Cell lines and culture conditions
The acute promyelocytic leukemia cell lines HL-60 and NB4, acute monocytic leukemia cell line THP-1, acute myelomonocytic leukemia cell line U937, acute erythrocytic leukemia cell line TF1α and acute myelogenous leukemia cell line KG1α were cryopreserved in the Hematological Laboratory of Zhujiang Hospital (Guangzhou, China). HL-60, NB4, TF1α and THP-1 cell lines were purchased from the cell bank of Sun Yet-san University (Guangzhou, China); the source of these cells was ATCC. U937 cell line was purchased from ATCC. The KG1α and HL-60/ADR cell lines were kindly provided by Tianjin Institute of Hematology (Tianjin, China). Normal PBMCs were obtained from 5 unrelated healthy donors at Southern Medical University (Guangzhou, China). All cell lines and the PBMCs were incubated in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum at 37 °C with 5% CO2.
EPS8 GEO expression profile in AML cells
The expression profiles of AML patients from the GSE13159 dataset, containing bone marrow samples from 501 AML patients at diagnosis and 72 healthy volunteers, were generated on Affymetrix Gene Chip HG-U133A arrays (Affymetrix, Santa Clara, CA, USA), and were extracted from CEL files using RMA normalization procedure and custom CDF annotation package. AML samples from GSE12417 and The Cancer Genome Atlas (TCGA) were used to test association between EPS8 expression and AML patient outcomes.
Creation and characterization of stable EPS8 knockdown cell lines
EPS8 expression was stably knocked down in U937 and KG1α cells via RNA interference. The annealed oligonucleotide fragments encoding short hairpin transcripts corresponding to EPS8 were as follows: TAGTGATTCAGGAGTGGAA and AACTTCTAATCGCCATATA. The non-targeting empty plasmid was used as the control shRNA plasmid. According to the manufacturer’s instructions, U937 and KG1α cells (2 × 105/well in six-well plates) were separately transfected separately with control shRNA plasmid or the EPS8 shRNA plasmid using Lipofectamine 2000 reagent (Invitrogen). After the dilution culture was limited under selection with puromycin, several clones in each transfection group were selected for further experiments and designated as U937/NC, U937/sh1, U937/sh2, KG1α/NC and KG1α/sh1.
RT2profiler™ PCR assay in KG1α/sh1 and KG1α/NC cells
An RT2profiler™ PCR assay (SuperArray, SABiosciences, a QIAGEN Company) was used to profile the expression of 84 EGF/PDGF signaling-specific genes plus 5 housekeeping genes according to the manufacturer’s protocol. The process in detail was described in previous work .
CP-EPS8-NLS, mutated CP-EPS8-NLS and penetratin were synthesized by the Chinese Peptide Company (Hangzhou, China). Peptides purity was greater than 95%. The peptides were dissolved in deionized water at a final concentration of 10 mg/ml and stored at − 20 °C until further use.
Cell viability assays
AML cell lines and normal PBMCs were plated in a 96-well plate (5 × 103 cells/well) and incubated for 24 h before treatment. All cells were then incubated with different concentrations of CP-EPS8-NLS (0, 35, 70, 105, 140 or 175 μM) for 24 h. U937 cells were also treated with mutated CP-EPS8-NLS and penetratin as controls. After treatment, 10 μl of CCK-8 reagent (Dojindo Laboratories, Japan) was added to each well, and cells were incubated for 3 h at 37 °C and 5% CO2. The optical density (OD) was analyzed at 450 nm. The data obtained are presented as percentage viability in best-fit (linear) dose response curves.
Soft agarose cloning assay
Low-melting agarose was dissolved in pure water at 1.2 and 0.7%, sterilized using an autoclave, and then warmed at 42 °C in a water bath. Then, 1 ml of 2× RPMI 1640 was transferred to each well of a 6-well plate, and 1 ml of 1.2% agarose was added. After these two solutions were mixed, 500 μl of 0.7% agarose and 500 μl of RPMI 1640 containing 500 cells were pipetted into each well and treated with CP-EPS8-NLS (0, 35, 70, 175 μM) on the following day. Two weeks later, the colony number was determined.
Cellular distribution of CP-EPS8-NLS in U937 cells
To examine the membrane penetration ability and the distribution of CP-EPS8-NLS, mutated CP-EPS8-NLS and penetratin in AML cells. Fluorescein isothiocyanate (FITC) was conjugated to the N-terminus of these peptides to form FITC-conjugated peptides. U937 cells (2 × 105 cells per plate) were placed in confocal microscope observation wells that had been pretreated with polylysine. Then, cells were treated with FITC-conjugated peptides (40 μM) in 1 ml medium of culture for 4 h and the cells were stained with propidium iodide (PI) for 20 mins to exclude the possibility that peptides penetrate dying cells and then washed twice with PBS. The cells were fixed for 30 mins and stained with DAPI (which produces blue fluorescence after binding to dsDNA). Cells were rinsed three times with PBS, and the fluorescence distribution was analyzed with a confocal laser scanning microscope (LSM 880 with Airyscan).
Analysis of apoptosis and cell cycle
U937, KG1α, HL-60, THP-1 and TF1α cells were seeded at 1 × 105 cells/well in 6-well plates in serum-containing media; cells were cultured for 12 h before treatment. CP-EPS8-NLS was added at concentrations ranging from 0 to 175 μM and incubated at 37 °C and 5% CO2 for 24 h and 48 h respectively in KG1α. U937, KG1α, HL-60, THP-1 and TF1α cells were added at 0 or 70 μM CP-EPS8-NLS for 24 h. CP-EPS8-NLS treated AML cells were collected and washed by PBS, suspended in binding buffer according to the manufacturer’s protocol (BD, Annexin-V-APC & PI Apoptosis Detection Kit). Cells were analyzed with CellQuest software and each measurement was repeated three times to ensure reproducibility. For cell cycle analysis, AML cells were cultured for 12 h before treatment. CP-EPS8-NLS was added to a final concentration of 0 or 70 μM and incubated at 37 °C and 5% CO2 for 24 h. Cells were collected, washed with PBS, and suspended in RNase A and PI for 1 h in the dark. Samples were analyzed on a FACSCalibur Flow Cytometer (Becton Dickinson, New Jersey, USA).
Daunorubicin (DNR), cytarabine (Ara-c), adriamycin (ADR) and perifosine were purchased from Selleckchem, dissolved in RPMI 1640 medium at a final concentration of 10 mg/ml and stored at − 20 °C.
Determination of combination index values and Chou-Talalay analysis
KG1α and U937 cells were seeded at 5 × 103 cells/well in 96-well plates for 24 h before treatment. Cells were treated with CP-EPS8-NLS and/or chemotherapeutic agents (DNR, Ara-c or ADR) at 37 °C and 5% CO2 for 24 h. Then, cell viability was measured using a CCK-8 assay. The assessment of synergy was performed using CompuSyn software. The combination index (CI) theorem of Chou-Talalay offers a quantitative definition for additive effect (CI = 1), synergism (CI < 1) or antagonism(CI > 1) in drug combinations . Isobolograms were also used to better investigate the combination effects.
Western blot analysis
All prepared cells were homogenized in protein lysate buffer, and debris was removed by centrifugation at 12,000 g for 10 min at 4 °C. The protein concentrations were determined using a Bradford protein assay kit (Beyotime, China). After addition of loading buffer, protein samples were electrophoresed, transferred to PVDF membranes (0.2 μm; Millipore, Bedford, MA), and subsequent blocked. The membranes were immunoblotted with rabbit anti-human primary antibody overnight at 4 °C. Antibodies to EPS8, Erk, p-Erk, Akt, p-Akt (473), p-Akt (450), p-Akt (308), p-Stat3, mTOR, p-mTOR, p38 MAPK, p-GSK3β, p-cRaf, Cyclin E, bcl-2 and GAPDH were obtained from Cell Signaling Technology. After three washes with TBST, the blots were incubated with horseradishperoxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h, and the HRP signal was detected using enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA).
In vivo study
All animal experiments complied with Southern Medical University’s Policy on the Care and Use of Laboratory Animals. Five-week-old athymic BALB/c nu/nu female mice (14–16 g) purchased from the experimental animal center of Southern Medical University (Guangzhou, China) were used for in vivo experiments. Animals were housed at a constant room temperature with a 12 h light/12 h dark cycle and fed a standard rodent diet and water. U937 cells were harvested and injected subcutaneously (5 × 106 cells in 100 μl of PBS) into mice. U937-injected mice were treated with CP-EPS8-NLS at the dose of 50 mg/kg body weight or with PBS as a control via intraperitoneal (i.p.) injection every other day. In addition, KG1α cells were harvested and injected subcutaneously (1 × 107 cells in 100 μl of PBS) into mice. KG1α-injected mice were treated with CP-EPS8-NLS (50 mg/kg) and/or DNR (20 mg/kg) every other day. Mutated CP-EPS8-NLS and PBS were injected as controls. The maximum tumor volume was not allowed to exceed 3000 mm3. At the end of the experiment, the animals were sacrificed, and the tumors were removed. The tumor volumes were determined by measuring tumor length (L) and width (W) and calculating the volume (V = 0.5 × L × W2) .
Statistical significance was evaluated using SPSS 11.0 software. P < 0.05 was considered statistically significant. * Represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001.