Reagents.
Anlotinib was purchased from Chia Tai Tianqing Pharmaceutical Group Co Ltdand dissolved in dimethyl sulfoxide (DMSO; Invitrogen, Carlsbad, CA, USA) as 10 mM stock solution for storage at -20℃. It was thawed and diluted to the required concentrations with cell culture medium prior to experiments.
Cell culture.
Human AML cell lines Molm-13 and MV4-11 carrying MLL-rearrangement were purchased from ATCC (Rockefeller, MD, USA) and cultured at 37 °C in a 5% CO2 incubator in Iscove’s modified Dulbecco’s medium (IMDM) and RPMI-1640 medium (HyClone, Thermo Scientific, Logan, UT, USA), respectively, and supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Scientific, Grand Island, NY, USA).
Cell viability analysis.
The assay of cell viability was performed as reported previously16. Briefly, 2 × 104/well cells were seeded in 100 µl medium on 96-well plates and treated with indicated concentrations of Anlotinib for 24, 48, and 72 h. Then, 10 µl of the Cell Counting Kit-8(CCK-8) reagent (Dojindo, Kumamoto, Japan) was added to each well. The cells were incubated for additional 2 h, followed by analyzing the absorbance at 450 nm with a microplate reader (ELx800, BioTek, Winooski,VT, USA). Data from three independent experiments in triplicate were presented as percentage of viable cells by comparing to untreated control. IC50 values were determined using the SPSS 20.0 software.
Analysis of apoptosis.
The assay was performed as reported previously16. In brief, cells were cultured and treated with Anlotinib for 24, 48, and 72 h in a same condition as described above. Cell apoptosis was determinated by double staining with Annexin V-FITC and PI (eBioscience, Thermo Scientific, San Diego, CA, USA) following the manufacturer’s instruction. The data were then analyzed by flow cytometry (FACS Fortessa, BD Biosciences, Franklin Lakes, NJ, USA) with using the FACS C6 software.
Analysis of cell cycle.
Molm-13 and MV4-11 cells were grown in 6-well plates and treated with Anlotinib for 8 h and24 h. Then cells were collected and washed with phosphate buffered saline (PBS), followed by fixation in ethanol (70%). After overnight incubation at 4 °C, the cells were stained with PI and examined by flow cytometry.
Western blot analysis.
Cells with 2 × 105/well were treated with Anlotinib for 24, and48 h, and then subjected to western blot analysis using indicated primary antibodies and secondary HRP-conjugated antibodies ( ab150121,Abcam, Cambridge, UK). Blots were detected by visualization using the ECL Western Blotting Detection Kit (GeneFlow, Staffordshire, UK). Antibodies included anti-POLD1(sc-17776, SantaCruz,USA), anti-POLD2(HPA026745, Atlas Antibodies AB,Sweden),anti-POLD3(A301-244A-M, Bethyl,USA), anti-SETD1A (ab70378, Abcam, Cambridge, UK), anti-AKT (ab8805, Cell Signaling Technology,USA),and anti-GAPDH(D16H11,Cell Signaling Technology,USA).
RNA sequencing.
Cells were incubated with Anlotinib for 24, after which total RNA was isolated as described previously24. RNA sequencing (RNA-seq) was then carried out via a commercially available service (Sangon Biotech, Shanghai). GSEA was used to enrich the signal pathways involved in differentially expressed genes. The gene set is c2.cp.kegg.v6.0.symbols.gmt[curated].
Animal study.
Animal studies were performed according to the Xiamen University Animal Guideline and approved by the Animal Welfare Committee. In brief, a total of 5 × 106 Molm-13 cells were inoculated subcutaneously into nude mice. After 3 days, mice were randomly divided into vehicle control and Anlotinib groups (n = 5, each group), and then treated respectively with either vehicle (PBS) or Anlotinib (6 mg/kg/day) by oral gavage for 9 days. Tumor size and body weight were measured daily. Mice were sacrificed at the end of drug treatment or tumor size reached 1000 mm3. All tumors was removed, measured, and weighted. Tumor volume was calculated using the formula: V = (L × W2)/2, where L is the longest and W the shortest diameter of the tumor.
Statistical analysis.
All statistical analyses were carried out using the SPSS 23.0 and GraphPad Prism 6.0 softwares. The differences between two groups were analyzed using the two-tailed Student’s t test. P < 0.05 was considered as statistically significant.