicular organelles (AVOs). In acridine orange-stained cells, the cytoplasm and nucleus exhibit bright green and dim red fluorescence, whereas acidic compartments exhibit bright red or orange fluorescence, as described previously . Following drug treatment, 5 µg/mL acridine orange (A1301; Invitrogen, Carlsbad, CA, USA) was added to the culture medium, and the cells were incubated at 37 °C for 15–30 min. The cells were then trypsinized, washed twice with cold PBS, and observed under a confocal microscope. Fluorescence imaging was performed using a blue bandpass filter with 490 nm excitation, and the fluorescence of the green and orange channels was recorded and merged.
Adenoviral GFP-LC3B was kindly provided by Dr. Xiao-Ming Yin (University of Pittsburgh School of Medicine, Pittsburgh, PA, USA). After the cells were washed with OPTI-MEM (Invitrogen), DNA was transfected into cells using Lipofectamine™ 2000 according to the manufacturer’s protocol (Invitrogen). After incubation for 4 h, the medium was replaced with complete medium containing 10% serum and antibiotics. The cells were incubated for 24 h and treated as indicated in the figure legends. Images were obtained using a confocal microscope (FluoView™ FV1000; Olympus, Tokyo, Japan).
Cells were harvested and lysed in radioimmunoprecipitation assay buffer [50 mM Tris–Cl (pH 7.4), 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/mL each of aprotinin and leupeptin, and 1 mM Na3VO4]. After centrifugation at 12,000×g for 30 min, the supernatant was collected, and the protein concentration was determined by the Bradford method (Bio-Rad Protein Assay; Hercules, CA, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and were subsequently transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk in TBS-T [25 mM Tris (pH 7.6), 138 mM NaCl, and 0.05% Tween-20] for 1 h and probed with primary antibodies (at 1:1000–1:5000). After washing, the membranes were incubated with the relevant HRP-conjugated secondary antibody (at 1:2000–1:10,000). Immunoreactive signals were detected using an ECL detection system.
Pooled small interfering RNA (siRNA) oligonucleotides against ATG5 were purchased from Cell Signaling Technology. siRNA against RIP1 was purchased from Ambion (Austin, TX, USA). Cells were transfected with 100 nM pooled oligonucleotide mixture using Lipofectamine2000 (Invitrogen) according to the manufacturer’s protocol. Twenty-four hours after transfection, media were removed and cells were treated with the indicated drugs. Gene silencing efficacy by siRNA was assessed by a western blot analysis.
Each experiment was performed at least three times, and all values are expressed as the mean ± SD of triplicate samples. The Student’s t test was used to determine statistical significance. Values of p < 0.05 were considered statistically significant.