APG-2575 was provided by Ascentage Pharma Group Co., Ltd. (Suzhou, China), and ABT-199 was purchased from Selleck Chemicals (Houston, TX, USA). HHT was purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies GAPDH, β-actin, PARP, Caspase3, Caspase7, Cleaved-Caspase3 (Cleaved-C3), Cleaved-Caspase7 (Cleaved-C7), PI3K (p85α), AKT, AKT (Ser473), GSK3β, GSK3β (Ser9), MCL-1, MCL-1 (Thr163) and MCL-1 (Thr163/Ser159) were purchased from Cell Signaling Technology (Beverly, MA, USA).
2.2 Cell lines and primary cells
MV4-11 cell line was kindly endowed by Professor R. Bhatia (City of Hope National Medical Center, Duarte, CA, USA). THP-1, HL-60, U937 and OCI-AML3 cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Kasumi-1 cell line was gifted by Professor Chen Saijuan (Shanghai Institute of Hematology, Shanghai, China). MV4-11 cells were maintained in Iscove’s modified Dulbecco’s medium (IMDM, Gibco, Billings, MT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), and THP-1, HL-60, U937 and OCI-AML3 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640, Gibco) medium supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO2.
Bone marrow samples were obtained from AML patients following written informed consent. Mononuclear cells were isolated by Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University, China.
2.3 Cell viability assay
20,000 AML cells (cell density of 2.0 ×105 cells/ml) or 100,000 primary AML cells (cell density of 1.0 ×106 cells/ml) were seeded in a 96-well plate and treated with designated drugs for the 24 or 48 h. Then 10 ul MTS solution (Promega CellTitre 96, Promega Corporation, Madison, WI, USA (5mg/ml) was added by incubation for additional 3–4 h at 37℃. The plates were measured at an absorbance of 490 nm. IC50 values and combination index (CI) were calculated by CalcuSyn Software (Biosoft, Cambridge, UK).
2.4 Flow-cytometric analysis of apoptosis
The extent of apoptosis was assessed through an apoptosis detection kit (Beyotime Institute of Biotechnology, Hangzhou, China). Cells were treated with drugs for 24 h, and then harvested and washed with phosphate buffered saline (PBS). According to the manufacturer’s instruction, after resuspending in binding buffer and staining with Annexin V-FITC (Annexin-V) and propidium iodide (PI) in dark at room temperature for 15 min, cells were subjected to FACScan™ flow cytometer (Becton Dickinson, San Diego, CA, USA). Data were presented as percentages of Annexin V + cells.
2.5 Western blot analysis
Cells were harvested and washed in PBS, and then lysed in radioimmunoprecipitation (RIPA) buffer in the presence of protease and phosphatase inhibitor (Cell Signaling Technology (CST), Beverly, MA, USA)) on ice for 30 min. Cells were centrifuged at 12,000 g for 15 min at 4°C to pellet cell debris. The protein concentration was determined using BCA reagent. Equal protein was electrophoresed in 10% SDS-PAGE (Life Technologies, Carlsbad, CA, USA) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The transblotted membranes were blocked with 5% non-fat milk in TBS-T for 1 h and then incubated with primary antibodies overnight at 4 ℃. Then the membranes were washed thrice with TBS-T buffer, 15min each time, followed by secondary HRP-conjugated antibody (CST, Beverly, MA, USA) for 1 h at room temperature. The target proteins were visualized using an ECL detection kit (Amersham, Little Chalfont, UK) and analyzed using IMAGE LABTM software (Bio-Rad Laboratories, Hercules, CA, USA).
2.6 AML xenograft model
Thirty-five BALB/c Nude mice and twenty NOD SCID mice (female, 6–8 weeks of age, weight 17–24 g) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Shanghai, China) and Beijing Vital River Laboratory Animal Technology Co., Ltd, respectively. MV4-11 cells were resuspended in PBS and Matrigel (1×106 cells/mouse), and then subcutaneously injected into the right back of BALB/c Nude mice to establish a subcutaneous xenograft tumor model. When the tumor volume reached to 100–200 mm3, the mice were randomly divided into 6 groups (vehicle control, 50 mg/kg APG-2575, 50 mg/kg ABT-199, 1 mg/kg HHT, 50 mg/kg APG-2575 + 1 mg/kg HHT, 50 mg/kg ABT-199 + 1 mg/kg HHT). The administration was started on the day of grouping (ie D1; APG-2575/1BT-199, PO, QDx21D; HHT, IP, QDx14D). The mouse body weight and tumor size were measured twice a week during the experiment. After the administration, all mice were killed by cervical dislocation. In addition, OCI-AML3 cells were injected subcutaneously on the right back of NOD SCID mice, and the other operations referred to the above.
Tumor volume (TV) was calculated as: TV = L⋅W2/2, Where L and W represent the length and width of the tumor, respectively. The relative tumor volume (RTV) was: RTV = Vt/V1. V1 is the TV on the first day of administration (Day1) and Vt is the TV at the time of measurement (Day t). The evaluation index of anti-leukemic effect was the relative tumor proliferation rate: (T/C (%) = (TRTV/CRTV) ⋅ 100%) [TRTV: the RTV of the treatment group, CRTV: the RTV of the vehicle control group]. Synergy analysis was measured by using the formula : expected value = (A/C) × (B/C) [actual value = (AB)/C; A: RTV of A drug; B: RTV of B drug; C = The RTV of the vehicle control group; AB = the RTV of the combined administration group]. The synergy index = expected value/actual value; ratio > 1, indicating that the two drugs have a synergistic effect; ratio = 1, indicating that the two drugs have an additive effect; ratio < 1, indicating that the two drugs have a weaker than additive effect. Change of body weight (%) = (measured weight-weight at grouping)/weight at grouping ⋅100%. In addition, complete regression (CR), partial regression (PR) and stable disease (SD) among the number of animals in each group represented the remission rate. T/C (%) ≤ 42% and statistical analysis P < 0.05 is effective. If the weight of the mouse drops by more than 20% or the number of drug-related death exceeds 20%, the drug dose is considered to be severely toxic.
All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Suzhou GenePharma (Approval No: IACUC-2021003).
2.7 Statistical analyses
Experiments were presented as the means ± standard deviation (SD) in three independent experiments. Student’s t-test and the one-way ANOVA test were used to assess statistical significance (P < 0.05 was considered statistically significant). Statistical analyses were conducted using Prism software v8.3.0 (GraphPad Software, La Jolla, CA, USA). The combination index (CI) was calculated using CalcuSyn software (Biosoft, Cambridge, UK).