1. Cell lines, primary tumor cells, and reagents
The AML cell lines OCI-AML3, THP-1, MV4;11, and MOLM13 were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 with 10% fetal bovine serum and penicillin (100 units/ml)/streptomycin (100 µg/ml). All cell lines were cultured in an incubator with 95% humidity at 37.5°C with an atmosphere of 5% CO2. Primary AML cells were isolated from peripheral blood containing >50% blasts. These specimens were collected before chemotherapy. Venetoclax powder was purchased from Selleck Company (USA) and dissolved in DMSO at a concentration of 10 mM. Chidamide was received as a gift from Shenzhen Microchip Biotechnology Co. Ltd. and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mM.
2. Cell viability assay
The cells were plated in 96-well plates (5000 cells/well) and treated with different doses of venetoclax and chidamide for 24, 48 and 72 hours. At different time points, the cell number was measured using a Cell-Counting Kit-8 (CCK8) proliferation assay kit (Tongren Institute of Chemistry, Japan). Ten microliters of CCK-8 solution was added to each well of the plate. After incubation for 2 hours at 37°C, the plates were measured at 450 nm using a microplate reader (Biotech, NY, USA).
3. Flow cytometry analysis
The AML cell lines were incubated with venetoclax and chidamide alone or in combination for 8 h and 24 h. Then, apoptosis was measured using Alexa Fluor 647-conjugated annexin V and propidium iodide (PI) (China Nanjing Kaiji Company) according to the manufacturer’s instructions. Briefly, the cells were harvested, washed twice with phosphate-buffered saline, and resuspended in 200 µL of binding buffer. Then, the cells were incubated with 2 µL Alexa Fluor 647-conjugated annexin V and 1 µL PI for 10 min. Apoptosis was analyzed using a Navios flow cytometer (Beckman Coulter, Brea CA, USA). The results are expressed as the percentage of Annexin V+ cells. AML cell line experiments were performed 3 independent times in triplicate, and the data presented are from one representative experiment; the patient sample experiments were performed once in triplicate due to the limited sample availability. The combination index (CI) values were determined using CompuSyn software. CI<1, CI=1, and CI>1 indicate synergistic, additive, and antagonistic effects, respectively.
3.2 Cell cycle analysis
The cells were treated with venetoclax alone or combined with chidamide for 72 h. Then, the cells were collected and washed with PBS and fixed overnight in 75% ice-cold ethanol at 4°C. The fixed cells were harvested, stained with propidium iodide/RNase (BD Pharmingen, San Diego, CA, USA) and incubated in the dark at room temperature for 15 min after being washed with PBS. The DNA content was analyzed by flow cytometry. ModFit software (Verity Software House, Inc., Topsham, ME) was used for data analysis.
3.3 Analysis of the mitochondrial membrane potential
First, the optimal concentrations of venetoclax and chidamide in the OCI-AML3, THP-1, MV4;11, and MOLM13 cell lines were determined according to the results of the above apoptosis assays. Second, AML cells were treated with venetoclax and chidamide alone or in combination for 24 h, and then JC-1, a cationic lipid fluorescent dye, was used to stain the cells. During the process of apoptosis, the mitochondrial transmembrane potential decreases, JC-1 exists in the cytoplasm in the form of monomers, and the number of polymers decreases. Flow cytometry was used to determine whether JC-1 existed in the form of monomers or polymers to detect changes in the mitochondrial transmembrane potential.
4. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays
Total cellular RNA was extracted from cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA was eluted with RNase-free water, quantified at an absorbance of 260/280 nm, and used for reverse transcription reactions. Total mRNA was reverse transcribed into cDNA using an RT reagent kit (TaKaRa, Dalian, China). Primer sequences of qRT-PCRI are shown in Table2. β-actin was used as an internal standard. qRT-PCR was performed with Fast Start Universal SYBR Green Master Mix (ROX) (Roche, Germany) following the instructions of the supplier. The qRT-PCR conditions were as follows: 1 cycle at 94°C for 10 min, 40 cycles at 94°C for 10 sec, 60°C for 30 sec, and one cycle at 72°C for 3 min. The results were analyzed using the 2-ΔΔCt method, in which ΔCt = Ct (target gene)-Ct (internal reference), and ΔΔCt = ΔCt (sample)ΔCt (control). Each sample was measured in triplicate.
5. Western blotting analysis
After treating cells with venetoclax and chidamide for 24 hours, cultured cells were harvested, washed with PBS and then lysed with ice-cold lysis buffer. The protein lysates were clarified by centrifugation at 14000 g for 15 min at 4°C, and the supernatant was collected. The protein level in each sample was quantified by a BCA (bicinchoninic acid) assay (Pierce). Equal amounts of proteins were separated by SDS-PAGE and then electrotransferred onto a PVDF membrane (Millipore). The membranes were blocked with 5% skim milk and incubated with primary antibodies at 4°C overnight in TBS-T (10 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.1% Tween 20). The primary antibodies against the following proteins were used: γ-H2AX, PARP, Caspase3, Bcl-2, Mcl-1, Bim, Bax, Bak, AKT, P-AKT, SOCS3, P-JAK2, and P-STAT3. β-actin (Cell Signaling, Herts, UK) was used as a loading control. The blots were washed, exposed for 1 hour to the corresponding HRP-conjugated secondary antibodies, and finally detected by chemiluminescence reagents (Millipore, Billerica, MA, USA). The target protein bands were visualized using ECL and exposed to X-ray film. Immunoreactive proteins were visualized using an Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE), as described by the manufacturer. Densitometry measurements were made using an Odyssey V3.0 (Li-Cor) and normalized to β-actin.
6. HDAC inhibitory activity of chidamide
The HDAC1 gene level was detected by qRT-PCR. Meanwhile, HDAC1 protein, acetylated histone H3 and histone H4 levels were detected by Western blotting to determine the ability of chidamide to inhibit histone deacetylation.
7. In vivo experiment
MOLM13 cells transfected with luciferase were injected into mice (5×106 cells) via the tail vein in mice with severe immunodeficiency (NOD/SCID). Four days after the injection, in vivo imaging was performed to confirm the successful establishment of an AML xenotransplantation mouse model. The mice were randomly divided into groups with 9 mice in each group. The mice were treated with venetoclax (100 mg/kg, intragastric administration, QD) and chidamide (25 mg/kg, intragastric administration, QD) alone or in combination. The untreated group was considered the control. The tumor growth was observed twice a week. Three mice in each group were sacrificed on the 10th day of treatment. The liver and spleen were collected for immunohistochemical staining with a human CD45 antibody (hCD45), and the expression of CD45+CD33+ cells in the bone marrow was analyzed by flow cytometry. The survival time of the remaining 5 mice in each group was observed, and the survival curve of tumor-bearing mice was generated. The weight of the mice in the four groups was measured every other day, and a weight curve was generated after 14 days of continuous monitoring.
8. Statistical analysis
Each experiment was performed at least three times, and the data are presented as the mean ± standard deviation for the indicated number of separate experiments. A t-test was used to compare the mean of each group with that of the control group in experiments. All analysis was performed with SPSS 22.0 System. The results were considered significant if the P-value was less than 0.05.