Apatinib (Houston, TX, USA). HHT (Zhejiang Minsheng Pharmaceutical, Zhejiang, China) was dissolved in dimethyl sulfoxide (DMSO) at 1 mg/mL and stored at -20°C.
HHT was diluted with a culture medium in subsequent experiments.
MV4-11, MOLM-13, OCL-AML2, and OCL-AML3. Were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MV4-11 and MOLM13 expressed MLL fusion oncoprotein with FLT3-ITD. OCL-AML2 and OCL-AML3 expressed mutant NPM1c+. THP1 without FLT3-ITD or NPM1c+ mutant, all cells were cultured in RPMI 1640 with 20%FBS.
Cell viability assay
AML cell lines (2×104 cells/well) were plated in 96-well plates and then treated with different concentrations of Apatinib and HHT for 24 h and 48 h. The cytotoxic effect was determined by cell counting kit-8 (CCK8; Dojindo, Japan) assay. IC50 (half-maximal inhibitory concentration) values were determined using a microplate reader (BIO-TEK EPOCH, USA).
To assess apoptosis, MV4-11, MOLM-13, OCL-AML2, and OCL-AML3 cells were cultured and presented with different doses of Apatinib or HHT alone or in combination for 24 h or 48 h then double-labeled with Annexin-V-FITC/PI (eBioscience). However, the same concentration did not work in non-FLT3-ITD mutations AML cell lines, for example, THP1. The stained cells were analyzed with a NovoCyte flow cytometer (ACEA Biosciences, Inc.) with NovoExpress software.
Annexin V-positive cells were defined as apoptotic cells.
In vitro clonogenicity assay
MV4-11, MOLM-13, OCL-AML2, and OCL-AML3 cells (2×105/well) were used to test colony-forming abilities. AML cells were seeded in 24-well plates and then, treated with 20 µM Apatinib or 16 nM HHT alone or both molecules. After 24 h, cells were washed and then cultured in complete methylcellulose medium at a cell density of 500 cells/well in 3.5-cm dishes for 10-14 days. The percentage of CFU was determined by counting colonies (≥50 cells). Data were presented as the mean ± S.D. of three independent experiments.
Cell cycle analysis
AML cells were treated with 24h, then cells were fixed overnight with 75% ethanol, PBS washes three times then incubation in buffer containing 50 µg/mL PI and 100 µg/mL RNase A for 30 min at room temperature. Cells were resuspended with DAPI saline solution and subjected to Flow Cytometer (NovoCyte™).
Analysis of mitochondrial membrane potential
AML cells (2×105/ml) were plated in 24-well plates treated with various concentrations of Apatinib and HHT. After 24 h, cells were stained with 2 μM Rhodamine 123 (Byeotime) for 30 min. Then washed, mitochondrial membrane potential (MMP) was presented by flow cytometry (FACS Fortessa).
Western Blot analysis
Cell protein was extracted with RIPA protein lysis buffer (Beyotime). MOLM-13 cells
(2×105/mL) were cultured with 20 µM Apatinib or 16 nM HHT alone or combined for
24 h and 48 h. Related antibodies included β-actin, VEGFR2, p-VEGFR2, PI3K, Akt, CyclinA2, CyclinD1, P21, and Bcl-2 (rabbit, 1:1000, Cell Signaling Technology). Blots were tested by the addition of horseradish peroxidase (HRP)-conjugated secondary antibody. Signals were visualized using the ECL Western blotting detection kit (Gene-Flow, Staffordshire, UK).
We used GraphPad Prism software v7.0 to analyze the data. All experiments performed at least three independent experiments. Multi-group comparisons were using a one-way test of variance (ANOVA). Statistical analyses were presented using SPSS 20.0 software (La Jolla, CA).