A drug targeting 5‑lipoxygenase enhances the activity of a JAK2 inhibitor in CD34+ bone marrow cells from patients with JAK2V617F‑positive polycythemia vera in vitro
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- Published online on: March 4, 2021 https://doi.org/10.3892/ol.2021.12612
- Article Number: 351
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Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis (1) are Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) that originate from hematopoietic stem cells (HSCs). In 2005, an activating point mutation in Janus kinase 2 (JAK2V617F) was identified in the majority of patients with MPNs (2–5), which provided critical insights into MPN pathogenesis and promoted the development of Janus kinase 2 (JAK2) inhibitors. According to clinical studies, JAK2 inhibitors effectively decrease spleen size and relieve MPN-associated symptoms; however, their disease-modifying activity is limited (6,7).
5-Lipoxygenase (5-LO) is an important dioxygenase, since it is the key enzyme that catalyzes the transformation of arachidonic acid into inflammatory leukotrienes (LTs) (8). Accumulating evidence has suggested that the 5-LO signaling pathway is directly involved in cancer development by promoting cell proliferation, angiogenesis, migration and invasion, and inhibiting apoptosis (9–13). As demonstrated in a study by Chen et al (14), 5-LO is upregulated in a mouse model of JAK2V617F-induced PV, and inhibition of 5-LO by zileuton, a selective 5-LO inhibitor, attenuates PV development by blocking JAK2V617F-expressing HSCs in mice. Therefore, it may be hypothesized that zileuton could potentially eliminate persistent malignant HSCs in patients with PV. However, to the best of our knowledge, no previous reports have described the role of 5-LO in patients with JAK2V617F-positive PV.
Based on the aforementioned evidence, the combination of zileuton with a JAK2 inhibitor may be a promising treatment strategy for patients with PV. The present study first analyzed 5-LO expression in CD34+ cells from the bone marrow of patients with JAK2V617F-positive PV using western blotting and reverse transcription-quantitative PCR (RT-qPCR). Subsequently, the effects of zileuton combined with ruxolitinib on colony formation, apoptosis and the cell cycle of CD34+ cells from patients with PV were analyzed in vitro.
Materials and methods
Patient specimens and cell preparation
Bone marrow and peripheral blood were donated by 18 patients who were newly diagnosed with PV and 10 healthy adult volunteers at the Affiliated Zhuzhou Hospital Xiangya Medical College CSU (Zhuzhou, China) between August 2017 and April 2019. All patients met the World Health Organization diagnostic criteria for PV (1). Patient characteristics are shown in Table I. The healthy volunteers were eligible if they were 18–69 years of age and in healthy condition without active infections, and serious liver, kidney, heart and other diseases. Bone marrow and peripheral blood from 10 healthy volunteers were used as normal controls. The volunteers included 6 women and 4 men. The mean age was 41.5 years, and the age ranged between 23 and 69 years. All participants provided written informed consent according to the protocol approved by the Medical Ethics Committees of the Affiliated Zhuzhou Hospital Xiangya Medical College CSU (Zhuzhou, China) and in accordance with the principles outlined in the Declaration of Helsinki. Mononuclear cells were separated from bone marrow samples at 440 × g for 30 min at room temperature using Ficoll-Hypaque density gradient centrifugation (GE Healthcare). An EasySep™ CD34-positive selection kit (Stemcell Technologies, Inc.) was used to enrich the CD34+ cell population according to the manufacturer's protocol. CD34+ cells with a purity ≥85% were used in each experiment.
Detection of leukotriene B4 (LTB4)
Plasma samples from patients with PV and healthy volunteers were collected to detect LTB4 levels using a leukotriene B4 Express ELISA kit (cat. no. 10009292; Cayman Chemical Company). Briefly, the standard or plasma sample, LTB4 AchE tracer and anti-LTB4 antibody were sequentially added to each well of a 96-well plate. The plates were then incubated for 60–90 min at room temperature before measuring the absorbance at 405 nm using an ELISA microplate reader (Thermo Fisher Scientific, Inc.).
RT-qPCR
Total RNA was extracted from the purified CD34+ cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA synthesis was performed using a PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (cat. no. RR047A; Takara Bio, Inc.) according to the manufacturer's protocols. Reactions were incubated at 37°C for 15 min followed by heat inactivation for 5 sec at 85°C for reverse transcription. The PCR amplification was performed using TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (cat. no. RR820A; Takara Bio, Inc.). The primer sequences of the human 5-LO gene and the GAPDH gene are listed in Table II. The thermocycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 31 sec. Amplification was performed using an ABI 7300 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The 2−ΔΔCq formula (15) was used to analyze the relative mRNA expression levels of 5-LO, which were normalized to the expression levels of GAPDH.
Western blotting
RIPA lysis solution (cat. no. P0013B; Beyotime Institute of Biotechnology) was used to extract total proteins from purified CD34+ cells. BCA assays (cat. no. 23227; Pierce; Thermo Fisher Scientific, Inc.) were used to determine the protein concentration. An equal amount of total proteins (30 µg/lane) was separated by 10% SDS-PAGE and then transferred onto PVDF membrane (cat. no. IPVH00010; EMD Millipore). The membranes were blocked using TBS with 0.05% Tween-20 (TBST) containing 5% non-fat milk at room temperature for 2 h, and then incubated with primary antibodies against 5-LO (dilution, 1:500; cat. no. sc-136195; Santa Cruz Biotechnology, Inc.) and β-actin (dilution, 1:1,000; cat. no. ab8226; Abcam) overnight at 4°C. After washing with TBST, the membranes were incubated with secondary antibodies (dilution, 1:2,000; cat. no. 7076; Cell Signaling Technology, Inc.) conjugated to horseradish peroxidase at room temperature for 1 h. Finally, the protein blots were visualized using a Hypersensitive ECL chemiluminescence kit (cat. no. P10018FS; Beyotime Institute of Biotechnology). The results were semi-quantified using ImageJ software (v1.52; National Institutes of Health).
Colony formation assay (CFA)
The colony-forming ability of the cells was estimated by inoculating 500 CD34+ cells in MethoCult H4435 (Stemcell Technologies, Inc.). Various concentrations of zileuton (50, 100, 250 and 500 µM; Cayman Chemical Company) and/or 50 nM ruxolitinib (Cayman Chemical Company) were added for 14 days at 37°C. After 14 days, the presence of colonies (>40 cells) was scored under a light microscope (Zeiss LSM 800 with Airyscan; magnification, ×25; Zeiss AG).
Flow cytometry
CD34+ cells (1×106 cells/well) from patients with PV and healthy volunteers were seeded into a 6-well plate and treated with 100 µM zileuton, 50 nM ruxolitinib or the combination of 100 µM zileuton and 50 nM ruxolitinib for 48 h at 37°C. After 2 days, 5×105 cells were collected and applied to the flow cytometry detection. The apoptosis assay was performed using an Annexin V-FITC Apoptosis Detection Kit (cat. no. 556547; BD Biosciences). In brief, cells were stained with Annexin V-FITC and PI for 15 min at room temperature, and detected using a flow cytometer. Stained cells were analyzed using a FACSCalibur instrument (BD Biosciences) using a 488 nm excitation wavelength and emission was detected at 530 nm (for FITC) and 575 nm (for PI). The data were analyzed using the BD FACSuite™ version 1.01 (BD Biosciences). The phases of the cell cycle and DNA synthesis activity of CD34+ cells were determined using a FITC BrdU Flow kit (cat. no. 559619; BD Biosciences) according to the manufacturer's protocol. Briefly, cells were incubated with BrdU, a nucleoside analogue of thymidine, and then stained with anti-human CD34-allophycocyanin (dilution, 1:5; cat. no. 560940; BD Biosciences). After fixing in BD Cytofix/Cytoperm Buffer for 30 min on ice and permeabilizing in BD Cytoperm Permeabilization Buffer Plus for 10 min on ice, cells were treated with DNase to expose BrdU epitopes and then incubated with a FITC-conjugated anti-BrdU antibody (provided in kit; BD Biosciences) for 20 min at room temperature. DNA was counterstained with 7-aminoactinomycin D (7AAD; provided in kit; BD Biosciences) for 15 min at room temperature. Stained cells were then analyzed using a FACSCalibur instrument using a 488 nm excitation wavelength and emission was detected at 530 nm (for FITC-conjugated anti-BrdU) and 610 nm (for 7AAD). The data were analyzed using BD FACSuite™ version 1.01.
Statistical analysis
All experiments were repeated three times. Data are presented as medians and interquartile ranges. Differences in 5-LO expression and LTB4 levels between groups were compared using a two-tailed Mann-Whitney test, while data derived from the same samples after different treatments were analyzed using the Friedman test, and the Nemenyi post hoc test was subsequently used for pairwise comparisons between groups. GraphPad Prism 7 software (GraphPad Software, Inc.) and SPSS 26.0 software (IBM Corp.) were utilized for statistical analyses. P<0.05 was considered to indicate a statistically significant difference.
Results
5-LO expression is increased in CD34+ cells from patients with PV
To determine the potential effects of 5-LO inhibitor zileuton in the treatment of human PV, the present study first assessed the basal protein and mRNA expression levels of 5-LO in CD34+ cells from the bone marrow of patients with PV and healthy volunteers. Western blot analysis revealed that CD34+ cells from patients with PV exhibited higher protein expression levels of 5-LO than those from healthy volunteers (Fig. 1A and B; Table SI). Consistent with the results observed for protein expression, 5-LO mRNA expression was also increased in CD34+ cells from patients with PV compared with in those from healthy volunteers, as demonstrated by the results of RT-qPCR (Fig. 1C; Table SII).
LTB4 levels are elevated in patients with PV
Plasma levels of LTB4, a metabolite of the 5-LO signaling pathway, were measured in 14 patients with PV and 10 healthy volunteers using ELISA. Higher LTB4 levels were observed in patients with PV compared with those in healthy volunteers (Fig. 2; Table SIII).
Zileuton suppresses the colony formation of CD34+ cells from patients with PV in a dose-dependent manner
A CFA was performed to assess the effect of zileuton treatment on the colony formation of primary CD34+ cells in vitro. As shown in Fig. 3, zileuton treatment inhibited granulocytes and monocytes (CFU-GM)- and burst-forming unit-erythroid (BFU-E)-derived colony formation by PV CD34+ cells in a dose-dependent manner, with an IC50 of 460.4 µM for CFU-GM and 233.5 µM for BFU-E (Fig. 3B and C; Table SIV). By contrast, zileuton treatment did not markedly alter the colony formation of the CD34+ cells from healthy volunteers (Fig. 3A and C; Table SIV).
Reduction of colony formation by zileuton treatment in PV CD34+ cells occurs through a reduction in 5-LO expression
To explore whether the effect of zileuton on colony formation of hematopoietic cells was associated with the levels of 5-LO, 5-LO protein expression in CD34+ cells from patients with PV and healthy volunteers was measured with and without treatment with increasing concentrations of zileuton using western blotting. As shown in the Fig. 4, zileuton dose-dependently decreased 5-LO protein expression in CD34+ cells from a patient with PV, which exhibited high 5-LO expression, but had no significant effects on 5-LO expression in CD34+ cells from a healthy volunteer.
Combination treatment with zileuton and ruxolitinib synergistically inhibits the colony formation of hematopoietic cells from patients with PV
The present study investigated the effect of combination treatment with zileuton and ruxolitinib on the formation of colonies of primary CD34+ cells from 10 patients with PV and 6 healthy volunteers in vitro. The doses selected for the present study were 100 µM zileuton and 50 nM ruxolitinib. In the CD34+ cells from healthy volunteers, treatment with 100 µM zileuton, 50 nM ruxolitinib or 100 µM zileuton combined with 50 nM ruxolitinib did not alter colony formation (Fig. 5A and C; Table SV). By contrast, the yields of CFU-GM and BFU-E in CD34+ cells from patients with PV treated with 50 nM ruxolitinib were decreased by 19 and 35%, respectively, and the yields of CFU-GM and BFU-E in CD34+ cells from patients with PV treated with 100 µM zileuton were decreased by 12 and 20%, respectively (Fig. 5B and C; Table SV). However, the combination of ruxolitinib with zileuton had a greater effect on the colony formation of CD34+ cells from patients with PV, with a 36% a reduction in CFU-GM and a 55% reduction in BFU-E (Fig. 5B and C; Table SV).
Combination treatment with zileuton and ruxolitinib induces apoptosis in CD34+ cells from patients with PV
The apoptosis rate of CD34+ cells from patients with PV and healthy volunteers after treatment with zileuton and/or ruxolitinib was detected using flow cytometry. The apoptosis rate of CD34+ cells from patients with PV was slightly increased following zileuton or ruxolitinib treatment (Fig. 6; Table SVI). However, combination treatment with zileuton and ruxolitinib induced apoptosis in a greater number of CD34+ cells from patients with PV than treatment with either individual drug. By contrast, neither ruxolitinib nor zileuton alone or in combination induced apoptosis of CD34+ cells from healthy volunteers (Fig. 6; Table SVI).
Treatment with zileuton and ruxolitinib arrests CD34+ cells from patients with PV at the G0/G1 phase of the cell cycle
Cell cycle arrest is an important effect of numerous anticancer agents. The present study investigated the effects of zileuton and/or ruxolitinib on the cell cycle of CD34+ cells from patients with PV in vitro. As shown in Fig. 7A and C, treatment with zileuton and ruxolitinib alone slightly increased the number of CD34+ cells from patients with PV that were arrested in the G0/G1 phase of the cell cycle, with a concomitant decrease in the percentage of cells in S phase (Table SVII). However, combination treatment with zileuton and ruxolitinib caused more CD34+ cells from patients with PV to be arrested in the G0/G1 phase and a significant decrease in the proportion of cells in S phase compared with either monotherapy. By contrast, the percentage of CD34+ cells from healthy volunteers in each phase of the cell cycle did not markedly change, regardless of whether they were treated with zileuton, ruxolitinib or the combination treatment (Fig. 7A and B; Table SVII).
Discussion
PV is the most common Philadelphia chromosome-negative MPN (16). Thrombosis and hemorrhage, as well as myelofibrotic and/or leukemic transformation (17,18), are potential complications occurring in patients with PV. The JAK2V617F mutation in the pseudokinase domain leads to constitutive phosphorylation of JAK2 and overactivation of downstream signaling pathways, ultimately resulting in uncontrolled myeloid cell proliferation (19,20). These findings motivated the clinical development of JAK kinase inhibitors for patients with MPNs. Ruxolitinib was the first selective JAK1/2 inhibitor that was demonstrated to be effective in patients with PV and myelofibrosis (21–23), and it provides substantial benefits, including a marked decrease in splenomegaly and disease symptoms (24). However, treatment with ruxolitinib rarely induces molecular or pathological remission in patients with MPNs (25–27). Furthermore, the dose-dependent hematological toxicity of ruxolitinib represents a major concern for a number of patients (28). Researchers have expressed an interest in identifying novel agents with different mechanisms of action other than targeting the JAK-STAT signaling pathway.
5-LO expression is upregulated in numerous different types of cancer, including pancreatic, breast, prostate, esophageal and colon cancer (29–33). The products of 5-LO, such as 5-hydroxyeicosatetraenoic acid (5-HETE) and LTs, are able to promote cell proliferation, suppress apoptosis, promote angiogenesis and enhance tumor cell invasion (34–36). According to previous studies, epidermal growth factors and neurotensin are involved in the 5-LO-mediated tumor progression in individuals with prostate cancer (37,38). A study of patients with colorectal cancer revealed that 5-HETE stimulates angiogenesis by inducing the expression of VEGF (39,40). Furthermore, increased activities of 5-LO and matrix metalloproteinases are associated with extracellular matrix stiffness (41) and enhance the invasiveness of cancer cells (42,43). The enzyme 5-LO is involved in the development of not only solid malignancies but also certain forms of leukemia (44,45). Recently, the 5-LO gene was shown to be a critical regulator for mouse leukemic stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML), and the combination of zileuton and imatinib extends the survival time of mice with CML (46). This finding motivated an initial clinical trial combining zileuton with imatinib as a treatment for CML (clinicaltrials.com; NCT02047149, NCT01130688) with no available efficacy data at present. Zileuton has been revealed to selectively deplete JAK2V617F-expressing HSCs, thereby preventing PV development in mice (14). The molecular mechanism of 5-LO in PV is associated with the β-catenin signaling pathway, which is required for the maintenance of both HSCs (47) and LSCs in individuals with CML (48–50). Based on these results, drugs targeting the 5-LO signaling pathway can eradicate human JAK2V617F+ malignant HSCs and thus are potentially curative treatments for MPNs. However, a previous study reported low expression levels of 5-LO and LTB4 receptor 1 in CD34+ cells from patients with BCR-ABL-positive CML compared with healthy donors (51). Another study reported that 5-LO expression was undetectable in more primitive CML LSCs (52). Therefore, 5-LO has distinct and important regulatory and functional roles in human and murine CML. To the best of our knowledge, no previous reports have described the role of 5-LO in human PV.
In the present study, 5-LO mRNA and protein expression was increased in CD34+ cells from patients with PV compared with in CD34+ cells from healthy volunteers. Higher LTB4 levels were detected in patients with PV compared with healthy volunteers. Based on these results, the 5-LO signaling pathway was upregulated in patients with JAK2V617F-positive PV, consistent with previous findings from PV mice (14), indicating that 5-LO may be involved in the pathogenesis of human JAK2V617F-positive PV. Zileuton treatment decreased the colony formation of CD34+ cells from patients with PV in a dose-dependent manner by reducing 5-LO expression in PV CD34+ cells. Furthermore, zileuton and ruxolitinib exerted their anticancer effects by suppressing the colony formation of hematopoietic cells, inducing apoptosis and blocking the cell cycle of CD34+ cells from patients with PV. The combination of the two drugs was more effective than either agent alone. Similar effects were not observed in CD34+ cells from healthy volunteers after treatment with zileuton or ruxolitinib, either alone or in combination. Therefore, zileuton enhanced the antitumor activity of ruxolitinib against hematopoietic progenitor cells from patients with PV, suggesting that zileuton and ruxolitinib may represent an effective therapeutic combination. Notably, activities of both 100 µM zileuton and 50 nM ruxolitinib were relatively specific for hematopoietic progenitor cells from patients with PV, while sparing CD34+ cells from healthy volunteers. The latter finding suggests that the combination treatment may exhibit improved safety and low hematological toxicity.
In conclusion, zileuton exerted a synergistic effect with ruxolitinib on CD34+ cells from patients with PV by suppressing cell proliferation, inducing apoptosis and arresting the cell cycle, which provides conceptual validation for further clinical applications of combination therapy with ruxolitinib and zileuton for patients with PV. However, because CD34+ cells collected from some patients were not amplified enough to complete all downstream experiments, only a portion of the samples were analyzed in some experiments. This was a limitation of the present study. Future studies should focus on investigating the molecular mechanisms by which 5-LO inhibitors block colony formation, and induce apoptosis and cell cycle arrest, in patients with PV.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
The present study was funded by the Clinical Medical Technology Innovation Guidance Project of Hunan Province (grant no. 2018SK52803).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contributions
GH and YC proposed and designed the current study. JL and YL selected patients and collected samples/clinical data. YC and HZ conducted the experiments. YC, HZ and KT were responsible for acquiring, analyzing and interpreting the data. YC and HZ drafted the initial manuscript. YC, HZ, JL and GH reviewed and edited the manuscript. GH, YC and HZ assessed the authenticity of all the raw data and ensured its legitimacy. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Medical Ethics Committees of The Affiliated Zhuzhou Hospital Xiangya Medical College CSU (Zhuzhou, China). Written informed consent was obtained from each individual.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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