Materials and cell culture
2′-HCA was kindly provided by Dr. Byoung-Mok Kwon (KRIBB). Primary antibodies against Bax, Bcl-2, caspase-3, PARP-1, ASK-1, α-tubulin, and β-actin were purchased from Santa Cruz Biotechnology Inc (Santa cruz, CA, U.S.A.). Antibody against cytochrome c and Annexin V-FITC apoptosis detection kit were purchased from BD Biosciences Pharmingen (San Jose, CA, USA). Antibodies against caspase-9, p-c-Jun, c-Jun, ASK-1, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38 MAPK, p38 MAPK, and COX-4 were purchased from Cell Signaling Technology (Danvers, MA, U.S.A). z-VAD-fmk, z-DEVD-fmk, and Ac-IETD-CHO were purchased from Calbiochem (Bad Soden, Germany). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 4',6-diamidino-2-phenylindole (DAPI), rhodamine 123, propidium iodide (PI), sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), RNase A, 2´,7´-dichlorofluorescein diacetate (DCFH-DA), and other chemicals were purchased from Sigma Chemical Co (St.Louis, MO, U.S.A.).
HL-60 human promyelocytic leukemia, Molt-4 human T lymphoblastic leukemia, U937 human histiocytic lymphoma, K562 human erythroleukemia, HepG2 human hepatoma, SNU-C5 human colorectal adenocarcinoma, A549 human lung adenocarcinoma, and KB human mouth epidermal carcinoma cells were obtained from the Korean Cell Line Bank (KCLB). Cells were cultured in RPMI 1640 or Dulbecco’s modified Eagle’s minimum essential medium (DMEM) with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin sulfate (100 µg/ml). Cells were maintained at 37 oC in a humidified atmosphere of 5% CO2.
Cytotoxicity was measured using the MTT assay. The MTT assay was performed for cytotoxicity measurement using a modified method described . Briefly, the cells (5 × 104) were seeded in each well containing 100 ml of the medium in 96-well plate. After incubation for 24 h, various concentrations of 2′-HCA were added to the 96-well plate. After 48 h, 50 ml of MTT (5 mg/ml stock solution in PBS) was added to each well for 4 h. The medium was discarded and the formazan blue which formed in the cells was dissolved with 100 mL DMSO. The optical density was measured at 540 nm.
Annexin V-FITC/ propidium iodide (PI) double staining analysis
After treatment with 2′-HCA, apoptotic cells were detected with Annexin V-FITC/PI kit (BD Bioscience Pharmingen, CA, USA), following the manufacturer’s instruction and analyzed by the fluorescence-activated cell sorting (FACS) cater-plus flow cytometry (Becton Dickinson Co, Heidelberg, Germany).
Detection of DNA fragmentation
DNA fragmentation was quantitated with DAPI assay as previously reported . In brief, cells were lysed in a solution containing 5 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 0.5% (w/v) Triton X-100 for 20 min on ice. After centrifugation at 27,000 ×g for 20 min, the lysate and supernatant were sonicated for 40 s, and the level of DNA was measured by a fluorometric method using DAPI. The amount of the fragmented DNA was calculated as the ratio of the amount of DNA in the supernatant to that in the lysate. The genomic DNA was prepared for gel electrophoresis as previously described . Electrophoresis was performed in a 1.5% (w/v) agarose gel in 40 mM Tris-acetate buffer (pH 7.4) at 50 V for 1 h. The fragmented DNA was visualized by staining with ethidium bromide after electrophoresis.
Preparation of mitochondrial and cytosolic fraction and total protein
HL-60 cells (2.5 × 107) were collected by centrifugation at 200 × g for 10 min at 4°C. The cells were washed twice with ice-cold PBS, followed by centrifugation at 200 × g for 5 min. The cell pellet was then resuspended in cell lysis buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 100 mM PMSF) for 30 min on ice. Cells were then homogenized with a glass dounce and a B-type pestle (80 strokes). Cell homogenates were spun at 15,000 × g for 15 min at 4°C and the supernatant (cytosolic fraction) was removed while taking care to avoid the pellet. The resulting pellet was resuspended in mitochondrial buffer. For total cell protein extracts, cells were washed with ice-cold PBS and extracted in protein lysis buffer (Intron, Seoul, Korea) for 30 min on ice. Cells were then spun at 15,000 ×g for 30 min at 4°C and the supernatant (total protein) was used to detection for protein expression.
Western blot analysis
For western blot analysis, protein concentration was determined by Bradford assay. Protein samples were mixed with 5 × SDS sample buffer, boiled for 4 min, and then separated by SDS-PAGE gels. After electrophoresis, proteins were transferred to polyvinylidene difluoride membrane. The membranes were incubated with the primary antibody in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) overnight at 4°C. The primary antibody of the membrane was removed by washing in TBS-T and the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Following washing in TBS-T, immune blots were visualized by ECL (GE Healthcare) and exposed to X-ray film (Amersham, NJ, USA).
Determination of mitochondrial membrane potential (DΨm)
Changes in the DΨm were examined by monitoring the cells after double staining with PI and rhodamine 123. After treatment of 40 mM 2′-HCA for 2 h, the cells were incubated with medium containing 5 mg/mL rhodamine 123 for 1 h to determine the mitochondrial membrane potential. The cells were resuspended in 1 mL of minimal essential medium containing 5 mg of PI to assess cell viability. The intensity of fluorescence from PI and rhodamine 123 was measured by flow cytometry. Fluorescence was measured after the cells staining for 30 min at 37 °C.
Detection of ROS generation
To measure 2′-HCA-induced intracellular ROS level, we used DCFH-DA, which is the most widely used fluorescent probe for the detection of intracellular oxidative stress . The 2′-HCA- treated cells were incubated with 20 mM DCFH-DA for 30 min at 37 oC. The intracellular ROS level was measured by flow cytometry.
Determination of the GSH level
Cells were washed twice with PBS and treated with 5% trichloroacetic acid (TCA) to extract cellular GSH. The mixture was centrifuged at 13,000 × g for 1 min to remove the denatured proteins. GSH was determined by the enzymatic method as previously described . To determine the glutathione disulfide (GSSG), the same DTNB recycling assay was performed after using 2-vinylpyridine to remove the reduced GSH . Briefly, 2 μL of 2-vinylpyridine and 6 μL of triethanolamine were simultaneously mixed with 100 μL of sample, followed by incubation in the dark at room temperature for 1 h before initiation of the recycling assay. The kinetics of the reaction was monitored for 10 min. The increment in absorbance at 412 nm was converted to GSH concentration using a standard curve with known amounts of GSH.
Measurement of intracellular protein thiols (PSH)
To measure the intracellular PSH, we performed the assay of intracellular PSH as previously report . Briefly, cells were treated with 5% TCA and then vortexed and kept on ice for 30 min to prepare complete protein precipitation. After centrifugation, the protein precipitate dissolved in 0.1 M Tris-HCl buffer (pH 7.5), containing 5 mM EDTA and 0.5% sodium dodecyl sulfate (SDS). One aliquot of this protein precipitate was reacted with a solution containing 0.1 M sodium phosphate buffer (pH 7.5), 5 μM EDTA, 0.6 mM DTNB, 0.2 mM NADPH, 1 unit/ml glutathione reductase, and another aliquot of the protein solution was treated with 5 mM N-ethylmaleimide (NEM) before the reaction to obtain the background value for subtraction. The concentration of intracellular protein thiol was expressed as nmol of SH equivalents/mg protein using GSH as a standard.
Caspase-3 activity assay
The caspase-3 activity was measured using a fluorogenic caspase-3 substrate (Ac-DEVD-AFC). Cells were washed once with PBS, resuspended in 400 μl of lysis buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 0.5% NP-40, 10 mM DTT) and incubated on ice for 30 min. After centrifugation (12,000 × g for 5 min), supernatants were collected and immediately measured for protein concentration and caspase activity, or stored at -70°C until assayed. For the activity assay, 100 μL of cell lysates were placed in a 96-well plate and a caspase substrate was added to each well. Plates were incubated at 37 °C for 1 h and caspase activity was determined from the fluorescence read at 505 nm induced by excitation at 400 nm.
Transfection for RNA interference
Control small interfering RNA (siRNA) and JNK targeting siRNA (5′-AAAAAGAATGTCCTACCTTCT-3′) specific for humans and mice were obtained from Santa Cruz Biotechnology (Santa cruz, CA, U.S.A.). HL-60 cells (2 × 106) were transfected with siRNA (200 nM) by RNAiFect transfection reagent (QIAGEN, Hilden, Germany) according to recommendations of the manufacturer. After transfection for 48h, cells were treated with 2′-HCA for 8 h and subjected to analysis.
c-Jun N-terminal kinase (JNK) kinase assay
To determine JNK kinase activity, JNK assay kit (Cell Signaling Technology, Danvers, MA, U.S.A.) was used according to the manufacture's instruction
Electrophoretic mobility shift assay (EMSA)
HL-60 cells (5 × 106 cells) were collected by centrifugation at 200 ×g for 10 min at 4 ºC. The cells were washed twice with ice-cold PBS, pH 7.2, followed by centrifugation at 200 ×g for 5 min. Nuclear extracts and EMSA assay were performed as described previously .
The male BALB/c nude mice (6-week-old, 20–23 g) were obtained from Nara Biotec Co. (Pyeongtaek, Republic of Korea). Mice were inhabited 6/cage/group and were had standard laboratory chow in an animal room with 12 h dark/light cycles at a constant temperature of 20 ± 5 ºC. All animal experiments were performed under university guidelines and were approved by the ethical committee for Animal Care and Use of Kyung Hee University according to the animal protocol (KHUASP(SE)-19-027).
Xenograft animal model
The male BALB/c nude mice were subjected to 150 mg/kg cyclophosphamide (CYP) by intraperitoneal (i.p.) injection three times per two days (Fig. 2). After CYP injection, HL-60 cells (1 × 106 per site) were inoculated subcutaneously into the right side of the flank of male BALB/c nude mice. Tumor size was checked with a caliper  once per 3 days in a week and calculated as V = π/6 × (length) × (width)2. When tumor volume reached around 300 mm3, mice were divided into 5 groups and treated with vehicle (DMSO: Cremophor: D.W. = 1:3:16, i.p.), paclitaxel (PTX; positive control, 5 mg/kg, i.p.) and 2′-HCA (5, 10, or 20 mg/kg, i.p.). During the treatment, tumor volume and body weight were measured once per 3 days. On day 21, mice were killed and tumors were obtained.
Tumor tissues were washed and cleaned by 1x PBS to fixation with 4% paraformaldehyde. After tumor fixation, the tissues were embedded in paraffin. Tissue section and IHC were performed by Korea Experimental Pathology Inc. (Gyeonggi-do, Korea) and the immune-stained slides were viewed under a light microscope (400×).
All data presented as means ± SD were analyzed by using GraphPad Prism 8.0 Software (San Diego, CA, USA). Student’s t-test analysis was applied for statistical analysis to compare all the different groups in the current study. The difference was considered to have statistical significance if P < 0.05.