Chronic myeloid leukemia (CML) accounts for approximately 15% of new adult leukemia cases. The fusion gene
Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder that affects the blood and bone marrow and accounts for about 15% of newly diagnosed adult leukemia cases (
The oncogenic BCR-ABL fusion protein has persistent kinase activity, resulting in the uncontrolled proliferation of myeloid cells through multiple downstream pathways (
CML therapy has seen impressive advances with the development of tyrosine kinase inhibitors (TKIs) against the BCR-ABL pathway (
ND-09 (4-methoxyl-3-[[4-(3-pyridyl)-2-pyrimidinyl]amino] benzoic acid pyrimidin-2-ylamino-benzamides) is a novel compound developed by our group (
ND-09 (
Thermal Cycle Dice Real time system, PrimeScript RT Master Mix Perfect Real Time kit, and SYBR® Premix Ex Taq™ II were obtained from Takara Biotechnology. Annexin V-FITC cell apoptosis detection kit was obtained from Pioneer Biotechnology Co., Ltd. RNase and propidium iodide (PI) were obtained from Sigma-Aldrich. Opti-MEM medium was purchased from Gibco. Lipofectamine 2000 reagent was obtained from Invitrogen. Phosphatase and protease inhibitors were obtained from Roche Tech. The BCA kit, RNAfast200 kit, and RIPA lysis buffer were obtained from Pioneer Biotechnology Co., Ltd.
The mcl-1 (monoclonal, 66026-1-Ig), Bad (monoclonal, 67830-1-Ig), Bcl-2 (monoclonal, 60178-1-Ig), Bak (polyclonal, 14673-1AP), Bax (monoclonal, 60267-1-Ig), CDC2 (monoclonal, 67575-1-Ig), Cyclin D1 (monoclonal, 60186-1-Ig), Cyclin E (polyclonal, 11554-1-AP), CDK2 (monoclonal, 60312-1-Ig), Cyclin A2 (monoclonal, 66391-1-Ig), and Cyclin B1 (monoclonal, 67686-1-Ig) antibodies were purchased from Protein Technology Group. The phospho-BCR-ABL (polyclonal, Tyr177), BCR-ABL (monoclonal, L99H4), phospho-ABL (monoclonal, 73E5), ABL (polyclonal, 2862), phospho-BCR (polyclonal, Tyr177), and BCR (polyclonal, 3902) antibodies were obtained from Cell Signaling Technology. The JAK2 (monoclonal, D2E12), phospho-JAK2 (monoclonal, D15E2), JAK3 (monoclonal, D1H3), phospho-JAK3 (monoclonal, D44E3), STAT3 (monoclonal, D3Z2G), phospho-STAT3 (monoclonal, D3A7), STAT5 (monoclonal, D3N2B), and phospho-STAT5 (monoclonal, D47E7) antibodies were obtained from Cell Signaling Technology. The p53 (polyclonal, 10442-1-AP), p38 (monoclonal, 66234-1-Ig), and PTEN (monoclonal, 60300-1-Ig) antibodies were obtained from the Protein Technology Group. The phospho-p38 (monoclonal, D3F9), PI3K-p110α (monoclonal, C73F8), PI3K-p110β (monoclonal, C33D4), PI3K-p110γ (monoclonal, D55D5), PI3K-p85 (monoclonal, 19H8), and phospho-PI3K-p85/p55 (monoclonal, E3U1H) antibodies were obtained from Cell Signaling Technology. The AKT (monoclonal, C67E7), phospho-AKT (monoclonal, D9E), PLCγ (monoclonal, D9H10), phospho-PLCγ (monoclonal, D6M9S), Erk1/2 (monoclonal, 137F5), phospho-Erk1/2 (monoclonal, Tyr202/204), MEK1/2 (monoclonal, D1A5), phospho-MEK1/2 (monoclonal, 166F8), mTOR (monoclonal, 7C10), and phospho-mTOR (monoclonal, D9C2) antibodies were obtained from Cell Signaling Technology. Rabbit anti-GAPDH (monoclonal, 60004-1-lg) was obtained from Protein Technology Group. The working solution for the primary antibodies was 1:1000, except for the Bad and Cyclin E antibodies, which was 1:500. HRP-conjugated goat anti-rabbit IgG (secondary antibody, dilution: 1:20,000) was obtained from Pierce Biotechnology. Enhanced Chemiluminescent Plus Reagent was obtained from Biotech Co., Ltd.
K562 and HUT78 cells were cultured in IMDM medium containing 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin. JURKAT cells were cultured in RPMI-1640 medium containing 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin. All cell lines were grown at 37°C in a 95% humidified atmosphere with 5% CO2.
K562, JURKAT and HUT78 cells were seeded in 96-well plates and cultivated in complete medium for 24 h. Then cells were treated with ND-09 (0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 µM) for 48 h. Then, 20 µl MTT solution (5 mg/ml) was added to each well and incubated at 37°C for 4 h. Subsequently, the cells were enriched at the bottom of each well using a plate spinner (4,192.5 × g for 5 min at 25°C) and the medium was slowly removed from each well. Then, 150 µl DMSO was added in each well for 15 min. The absorbance was recorded by a microplate reader (Bio-Rad) at 490 nm.
Cells were seeded in 6-well plates and treated with ND-09 for 48 h, after which the cells were collected by centrifugation (4,192.5 × g for 5 min at 25°C), washed, and resuspended in PBS. According to the instructions, Annexin V-FITC and PI double staining were used to detect the apoptotic rate. Then, Cell Quest software was used for flow cytometry and data analysis.
Cells were seeded in 6-well plates and treated with ND-09 for 48 h, after which the cells were washed with complete medium, followed by incubation with 1 mM Rhodamine 123 at 37°C in the dark for 30 min. Then BD FACSCalibur Flow cytometer was used to perform flow cytometric analysis and analyze data.
Cells were seeded in 6-well plates, treated with ND-09 for 48 h, and then fixed in ice-cold 70% ethanol at 4°C overnight. Then, the cells were washed with cold PBS and stained with RNase and PI for 30 min in the dark. Cell Quest software was used to perform flow cytometric analysis and analyze data.
Cells were seeded in 6-well plates and treated with ND-09 for 48 h. According to the manufacturer's protocol, total RNA from K562, JURKAT and HUT78 cells was extracted using RNAfast200 kit (Pioneer Biotechnology Co., Ltd). RT-PCR was performed using PrimeScript RT Master Mix Perfect Real Time kit and was performed using SYBR® Premix Ex Taq™ II and Thermal Cycle Dice Real time system.
Threshold cycle (Ct) values of
K562 cells were seeded in cell culture dishes at a density of approximately 50%. According to the manufacturer's protocol, Lipofectamine 2000 reagent was used to transfect anti-BCR-ABL siRNA or control siRNA (100 nM) into cells for 24 h. Then, transfected cells were immediately seeded to perform subsequent assays.
K562 cells were seeded into 6-well plates at the density of 2×105 per well. After 24 h, transfection of EphB4 plasmid into
SYBYL-X 1.1 was used to conduct docking studies to understand the binding mode of ND-09 and BCR-ABL domain (PDB ID: 1IEP). The substrate was constructed by Sybyl/Sketch modul and optimized by Powell's method. Tripos force field and Gasteiger-Hückel charges were used to minimize energy, the convergence criterion was set at 0.005 kcal/(Å mol), and the maximum value was set at 1,000 iterations. To explore intramolecular interactions, the non-bonded cut-off distance was set to 8 Å.
Firstly, BCR-ABL kinase (2 µl, 5 ng/µl) and substrate (2 µl, 10 µM) were added to 384-well plates, and then drugs (4 µl) were added to the test plate at different concentrations (0.016, 0.008, 0.040, 0.20, 1.02, 5.12, 25.60, 128.00, and 640 nM). ATP solution (2 µl, 1 mM) was added and the system was incubated at 37°C for 30 min. TK-Antibody (5 µl, 1,000 tests, reconstituted with 5 ml of HTRF® detection buffer) and streptavidin-XL665 (5 µl, 125 nM) were then added to the test plate at room temperature for 1 h.
Cells treated with ND-09 were seeded in 6-well plates. RIPA buffer with protease and phosphatase inhibitor were used to extract protein from cells. The cell lysate was then concentrated at 12,000 × g for 10 min at 4°C. Protein (40 µg) quantified by using BCA kit was then loaded to 10% SDS-PAGE gel, after which protein was transferred to polyvinylidene difluoride membranes. The membranes with protein were blocked with Tris-buffered saline for 1 h at room temperature. Subsequently, the primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated at room temperature for 1 h. The blot was then exposed to the Enhanced Chemiluminescent substrate. Band intensity was quantified by densitometric analysis using an image quantitative analysis system (Image-Pro Plus 5.1; Media Cybernetics Inc.).
Statistical analyses were performed using SPSS 18.0. All the experiments were performed at least three times, and the results are expressed as mean ± SD. Statistical analyses of differences between the groups were performed with ANOVA followed by the Tukey post-hoc test. P<0.05 was considered statistically significant, and P<0.01 was considered extremely significant.
In order to evaluate the inhibitory effect of ND-09 on hematologic tumor cell growth, CML K562 cells, cutaneous T-cell lymphoma HUT78 cells, and acute T-cell lymphoma JURKAT cells were treated with ND-09. As BCR-ABL is a key factor in the myeloproliferative disorder of CML,
Following ND-09 treatment of 1.56, 3.12, and 6.25 µM, the percentage of apoptotic K562 cells increased to 10.17±1.03, 16.40±1.41, and 49.50±1.25%, respectively, compared to control cells (5.55±0.93%) (
Subsequently, the effect of ND-09 on mitochondrial transmembrane potential was examined. Rhodamine 123 fluorescence intensity of K562 cells exposed to ND-09 decreased significantly from 74.82±2.58% in the control group to 64.26±2.15% (1.56 µΜ), 49.98±1.69% (3.12 µΜ), and 23.25±1.76% (6.25 µΜ) (
To explore the mechanism of ND-09-induced cell apoptosis, western blot analysis was performed to detect the levels of apoptosis-related proteins. Results indicated that Bak, Bax, and Bad levels significantly increased after ND-09 treatment, while Bcl-2 and Mcl-1 levels decreased in a dose-dependent manner (
ND-09 treatment led to a significant increase in G0/G1 phase cells (
As ND-09 arrested the K562 cell cycle in the G0/G1 phase, levels of CDK/cyclin proteins were evaluated through western blot analysis.
A siRNA assay was carried out to determine whether
Then, to assess the role of BCR-ABL in ND-09-induced cell growth inhibition, wild-type and
An MD assay was performed to evaluate the affinity characteristics of ND-09 binding to the active site of BCR-ABL. The binding energy of ND-09 with BCR-ABL was −7.07. ND-09 bound to the BCR-ABL ATP-binding pocket through an electrostatic interaction (
Accordingly, we examined the inhibitory effect of ND-09 on BCR-ABL kinase activity. ND-09 inhibited BCR-ABL kinase activity with an IC50 value of 0.57 nM (
As shown in
CML, a myeloproliferative malignancy driven by the constitutively active BCR-ABL1 tyrosine kinase, is currently treated with TKIs that have proven to be clinically effective (
K562 cells, which express high levels of
As ND-09 targets BCR-ABL to inhibit cell proliferation, MD was used to simulate the binding properties of ND-09 and BCR-ABL. Notably, ND-09 fit well within BCR-ABL and occupied the BCR-ABL ATP-binding pocket (
Apoptosis is a major mechanism of programmed cell death. Findings have shown that BCR-ABL exerts anti-apoptotic effects that play an important role in the development of CML (
Previous findings have shown that the BCR-ABL oncoprotein plays an important role in regulating the cell cycle of cancer cells (
As BCR-ABL is a key factor for K562 cell growth, major downstream molecules of the BCR-ABL pathway were evaluated. BCR-ABL activates a number of signaling pathways (JAK/STAT, PI3K/AKT, and MAPK), promotes the proliferation of hematopoietic stem cells, and prevents cell apoptosis (
In conclusion, our study has established ND-09 as a selective inhibitor of BCR-ABL. The findings suggest that BCR-ABL pathway downregulation by ND-09 drives growth arrest in CML.
Not applicable.
This study was supported by the Natural Science Basic Research Program of Shaanxi Province (grant no. 2018JQ8019), the Fundamental Research Funds for the Central Universities (grant no. xzy012019078).
The authors declare that all the data supporting the results in this study are available within the article.
WNM was responsible for the conceptualization, collection of material, and writing and reviewing the manuscript. YHL was responsible for data collection, curation and analysis, as well as writing the original draft. MZ contributed to the curation and interpretation of data. PPL contributed to the design and data analysis, and writing of the manuscript. XYP was responsible for the synthesis of compound ND-09. All authors read and approved the study.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
chronic myeloid leukemia
breakpoint cluster region
Abelson murine leukemia
mitogen-activated protein kinase
Janus-activated kinase/signal transducer and activator of transcription
phosphatidylinositide 3-kinase/protein kinase B
tyrosine kinase inhibitors
Iscove's modified Dulbecco's medium
3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide
fetal bovine serum
propidium iodide
The effect of ND-09 on hematologic tumor cell proliferation. (A) Chemical structure of ND-09. (B) mRNA level of
Effect of ND-09 treatment on cell apoptosis. The proportion of apoptotic cells was determined by double staining with Annexin V/FITC and PI in (A) K562, (B) JURKAT, and (C) HUT78 cells after treatment with ND-09 (0, 1.56, 3.12, and 6.25 µM). (D) Effect of ND-09 on mitochondrial membrane potential (Δψm). Δψm was assessed through flow cytometry following treatment of K562 cells with ND-09 (0, 1.56, 3.12, and 6.25 µΜ) for 48 h. (E) Quantitative analysis of flow cytometry data. (F) Effects of ND-09 on apoptosis-related protein expression in K562 cells. All results were quantified by densitometric analysis of the bands and were normalized to GAPDH (internal control). Samples were derived from the same experiment, and blots were processed in parallel. Values represent the average of three independent experiments. Data are presented as mean ± SEM (n=3). *P<0.05, **P<0.01 compared to the untreated control group.
Effect of ND-09 treatment on the cell cycle. Representative flow cytometry DNA content histogram of (A) K562, (B) JURKAT, and (C) HUT78 cells after treatment with ND-09 (0, 1.56, 3.12 and 6.25 µM). (D) Effects of ND-09 on cell cycle-related protein expression in K562 cells. All results were quantified by densitometric analysis of the bands and were normalized to GAPDH (internal control). Samples were derived from the same experiment, and blots were processed in parallel. Values represent the average of three independent experiments. Data are presented as mean ± SEM (n=3). *P<0.05, **P<0.01 compared to the untreated control group.
The role of BCR-ABL in the biological activity of ND-09 treatment. (A)
Effects of ND-09 on JAK/STAT signaling protein levels. (A) Protein levels of JAK2, p-JAK2, JAK3, and p-JAK3 in K562 cells treated with ND-09 were evaluated by western blot analysis. (B) Protein levels of STAT3, p-STAT3, STAT5, and p-STAT5 in K562 cells treated with ND-09 were evaluated by western blot analysis. Results were quantified by densitometry analysis of the bands and were normalized to GAPDH (internal control). Samples were derived from the same experiment, and blots were processed in parallel. Values represent the average of three independent experiments. Data are presented as mean ± SEM (n=3). *P<0.05, **P<0.01 compared to the untreated control group.
Effects of ND-09 on PI3K/AKT signaling protein levels. (A) Protein levels of PTEN, PI3K-p110α, PI3K-p110β, PI3K-p110γ, p-PI3K p85/p55, and PI3K-p85 in K562 cells treated with ND-09 were evaluated by western blot analysis. (B) Protein levels of AKT, p-AKT, mTOR, and p-mTOR in K562 cells treated with ND-09 were evaluated by western blot analysis. Results were quantified by densitometric analysis of the bands and were normalized to GAPDH (internal control). Samples were derived from the same experiment, and blots were processed in parallel. Values represent the average of three independent experiments. Data are presented as mean ± SEM (n=3). *P<0.05, **P<0.01 compared to the untreated control group.
Effects of ND-09 on MAPK signaling protein levels. (A) Protein levels of PLCγ and p-PLCγ in K562 cells treated with ND-09 were examined by western blot analysis. (B) Protein levels of MEK1/2, p-MEK1/2, Erk1/2, and p-Erk1/2 in K562 cells treated with ND-09 were examined by western blot analysis. (C) Protein levels of p38, p-p38, and p53 in K562 cells treated with ND-09 were examined by western blot analysis. Results were quantified by densitometric analysis of the bands and were normalized to GAPDH (internal control). Samples were derived from the same experiment, and blots were processed in parallel. The values represent the average of three independent experiments. Data are presented as mean ± SEM (n=3). *P<0.05, **P<0.01 compared to the untreated control group.