Med Oncol (2014) 31:836 DOI 10.1007/s12032-013-0836-9
ORIGINAL PAPER
Homoharringtonine contributes to imatinib sensitivity by blocking the EphB4/RhoA pathway in chronic myeloid leukemia cell lines Bin-Tao Huang • Qing-Chun Zeng Wei-Hong Zhao • Yan Tan
•
Received: 31 October 2013 / Accepted: 27 December 2013 / Published online: 11 January 2014 Ó Springer Science+Business Media New York 2014
Abstract The purpose was to investigate the role of EphB4 in imatinib (IM) resistance and the mechanism responsible for homoharringtonine (HHT) contributing to imatinib sensitivity for a chronic myeloid leukemia (CML) cell lines. We established cell lines from a patient with CML at the time of first diagnosis and relapsed phase and designated them as NPhA1 and NPhA2, respectively. Stable underexpressing EphB4 cells (NPhA2-sh) were obtained. The activated signal proteins in cells were tested by Western blot. The EphB4 was overexpressed in IMresistant NPhA2 in comparison with the NPhA1 cell line, but the expression of EphB4 mRNA and protein significantly decreased in knockdown NPhA2-EphB4-sh cells compared with NPhA2 and NPhA1 (P \ 0.001) cell lines. NPhA2-EphB4-sh cells were sensitive to IM (IC50 Bin-Tao Huang and Qing-Chun Zeng have contributed equally to this work. B.-T. Huang (&) Department of Hematology, The Affiliated Hospital of Inner Mongolia Medical University, 1 TongDao Avenue North, Hohhot 010059, People’s Republic of China e-mail:
[email protected] Q.-C. Zeng Department of Cardiology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Ave, Guangzhou 510515, Guangdong, People’s Republic of China W.-H. Zhao Department of Gastroenterology, The Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010059, People’s Republic of China Y. Tan Department of Neurology, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Ave, Guangzhou 510515, Guangdong, People’s Republic of China
0.93 mg/L), and NPhA2 showed IM resistance (IC50 5.45 mg/L) (P \ 0.001). Meanwhile, phospho-Rac1/cdc42 was significantly increased in NPhA2 cells compared to NPhA2-EphB4-sh (P \ 0.001). The apoptosis rate reached 58.71 ± 2.39 % with NPhA2 cells incubated with HHT ? IM, which was higher than NPhA2 cells incubated with IM alone (P = 0.002). IC50 of NPhA2 cells incubated with IM was 5.45 mg/L. However, co-stimulation with HHT ? IM decreased the IC50 of NPhA2 cells from 5.45 to 1.17 mg/L (P \ 0.001). Furthermore, HHT blocked the expressions of EphB4/RhoA, but did not down-regulate the phospho-MEK/ERK in NPhA2 cells. The overexpression of EphB4 contributed to IM resistance in CML line cells. EphB4/RhoA may be a new marker of IM resistance. HHT ? IM gained more treatment advantages than IM alone by blocking EphB4/RhoA pathways in CML cell lines. Keywords Homoharringtonine Imatinib Chronic myeloid leukemia
Introduction Imatinib (IM) is currently the standard therapy for patients with early chronic phase chronic myeloid leukemia (CML), resulting in an improved 5-year survival rate and a decreased rate of transformation to blast crisis [1]. Although most patients with chronic phase CML treated with IM have been well controlled, some patients relapse and/or progress to an accelerated phase or blast crisis [2–4]. The erythropoietin-producing hepatocyte receptors (Eph) are the largest family of PTKs [5]. Well-known effects of Eph receptor activation include the regulation of cell shape and movement. EphB4, a member of the Eph
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family, plays an important role in tumorigenesis and regulating diverse cell functions including cell-to-cell contact, cell adhesion, migration, and repulsion [6, 7]. Recent studies showed that the overexpression of EphB4 was associated with drug resistance in Ph? acute lymphocytic leukemia (ALL). Activation of the MEK/ERK pathway was suggested as an explanation for the incomplete effect of IM on Ph ? ALL [8]. In 2003, Ohmine et al. [9] suggested that the disturbance of RhoA kinase signaling was involved in imatinib resistance. Thus, in order to determine the mechanism of EphB4 receptor contributing to IM resistance in CML, we examined RhoA or MEK/ERKrelated protein activity in regulating the expression of EphB4. Homoharringtonine (HHT) is a plant alkaloid that has been widely used for the past 30 years for the treatment for myelogenous leukemia. Both inhibition of protein synthesis and inducing cell apoptosis appear to be involved in the anti-leukemic mechanism of HHT [10, 11]. In 2004, Li et al. [12] found that HHT enhanced the expression of DJ-1 in K562 cell by proteomics and spectrometry identification. The online string software analyzed that DJ-1 down-regulated the EphB4 expression. This meant that HHT could potentially decrease EphB4 in CML. Our purpose is to identify the role and mechanism of EphB4 in IM resistance to CML. Furthermore, we determined that HHT contributed to blocking the EphB4-mediated IM resistance pathway.
Materials and methods Patient and cell culture The patient was a 76-year-old man who was diagnosed with Ph ? CML in 2010. He received IM and achieved remission but relapsed 2 years later. The karyotype was 46,XX,t(9;22)(q34;q11) at first diagnosis and relapsed phase. Heparinized BM samples were obtained with informed consent at initial diagnosis and during the relapse phase. Mononuclear cells were separated by Ficoll–Conray gradient centrifugation. The cells were cultured in RPMI1640 medium supplemented with 10 % FCS, 2 mM L-glutamine, 100 U/mL penicillin, and 0.1 mg/ml streptomycin at 37 °C and a humidified atmosphere of 5 % CO2. Continuously growing cells were obtained from BM samples at first diagnosis and in the relapse phase and were named NPhA1 and NPhA2, respectively. Knockdown of EphB4 by RNA interference (RNAi) Oligonucleotides for shRNA were designed using ABI online software. The oligonucleotides for EphB4-sh were sense
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(50 -AACTGTTCACTTCCGCCAGTG ATTTCAAGAGAA TCACTGGCGGAAGTGAACTTTTTTC-30 ) and antisense (50 -TCGAGAAAAAAGTTCACTTCCGCCAGTGATTCT TGAAATCACTGGCGGAAGTGAACAGTT-30 ). Lentiviruses were generated using 5 lg of lentiviral vector and 2.5 lg of each packaging vector co-transfected in 293T cells using the lipofectamine 2000 reagent (Invitrogen, US). Supernatants were collected 36–48 h after transfection, filtered through a 0.4 lm filter, and used directly to infect NPhA2 cells. GFP-positive cells were sorted 3–4 days after infection by flow cytometry, and the expression of EphB4 was detected by Western blot. Then, stable underexpressing EphB4 cells (NPhA2-EphB4-sh) were obtained. PE annexin V apoptosis detection The apoptosis rate of NPhA2 cells (HHT, IM, or HHT ? IM incubated) was detected with PE annexin V in a buffer containing 7-amino-actinomycin (7-AAD) and analyzed by flow cytometry. MTT assay MTT was used to analyze the tolerance dose of IM in NPhA2-EphB4-sh cells. NPhA1, NPhA2, and NPhA2EphB4-sh cells were plated into 96-well flat-bottomed microtiter plates at 5 9 104 cells/well in 100 lL media. After 24 h of pre-incubation, gradient concentrations of IM (0–8 lM) were added. Control cells treated with an equal concentration of DMSO were included in each experiment. Untreated cells were also included as controls. The inhibition rate was quantified as [1-(OD treated-OD blank)/ (OD control-OD blank] 9 100 %. Semi-quantitative PCR (SQ-PCR) analysis All cell lines used in experiments were harvested in the exponential growth phase. Total RNA was isolated with Trizol reagent (Invitrogen, USA). SQ-PCR was performed on ABI 9700 system. Primers for gene amplification were designed with Primer 5.0 and sequences are as follows: EphB4-upstream primer: 50 -ACTCCTTCCTGCGGCT AA-30 ; EphB4-downstream primer: 50 -AGACGAGGTTGCTG TTGACT-3. Western blot analysis Total protein was isolated by RIPA lysis buffer, and protein concentrations were determined by the Bradford method. Proteins (80 lg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes with the Bio-Rad
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Fig. 1 Expressions of EphB4 mRNA and protein in knockdown NPhA2-EphB4-sh cells comparing with NPhA2. Expression of EphB4 mRNA transcripts was compared by reverse transcription PCR among NPhA1, NPhA2, and NPhA2-EphB4-sh cells. Band intensity on gel electrophoresis was quantified using computer software. Expression of EphB4 proteins were analyzed by Western blotting. Whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes with the Bio-Rad Transblot system and detected with the indicated antibodies. The experiments were repeated three times and showed similar results Table 1 IM resistance to change for NPhA cell (Mean ± SD)
Fig. 2 Phosphorylation of signaling protein in the IM-resistant NPhA2 and NPhA2-EphB4-sh cells compared to NPhA1 cells. Phosphorylation and expression of signal molecules were analyzed by Western blotting. Phosphorylation of Rac1/cdc42, MEK, and ERK were detected by phospho-specific antibodies. The experiments were repeated three times and showed similar results
Table 2 Effect of HHT (20 lg/L) on apoptosis of NPhA2 cell lines (Mean ± SD) N = 3/group
Total apoptosis rate (%)
NPhA1 cell IM (1.2 mg/L)
71.82 ± 1.94
NPhA2 cell-blank control
2.79 ± 0.18
NPhA2 cell IM (1.2 mg/L)
2.35 ± 0.111,2,3
N = 3/ group
NPhA1
NPhA2
NPhA2EphB4-sh
P value
NPhA2 cell IM (1.2 mg/L) ? HHT (20 lg/L)
58.71 ± 2.39
P value
\0.001
IC50 (mg/ L)
0.16 ± 0.015
5.45 ± 0.46
0.93 ± 0.13
\0.001
1
Trans blot system. After blocking in 5 % bovine serum albumin (BSA) for 2 h, membranes were incubated overnight in primary antibody diluted in 5 % BSA at 4 °C. The following primary antibodies were used: antiEphB4 (R&D, USA), anti-phospho-Rac1/cdc42 (Cell Signaling Technology, USA), anti-phospho-ERK1/2 (Cell Signaling Technology, USA), anti-phospho-MEK1/2(Cell Signaling Technology, USA), and anti-b-actin (Santa Cruz Biotechnology, USA). After washing, membranes were incubated for 1 h with HRP-conjugated cow anti-goat (or rabbit) immunoglobulin (1/5000) (from Santa Cruz Biotechnology, USA), diluted in 5 % BSA. After washing, the enhanced chemiluminescence kit (Santa Cruz Biotechnology, USA) was used to visualize the secondary antibody. Statistical analysis Statistical analysis was performed with SPSS 13.0 statistical software. Values represented the mean of 3 independent experiments and were expressed as mean ± SD. The expressions of phosphorylated protein and mRNA were
P \ 0.001, NPhA2 cell IM (1.2 mg/L) versus NPhA1 cell IM (1.2 mg/L); 2 P = 0.121, NPhA2 cell IM (1.2 mg/L) versus NPhA2 cell-blank control; 3 P = 0.002, K562-R cell IM (1.2 mg/L) versus K562-R cell IM (1.2 mg/L) ? HHT (20 lg/L)
evaluated by ANOVA analysis. Paired t test was used in the comparison of phosphorylated proteins of cell by IM or HHT intervention. P value \ 0.05 was considered statistically significant.
Results NPhA2-EphB4-sh cells were was sensitive to IM SQ-PCR and Western blot were used to demonstrate the decreased expression of the EphB4 mRNA and protein in knockdown cells. The EphB4 mRNA and protein were overexpressed in the IM-resistant NPhA2 cell line in comparison with the NPhA1 cell line. But the expression of EphB4 mRNA and protein significantly decreased in knockdown NPhA2EphB4-sh cells compared with NPhA2 (P \ 0.001, Fig. 1) and NPhA1 (Fig. 1, P \ 0.001) cell lines. The NPhA2EphB4-sh cell line was well established. NPhA2-EphB4-sh
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Fig. 3 PE annexin V apoptosis detection in showed the apoptosis rate. NPhA1 (sensitivity to IM) was treated with IM (1.2 mg/mL) for 24 h as positive control. NPhA2 (resistance to IM) was treated with IM (1.2 mg/mL) or IM (1.2 mg/mL) ? HHT (20 lg/ mL) for 24 h as study control. The experiments were repeated three times and showed similar results
Table 3 IC50 of NPhA2 cell by HHT intervention Imatinib IC50 (mg/L) (N = 3)
Before HHT incubation
After HHT incubation
P value
NPhA2 cell
5.45 ± 0.46
1.17 ± 0.17
0.007
cells were sensitive to IM (IC50 0.93 mg/L), and NPhA2 showed IM resistance (IC50 5.45 mg/L) (P \ 0.001, Table 1). Activated RhoA was involved in NPhA2-EphB4-sh cells We examined the phosphorylation of molecules (MEK/ ERK, RhoA) in NPhA1, NPhA2, and NPhA2-EphB4-sh cells. Phospho-MEK/ERK was higher in the IM-resistant NPhA2 and NPhA2-EphB4-sh cells compared to NPhA1 type cells (P \ 0.001, Fig. 2). However, there was no difference in the expression of phospho-MEK/ERK between NPhA2 and EphB4-sh cells and NPhA1 cell lines (P [ 0.05, Fig. 2). In contrast, phospho-Rac1/cdc42 was significantly increased in NPhA2 cells compared to
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NPhA2-EphB4-sh and NPhA1 cell lines (P \ 0.001, Fig. 2). HHT enhanced NPhA2 cells to IM sensitivity The result of PE annexin V apoptosis detection showed that the apoptosis rate of NPhA2 was only 2.35 ± 0.11 %, which was significantly lower than (P \ 0.001, Table 2, Fig. 3) NPhA1 (71.82 ± 1.94 %) when they were co-cultured with IM (1.2 mg/L). But the apoptosis rate reached 58.71 ± 2.39 % with NPhA2 incubated with HHT (20 lg/ L) ? IM, which was higher than NPhA2 incubated with IM (P = 0.002, Table 2, Fig. 3). MTT assay showed that IC50 of NPhA2 treated with IM was 5.45 mg/L. But under the stimulation of HHT, IC50 of NPhA2 decreased from 5.45 to 1.17 mg/L (P \ 0.001, Table 3). HHT blocked the EphB4/RhoA pathway We examined the expressions of EphB4-related pathway proteins. The expressions of EphB4 and phospho-Rac1/ cdc42 were significantly decreased after HHT incubation in
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Fig. 4 Expressions of pathway protein after HHT incubation in NPhA2 cell. Expressions of EphB4 and phospho-Rac1/cdc42, phospho-MEK/ERK were analyzed by Western blotting. Whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes with the Bio-Rad Trans blot system and detected with the indicated antibodies. The experiments were repeated three times and showed similar results
NPhA2 cells (P \ 0.001, Fig. 4), but there were no differences in phospho-MEK/ERK (P = 0.324 and P = 0.370, Fig. 4).
Discussion In 2010, Suzuki et al. [8] reported a new mechanism of IM resistance mediated by the activation of MEK/ERK pathway and EphB4 in Philadelphia chromosome-positive ALL. Our findings show that EphB4 is overexpressed in IM-resistant NPhA2 cells. Knockdown of EphB4 in NPhA2 cells confers sensitivity to IM in vitro. These data indicate that overexpression of EphB4 contributes to IM resistance in CML. The small GTPases of the RhoA are known regulators of the actin cytoskeleton [13]. In 2003, Ohmine et al. [9] found that the disturbance of RhoA protein kinase signaling was involved in imatinib resistance. Another study indicated that Rho kinase was related to Bcr-Abl nonkinase activity, as a potential protein target [14]. In the study, we examine phospho-Rac1/cdc42 to recognize phospho-RhoA activity. The phospho-Rac1/cdc42 is simultaneously decreased in the NPhA2-EphB4-sh cell line compared with the NPhA2 cell line. Our data indicate that EphB4 overexpression is associated with IM resistance, but it is through an EphB4/RhoA mechanism other than MEK/ ERK pathway activation. HHT, alone and in combination with other drugs, has been used effectively for more than 20 years for therapy in
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acute myeloid leukemia. IM combined with HHT may improve the prognosis in CML [15]. In 2010, Fang et al. [16] reported that the HHT ? IM regimen reinduced hematologic responses or improved the cytogenetic responses in advanced CML patients with disease-poor response to conventional doses of imatinib. In our study, HHT significantly enhanced the apoptosis rate and proliferative inhibition of NPhA2 cells treated by IM. We suggest that EphB4/RhoA was simultaneously decreased after HHT intervention in NPhA2 cells. Meanwhile, no notable changes were observed in phosphoMEK/ERK. In summary, our understanding of the complex roles of EphB4 and other Eph receptors in leukemia is still evolving, and more information is needed to resolve these confusing and controversial issues. We present the induction of RhoA activation associated with EphB4 receptor overexpression as a new marker of IM resistance. We can make a theoretical explanation for the efficacy of HHT ? IM regimen over that of HHT and/or IM alone in the treatment for myeloid leukemia. Acknowledgments This work was supported by the Natural Science Foundation of Inner Mongolia (2013MS1157). Conflict of interest I would like to declare on behalf of my co-authors that this work is original research that has not been published previously and is not under consideration for publication elsewhere, either in part or in whole. All the authors listed have approved the manuscript that is enclosed.
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