VEGF depletion enhances bcr-abl-specific sensitivity of arsenic trioxide in chronic myelogenous leukemia Xiaochuang Luo 1, Maoxiao Feng1, Xuejiao Zhu1, Yumin Li 1, Jia Fei1, Yuan Zhang 2 1
Department of Biochemistry and Molecular Biology, Medical College of Jinan University, Guangzhou, China, Institute of Hematology, Medical College of Jinan University, Guangzhou, China
2
The development of resistance to imatinib mesylate may partly depend on high bcr-abl expression levels or point mutation(s). Arsenic trioxide (ATO) has bcr-abl suppressing activity in vitro, without cross-resistance to imatinib. Meanwhile, bcr-abl also induces expression of vascular endothelial growth factor (VEGF), which is associated with tumor-related angiogenesis and is involved in chronic myelogenous leukemia (CML) pathogenesis. Here, we investigated ways to improve ATO activity in CML by modulating cellular VEGF levels. K562 and primary CML cells were transfected with a VEGF antisense sequence. Cell viability and survival were assessed using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and trypan blue exclusion assays. Apoptotic cells were detected by flow cytometry following annexin V and propidium iodide staining. The results showed that VEGF depletion effectively promotes enhanced ATO antileukemic activity by repressing bcr-abl protein levels. These data provide a rationale for the clinical development of optimized ATO-based regimens that incorporate VEGF modulator for CML treatment. Keywords: CML, VEGF, bcr-abl, Arsenic trioxide, Sensitivity
Introduction Chronic myelogenous leukemia (CML) is a type of clonal myeloproliferative neoplasm initiated by bcrabl-mediated transformation of hematopoietic stem cells.1 The main target of the drug imatinib in CML is bcr-abl, an oncoprotein with greatly enhanced tyrosine kinase activity. Imatinib mesylate induces cytogenetic remission in over 75% of CML patients and is a first-line therapy. While responses in chronic phase CML are generally durable, resistance develops in many patients with advanced disease. The development of imatinib mesylate resistance may partly depend on high bcr-abl expression levels2 or its mutation.3 Arsenic trioxide (ATO) induces apoptosis and was successfully used to treat acute promyelocytic leukemia.4 Meanwhile, ATO has been found to be effective in some CML patients, and there seem to be synergistic effects between ATO and imatinib.5–7 Further research demonstrated that ATO has bcr-abl suppressing activity in vitro,8,9 without cross-resistance to imatinib.10 However, ATO use as a single agent at higher concentrations causes many side effects, including ventricular arrhythmia, skin reaction, peripheral neuropathy, electrolyte changes, leukocytosis, hepatic Correspondence to: Jia Fei, Department of Biochemistry and Molecular Biology, Medical College of Jinan University, 601 Western Huangpu Avenue, Guangzhou 510632, China. Email: [email protected]
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dysfunction, and gastrointestinal reactions. Therefore, low-dose combination therapy is required.4,11,12 bcrabl exhibits constitutive tyrosine kinase activity, which leads to autophosphorylation and activation of multiple signaling molecules.13–15 For example, bcrabl can induce vascular endothelial growth factor (VEGF) expression through a pathway involving phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR). VEGF expression induced by bcr-abl may contribute to CML pathogenesis and increased angiogenesis.16–18 Recent studies have shown that leukemia cells release VEGF and express functional VEGF receptors (VEGFRs), which results in an autocrine loop that facilitates tumor growth and propagation.18–23 We asked whether ATO might modulate cellular VEGF. We previously used RNA structure software to predict the accessible sites on VEGF mRNA, and high efficacy antisense sequences (ASs) were screened with cell culture assays.24 Here, we attempt to enhance ATO activity against CML cells by employing ASs to block the VEGF/VEGFR autocrine loop.
Materials and methods Design and synthesis of AS VEGF The RNA structure software update was kindly provided by Prof. Mathews DH (Department of
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Chemistry, University of Rochester, NY, USA) after our registration. The complete human VEGF mRNA sequence was obtained from GenBank (entry code: X62568). The VEGF AS sequence, 5′ GGGTGCAGCCTGGGACCACT-3′ (20 bp), was the most effective antisense phosphorothioate oligodeoxynucleotide (PS-ODN) by experimental screening. Scramble (SCR): 5′ -CATTAATGTCGGACA ACTCA-3′ (20 bp). All sequences were synthesized by Sangon Bioengineering Company (Shanghai, China).
Cell line and primary leukemia culture The K562 (CML) cell line (from the Institute of Cell Biology, Shanghai, China) was grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS) in a 37°C, 5% CO2 humidified atmosphere (Thermo FORMA 3110, Thermo Fisher Scientific, Waltham, MA, USA). Bone marrow (BM) or peripheral blood samples from four patients with untreated chronic phase CML (from Department of Hematology, the first affiliated hospital of Jinan University) were obtained before chemotherapy. CML diagnoses were based on BM aspirates and special staining according to the French– American–British classification criteria and on leukemia cell immunophenotyping. Normal cells (four peripheral blood and three BM samples) were obtained from healthy volunteers. Mononuclear cells from about 2 ml BM were separated and overlaid on 4 ml of Lymphoprep (Shenneng Inc., Shanghai, China). Each sample was spun at 2000 g for 30 minutes, and the mononuclear interphase was collected into a fresh tube and washed twice with phosphate-buffered saline (PBS) for 5 minutes at 780 g. The resulting cell pellet was cultured in DMEM with 10% FCS in a 37°C, 5% CO2 humidified atmosphere.
Cell proliferation, survival, and arsenicsensitivity assay K562 cells were seeded at a density of 1 × 105/ml in 96well plates (Costar, Corning, NY, USA), 100 μl/well, and transfected with 0.4 μM VEGF AS with Lipofectin (Lipofectin:oligonucleotides, 2:1; Invitrogen, Carlsbad, CA, USA) in serum-free DMEM conditions for 6 hours. Next, 100 μl of the appropriate growth medium containing 20% FCS was added to each well. Viable cells were counted with the trypan blue exclusion assay 24, 48, 72, and 96 hours after transfection. ATO of various concentrations (0.5, 1, 2, or 4 μmol/l) were added to corresponding wells for 48 hours. Then, 20 μl 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) stock solution (5 mg/ml) was added to each well at final MTT concentration of 0.45 mg/ml,
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and the plate was incubated for 4 hours at 37°C. The medium was then removed, and dimethyl sulfoxide (DMSO, 150 μl) was added to dissolve the blue formazan crystals at room temperature for 30 minutes. Cell viability was assessed by absorbance at 570 nm on a Bio-Rad microtiter plate reader (Hercules, CA, USA). The IC50 values (μM) were determined by ICp software (PerkinElmer, Waltham, MA, USA). Primary CML cells were seeded at a density of 2 × 105/ml in 96-well plates (Costar), 100 μl/well, and incubated with 0.4 μM AS VEGF transfected with Lipofectin, as described above. Then, 100 μl of the appropriate growth medium containing 20% FCS in the presence of ATO (2 μmol/l) was added to corresponding wells for 72 hours. Surviving CML cells were assessed using the trypan blue dye exclusion assay.
Analysis of apoptosis by flow cytometry K562 cells were seeded at a density of 5 × 105/ml in 24-well plates (Costar), 500 μl/well, transfected with 0.4 μmol/l VEGF AS by Lipofectin, as described above. Next, 500 μl of the appropriate growth medium containing 20% FCS were added to each well. ATO (2 μM) was added to the corresponding wells and incubated for 48 hours. Viable cells were collected and double stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI). For each sample, data from approximately 10 000 cells were recorded in the list mode on logarithmic scales. Apoptosis was analyzed with quadrant statistics on PI-negative, annexin V-positive cells by flow cytometry (Coulter Elite, Beckman Coulter, Brea, CA, USA).
Determination of VEGF protein levels in supernatants by enzyme-linked immunosorbent assay Cell culture was performed as described above. ATO (2 μM) was added to the corresponding wells for 48 hours. The supernatants were collected, centrifuged to remove all cell debris, and stored at −80°C until analysis using enzyme-linked immunosorbent assay (ELISA) kits (Jin Mei, Zhengzhou, China) according to the manufacturer’s instructions.
Detection of VEGF mRNA expression levels by real-time polymerase chain reaction Pretreatment of K562 cells was performed as described above. Total RNA from treated cells was extracted in TRIzol (Invitrogen) and quantified by an ultraviolet spectrophotometer (UVP, Upland, CA, USA) at a wavelength of 260 nm. VEGF mRNA was reverse transcribed with an moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA) and determined by SYBR-Green real-time
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polymerase chain reaction (PCR) assay conducted by 40 cycles of 10 seconds at 95°C, 30 seconds at 60°C, and 45 seconds at 72°C. Melting curve analysis was performed. VEGF mRNA levels were normalized to the housekeeping gene glyceraldehyde3-phosphate dehydrogenase. The fold-change for VEGF mRNA expression levels were calculated using 2−ΔΔCT.
Western blot K562 cells (48 hours after transfection) were lysed with RIPA buffer in the presence of proteinase inhibitor (Biocolor BioScience & Technology Co., Shanghai, China). Protein concentrations were determined by bicinchoninic acid assay (Bioss, Beijing, China), and the samples were denatured in Laemmli sample buffer (Bio-Rad) for 5 minutes at 95.1°C. The total protein extracts (30 μg) were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane blocked with 5% (w/v) fat-free milk powder in PBS and 0.5% (v/v) Tween-20 for 1 hour. The blots were incubated first with a primary antibody, anti-abl antibody (Abcam, Cambridge, UK) at room temperature for 2 hours, and then with horseradish peroxidase-conjugated secondary antibody at 1:1000 dilution. The signals were visualized with enhanced chemiluminescence (BeyoECL Plus, Beyotime Company, Haimen, China), and analyzed using a BI-2000 system (Taimeng Inc, Chengdu, Sichuang Province, China).
KEGG analysis of VEGF pathways For VEGF pathway analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used. KEGG is available at http://www.genome.jp.
Statistical analysis All experiments were carried out in triplicate, and all results are reported as mean ± standard deviation. Differences among groups were analyzed using analysis of variance with SPSS10 software (Chicago, IL, USA). Statistical significance was defined as P < 0.05.
Results VEGF AS inhibited K562 cell growth and increased sensitivity to ATO To explore the inhibitory effects of VEGF AS on cell growth, K562 cells were transfected with VEGF AS. The results showed that the IC50 value of ATO was markedly decreased by VEGF AS in K562 cells. VEGF AS PS-ODN was able to enhance the sensitivity of K562 to ATO (Fig. 1A). Meanwhile, VEGF AS effectively inhibited cell growth 72 hours posttransfection, and this effect was time dependent (*P < 0.01, Fig. 1B).
VEGF AS modulated survival of primary CML cells and ATO sensitivity To further study the influence of VEGF AS on primary CML, cells from CML patients were seeded at a density of 1 × 105 cells/ml in 96-well plates. Cell viability was determined by the trypan blue exclusion
Figure 1 Depletion of VEGF-regulated K562 cell proliferation and ATO sensitivity. K562 cells were seeded at a density of 1 × 105/ ml in 96-well plates and transfected with 0.4 μM VEGF AS for 6 hours. (A) ATO (0.5, 1, 2, or 4 μmol/l) was added, and the cells were incubated for 48 hours. Next, 20 μl MTT stock solution (5 mg/ml) was added to each well for a final MTT concentration of 0.45 mg/ ml and incubated for 4 hours at 37°C. The medium was then removed, and DMSO (150 μl) was added to dissolve the blue formazan crystals at room temperature for 30 minutes. Cell viability was assessed by absorbance at 570 nm on a microplate reader, and the IC50 values (μM) were determined. (B) Viable cells were counted by trypan blue exclusion assay at 24, 48, 72, and 96 hours after transfection. The data indicated that VEGF AS effectively suppressed K562 cell proliferation and reduced ATO IC50. *P < 0.01 vs. SCR control or SCR/ATO control; B, blank.
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Figure 2 VEGF AS modulated primary CML cell survival following ATO treatment. Primary CML cells were seeded at a density of 2 × 105/ml in 96-well plates and transfected with 0.4 μM VEGF AS with Lipofectin in low serum (2% FCS) DMEM conditions for 6 hours. The appropriate growth medium in the presence of ATO (2 μM) was added to corresponding wells for 72 hours. CML cell survival was assessed using the trypan blue exclusion assay. The results demonstrated that VEGF AS significantly inhibited ATOinduced CML cell survival. *P < 0.01 vs. SCR control, #P < 0.01 vs. SCR/ATO control; B, blank.
assay 24, 48, and 72 hours post-transfection. The data showed that VEGF AS significantly inhibited CML cell survival and increased arsenic sensitivity. We observed a synergistic effect between AS and ATO (Fig. 2).
VEGF AS increased arsenic-induced apoptosis We also assessed the effect of VEGF AS on cell apoptosis (alone or in combination with ATO). As shown in Fig. 3, apoptotic K562 cells were detected by flow cytometry using double staining with annexin V and
Figure 3 VEGF AS-induced apoptosis and increased arsenic-induced apoptosis. K562 cells were seeded at a density of 5 × 105/ ml in 24-well plates and transfected with 0.4 μM VEGF AS for 6 hours. ATO was added to the corresponding well at a final concentration of 2 μM and incubated for another 48 hours. Viable cells were collected and double stained with FITC-conjugated annexin V and PI. Apoptosis and necrosis were analyzed by quadrant statistics on PI-negative, annexin V-positive cells. The results showed that VEGF AS-induced K562 cell apoptosis and promoted ATO sensitivity. B, blank.
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Figure 4 VEGF AS downregulated VEGF protein levels in (A) K562 and (B) primary CML cell supernatants. K562 and primary CML cells were transfected with 0.4 μM VEGF AS in serum-free or 2% FBS DMEM conditions for 6 hours. Then, media were replaced with DMEM supplemented with 10% FCS. Next ATO (2 μM) was added to each well for 48 hours. VEGF protein concentrations in the supernatants were detected with ELISAs. The results showed that AS VEGF downregulated VEGF protein levels. *P < 0.01 vs. SCR control; B, blank.
PI. The results demonstrated that VEGF AS alone induced cell apoptosis and promoted ATO-induced apoptosis compared with controls.
VEGF AS downregulated VEGF protein level in supernatants of K562 and primary CML cells To further elucidate the inhibitory effect of VEGF AS on VEGF expression, we assessed VEGF protein concentrations in supernatants using ELISAs. As shown in Fig. 4A and B, both VEGF AS and ATO downregulated VEGF protein levels in the supernatants.
VEGF AS downregulated VEGF mRNA expression levels by real-time PCR We evaluated whether VEGF AS downregulated intracellular VEGF mRNA levels in K562 cells with SYBR-Green real-time PCR assays. The results indicated that VEGF AS effectively downregulated intracellular VEGF mRNA levels (Fig. 5).
ATO downregulates bcr-abl protein level by western blot CML develops following the transformation of a primitive hematopoietic cell by the bcr-abl gene. Therefore, we performed western blotting to explore whether ATO affected bcr-abl protein levels. The results showed that ATO effectively downregulated bcr-abl protein levels in K562 cells (Fig. 6).
VEGF pathways Using the KEGG database and information in recent publications, we found multiple signaling pathways downstream of VEGF, including PI3K/PIP3/Akt/ PKB, MAPKAPK2/HSP27, and Ras/MEK/ERK, which are involved in regulating cell survival, proliferation, apoptosis, and migration (Fig. S1).
Discussion Figure 5 VEGF AS downregulated VEGF mRNA expression levels. K562 cells were seeded at a density of 5 × 105/ml in 24well plates and transfected with 0.4 μmol/l VEGF AS in serumfree DMEM conditions for 6 hours. Media were replaced with DMEM supplemented with 10% FCS and incubated for another 48 hours. Total RNA was extracted, and VEGF mRNA was determined by SYBR-Green real-time PCR. VEGF AS effectively downregulated cellular VEGF mRNA levels. *P < 0.01 vs. SCR control.
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Mayerhofer et al. 16 demonstrated that the CMLassociated oncogene bcr-abl induces VEGF gene expression in growth factor-dependent Ba/F3 cells. Starved cells were found to contain only baseline levels of VEGF mRNA, whereas bcr-abl-induced Ba/F3 cells exhibited substantial amounts of VEGF mRNA. bcr-abl also induced VEGF promoter activity and increased VEGF protein levels in Ba/F3 cells.
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Figure 6 ATO downregulated bcr-abl protein level by western blot. K562 cells were treated with ATO (2.0 μM) for 48 hours, and the bcr-abl protein level was determined by western blot. The data represents the mean value of three independent experiments. ATO significantly reduced bcr-abl protein levels in CML cell. *P < 0.01, compared with blank controls.
Moreover, bcr-abl induces VEGF gene expression through a pathway involving PI3K and mTOR signaling.16 Recent studies have shown that VEGF is also overexpressed in neoplastic myeloid cells and may play a role in human leukemia. In particular, VEGF has been implicated in leukemia-associated angiogenesis and has been described as an autocrine growth factor for leukemic cells.16,17 Increased serum levels of VEGF and greater VEGF expression in affected BM have been reported in CML.16,18 Further studies demonstrated that VEGF overexpression is associated with aggressive clinical course, chemotherapy and radiotherapy resistance,25–28 and poor prognosis in patients with leukemia.20–23 High VEGF levels correlated with shorter survival of chronic CML patients. Thus, the use of VEGF inhibitors should be investigated in CML.21 In this study, VEGF AS significantly downregulated VEGF protein level in supernatants of K562 and primary CML cells (Fig. 4), and VEGF mRNA levels in K562 cells (Fig. 5). Previous studies have shown that leukemia cells release VEGF and express functional VEGFR. This seems to be isoform specific; VEGFR-1 is highly expressed in K562 cells, whereas VEGFR-2 is not expressed or is very weakly expressed in K562 cells.18,19 It is known that the VEGF/VEGFR-2 autocrine loop is more important for mediating leukemia cell proliferation and survival than VEGF/VEGFR-1, which is primarily involved in leukemia cell migration.29 Interestingly, we observed that VEGF AS expression decreased K562 cell proliferation and primary CML survival and increased the number of apoptotic cells. As shown in Fig. 1, VEGF AS effectively inhibited K562 cell proliferation (Fig. 1B) and primary CML cell survival (Fig. 2), and reduced the IC50 value of ATO (Fig. 1A). A study indicated that ATO could also induce bcr-abl ubiquitination without affecting bcr-abl mRNA.5 Recent findings revealed that autophagic degradation of bcr-abl is critical for the induction of the antileukemic effects of ATO.30 Moreover, resistance to imatinib mesylate does not induce
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cross-resistance to ATO.10 The combination of imatinib mesylate and ATO is being investigated in a phase I/II multicenter study in patients with chronic phase CML who did not attain complete remission with imatinib mesylate as a single agent.7 Our findings show that ATO effectively downregulated the bcr-abl protein level (Fig. 6). Therefore, ATO is a potential treatment for CML. Our data indicated that VEGF AS alone induced cell apoptosis and promoted ATOinduced apoptosis in K562 cells, suggesting synergistic inhibitory effects between AS and ATO (Fig. 3). The mechanisms of drug insensitivity and resistance mainly involve the ability of cancer cells to evade apoptosis.31 Almost all cytotoxic antitumor drugs in clinical use exert their antitumor activity by inducing apoptosis.32 Extensive work has established that ATO-dependent apoptosis includes induction of reactive oxygen species, which initiate downstream proapoptotic pathways such as, activation of caspase-3 and -9.33 Many tumors may develop by escaping apoptotic death signals through expressing antiapoptotic proteins, such as Bcl-2.31,32 Thus, VEGF AS contributed to the sensitization of K562 cells to ATO by promoting apoptosis. KEGG pathway mapping shows multiple signaling pathways downstream of VEGF, including PI3K/PIP3/Akt/PKB, MAPKAPK2/HSP27, and Ras/MEK/ERK, which are involved in regulating cell survival, proliferation, apoptosis, and migration (Fig. S1). Many of the antisense drugs in our pipeline bind to mRNAs and inhibit the production of disease-causing proteins through the RNase H mechanism. Recently, the scientific community has discovered many new types of RNAs, including microRNAs, which are involved in the regulation of protein production. Antisense technology prevents the production of proteins involved in disease processes, which provides a therapeutic benefit to patients. Currently, several antisense studies are being carried out in phase III clinical trials.34,35 The work described here provides proof of therapy by a combination of ATO and antisense oligonucleotide for leukemia treatment. VEGF AS may have dual inhibitory effects: direct inhibition of tumor cell growth and inhibition of angiogenesis by blocking the autocrine and paracrine loops. Therefore, targeted VEGF inhibition could modulate survival and bcr-abl-specific sensitivity of ATO in CML.
Acknowledgements Xiaochuang Luo and Maoxiao Feng contributed equally to this work. This study was supported by grants from the Fundamental Research Funds for the Central Universities (no. 21609406), the Guangdong Administration of Traditional Chinese Medicine Research Project (no. 2010364), and the National Natural Science Foundation of China (no. 81170496).
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Xiaochuang Luo and Maoxiao Feng contributed equally to this study.
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18 Bellamy WT, Richter L, Frutiger Y, Grogan TM. Expression of VEGF and its receptor in hematopoietic malignancies. Cancer Res. 1999;59:728–33. 19 Dias S, Hattori K, Zhou Z, Heissig B, Choy M, Lane W, et al. Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration. J Clin Invest. 2000;106:511–21. 20 Aguayo A, Estey E, Kantarjian H, Mansouri T, Gidel C, Keating M, et al. Cellular vascular endothelial growth factor is a predictor of outcome in patients with acute myeloid leukemia. Blood. 1999;94(11):3717–21. 21 Verstovsek S, Kantarjian H, Manshouri T, Cortes J, Giles FJ, Rogers A, et al. Prognostic significance of cellular vascular endothelial growth factor expression in chronic phase chronic myeloid leukemia. Blood. 2002;99(6):2265–7. 22 Molica S, Vitell G, Levato D, Ricciotti A, Digiesi G. Clinicoprognostic implications of increased serum level of vascular endothelial growth factor and basic fibroblastic growth factor in early B-cell chronic lymphocytic leukaemia. Br J Cancer. 2002;86(1):31–5. 23 Niitsu N, Okamato M, Nakamine H, Yoshino T, Tamaru J, Nakamura S, et al. Simultaneous elevation of the serum concentrations of vascular endothelial growth factor and interleukin-6 as independent predictors of prognosis in aggressive non-Hodgkin’s lymphoma. Eur J Haematol. 2002;68(2): 91–100. 24 Fei J, Zhang Y. Prediction of VEGF mRNA antisense oligodeoxynucleotides by RNA structure software and their effects on HL60 and K562 cells. Cell Biol Int. 2005;29(9):737–41. 25 Dias S, Shmelkov SV, Lam G, Rafii S. VEGF(165) promotes survival of leukemic cells by Hsp9 mediated induction of Bcl-2 expression and apoptosis inhibition. Blood. 2002;99: 2532–40. 26 Dias S, Choy M, Alitalo K, Rafii S. Vascular endothelial growth factor (VEGF)-C signaling through FLT-4(VEGFR-3) mediates leukemic cell proliferation, survival, and resistance to chemotherapy. Blood. 2002;99(6):2179–84. 27 Kautoh O, Takahashi T, Oguri T, Kuramoto K, Mihara K, Kobayashi M, et al. VEGF inhibits apoptotic cell death in hematopoietic cells after exposure to chemotherapeutic drugs by inducing MCL1 acting as an antiapoptotic factor. Cancer Res. 1998; 58:5565–9. 28 Kuramoto K, Uesaka T, Kimura A, Kobayashi M, Watanabe H, Katoh O. ZK7, a novel zinc finger gene, is induced by vascular endothelial growth factor and inhibits apoptotic death in hematopoietic cell. Cancer Res. 2000;60:425–30. 29 Dias S, Hattori K, Heissig B, Zhu Z, Wu Y, Witte L, et al. Inhibition of both paracrine and autocrine VEGF/VEGFR2 signaling pathways is essential to induce long-term remission of xenotransplanted human leukemias. Proc Natl Acad Sci USA. 2001;98(19):10857–62. 30 Goussetis DJ, Gounaris E, Wu EJ, Vakana E, Sharma B, Bogyo M, et al. Autophagic degradation of the BCR-ABL oncoprotein and generation of antileukemic responses by arsenic trioxide. Blood. 2012;120(17):3555–62. 31 Fraser M, Leung BM, Yan X, Dan HC, Cheng JQ, Tsang BK. p53 is a determinant of X-linked inhibitor of apoptosis protein/ Akt-mediated chemoresistance in human ovarian cancer cells. Cancer Res. 2003;63:7081–8. 32 Fraser M, Leung B, Jahani-Asl A, Yan X, Thompson WE, Tsang BK. Chemoresistance in human ovarian cancer: the role of apoptotic regulators. Reprod Biol Endocrinol. 2003;1:66. 33 Wang R, Liu C, Xia L, Zhao G, Gabrilove J, Waxman S, et al. Ethacrynic acid and a derivative enhance apoptosis in arsenic trioxide-treated myeloid leukemia and lymphoma cells: the role of glutathione s-transferase p1–1. Clin Cancer Res. 2012;18(24): 6690–701. 34 Esau CC. Inhibition of microRNA with antisense oligonucleotides. Methods. 2008;44(1):55–60. 35 Cheng AM, Byrom MW, Shelton J, Ford LP. Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res. 2005;33(4):1290–7.
Department of Biochemistry and Molecular Biology, Medical College of Jinan University, Guangzhou, China, Institute of Hematology, Medical College of Jinan University, Guangzhou, China
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The development of resistance to imatinib mesylate may partly depend on high bcr-abl expression levels or point mutation(s). Arsenic trioxide (ATO) has bcr-abl suppressing activity in vitro, without cross-resistance to imatinib. Meanwhile, bcr-abl also induces expression of vascular endothelial growth factor (VEGF), which is associated with tumor-related angiogenesis and is involved in chronic myelogenous leukemia (CML) pathogenesis. Here, we investigated ways to improve ATO activity in CML by modulating cellular VEGF levels. K562 and primary CML cells were transfected with a VEGF antisense sequence. Cell viability and survival were assessed using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and trypan blue exclusion assays. Apoptotic cells were detected by flow cytometry following annexin V and propidium iodide staining. The results showed that VEGF depletion effectively promotes enhanced ATO antileukemic activity by repressing bcr-abl protein levels. These data provide a rationale for the clinical development of optimized ATO-based regimens that incorporate VEGF modulator for CML treatment. Keywords: CML, VEGF, bcr-abl, Arsenic trioxide, Sensitivity
Introduction Chronic myelogenous leukemia (CML) is a type of clonal myeloproliferative neoplasm initiated by bcrabl-mediated transformation of hematopoietic stem cells.1 The main target of the drug imatinib in CML is bcr-abl, an oncoprotein with greatly enhanced tyrosine kinase activity. Imatinib mesylate induces cytogenetic remission in over 75% of CML patients and is a first-line therapy. While responses in chronic phase CML are generally durable, resistance develops in many patients with advanced disease. The development of imatinib mesylate resistance may partly depend on high bcr-abl expression levels2 or its mutation.3 Arsenic trioxide (ATO) induces apoptosis and was successfully used to treat acute promyelocytic leukemia.4 Meanwhile, ATO has been found to be effective in some CML patients, and there seem to be synergistic effects between ATO and imatinib.5–7 Further research demonstrated that ATO has bcr-abl suppressing activity in vitro,8,9 without cross-resistance to imatinib.10 However, ATO use as a single agent at higher concentrations causes many side effects, including ventricular arrhythmia, skin reaction, peripheral neuropathy, electrolyte changes, leukocytosis, hepatic Correspondence to: Jia Fei, Department of Biochemistry and Molecular Biology, Medical College of Jinan University, 601 Western Huangpu Avenue, Guangzhou 510632, China. Email: [email protected]
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dysfunction, and gastrointestinal reactions. Therefore, low-dose combination therapy is required.4,11,12 bcrabl exhibits constitutive tyrosine kinase activity, which leads to autophosphorylation and activation of multiple signaling molecules.13–15 For example, bcrabl can induce vascular endothelial growth factor (VEGF) expression through a pathway involving phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR). VEGF expression induced by bcr-abl may contribute to CML pathogenesis and increased angiogenesis.16–18 Recent studies have shown that leukemia cells release VEGF and express functional VEGF receptors (VEGFRs), which results in an autocrine loop that facilitates tumor growth and propagation.18–23 We asked whether ATO might modulate cellular VEGF. We previously used RNA structure software to predict the accessible sites on VEGF mRNA, and high efficacy antisense sequences (ASs) were screened with cell culture assays.24 Here, we attempt to enhance ATO activity against CML cells by employing ASs to block the VEGF/VEGFR autocrine loop.
Materials and methods Design and synthesis of AS VEGF The RNA structure software update was kindly provided by Prof. Mathews DH (Department of
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Chemistry, University of Rochester, NY, USA) after our registration. The complete human VEGF mRNA sequence was obtained from GenBank (entry code: X62568). The VEGF AS sequence, 5′ GGGTGCAGCCTGGGACCACT-3′ (20 bp), was the most effective antisense phosphorothioate oligodeoxynucleotide (PS-ODN) by experimental screening. Scramble (SCR): 5′ -CATTAATGTCGGACA ACTCA-3′ (20 bp). All sequences were synthesized by Sangon Bioengineering Company (Shanghai, China).
Cell line and primary leukemia culture The K562 (CML) cell line (from the Institute of Cell Biology, Shanghai, China) was grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS) in a 37°C, 5% CO2 humidified atmosphere (Thermo FORMA 3110, Thermo Fisher Scientific, Waltham, MA, USA). Bone marrow (BM) or peripheral blood samples from four patients with untreated chronic phase CML (from Department of Hematology, the first affiliated hospital of Jinan University) were obtained before chemotherapy. CML diagnoses were based on BM aspirates and special staining according to the French– American–British classification criteria and on leukemia cell immunophenotyping. Normal cells (four peripheral blood and three BM samples) were obtained from healthy volunteers. Mononuclear cells from about 2 ml BM were separated and overlaid on 4 ml of Lymphoprep (Shenneng Inc., Shanghai, China). Each sample was spun at 2000 g for 30 minutes, and the mononuclear interphase was collected into a fresh tube and washed twice with phosphate-buffered saline (PBS) for 5 minutes at 780 g. The resulting cell pellet was cultured in DMEM with 10% FCS in a 37°C, 5% CO2 humidified atmosphere.
Cell proliferation, survival, and arsenicsensitivity assay K562 cells were seeded at a density of 1 × 105/ml in 96well plates (Costar, Corning, NY, USA), 100 μl/well, and transfected with 0.4 μM VEGF AS with Lipofectin (Lipofectin:oligonucleotides, 2:1; Invitrogen, Carlsbad, CA, USA) in serum-free DMEM conditions for 6 hours. Next, 100 μl of the appropriate growth medium containing 20% FCS was added to each well. Viable cells were counted with the trypan blue exclusion assay 24, 48, 72, and 96 hours after transfection. ATO of various concentrations (0.5, 1, 2, or 4 μmol/l) were added to corresponding wells for 48 hours. Then, 20 μl 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) stock solution (5 mg/ml) was added to each well at final MTT concentration of 0.45 mg/ml,
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and the plate was incubated for 4 hours at 37°C. The medium was then removed, and dimethyl sulfoxide (DMSO, 150 μl) was added to dissolve the blue formazan crystals at room temperature for 30 minutes. Cell viability was assessed by absorbance at 570 nm on a Bio-Rad microtiter plate reader (Hercules, CA, USA). The IC50 values (μM) were determined by ICp software (PerkinElmer, Waltham, MA, USA). Primary CML cells were seeded at a density of 2 × 105/ml in 96-well plates (Costar), 100 μl/well, and incubated with 0.4 μM AS VEGF transfected with Lipofectin, as described above. Then, 100 μl of the appropriate growth medium containing 20% FCS in the presence of ATO (2 μmol/l) was added to corresponding wells for 72 hours. Surviving CML cells were assessed using the trypan blue dye exclusion assay.
Analysis of apoptosis by flow cytometry K562 cells were seeded at a density of 5 × 105/ml in 24-well plates (Costar), 500 μl/well, transfected with 0.4 μmol/l VEGF AS by Lipofectin, as described above. Next, 500 μl of the appropriate growth medium containing 20% FCS were added to each well. ATO (2 μM) was added to the corresponding wells and incubated for 48 hours. Viable cells were collected and double stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI). For each sample, data from approximately 10 000 cells were recorded in the list mode on logarithmic scales. Apoptosis was analyzed with quadrant statistics on PI-negative, annexin V-positive cells by flow cytometry (Coulter Elite, Beckman Coulter, Brea, CA, USA).
Determination of VEGF protein levels in supernatants by enzyme-linked immunosorbent assay Cell culture was performed as described above. ATO (2 μM) was added to the corresponding wells for 48 hours. The supernatants were collected, centrifuged to remove all cell debris, and stored at −80°C until analysis using enzyme-linked immunosorbent assay (ELISA) kits (Jin Mei, Zhengzhou, China) according to the manufacturer’s instructions.
Detection of VEGF mRNA expression levels by real-time polymerase chain reaction Pretreatment of K562 cells was performed as described above. Total RNA from treated cells was extracted in TRIzol (Invitrogen) and quantified by an ultraviolet spectrophotometer (UVP, Upland, CA, USA) at a wavelength of 260 nm. VEGF mRNA was reverse transcribed with an moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA) and determined by SYBR-Green real-time
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polymerase chain reaction (PCR) assay conducted by 40 cycles of 10 seconds at 95°C, 30 seconds at 60°C, and 45 seconds at 72°C. Melting curve analysis was performed. VEGF mRNA levels were normalized to the housekeeping gene glyceraldehyde3-phosphate dehydrogenase. The fold-change for VEGF mRNA expression levels were calculated using 2−ΔΔCT.
Western blot K562 cells (48 hours after transfection) were lysed with RIPA buffer in the presence of proteinase inhibitor (Biocolor BioScience & Technology Co., Shanghai, China). Protein concentrations were determined by bicinchoninic acid assay (Bioss, Beijing, China), and the samples were denatured in Laemmli sample buffer (Bio-Rad) for 5 minutes at 95.1°C. The total protein extracts (30 μg) were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane blocked with 5% (w/v) fat-free milk powder in PBS and 0.5% (v/v) Tween-20 for 1 hour. The blots were incubated first with a primary antibody, anti-abl antibody (Abcam, Cambridge, UK) at room temperature for 2 hours, and then with horseradish peroxidase-conjugated secondary antibody at 1:1000 dilution. The signals were visualized with enhanced chemiluminescence (BeyoECL Plus, Beyotime Company, Haimen, China), and analyzed using a BI-2000 system (Taimeng Inc, Chengdu, Sichuang Province, China).
KEGG analysis of VEGF pathways For VEGF pathway analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used. KEGG is available at http://www.genome.jp.
Statistical analysis All experiments were carried out in triplicate, and all results are reported as mean ± standard deviation. Differences among groups were analyzed using analysis of variance with SPSS10 software (Chicago, IL, USA). Statistical significance was defined as P < 0.05.
Results VEGF AS inhibited K562 cell growth and increased sensitivity to ATO To explore the inhibitory effects of VEGF AS on cell growth, K562 cells were transfected with VEGF AS. The results showed that the IC50 value of ATO was markedly decreased by VEGF AS in K562 cells. VEGF AS PS-ODN was able to enhance the sensitivity of K562 to ATO (Fig. 1A). Meanwhile, VEGF AS effectively inhibited cell growth 72 hours posttransfection, and this effect was time dependent (*P < 0.01, Fig. 1B).
VEGF AS modulated survival of primary CML cells and ATO sensitivity To further study the influence of VEGF AS on primary CML, cells from CML patients were seeded at a density of 1 × 105 cells/ml in 96-well plates. Cell viability was determined by the trypan blue exclusion
Figure 1 Depletion of VEGF-regulated K562 cell proliferation and ATO sensitivity. K562 cells were seeded at a density of 1 × 105/ ml in 96-well plates and transfected with 0.4 μM VEGF AS for 6 hours. (A) ATO (0.5, 1, 2, or 4 μmol/l) was added, and the cells were incubated for 48 hours. Next, 20 μl MTT stock solution (5 mg/ml) was added to each well for a final MTT concentration of 0.45 mg/ ml and incubated for 4 hours at 37°C. The medium was then removed, and DMSO (150 μl) was added to dissolve the blue formazan crystals at room temperature for 30 minutes. Cell viability was assessed by absorbance at 570 nm on a microplate reader, and the IC50 values (μM) were determined. (B) Viable cells were counted by trypan blue exclusion assay at 24, 48, 72, and 96 hours after transfection. The data indicated that VEGF AS effectively suppressed K562 cell proliferation and reduced ATO IC50. *P < 0.01 vs. SCR control or SCR/ATO control; B, blank.
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Figure 2 VEGF AS modulated primary CML cell survival following ATO treatment. Primary CML cells were seeded at a density of 2 × 105/ml in 96-well plates and transfected with 0.4 μM VEGF AS with Lipofectin in low serum (2% FCS) DMEM conditions for 6 hours. The appropriate growth medium in the presence of ATO (2 μM) was added to corresponding wells for 72 hours. CML cell survival was assessed using the trypan blue exclusion assay. The results demonstrated that VEGF AS significantly inhibited ATOinduced CML cell survival. *P < 0.01 vs. SCR control, #P < 0.01 vs. SCR/ATO control; B, blank.
assay 24, 48, and 72 hours post-transfection. The data showed that VEGF AS significantly inhibited CML cell survival and increased arsenic sensitivity. We observed a synergistic effect between AS and ATO (Fig. 2).
VEGF AS increased arsenic-induced apoptosis We also assessed the effect of VEGF AS on cell apoptosis (alone or in combination with ATO). As shown in Fig. 3, apoptotic K562 cells were detected by flow cytometry using double staining with annexin V and
Figure 3 VEGF AS-induced apoptosis and increased arsenic-induced apoptosis. K562 cells were seeded at a density of 5 × 105/ ml in 24-well plates and transfected with 0.4 μM VEGF AS for 6 hours. ATO was added to the corresponding well at a final concentration of 2 μM and incubated for another 48 hours. Viable cells were collected and double stained with FITC-conjugated annexin V and PI. Apoptosis and necrosis were analyzed by quadrant statistics on PI-negative, annexin V-positive cells. The results showed that VEGF AS-induced K562 cell apoptosis and promoted ATO sensitivity. B, blank.
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Figure 4 VEGF AS downregulated VEGF protein levels in (A) K562 and (B) primary CML cell supernatants. K562 and primary CML cells were transfected with 0.4 μM VEGF AS in serum-free or 2% FBS DMEM conditions for 6 hours. Then, media were replaced with DMEM supplemented with 10% FCS. Next ATO (2 μM) was added to each well for 48 hours. VEGF protein concentrations in the supernatants were detected with ELISAs. The results showed that AS VEGF downregulated VEGF protein levels. *P < 0.01 vs. SCR control; B, blank.
PI. The results demonstrated that VEGF AS alone induced cell apoptosis and promoted ATO-induced apoptosis compared with controls.
VEGF AS downregulated VEGF protein level in supernatants of K562 and primary CML cells To further elucidate the inhibitory effect of VEGF AS on VEGF expression, we assessed VEGF protein concentrations in supernatants using ELISAs. As shown in Fig. 4A and B, both VEGF AS and ATO downregulated VEGF protein levels in the supernatants.
VEGF AS downregulated VEGF mRNA expression levels by real-time PCR We evaluated whether VEGF AS downregulated intracellular VEGF mRNA levels in K562 cells with SYBR-Green real-time PCR assays. The results indicated that VEGF AS effectively downregulated intracellular VEGF mRNA levels (Fig. 5).
ATO downregulates bcr-abl protein level by western blot CML develops following the transformation of a primitive hematopoietic cell by the bcr-abl gene. Therefore, we performed western blotting to explore whether ATO affected bcr-abl protein levels. The results showed that ATO effectively downregulated bcr-abl protein levels in K562 cells (Fig. 6).
VEGF pathways Using the KEGG database and information in recent publications, we found multiple signaling pathways downstream of VEGF, including PI3K/PIP3/Akt/ PKB, MAPKAPK2/HSP27, and Ras/MEK/ERK, which are involved in regulating cell survival, proliferation, apoptosis, and migration (Fig. S1).
Discussion Figure 5 VEGF AS downregulated VEGF mRNA expression levels. K562 cells were seeded at a density of 5 × 105/ml in 24well plates and transfected with 0.4 μmol/l VEGF AS in serumfree DMEM conditions for 6 hours. Media were replaced with DMEM supplemented with 10% FCS and incubated for another 48 hours. Total RNA was extracted, and VEGF mRNA was determined by SYBR-Green real-time PCR. VEGF AS effectively downregulated cellular VEGF mRNA levels. *P < 0.01 vs. SCR control.
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Mayerhofer et al. 16 demonstrated that the CMLassociated oncogene bcr-abl induces VEGF gene expression in growth factor-dependent Ba/F3 cells. Starved cells were found to contain only baseline levels of VEGF mRNA, whereas bcr-abl-induced Ba/F3 cells exhibited substantial amounts of VEGF mRNA. bcr-abl also induced VEGF promoter activity and increased VEGF protein levels in Ba/F3 cells.
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Figure 6 ATO downregulated bcr-abl protein level by western blot. K562 cells were treated with ATO (2.0 μM) for 48 hours, and the bcr-abl protein level was determined by western blot. The data represents the mean value of three independent experiments. ATO significantly reduced bcr-abl protein levels in CML cell. *P < 0.01, compared with blank controls.
Moreover, bcr-abl induces VEGF gene expression through a pathway involving PI3K and mTOR signaling.16 Recent studies have shown that VEGF is also overexpressed in neoplastic myeloid cells and may play a role in human leukemia. In particular, VEGF has been implicated in leukemia-associated angiogenesis and has been described as an autocrine growth factor for leukemic cells.16,17 Increased serum levels of VEGF and greater VEGF expression in affected BM have been reported in CML.16,18 Further studies demonstrated that VEGF overexpression is associated with aggressive clinical course, chemotherapy and radiotherapy resistance,25–28 and poor prognosis in patients with leukemia.20–23 High VEGF levels correlated with shorter survival of chronic CML patients. Thus, the use of VEGF inhibitors should be investigated in CML.21 In this study, VEGF AS significantly downregulated VEGF protein level in supernatants of K562 and primary CML cells (Fig. 4), and VEGF mRNA levels in K562 cells (Fig. 5). Previous studies have shown that leukemia cells release VEGF and express functional VEGFR. This seems to be isoform specific; VEGFR-1 is highly expressed in K562 cells, whereas VEGFR-2 is not expressed or is very weakly expressed in K562 cells.18,19 It is known that the VEGF/VEGFR-2 autocrine loop is more important for mediating leukemia cell proliferation and survival than VEGF/VEGFR-1, which is primarily involved in leukemia cell migration.29 Interestingly, we observed that VEGF AS expression decreased K562 cell proliferation and primary CML survival and increased the number of apoptotic cells. As shown in Fig. 1, VEGF AS effectively inhibited K562 cell proliferation (Fig. 1B) and primary CML cell survival (Fig. 2), and reduced the IC50 value of ATO (Fig. 1A). A study indicated that ATO could also induce bcr-abl ubiquitination without affecting bcr-abl mRNA.5 Recent findings revealed that autophagic degradation of bcr-abl is critical for the induction of the antileukemic effects of ATO.30 Moreover, resistance to imatinib mesylate does not induce
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cross-resistance to ATO.10 The combination of imatinib mesylate and ATO is being investigated in a phase I/II multicenter study in patients with chronic phase CML who did not attain complete remission with imatinib mesylate as a single agent.7 Our findings show that ATO effectively downregulated the bcr-abl protein level (Fig. 6). Therefore, ATO is a potential treatment for CML. Our data indicated that VEGF AS alone induced cell apoptosis and promoted ATOinduced apoptosis in K562 cells, suggesting synergistic inhibitory effects between AS and ATO (Fig. 3). The mechanisms of drug insensitivity and resistance mainly involve the ability of cancer cells to evade apoptosis.31 Almost all cytotoxic antitumor drugs in clinical use exert their antitumor activity by inducing apoptosis.32 Extensive work has established that ATO-dependent apoptosis includes induction of reactive oxygen species, which initiate downstream proapoptotic pathways such as, activation of caspase-3 and -9.33 Many tumors may develop by escaping apoptotic death signals through expressing antiapoptotic proteins, such as Bcl-2.31,32 Thus, VEGF AS contributed to the sensitization of K562 cells to ATO by promoting apoptosis. KEGG pathway mapping shows multiple signaling pathways downstream of VEGF, including PI3K/PIP3/Akt/PKB, MAPKAPK2/HSP27, and Ras/MEK/ERK, which are involved in regulating cell survival, proliferation, apoptosis, and migration (Fig. S1). Many of the antisense drugs in our pipeline bind to mRNAs and inhibit the production of disease-causing proteins through the RNase H mechanism. Recently, the scientific community has discovered many new types of RNAs, including microRNAs, which are involved in the regulation of protein production. Antisense technology prevents the production of proteins involved in disease processes, which provides a therapeutic benefit to patients. Currently, several antisense studies are being carried out in phase III clinical trials.34,35 The work described here provides proof of therapy by a combination of ATO and antisense oligonucleotide for leukemia treatment. VEGF AS may have dual inhibitory effects: direct inhibition of tumor cell growth and inhibition of angiogenesis by blocking the autocrine and paracrine loops. Therefore, targeted VEGF inhibition could modulate survival and bcr-abl-specific sensitivity of ATO in CML.
Acknowledgements Xiaochuang Luo and Maoxiao Feng contributed equally to this work. This study was supported by grants from the Fundamental Research Funds for the Central Universities (no. 21609406), the Guangdong Administration of Traditional Chinese Medicine Research Project (no. 2010364), and the National Natural Science Foundation of China (no. 81170496).
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Xiaochuang Luo and Maoxiao Feng contributed equally to this study.
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