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Dacomitinib, a new pan-EGFR inhibitor, is effective in attenuating pulmonary vascular remodeling and pulmonary hypertension
European Journal of Pharmacology 850 (2019) 97–108
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Dacomitinib, a new pan-EGFR inhibitor, is effective in attenuating pulmonary vascular remodeling and pulmonary hypertension
T
Xiufeng Yua,b,c,d,1, Xijuan Zhaoa,b,c,1, Junting Zhangb,c, YiYing Lib,c, Ping Shengd, Cui Maa,b,c, ⁎ Lixin Zhanga,b,c, XueWei Haoa,b,c, XiaoDong Zhengb,e, Yan Xingb,e, Hui Qiaob,e, Lihui Qub,e, , ⁎⁎ Daling Zhub,c, a
College of Medical Laboratory Science and Technology, Harbin Medical University (Daqing), Daqing 163319, PR China Central Laboratory of Harbin Medical University (Daqing), Daqing 163319, PR China c Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical University, Harbin 150081, PR China d Department of Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin 150081, PR China e Department of Basic Medical College, Harbin Medical University (Daqing), Daqing 163319, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Dacomitinib EGFR Proliferation PASMCs Pulmonary artery hypertension
Accumulating evidence suggests that epidermal growth factor receptor (EGFR) plays a role in the progression of pulmonary arterial hypertension (PAH). Clinically-approved epidermal growth factor inhibitors such as gefitinib, erlotinib, and lapatinib have been explored for PAH. However, None of them were able to attenuate PAH. So, we explored the role of dacomitinib, a new pan-EGFR inhibitor, in PAH. Adult male Sprague–Dawley rats were used to study hypoxia- or monocrotaline-induced right ventricular remodeling as well as systolic function and hemodynamics using echocardiography and a pressure-volume admittance catheter. Morphometric analyses of lung vasculature and pressure-volume vessels were performed. Immunohistochemical staining, flow cytometry, and viability, as well as scratch-wound, and Boyden chamber migration assays were used to identify the roles of dacomitinib in pulmonary artery smooth muscle cells (PASMCs). The results revealed that dacomitinib has a significant inhibitory effect on the thickening of the media, adventitial collagen increased. Dacomitinib also has a significant role in attenuating pulmonary artery pressure and right ventricular hypertrophy. Additionally, dacomitinib inhibits hypoxia-induced proliferation, migration, autophagy and cell cycle progression through PI3K-AKT-mTOR signaling in PASMCs. Our study indicates that dacomitinib inhibited hypoxiainduced cell cycle progression, proliferation, migration, and autophagy of PASMCs, thereby attenuating pulmonary vascular remodeling and development of PAH via the PI3K-AKT-mTOR signaling pathway. Overall, dacomitinib may serve as new potential therapeutic for the treatment of PAH.
1. Introduction Pulmonary artery hypertension (PAH) is a progressive pulmonary vascular disease characterized by vasoconstriction, vascular remodeling, and increased vascular resistance; PAH leads to right heart failure and death (Chan and Loscalzo, 2008; Humbert et al., 2004; Rubin, 1997). To date, there is no cure for the disease, and the mortality rate in PAH patients is fairly high. Therefore, elucidating the molecular and cellular basis underlying the pathogenesis of PAH and exploring new therapeutic strategies are necessary (Thenappan et al., 2010). Pulmonary vascular remodeling, the key pathological feature of
PAH, is attributable to the increased proliferation, resistance to apoptosis, and migration of pulmonary vascular cells (Chan and Loscalzo, 2008; Humbert et al., 2004). Such cellular events are mediated by growth factors such as platelet-derived growth factor (PDGF) (Heldin et al., 1998; Liang et al., 2017), vascular endothelial growth factor (VEGF) (Liang et al., 2017)and epidermal growth factor (EGF) through activation of their receptor tyrosine kinases (RTKs) and downstream signaling pathways (Dahal et al., 2010). In recent years, EGF (Dahal et al., 2010; Merklinger et al., 2005; Ushio-Fukai et al., 2001) is emerging as important players in the pathomechanism of PAH (Izikki et al., 2013), suggesting that the field of
Corresponding author at: Central Laboratory of Harbin Medical University (Daqing), Daqing 163319, PR China. Correspondence to: College of Pharmacy, Harbin Medical University (Daqing), Xinyang Road, Daqing, Heilongjiang 163319, PR China. E-mail addresses: [email protected] (L. Qu), [email protected] (D. Zhu). 1 These authors contributed equally to this work. ⁎
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https://doi.org/10.1016/j.ejphar.2019.02.008 Received 23 July 2018; Received in revised form 1 February 2019; Accepted 8 February 2019 Available online 10 February 2019 0014-2999/ © 2019 Published by Elsevier B.V.
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growth factors and their RTKs as targets for treatment of cardiopulmonary diseases is expanding. Researches have confirmed that used two independent animal models of PAH to investigate the epidermal growth factor receptor (EGFR) inhibitors gefitinib, erlotinib, and lapatinib, which found that gefitinib and erlotinib reduced the right ventricular systolic pressure (RVSP), total systemic resistance, and right heart hypertrophy in rats with monocrotaline (MCT)-induced PAH. Moreover, measurements of medial wall thickness and degree of muscularization of small peripheral pulmonary arteries revealed that the pulmonary vascular remodeling was improved (Dahal et al., 2010). Despite the in vitro efficacy, lapatinib did not provide therapeutic benefit with respect to significantly attenuating the right heart hypertrophy and pulmonary vascular remodeling (Dahal et al., 2010). Dacomitinib (PF-00299804), a second generation EGFR tyrosine kinases inhibitors (TKIs), is an irreversible tyrosine kinases inhibitors of the human EGFRs (HERs), including HER-1/EGFR, HER-2, and HER-4 (Engelman et al., 2007). Previous studies have found that gefitinib, erlotinib, and lapatinib did not provide any significant therapeutic benefit, either in hemodynamic or in pulmonary vascular remodeling, suggesting that an inhibition of EGFR signaling did not impair the progression of chronic hypoxic PAH and pulmonary vascular remodeling (Dahal et al., 2010). However, None of them were able to attenuate PAH (Dahal et al., 2010). We want to explore the new panEGFR inhibitor in pulmonary hypertension. Therefore, we examined the role of dacomitinib in MCT-induced PAH and chronic hypoxic PAH.
the right ventricular hypertrophy index ratio of right ventricular free wall weight over a sum of septum plus left ventricular free wall weight: (RV/LV+S). 2.4. Echocardiography Rats were anesthetized by 4% chloral hydrate (0.1 ml/kg, i.p.) before echocardiography by Vevo 2100 imaging system (Visual Sonics Inc., Toronto, Ontario, Canada)with a 30 MHZ probe. Stable images were obtained in M, B, and Doppler Mode. right ventricular inner dimension, right ventricular stroke volume, right ventricular fractional shortening, tricuspid valve early and late diastolic filling velocities, pulmonary arterial velocity time integral, pulmonary arterial pre-ejection time, and pulmonary arterial ejection time were measured. 2.5. Histological and histomorphometric evaluation The lung tissues were sliced into tissue blocks and were immersed in 4% paraformaldehyde for overnight fixation. Then fixed tissues were dehydrated, cleared, and embedded in the paraffin base. The paraffin blocks were cut into sections of 5 µm thick. Some sections were stained with hematoxylin and eosin (H&E), Elastica van Gieson's stain and Masson staining. Histopathological analysis was performed as previously described (Zhang et al., 2016). The total area of the vascular walls was quantified with high-resolution images of individual vessels using a color-recognition algorithm in Image-Pro Plus 6.0 software. Linear micrometer measurements of medial thickness were achieved in ten images at 10x magnification (100 µm per panel). The thickest part of the media was selected as the center point, and then, the arterial cross-section was divided into five segments from the center point. The average thickness of the media was measured at five points, including the center point.
2. Materials and methods 2.1. Animal used Animal care and use conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, revised 1996). Sprague–Dawley rats (200 g) were used in the study which was bought from the Experimental Animal Center of Harbin Medical University and was approved by the Institutional Animal Care and Use Committee. This study was also approved by the ethics review board of Harbin Medical University ([2015]-016).
2.6. Cell culture The primary rat pulmonary smooth muscle cells (PASMCs) were obtained from Procell, China. Smooth Muscle Cell Medium was supplied with SMC Bullet Kit from Science, the USA which was used as the growth medium. 13% Bovine Fetal Serum Gold from Clark Bioscience was added to the growth medium to maintain the proliferation of the cells. The cells were cultured at 37 °C with 5% CO2 in humidified conditions. Cell passage to 2–3 generations for experiments, before the experiment, the PASMCs were starvation for 24 hin Smooth Muscle Cell Medium without serum. Then treated with dacomitinib (50 nM) or dacomitinib (50 nM) plus EGF (50 ng/ml）in 5% FBS-DMEM.
2.2. Hypoxia-induced or MCT-induced PAH model 6-week-old male Sprague–Dawley rats weighing approximately 200 g at protocol onset were divided into the following groups: (A) Animals into normoxic groups (n = 5) were received a single subcutaneous injection of 0.9% saline housed at ambient barometric pressure (FiO2) 0.21 for 21days. (B) Animals in hypoxic groups were (n = 5) were received a single subcutaneous injection of 0.9% saline housed at inspired oxygen (FiO2) 0.12 for 21 days. (C) 3rd group of rats (n = 5) were administered daily with Dacomitinib (500 nmol/kg/day by subcutaneous injection) from 2-weeks after the hypoxic treated until the terminal experiment. (D) PAH rats received a single subcutaneous injection of 60 mg/kg monocrotaline(n = 5) to induce severe PAH. (E) To further assess the role of dacomitinib (respectively, 100 nm/kg/day, 250 nm/kg/day, 500 nmol/kg/day by subcutaneous injection) (n = 15) in inducing regional RV myocardial remodeling the 5th group of rats was administered from 2-weeks after the monocrotaline injection, for a duration of 3-weeks, until the terminal experiment. Four-weeks after saline or monocrotaline injection (Nielsen et al., 2017).
2.7. Cell proliferation assay Cell proliferation was analyzed by the WST-1 Cell Proliferation and Cytotoxicity Assay kit (Roche, USA). Briefly, PASMCs were seeded onto 96-well plates at a density of 2 × 103 cells per well and cultured overnight. The cells continued to grow at 37 °C in a humidified incubator. The cells were subjected to different treatments. After 24 h, 20 μl of WST-1 was added to each well, and the cells were incubated for another 4 h at 37 °C. Absorbance was measured at 490 nm. All experiments were performed in triplicate. 2.8. Cell cycle and DNA analysis
2.3. The Hemodynamic Evaluation of PAH–model
The Cycle TEST PLUS DNA Reagent Kit was used to examine whether the cell cycle was influenced by dacomitinib. In brief, prepared PASMCs in groups were trypsinized, harvested and fixed in 70% cold ethanol at 4 ℃. After discarding the ethanol. cells were resuspended in 200μl propidium iodide at 4 ℃. Finally, the stained cells were filtered and DNA fluorescence was measured by flow cytometry using BD FACS Calibur Flow Cytometer (Bedford, MA). We analyzed the proportion of
A 1.2 French Pressure Catheter (Scisense Inc.) was connected to the Science FA-404 recorder. When the right jugular vein was exposed, the catheter was inserted into the vein, then was advanced into the superior vena cava, and finally into the right ventricular vein. RVSP was continuously recorded for 45 min. After measurement of RVSP, the thorax was opened and the heart was dissected and weighed for calculation of 98
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cells in the G0/G1, S and G2/M phases by flow cytometry as reported recently (Ma et al., 2011).
equal to 0.05. 2.14. Materials
2.9. Migration assays
Dacomitinib (PF299804, PF299), Gefitinib (ZD1839), erlotinib, lapatinib, were purchased from Selleck Chemicals (Houston, TX, USA). Recombinant rat EGF Protein was purchased from R&D Systems (Minneapolis, MN, USA). LC3B were purchased from Abcam (Cambridge, MA, USA). AKT, p-AKT, p-mTOR were purchased from Cell Signal Technology (Beverly, MA, USA). Cyclin A was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Beclin, SQSTM-1, EGFR, PCNA antibody were obtained from Boster Biological Technology Co. Ltd (Wuhan, China). Cyclin D, CDK1, CDK2 antibodies were purchased from Beyotime Institute of Biotechnology (Haimen, China).
Migration in vitro was assayed using a Transwell chamber (Costar, Corning, NY, USA) with a polycarbonic membrane (6.5 mm in diameter and 8 µm pore size). PASMCs treated with dacomitinib (50 nM) in Smooth Muscle Cell medium supplemented with 5% FBS for 24 h. After dacomitinib treatment, PASMCs were trypsinized and suspended into single cells with a serum-free medium at the density of 5 × 105 cells/ ml. 100 μl of the cell suspension was added to the upper chamber, and 600 ml of Smooth Muscle Cell medium supplemented with 10% FBS was added to the lower chamber. Cells were incubated for 24 h at 37 °C. Non-migrating cells on the top surface of the membrane were removed with cotton swabs. Cells were measured after the fixing with 4% formaldehyde solution for 10 min and then staining of 0.4% crystal violet in 10% ethanol for 5 min. Migrated cell number was measured by counting the number of stained nuclei per high-power field in a microscope (Olympus, Japan), using Image Pro-Plus 6.0.
3. Result 3.1. Effects of dacomitinib on hemodynamics and right cardiac function Hypoxia- and MCT-induced PAH rat models were used to test whether EGFR-tyrosine kinase inhibitor dacomitinib was involved in the pathogenesis of PAH. We characterized the PAH parameters in detail, including right ventricular hypertrophy (RVH), RVSP, hemodynamics, cardiac function and pulmonary vascular remodeling. RVH and RVSP were used to evaluate the effects of the dacomitinib on the development of Hypoxia or MCT- induced PAH. Hypoxia or MCT-significantly elevated RVSP and RVH in rats, while this effect was attenuated by dacomitinib (Fig. 1A–C). An echocardiography was performed on rats (exposed to hypoxia or MCT) to characterize the effect of dacomitinib on right ventricular function (Fig. 1D). The results showed that dacomitinib inhibited hypoxia-induced PAH or MCT-induced PAH development assessed by pulmonary artery acceleration time, pulmonary arterial velocity time integral, right ventricular anterior wall; diastole, and right ventricular anterior wall systole (Fig. 1E-H).
2.10. Scratch-wound assay For Scratch-wounding cell migration assay, the PASMCs cultured in 6-well plates were wounded by pipette tips, given rise to one acellular 1-mm-wide lane per well, and the ablated cells were washed with PBS. After that, cells were treated with dacomitinib (50 nM) in 5% FBS DMEM. Migration was calculated based on the same wounded areas at time zero and 24 h. After 24 h of incubation, photos were taken from the same areas as those recorded at zero time. the closure area of the wound was calculated as follow: migration area(%）=（A0-An)/A0 × 100, where A0 represents the area of the original wound area, An represents the remaining area of the wound at the metering point. 2.11. Relative mRNA quantification by real-time polymerase chain reaction Total RNA was extracted from cultured PASMCs using the Trizol reagent according to the manufacturer’s instructions. Extracted total RNAs were reverse-transcribed using the SuperScript First-strand cDNA Synthesis Kit (Invitrogen). cDNA samples were amplified in a DNA thermal cycler (Thermo Scientific, Waltham, MD, USA). The gene-specific primers were designed using the coding regions, and the nucleotide sequences of the primers are as follows: EGFR (Gen Bank accession no; sense: NM_031507.1 5′-AGTACCTGTTCTGGCTAATGG-3′ and antisense: 5′ -TCACTTTCGTGCGCTCGTAG-3′, 119 bp). Real-time polymerase chain reaction (RT-PCR) was used for relative quantification of EGFR mRNA. The reactions were performed in an ABI 7300 Sequence Detection System (Applied Biosystems, Foster City, Calif).
3.2. Effects of dacomitinib on pulmonary vascular remodeling H&E staining showing that hypoxia or MCT significantly increased the thickness of distal blood vessels, which was attenuated by dacomitinib treatment (Fig. 2A–B). Elastica van Gieson's stain showed the elastic membrane(Fig. 2A). The Masson staining showed that the deposition of collagen in rat lungs was increased in hypoxia and MCT model, which was attenuated by dacomitinib (Fig. 2A). Immuno-histochemical evaluation of the expression of EGFR was increased in the pulmonary vascular wall in hypoxia and MCT model and mainly distributed in PASMCs. Dacomitinib attenuates hypoxia or MCT -induced increased expression of EGFR on PASMCs (Fig. 2C). Next, we explored whether dacomitinib affected the expression of EGFR was involved in the progression of pulmonary vascular remodeling. We observed that the expression of EGFR was increased in pulmonary arteries from rats exposed to hypoxia or MCT, dacomitinib reduced the EGFR expression induced by hypoxia or MCT at both the mRNA and protein level (Fig. 2D–E). These results indicated that the beneficial effect of dacomitinib could alter the protein and mRNA level of EGFR and through blocking EGFR activating inhibited hypoxia-induced or MCT-induced pulmonary vascular remodeling. We used different concentrations of dacomitinib to evaluate the proliferation of PASMCs under normoxia and hypoxia. Our results indicate that dacomitinib (50 nM) can significantly inhibit PASMCs proliferation under normoxia or hypoxia (Fig. 3A-B). Previous studies have confirmed that several EGFR inhibitors including gefitinib, erlotinib, and lapatinib significantly inhibited the proliferation of PASMCs (Dahal et al., 2010). To compare the effects of dacomitinib and gefitinib, erlotinib, and lapatinib on the proliferation of PASMCs, EGF or hypoxia
2.12. Western blot analysis The protein samples were extracted from PAECs with the procedures essentially the same as described in details previously (Masri et al., 2005). Protein samples (20–50 μg) were fractionated by SDSPAGE (7.5–10% polyacrylamide gels). The primary antibodies against EGFR (1:500), Cyclin A (1:250), Cyclin D1 (1:200), CDK1(1:400), CDK2 (1:500), PCNA (1:200), p-AKT (1:1000), AKT(1:500), p-S6 (1:500), LC3BⅠⅡ(1:4000), Beclin (1:400), SQSTM-1 (1:400), β-actin (1:2000) were used, with β-actin as an internal control. 2.13. Data analysis The composite data are expressed as means ± standard errors of the means. Statistical analysis was performed with one-way analysis of variance followed by Dunnett’s test where appropriate. Differences were considered to be significant when the P values were less than or 99
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Fig. 1. Effect of dacomitinib on hemodynamics, RV function. A: Bar graph (mean ± S.E.M.) shows the RVSP in the hypoxia model or the MCT model (** P < 0.01; n = 5). B: Bar graph (mean ± S.E.M.) shows the mPAP in the hypoxia model or the MCT model (** P < 0.01; n = 5). C: Bar graph (mean ± S.E.M.) shows the RV/ LV+S weight ratio in rat exposed to hypoxia or MCT in the absence or presence of dacomitinib (** P < 0.01; n = 5). D: Echocardiographic evaluation of the right ventricle and the pulmonary arteries of hypoxia model or the MCT model (** P < 0.01; n = 5). E: Bar graph (mean ± S.E.M.) shows the PAAT in the hypoxic model or the MCT model(** P < 0.01; n = 5). F:Bar graph (mean ± S.E.M.) shows the PAVTI in the hypoxic model or in the MCT mode(** P < 0.01; n = 5). G: Bar graph (mean ± S.E.M.) shows the RVAW; d in the hypoxic model or in the MCT model (** P < 0.01; n = 5). H: Bar graph (mean ± S.E.M.) shows the RVAW; s in the hypoxic model or in the MCT model (** P < 0.01; n = 5). Con, control; Hyp, hypoxia; MCT, monocrotaline; Dac, Dacomitinib. PAAT; pulmonary artery acceleration time, PAVTI; pulmonary arterial velocity time integral, RVAW; d, right ventricular anterior wall; diastole, RVAW; s, and right ventricular anterior wall; systole.
significantly increased proliferation of rat PASMC and the EGFR inhibitors dacomitinib, gefitinib, erlotinib, and lapatinib significantly inhibited the proliferation as determined by WST-1 assay. All four EGFR inhibitors displayed comparable efficacy of inhibiting the PASMCs proliferation. These results indicated that dacomitinib do not show more significant inhibition than other inhibitors in suppressing PASMCs proliferation.(Fig. 3C–D). At the same time, the data showed that EGFR protein expression was significantly increased in PASMCs exposed to hypoxia alone and that dacomitinib reversed this increased expression (Fig. 3E). In addition, real-time-PCR results showed that the mRNA level of EGFR was also decreased in PASMCs after exposure to hypoxia (Fig. 3F). These results indicated that the beneficial effect of dacomitinib could be through altered EGFR protein and mRNA expression and blocking EGFR activation lead to inhibition of hypoxia-induced PASMCs proliferation.
pathogenesis (Sarkar et al., 1995). To elucidate the effect of dacomitinib on PASMCs proliferation (a key component of pulmonary vascular remodeling). A monoclonal antibody, Ki-67, that reacts with cells in the proliferative phases (G1, G2, S, and M) of the cell cycle was used in an immunohistochemical labeling reaction to examine the hypoxia treatment PASMCs with or without dacomitinib. Our results showed that dacomitinib markedly reduced Ki67 staining (Fig. 4A). On analyzing proliferating cell nuclear antigen (PCNA) expression in PASMCs, we found that hypoxia increased this expression and that the effect was decreased in the presence of dacomitinib (Fig. 4B). These results demonstrate that dacomitinib regulates the proliferation of PASMCs under hypoxic conditions. In the scratch-wound assay, the PASMCs migration induced by hypoxia was significantly inhibited by dacomitinib (Fig. 4C). In addition, we examined the effects of dacomitinib during PASMCs migration in a modified Boyden chamber. Accordingly, the migration of PASMCs induced by hypoxia was significantly abolished dacomitinib (Fig. 4D). These results demonstrate that the dacomitinib regulates the migration of PASMCs under hypoxic conditions.
3.3. Effect of dacomitinib on PASMCs proliferation and migration PASMCs proliferation is an important factor contributing to PAH 100
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Fig. 2. Effect of dacomitinib on pulmonary vascular remodeling. A: (a) H& E staining showing that hypoxia or MCT significantly increased the thickness of arteries close to bronchi, which was reversed by dacomitinib treatment. (b) EVG staining showing the elastic membrane (c) The Masson staining showed that the deposition of collagen in rat lungs was increased in hypoxia and MCT model, which was reversed by dacomitinib. (d)Immunohistochemical evaluation of the expression of EGFR was increased in the pulmonary vascular wall in hypoxia or MCT model. B: Bar graph (mean ± S.E.M.) shows the arteries wall of thickness in the hypoxia model or the MCT model. (** P < 0.01; n = 5). C: Bar graph (mean ± S.E.M.) shows the vascular EGFR positive area in the hypoxia model or the MCT model. (** P < 0.01; n = 5). D: The mRNA expression of EGFR in pulmonary vascular from hypoxia or MCT model (** P < 0.01; n = 5). E: The protein expression of EGFR in pulmonary vascular from hypoxia or MCT model (** P < 0.01; n = 5). All values are presented as the mean ± S.E.M. . Con, control; Hyp, hypoxia; MCT, monocrotaline; Dac, Dacomitinib.
3.4. A pivotal role of dacomitinib in cell cycle progression in PASMCs
may play a role in cell cycle progression; therefore, the effects of dacomitinib on cell cycle progression were evaluated. The percentage of S phase cells under hypoxic increased from 18.51% to 31.13%, accompanied by a reduction in cells in the G0/G1 phase from 72.96% to 54.30%, as compared to normoxic cells. However, Dacomitinib attenuated the effect of hypoxia-induced PASMCs from G0/G1 phase into S
To investigate the potential effects of dacomitinib under hypoxia, our finding that dacomitinib regulated PASMCs proliferation effect the expression of Ki-67, that reacts with cells in the proliferative phases (G1, G2, S, and M) of the cell cycle. These suggested that dacomitinib 101
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Fig. 3. Dacomitinib inhibited EGFR expression and Effect on PASMCs proliferation. A：Effect of dacomitinib on PASMCs proliferation in normoxia condition (* P < 0.05; n = 6). B: Effect of dacomitinib on PASMCs proliferation in hypoxia condition (* P < 0.05 compared with Normoxia, # P < 0.05 compared with Hypoxia, n = 6) C: Compared the effect of dacomitinib, gefitinib, erlotinib, and lapatinib on epidermal growth factor (EGF)-induced proliferation of PASMCs (* P < 0.05 compared with Normoxia, # P < 0.05 compared with EGF, n = 6). D: Compared the effect of dacomitinib, gefitinib, erlotinib, and lapatinib on the hypoxia-induced proliferation of PASMCs (* P < 0.05 compared with Normoxia, # P < 0.05 compared with Hypoxia, n = 6). E: The protein expression of EGFR in PASMCs treated with hypoxia in the absence or presence of dacomitinib (** P < 0.01; n = 6). F: The mRNA expression of EGFR in PASMCs treated with hypoxia in the absence or presence of dacomitinib (** P < 0.01; n = 6). All values are presented as the mean ± S.E.M. Con, control; Nor, normoxia; Hyp, hypoxia; MCT, monocrotaline; Dac, Dacomitinib.
phase (Fig. 5A). We analyzed the protein levels of the cell cycle-related proteins cyclin A, cyclin D and of the cyclin-dependent kinase CDK1 and CDK2 in PASMCs. The expression levels of cyclin A, cyclin D, CDK1, and CDK2 were significantly increased after hypoxia, but these effects were attenuated by dacomitinib (Fig. 5B-C). These results confirmed that dacomitinib played an important role in cell cycle progression during hypoxia.
3.5. Dacomitinib promotes protective autophagy in PASMCs Our previous study has shown that autophagy-associated accumulation of LC3B-Ⅱ, Beclin (BECN-1) and degradation of SQSTM-1 were markedly increased in the lung tissues from hypoxic rats compared with the normal group (Mao et al., 2017). Previous studies suggested that TKIs including erlotinib and gefitinib, first generation EGFR TKIs, 102
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Fig. 4. Dacomitinib inhibited hypoxic-induced PASMCs proliferation and migration. A: Cultured PASMCs were exposed to dacomitinib treatment, Ki67 labeled fluorescence images were obtained through immunocytochemistry (n = 3). B: The protein expression of PCNA in PASMCs treated with hypoxia in the absence or presence of dacomitinib (* P < 0.05; ** P < 0.01; n = 6). C: Scratch-wounding cell migration assay. The migration induced by hypoxia was blocked by dacomitinib in PASMCs of human (** P < 0.01; n = 6). D: Boyden chamber migration assays were performed, and the number of migratory cells under normoxic and hypoxic conditions was assessed by crystal violet staining (** P < 0.01; n = 6). All values are presented as the mean ± S.E.M. Nor, normoxia; Hyp, hypoxia; Dac, Dacomitinib.
induce a protective autophagy to resist proliferation and cell cycle progression effects (Li et al., 2013). So, we speculated dacomitinib effecting in autophagy of PAH. First, we evaluated the expression of BECN-1, LC3B-Ⅱand SQSTM-1 in pulmonary arteries from animal models of experimental PAH (hypoxia-induced PAH or MCT-induced PAH) treated with dacomitinib. Hypoxia-induced or MCT-induced
highly expressed in BECN-1 and LC3B-Ⅱ was attenuated by dacomitinib and degradation of SQSTM-1 was markedly increased by dacomitinib (Fig. 6A–B). In PASMCs, hypoxia-induced elevated expressed of BECN-1 and LC3B-Ⅱ which was attenuated by dacomitinib, and degradation of SQSTM-1 was markedly increased by dacomitinib in PASMCs (Fig. 6C). To elucidate the inductive role of dacomitinib in autophagy, expression 103
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Fig. 5. Dacomitinib play an important role in PASMCs cell cycle progression. A: The number of cells in each phase of the cell cycle was examined by FACS analysis (n = 3). The Result showed that dacomitinib reduced the percentage of cells phase accompanied by a concomitant increase of cells at G0/G1 phase. B: The expression of cyclin A and cyclin D1 of PASMCs was inhibited by dacomitinib (** P < 0.01; n = 6). C: hypoxia-induced the increased the expression of CDK1 and CDK2 of PASMCs was inhibited by dacomitinib (** P < 0.01; n = 6).
cell cycle progression and promoted autophagy. (3) The analysis of dacomitinib affects the proliferation and migration, the cell cycle progression and autophagy of PASMCs are closely related to the activation of PI3K-AKT-mTOR signaling. Chronic hypoxia and MCT-induced PAH were the mainly models for studying human PAH (Pugliese et al., 2015). Both chronic hypoxia and MCT-induced PAH can promote pulmonary vascular remodeling, causing PAH and even heart faiure (Pugliese et al., 2015; Sakao and Tatsumi, 2011). MCT induced PAH involving inflammatory response, and closer to the pathogenesis of PAH associated with connective tissue disease (Gomez-Arroyo et al., 2012). PAH and cancer share growth factor and protein kinase signaling pathways that result in smooth muscle cell proliferation and vasculopathy((Alkhatib et al., 2016). Therefore, Our research with other laboratories have apply anti-tumor drugs (kinase inhibors) to experimental treatment of PAH models (Chhina et al., 2010; Dahal et al., 2010; Liang et al., 2017; Yu et al., 2018, 2015). Kinase inhibitors inhibited the proliferation of PAECs or PASMCs by blocking the signaling pathway of growth factors (Chhina et al., 2010; Dahal et al., 2010; Liang et al., 2017; Nielsen et al., 2017; Yu et al., 2018, 2015). However, apparent lack of translation of these two models for the human disease as kinase inhibitors are concerned. An accumulating of evidence has revealed that the cell differentiation, proliferation, survival, and migration mediated by EGFR/ErbB signaling are crucially involved in the pathobiology of PAH and thus this signaling is a major target for PAH therapy (Normanno et al., 2006; Wieduwilt and Moasser, 2008). Research has confirmed that gefitinib (Iressa) and erlotinib (Tarceva) reduced the RVSP, right heart hypertrophy and medial wall thickness and degree of muscularization of small peripheral pulmonary arteries revealed that the pulmonary vascular remodeling was improved in MCT-induced PAH, but none of the two inhibitors provided significant therapeutic benefit, either in hemodynamic or in pulmonary vascular remodeling in chronic hypoxic PAH (Dahal et al., 2010). The therapeutic effect of lapatinib (Tykerb) in the field of pulmonary hypertension is controversial (Dahal et al., 2010). Studies have shown that in two experimental model of PAH (hypoxia- or MCT-induced PAH), lapatinib did not reverse PAH and pulmonary vascular remodeling (Dahal et al., 2010). Some studies have shown experiments on animal models has shown an effect of lapatinib
of EGFR was knocked down with its inhibitor, dacomitinib. Further experiments confirmed the altered autophagy markers and the appearance of the eGFP-mRFP-LC3 adenoviruses under hypoxia were inhibited by dacomitinib (Fig. 6D). These results confirmed that dacomitinib played an important role in autophagy during hypoxia. 3.6. Dacomitinib attenuated pulmonary vascular remodeling and PASMCs proliferation involved PI3K-AKT-mTOR signaling The PI3K-AKT-mTOR pathway signaling pathway is critical in the regulation of cell growth, metabolism and survival, angiogenesis, tumor invasion, cell cycle regulation, and DNA repair (Courtney et al., 2010; Shen et al., 2013). Previous studies have confirmed that the EGF/EGFR regulation of autophagy through PI3K involves Akt and mTOR (Sobolewska et al., 2009). Interesting is that EGFR inhibitors, dacomitinib can affect autophagy (Brana et al., 2017). So, we investigated the role of dacomitinib affect PI3K-AKT-mTOR signaling pathway in animal models of experimental PAH and PASMCs. We evaluated the expression of PI3K-AKT-mTOR in pulmonary arteries from animal models of experimental PAH (hypoxia-induced PAH or MCT-induced PAH) treated with dacomitinib. The results showed that dacomitinib decreased the expression of p-Akt (Ser 473) and p-mTOR (Ser 248), without altering the total protein levels of Akt and mTOR (Fig. 7A–B). We observed similar results of p-Akt (Ser 473) and p-mTOR (Ser 248) in PASMCs (Fig. 7C–D). The above results indicated that dacomitinib attenuated pulmonary vascular remodeling and PASMCs proliferation involved PI3K-AKT-mTOR signaling. Fig. 8 4. Discussion and conclusions The major findings of this study are (1) the EGFR antagonists dacomitinib exhibit therapeutic efficacy in animal models of experimental PAH (including chronic hypoxia and MCT-induced PAH). Dacomitinib has a significant role in attenuating pulmonary artery pressure and RVH, and dacomitinib has a significant inhibitory effect on vascular remodeling. (2) Dacomitinib showed no significant difference from gefitinib, erlotinib, and lapatinib in inhibit of PASMCs proliferation, however, dacomitinib inhibited hypoxia-induced PASMCs migration, 104
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Fig. 6. Dacomitinib promotes protective autophagy in PASMCs. A-B: Autophagy-associated alteration of LC3B-, BECN-1, SQSTM1 were analyzed in the pulmonary vascular from hypoxia-induced PAH rats and MCT-induced PAH rats (* P < 0.05; ** P < 0.01; n = 6). C: Autophagy-associated alteration of LC3B-, BECN-1, SQSTM1 were analyzed in PASMCs were treated with dacomitinib (* P < 0.05; ** P < 0.01; n = 6). D: PASMCs was treated with the eGFP-mGFP-LC3B plasmid. Yellow and red dots refer to autolysosomes and autophagosomes, respectively (** P < 0.01; n = 4). All values are presented as the mean ± S.E.M. Nor, normoxia; Hyp, hypoxia; Dac, Dacomitinib.
in attenuating induced PAH in rats (Alkhatib et al., 2016). Lapatinib chronic use might be associated with the development of PAH (Alkhatib et al., 2016). Stopping treatment in cases where no other reasons were identified might be associated with reversibility of the elevated pulmonary artery pressure (Dahal et al., 2010). Our research first time showed that dacomitinib exhibit therapeutic efficacy in animal models of experimental PAH (including chronic hypoxia PAH and MCT-induced PAH). Dacomitinib has a significant role in attenuating pulmonary artery pressure and RVH, and dacomitinib has a significant inhibitory effect on vascular remodeling. Our study
provides a new indication of dacomitinib and provides a new idea for the treatment of PAH. However, the study has some limitations. Dacomitinib is still in clinical trials, the study of patients with PAH is currently unable to carry out, and could not determine whether dacomitinib has other adverse reactions, which we need in the follow-up experiments in the exploration. PASMCs proliferation and migration constitutes an important incentive for pulmonary vascular remodeling. Our results have confirmed that effect of dacomitinib could significantly alter the protein and mRNA level of EGFR and through blocking EGFR activation, attenuated 105
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Fig. 7. Dacomitinib attenuated pulmonary vascular remodeling and PASMCs proliferation involved PI3K-AKT-mTOR signaling. A–B: The expression of p-Akt (Ser 473) and p-mTOR (Ser 248) were analyzed by western-blot in pulmonary vascular from hypoxia-induced PAH rats and MCT-induced PAH rats (* P < 0.05; ** P < 0.01; n = 6). C-D: The expression of p-Akt (Ser 473) and p-mTOR (Ser 248) were analyzed by western-blot in PASMCs (* P < 0.05; ** P < 0.01; n = 6). All values are presented as the mean ± S.E.M. Nor, normoxia; Hyp, hypoxia; Dac, Dacomitinib.
the pulmonary vascular remodeling also reduced the positive rate of EGFR expression in PASMCs. Previous studies have shown that EGFR signaling contributes to vascular SMC proliferation and migration (Chan et al., 2003; Kalmes et al., 2000; Zhou et al., 2007). In addition, EGFR signaling has also been implicated in the survival of PASMCs (Merklinger et al., 2005). There are results showed that dacomitinib has an effect on cell viability, self-renewal, and proliferation both in EGFRamplified ± EGFRvIII GBM cells and H1975 human NSCLC line (Zahonero et al., 2015). Our results proved the solid evidence that dacomitinib through blocking EGFR activating mediated in hypoxiainduced proliferation and migration of PASMCs, resulting in reduced pulmonary vascular remodeling. Disorders of cell cycle components may lead to cell proliferation. The percentage of cancer cells in the S and G2 phases of the cell cycle significantly decreased, and Cdk1 and Cdk2 protein levels declined after dacomitinib treatment in SKOV3 and OV4 cells (Xu et al., 2016).
Dacomitinib can significantly regulate the cell cycle of cancer cells (Xu et al., 2016). In our research, the percentage of PASMCs in the S and G2 phases of the cell cycle significantly decreased, and cyclin A; cyclin D; Cdk1 and Cdk2 protein levels declined after dacomitinib treatment in PASMCs. This is the first time confirmed that dacomitinib regulates the cell cycle in PASMCs. Autophagy is a major intracellular degradation and recycling process that maintains cellular homeostasis, which is involved in structural and functional abnormalities of PAH (Mao et al., 2017). Previous studies suggested that EGFR tyrosine kinases inhibitors including erlotinib and gefitinib, the first generation EGFR tyrosine kinases inhibitors, induce a protective autophagy to resist theirs in proliferation and cell cycle progression effects (Li et al., 2013). Results showed that a novel autophagic inhibitor cepharanthine enhanced the anti-cancer property of dacomitinib in non-small cell lung cancer (Tang et al., 2018). Furthermore, some studies have confirmed that dacomitinib in phase III
Fig. 8. A schematic model showing the proposed mechanism for dacomitinib attenuated pulmonary vascular remodeling and pulmonary hypertension. 106
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clinical trials for NSCLC treatment induced a protective autophagy to decrease its anti-cancer effect (Tang et al., 2018). Combined treatment with cepharanthine increased the anti-proliferative and apoptotic effects of dacomitinib in vitro and enhanced the anti-cancer effect of dacomitinib in NCI-H1975 x-enograft mice (Tang et al., 2018). In view of the above studies, we explored the effect of dacomitinib on autophagy in PASMCs. Our study found that dacomitinib induced a protective autophagy in two experimental model of PAH (hypoxia or MCT induced PAH) and PASMCs, which effect may be an important mechanism that causes dacomitinib to attenuate pulmonary vascular remodeling. PI3K-AKT-mTOR signaling pathway is critical in the regulation of cell growth, metabolism and survival, angiogenesis, tumor invasion, cell cycle regulation and DNA repair (Courtney et al., 2010; Shen et al., 2013). Dacomitinib has been reported to be involved regulated the PI3K-AKT-mTOR signaling pathway in PTEN-deficient patient-derived tumor xenografts (Brana et al., 2017). Here, we found that PI3K/AKT/ mTOR signaling pathway involved two experimental model of PAH (hypoxia or MCT induced PAH) and PASMCs treated with dacomitinib. This is the first time confirmed that dacomitinib inhibited PI3K-AKTmTOR signaling pathway in PASMCs and animal models of hypoxiainduced PAH and MCT- induced PAH. In conclusion, this study indicates that dacomitinib inhibits hypoxia-induced proliferation, migration, autophagy and Cell cycle progression involved PI3K-AKT-mTOR signaling pathway. Moreover, dacomitinib has a significant inhibitory effect on the thickening of the media; fibrosis of the adventitia. Dacomitinib has a significant role in attenuating pulmonary artery pressure and right ventricular hypertrophy andmay serve as new potential therapeutic for the treatment of PAH.
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Acknowledgments This work was supported by National Natural Science Foundation of China (contract grant numbers: 31820103007 and 31771276 to D.Z.; 81800047 to X.Y.) University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (contract grant numbers: UNPYSCT-2018067 to X.Y.); Youth Science Foundation of Heilongjiang Province (contract grant number: QC2016111 to X.Y.); Postdoctoral Foundation of Heilongjiang Province (contract grant number: LBHZ16241 to X.Y.); Wu Liande Young Scientific Research Foundation of Harbin Medical University Daqing (contract grant number: DQWLD20 to X.Y.); China Postdoctoral Science Foundation Funded Project (contract grant number: 2016M591557 to X.Y.); and Academician Mr. Yu Weihan Foundation for Distinguished Young Scholars (contract grant numbers: DQYWH201601 to L.Q.). Author contributions Daling Zhu, Lihui Qu, Xiufeng Yu created this rearch design. Xiufeng Yu and Xijuan Zhao analysed data and drafeted manuscript. Junting Zhang performed animal experiments and histological experiments. Yiying Li, Ping Sheng, Cui Ma, Xuewei Hao and Qiao Hui performed the cell culture and molecular biology experiments. Lixin Zhang performed Echocardiography and cell cycle DNA analysis experiments. Xiaodong Zheng and Yan Xing performed The Hemodynamic Evaluation of PAH–model. All authors read and approved the manuscript. Conflict of interests There is no conflict of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ejphar.2019.02.008. 107
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Dacomitinib, a new pan-EGFR inhibitor, is effective in attenuating pulmonary vascular remodeling and pulmonary hypertension
T
Xiufeng Yua,b,c,d,1, Xijuan Zhaoa,b,c,1, Junting Zhangb,c, YiYing Lib,c, Ping Shengd, Cui Maa,b,c, ⁎ Lixin Zhanga,b,c, XueWei Haoa,b,c, XiaoDong Zhengb,e, Yan Xingb,e, Hui Qiaob,e, Lihui Qub,e, , ⁎⁎ Daling Zhub,c, a
College of Medical Laboratory Science and Technology, Harbin Medical University (Daqing), Daqing 163319, PR China Central Laboratory of Harbin Medical University (Daqing), Daqing 163319, PR China c Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical University, Harbin 150081, PR China d Department of Hepatic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin 150081, PR China e Department of Basic Medical College, Harbin Medical University (Daqing), Daqing 163319, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Dacomitinib EGFR Proliferation PASMCs Pulmonary artery hypertension
Accumulating evidence suggests that epidermal growth factor receptor (EGFR) plays a role in the progression of pulmonary arterial hypertension (PAH). Clinically-approved epidermal growth factor inhibitors such as gefitinib, erlotinib, and lapatinib have been explored for PAH. However, None of them were able to attenuate PAH. So, we explored the role of dacomitinib, a new pan-EGFR inhibitor, in PAH. Adult male Sprague–Dawley rats were used to study hypoxia- or monocrotaline-induced right ventricular remodeling as well as systolic function and hemodynamics using echocardiography and a pressure-volume admittance catheter. Morphometric analyses of lung vasculature and pressure-volume vessels were performed. Immunohistochemical staining, flow cytometry, and viability, as well as scratch-wound, and Boyden chamber migration assays were used to identify the roles of dacomitinib in pulmonary artery smooth muscle cells (PASMCs). The results revealed that dacomitinib has a significant inhibitory effect on the thickening of the media, adventitial collagen increased. Dacomitinib also has a significant role in attenuating pulmonary artery pressure and right ventricular hypertrophy. Additionally, dacomitinib inhibits hypoxia-induced proliferation, migration, autophagy and cell cycle progression through PI3K-AKT-mTOR signaling in PASMCs. Our study indicates that dacomitinib inhibited hypoxiainduced cell cycle progression, proliferation, migration, and autophagy of PASMCs, thereby attenuating pulmonary vascular remodeling and development of PAH via the PI3K-AKT-mTOR signaling pathway. Overall, dacomitinib may serve as new potential therapeutic for the treatment of PAH.
1. Introduction Pulmonary artery hypertension (PAH) is a progressive pulmonary vascular disease characterized by vasoconstriction, vascular remodeling, and increased vascular resistance; PAH leads to right heart failure and death (Chan and Loscalzo, 2008; Humbert et al., 2004; Rubin, 1997). To date, there is no cure for the disease, and the mortality rate in PAH patients is fairly high. Therefore, elucidating the molecular and cellular basis underlying the pathogenesis of PAH and exploring new therapeutic strategies are necessary (Thenappan et al., 2010). Pulmonary vascular remodeling, the key pathological feature of
PAH, is attributable to the increased proliferation, resistance to apoptosis, and migration of pulmonary vascular cells (Chan and Loscalzo, 2008; Humbert et al., 2004). Such cellular events are mediated by growth factors such as platelet-derived growth factor (PDGF) (Heldin et al., 1998; Liang et al., 2017), vascular endothelial growth factor (VEGF) (Liang et al., 2017)and epidermal growth factor (EGF) through activation of their receptor tyrosine kinases (RTKs) and downstream signaling pathways (Dahal et al., 2010). In recent years, EGF (Dahal et al., 2010; Merklinger et al., 2005; Ushio-Fukai et al., 2001) is emerging as important players in the pathomechanism of PAH (Izikki et al., 2013), suggesting that the field of
Corresponding author at: Central Laboratory of Harbin Medical University (Daqing), Daqing 163319, PR China. Correspondence to: College of Pharmacy, Harbin Medical University (Daqing), Xinyang Road, Daqing, Heilongjiang 163319, PR China. E-mail addresses: [email protected] (L. Qu), [email protected] (D. Zhu). 1 These authors contributed equally to this work. ⁎
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https://doi.org/10.1016/j.ejphar.2019.02.008 Received 23 July 2018; Received in revised form 1 February 2019; Accepted 8 February 2019 Available online 10 February 2019 0014-2999/ © 2019 Published by Elsevier B.V.
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growth factors and their RTKs as targets for treatment of cardiopulmonary diseases is expanding. Researches have confirmed that used two independent animal models of PAH to investigate the epidermal growth factor receptor (EGFR) inhibitors gefitinib, erlotinib, and lapatinib, which found that gefitinib and erlotinib reduced the right ventricular systolic pressure (RVSP), total systemic resistance, and right heart hypertrophy in rats with monocrotaline (MCT)-induced PAH. Moreover, measurements of medial wall thickness and degree of muscularization of small peripheral pulmonary arteries revealed that the pulmonary vascular remodeling was improved (Dahal et al., 2010). Despite the in vitro efficacy, lapatinib did not provide therapeutic benefit with respect to significantly attenuating the right heart hypertrophy and pulmonary vascular remodeling (Dahal et al., 2010). Dacomitinib (PF-00299804), a second generation EGFR tyrosine kinases inhibitors (TKIs), is an irreversible tyrosine kinases inhibitors of the human EGFRs (HERs), including HER-1/EGFR, HER-2, and HER-4 (Engelman et al., 2007). Previous studies have found that gefitinib, erlotinib, and lapatinib did not provide any significant therapeutic benefit, either in hemodynamic or in pulmonary vascular remodeling, suggesting that an inhibition of EGFR signaling did not impair the progression of chronic hypoxic PAH and pulmonary vascular remodeling (Dahal et al., 2010). However, None of them were able to attenuate PAH (Dahal et al., 2010). We want to explore the new panEGFR inhibitor in pulmonary hypertension. Therefore, we examined the role of dacomitinib in MCT-induced PAH and chronic hypoxic PAH.
the right ventricular hypertrophy index ratio of right ventricular free wall weight over a sum of septum plus left ventricular free wall weight: (RV/LV+S). 2.4. Echocardiography Rats were anesthetized by 4% chloral hydrate (0.1 ml/kg, i.p.) before echocardiography by Vevo 2100 imaging system (Visual Sonics Inc., Toronto, Ontario, Canada)with a 30 MHZ probe. Stable images were obtained in M, B, and Doppler Mode. right ventricular inner dimension, right ventricular stroke volume, right ventricular fractional shortening, tricuspid valve early and late diastolic filling velocities, pulmonary arterial velocity time integral, pulmonary arterial pre-ejection time, and pulmonary arterial ejection time were measured. 2.5. Histological and histomorphometric evaluation The lung tissues were sliced into tissue blocks and were immersed in 4% paraformaldehyde for overnight fixation. Then fixed tissues were dehydrated, cleared, and embedded in the paraffin base. The paraffin blocks were cut into sections of 5 µm thick. Some sections were stained with hematoxylin and eosin (H&E), Elastica van Gieson's stain and Masson staining. Histopathological analysis was performed as previously described (Zhang et al., 2016). The total area of the vascular walls was quantified with high-resolution images of individual vessels using a color-recognition algorithm in Image-Pro Plus 6.0 software. Linear micrometer measurements of medial thickness were achieved in ten images at 10x magnification (100 µm per panel). The thickest part of the media was selected as the center point, and then, the arterial cross-section was divided into five segments from the center point. The average thickness of the media was measured at five points, including the center point.
2. Materials and methods 2.1. Animal used Animal care and use conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, revised 1996). Sprague–Dawley rats (200 g) were used in the study which was bought from the Experimental Animal Center of Harbin Medical University and was approved by the Institutional Animal Care and Use Committee. This study was also approved by the ethics review board of Harbin Medical University ([2015]-016).
2.6. Cell culture The primary rat pulmonary smooth muscle cells (PASMCs) were obtained from Procell, China. Smooth Muscle Cell Medium was supplied with SMC Bullet Kit from Science, the USA which was used as the growth medium. 13% Bovine Fetal Serum Gold from Clark Bioscience was added to the growth medium to maintain the proliferation of the cells. The cells were cultured at 37 °C with 5% CO2 in humidified conditions. Cell passage to 2–3 generations for experiments, before the experiment, the PASMCs were starvation for 24 hin Smooth Muscle Cell Medium without serum. Then treated with dacomitinib (50 nM) or dacomitinib (50 nM) plus EGF (50 ng/ml）in 5% FBS-DMEM.
2.2. Hypoxia-induced or MCT-induced PAH model 6-week-old male Sprague–Dawley rats weighing approximately 200 g at protocol onset were divided into the following groups: (A) Animals into normoxic groups (n = 5) were received a single subcutaneous injection of 0.9% saline housed at ambient barometric pressure (FiO2) 0.21 for 21days. (B) Animals in hypoxic groups were (n = 5) were received a single subcutaneous injection of 0.9% saline housed at inspired oxygen (FiO2) 0.12 for 21 days. (C) 3rd group of rats (n = 5) were administered daily with Dacomitinib (500 nmol/kg/day by subcutaneous injection) from 2-weeks after the hypoxic treated until the terminal experiment. (D) PAH rats received a single subcutaneous injection of 60 mg/kg monocrotaline(n = 5) to induce severe PAH. (E) To further assess the role of dacomitinib (respectively, 100 nm/kg/day, 250 nm/kg/day, 500 nmol/kg/day by subcutaneous injection) (n = 15) in inducing regional RV myocardial remodeling the 5th group of rats was administered from 2-weeks after the monocrotaline injection, for a duration of 3-weeks, until the terminal experiment. Four-weeks after saline or monocrotaline injection (Nielsen et al., 2017).
2.7. Cell proliferation assay Cell proliferation was analyzed by the WST-1 Cell Proliferation and Cytotoxicity Assay kit (Roche, USA). Briefly, PASMCs were seeded onto 96-well plates at a density of 2 × 103 cells per well and cultured overnight. The cells continued to grow at 37 °C in a humidified incubator. The cells were subjected to different treatments. After 24 h, 20 μl of WST-1 was added to each well, and the cells were incubated for another 4 h at 37 °C. Absorbance was measured at 490 nm. All experiments were performed in triplicate. 2.8. Cell cycle and DNA analysis
2.3. The Hemodynamic Evaluation of PAH–model
The Cycle TEST PLUS DNA Reagent Kit was used to examine whether the cell cycle was influenced by dacomitinib. In brief, prepared PASMCs in groups were trypsinized, harvested and fixed in 70% cold ethanol at 4 ℃. After discarding the ethanol. cells were resuspended in 200μl propidium iodide at 4 ℃. Finally, the stained cells were filtered and DNA fluorescence was measured by flow cytometry using BD FACS Calibur Flow Cytometer (Bedford, MA). We analyzed the proportion of
A 1.2 French Pressure Catheter (Scisense Inc.) was connected to the Science FA-404 recorder. When the right jugular vein was exposed, the catheter was inserted into the vein, then was advanced into the superior vena cava, and finally into the right ventricular vein. RVSP was continuously recorded for 45 min. After measurement of RVSP, the thorax was opened and the heart was dissected and weighed for calculation of 98
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cells in the G0/G1, S and G2/M phases by flow cytometry as reported recently (Ma et al., 2011).
equal to 0.05. 2.14. Materials
2.9. Migration assays
Dacomitinib (PF299804, PF299), Gefitinib (ZD1839), erlotinib, lapatinib, were purchased from Selleck Chemicals (Houston, TX, USA). Recombinant rat EGF Protein was purchased from R&D Systems (Minneapolis, MN, USA). LC3B were purchased from Abcam (Cambridge, MA, USA). AKT, p-AKT, p-mTOR were purchased from Cell Signal Technology (Beverly, MA, USA). Cyclin A was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Beclin, SQSTM-1, EGFR, PCNA antibody were obtained from Boster Biological Technology Co. Ltd (Wuhan, China). Cyclin D, CDK1, CDK2 antibodies were purchased from Beyotime Institute of Biotechnology (Haimen, China).
Migration in vitro was assayed using a Transwell chamber (Costar, Corning, NY, USA) with a polycarbonic membrane (6.5 mm in diameter and 8 µm pore size). PASMCs treated with dacomitinib (50 nM) in Smooth Muscle Cell medium supplemented with 5% FBS for 24 h. After dacomitinib treatment, PASMCs were trypsinized and suspended into single cells with a serum-free medium at the density of 5 × 105 cells/ ml. 100 μl of the cell suspension was added to the upper chamber, and 600 ml of Smooth Muscle Cell medium supplemented with 10% FBS was added to the lower chamber. Cells were incubated for 24 h at 37 °C. Non-migrating cells on the top surface of the membrane were removed with cotton swabs. Cells were measured after the fixing with 4% formaldehyde solution for 10 min and then staining of 0.4% crystal violet in 10% ethanol for 5 min. Migrated cell number was measured by counting the number of stained nuclei per high-power field in a microscope (Olympus, Japan), using Image Pro-Plus 6.0.
3. Result 3.1. Effects of dacomitinib on hemodynamics and right cardiac function Hypoxia- and MCT-induced PAH rat models were used to test whether EGFR-tyrosine kinase inhibitor dacomitinib was involved in the pathogenesis of PAH. We characterized the PAH parameters in detail, including right ventricular hypertrophy (RVH), RVSP, hemodynamics, cardiac function and pulmonary vascular remodeling. RVH and RVSP were used to evaluate the effects of the dacomitinib on the development of Hypoxia or MCT- induced PAH. Hypoxia or MCT-significantly elevated RVSP and RVH in rats, while this effect was attenuated by dacomitinib (Fig. 1A–C). An echocardiography was performed on rats (exposed to hypoxia or MCT) to characterize the effect of dacomitinib on right ventricular function (Fig. 1D). The results showed that dacomitinib inhibited hypoxia-induced PAH or MCT-induced PAH development assessed by pulmonary artery acceleration time, pulmonary arterial velocity time integral, right ventricular anterior wall; diastole, and right ventricular anterior wall systole (Fig. 1E-H).
2.10. Scratch-wound assay For Scratch-wounding cell migration assay, the PASMCs cultured in 6-well plates were wounded by pipette tips, given rise to one acellular 1-mm-wide lane per well, and the ablated cells were washed with PBS. After that, cells were treated with dacomitinib (50 nM) in 5% FBS DMEM. Migration was calculated based on the same wounded areas at time zero and 24 h. After 24 h of incubation, photos were taken from the same areas as those recorded at zero time. the closure area of the wound was calculated as follow: migration area(%）=（A0-An)/A0 × 100, where A0 represents the area of the original wound area, An represents the remaining area of the wound at the metering point. 2.11. Relative mRNA quantification by real-time polymerase chain reaction Total RNA was extracted from cultured PASMCs using the Trizol reagent according to the manufacturer’s instructions. Extracted total RNAs were reverse-transcribed using the SuperScript First-strand cDNA Synthesis Kit (Invitrogen). cDNA samples were amplified in a DNA thermal cycler (Thermo Scientific, Waltham, MD, USA). The gene-specific primers were designed using the coding regions, and the nucleotide sequences of the primers are as follows: EGFR (Gen Bank accession no; sense: NM_031507.1 5′-AGTACCTGTTCTGGCTAATGG-3′ and antisense: 5′ -TCACTTTCGTGCGCTCGTAG-3′, 119 bp). Real-time polymerase chain reaction (RT-PCR) was used for relative quantification of EGFR mRNA. The reactions were performed in an ABI 7300 Sequence Detection System (Applied Biosystems, Foster City, Calif).
3.2. Effects of dacomitinib on pulmonary vascular remodeling H&E staining showing that hypoxia or MCT significantly increased the thickness of distal blood vessels, which was attenuated by dacomitinib treatment (Fig. 2A–B). Elastica van Gieson's stain showed the elastic membrane(Fig. 2A). The Masson staining showed that the deposition of collagen in rat lungs was increased in hypoxia and MCT model, which was attenuated by dacomitinib (Fig. 2A). Immuno-histochemical evaluation of the expression of EGFR was increased in the pulmonary vascular wall in hypoxia and MCT model and mainly distributed in PASMCs. Dacomitinib attenuates hypoxia or MCT -induced increased expression of EGFR on PASMCs (Fig. 2C). Next, we explored whether dacomitinib affected the expression of EGFR was involved in the progression of pulmonary vascular remodeling. We observed that the expression of EGFR was increased in pulmonary arteries from rats exposed to hypoxia or MCT, dacomitinib reduced the EGFR expression induced by hypoxia or MCT at both the mRNA and protein level (Fig. 2D–E). These results indicated that the beneficial effect of dacomitinib could alter the protein and mRNA level of EGFR and through blocking EGFR activating inhibited hypoxia-induced or MCT-induced pulmonary vascular remodeling. We used different concentrations of dacomitinib to evaluate the proliferation of PASMCs under normoxia and hypoxia. Our results indicate that dacomitinib (50 nM) can significantly inhibit PASMCs proliferation under normoxia or hypoxia (Fig. 3A-B). Previous studies have confirmed that several EGFR inhibitors including gefitinib, erlotinib, and lapatinib significantly inhibited the proliferation of PASMCs (Dahal et al., 2010). To compare the effects of dacomitinib and gefitinib, erlotinib, and lapatinib on the proliferation of PASMCs, EGF or hypoxia
2.12. Western blot analysis The protein samples were extracted from PAECs with the procedures essentially the same as described in details previously (Masri et al., 2005). Protein samples (20–50 μg) were fractionated by SDSPAGE (7.5–10% polyacrylamide gels). The primary antibodies against EGFR (1:500), Cyclin A (1:250), Cyclin D1 (1:200), CDK1(1:400), CDK2 (1:500), PCNA (1:200), p-AKT (1:1000), AKT(1:500), p-S6 (1:500), LC3BⅠⅡ(1:4000), Beclin (1:400), SQSTM-1 (1:400), β-actin (1:2000) were used, with β-actin as an internal control. 2.13. Data analysis The composite data are expressed as means ± standard errors of the means. Statistical analysis was performed with one-way analysis of variance followed by Dunnett’s test where appropriate. Differences were considered to be significant when the P values were less than or 99
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Fig. 1. Effect of dacomitinib on hemodynamics, RV function. A: Bar graph (mean ± S.E.M.) shows the RVSP in the hypoxia model or the MCT model (** P < 0.01; n = 5). B: Bar graph (mean ± S.E.M.) shows the mPAP in the hypoxia model or the MCT model (** P < 0.01; n = 5). C: Bar graph (mean ± S.E.M.) shows the RV/ LV+S weight ratio in rat exposed to hypoxia or MCT in the absence or presence of dacomitinib (** P < 0.01; n = 5). D: Echocardiographic evaluation of the right ventricle and the pulmonary arteries of hypoxia model or the MCT model (** P < 0.01; n = 5). E: Bar graph (mean ± S.E.M.) shows the PAAT in the hypoxic model or the MCT model(** P < 0.01; n = 5). F:Bar graph (mean ± S.E.M.) shows the PAVTI in the hypoxic model or in the MCT mode(** P < 0.01; n = 5). G: Bar graph (mean ± S.E.M.) shows the RVAW; d in the hypoxic model or in the MCT model (** P < 0.01; n = 5). H: Bar graph (mean ± S.E.M.) shows the RVAW; s in the hypoxic model or in the MCT model (** P < 0.01; n = 5). Con, control; Hyp, hypoxia; MCT, monocrotaline; Dac, Dacomitinib. PAAT; pulmonary artery acceleration time, PAVTI; pulmonary arterial velocity time integral, RVAW; d, right ventricular anterior wall; diastole, RVAW; s, and right ventricular anterior wall; systole.
significantly increased proliferation of rat PASMC and the EGFR inhibitors dacomitinib, gefitinib, erlotinib, and lapatinib significantly inhibited the proliferation as determined by WST-1 assay. All four EGFR inhibitors displayed comparable efficacy of inhibiting the PASMCs proliferation. These results indicated that dacomitinib do not show more significant inhibition than other inhibitors in suppressing PASMCs proliferation.(Fig. 3C–D). At the same time, the data showed that EGFR protein expression was significantly increased in PASMCs exposed to hypoxia alone and that dacomitinib reversed this increased expression (Fig. 3E). In addition, real-time-PCR results showed that the mRNA level of EGFR was also decreased in PASMCs after exposure to hypoxia (Fig. 3F). These results indicated that the beneficial effect of dacomitinib could be through altered EGFR protein and mRNA expression and blocking EGFR activation lead to inhibition of hypoxia-induced PASMCs proliferation.
pathogenesis (Sarkar et al., 1995). To elucidate the effect of dacomitinib on PASMCs proliferation (a key component of pulmonary vascular remodeling). A monoclonal antibody, Ki-67, that reacts with cells in the proliferative phases (G1, G2, S, and M) of the cell cycle was used in an immunohistochemical labeling reaction to examine the hypoxia treatment PASMCs with or without dacomitinib. Our results showed that dacomitinib markedly reduced Ki67 staining (Fig. 4A). On analyzing proliferating cell nuclear antigen (PCNA) expression in PASMCs, we found that hypoxia increased this expression and that the effect was decreased in the presence of dacomitinib (Fig. 4B). These results demonstrate that dacomitinib regulates the proliferation of PASMCs under hypoxic conditions. In the scratch-wound assay, the PASMCs migration induced by hypoxia was significantly inhibited by dacomitinib (Fig. 4C). In addition, we examined the effects of dacomitinib during PASMCs migration in a modified Boyden chamber. Accordingly, the migration of PASMCs induced by hypoxia was significantly abolished dacomitinib (Fig. 4D). These results demonstrate that the dacomitinib regulates the migration of PASMCs under hypoxic conditions.
3.3. Effect of dacomitinib on PASMCs proliferation and migration PASMCs proliferation is an important factor contributing to PAH 100
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Fig. 2. Effect of dacomitinib on pulmonary vascular remodeling. A: (a) H& E staining showing that hypoxia or MCT significantly increased the thickness of arteries close to bronchi, which was reversed by dacomitinib treatment. (b) EVG staining showing the elastic membrane (c) The Masson staining showed that the deposition of collagen in rat lungs was increased in hypoxia and MCT model, which was reversed by dacomitinib. (d)Immunohistochemical evaluation of the expression of EGFR was increased in the pulmonary vascular wall in hypoxia or MCT model. B: Bar graph (mean ± S.E.M.) shows the arteries wall of thickness in the hypoxia model or the MCT model. (** P < 0.01; n = 5). C: Bar graph (mean ± S.E.M.) shows the vascular EGFR positive area in the hypoxia model or the MCT model. (** P < 0.01; n = 5). D: The mRNA expression of EGFR in pulmonary vascular from hypoxia or MCT model (** P < 0.01; n = 5). E: The protein expression of EGFR in pulmonary vascular from hypoxia or MCT model (** P < 0.01; n = 5). All values are presented as the mean ± S.E.M. . Con, control; Hyp, hypoxia; MCT, monocrotaline; Dac, Dacomitinib.
3.4. A pivotal role of dacomitinib in cell cycle progression in PASMCs
may play a role in cell cycle progression; therefore, the effects of dacomitinib on cell cycle progression were evaluated. The percentage of S phase cells under hypoxic increased from 18.51% to 31.13%, accompanied by a reduction in cells in the G0/G1 phase from 72.96% to 54.30%, as compared to normoxic cells. However, Dacomitinib attenuated the effect of hypoxia-induced PASMCs from G0/G1 phase into S
To investigate the potential effects of dacomitinib under hypoxia, our finding that dacomitinib regulated PASMCs proliferation effect the expression of Ki-67, that reacts with cells in the proliferative phases (G1, G2, S, and M) of the cell cycle. These suggested that dacomitinib 101
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Fig. 3. Dacomitinib inhibited EGFR expression and Effect on PASMCs proliferation. A：Effect of dacomitinib on PASMCs proliferation in normoxia condition (* P < 0.05; n = 6). B: Effect of dacomitinib on PASMCs proliferation in hypoxia condition (* P < 0.05 compared with Normoxia, # P < 0.05 compared with Hypoxia, n = 6) C: Compared the effect of dacomitinib, gefitinib, erlotinib, and lapatinib on epidermal growth factor (EGF)-induced proliferation of PASMCs (* P < 0.05 compared with Normoxia, # P < 0.05 compared with EGF, n = 6). D: Compared the effect of dacomitinib, gefitinib, erlotinib, and lapatinib on the hypoxia-induced proliferation of PASMCs (* P < 0.05 compared with Normoxia, # P < 0.05 compared with Hypoxia, n = 6). E: The protein expression of EGFR in PASMCs treated with hypoxia in the absence or presence of dacomitinib (** P < 0.01; n = 6). F: The mRNA expression of EGFR in PASMCs treated with hypoxia in the absence or presence of dacomitinib (** P < 0.01; n = 6). All values are presented as the mean ± S.E.M. Con, control; Nor, normoxia; Hyp, hypoxia; MCT, monocrotaline; Dac, Dacomitinib.
phase (Fig. 5A). We analyzed the protein levels of the cell cycle-related proteins cyclin A, cyclin D and of the cyclin-dependent kinase CDK1 and CDK2 in PASMCs. The expression levels of cyclin A, cyclin D, CDK1, and CDK2 were significantly increased after hypoxia, but these effects were attenuated by dacomitinib (Fig. 5B-C). These results confirmed that dacomitinib played an important role in cell cycle progression during hypoxia.
3.5. Dacomitinib promotes protective autophagy in PASMCs Our previous study has shown that autophagy-associated accumulation of LC3B-Ⅱ, Beclin (BECN-1) and degradation of SQSTM-1 were markedly increased in the lung tissues from hypoxic rats compared with the normal group (Mao et al., 2017). Previous studies suggested that TKIs including erlotinib and gefitinib, first generation EGFR TKIs, 102
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Fig. 4. Dacomitinib inhibited hypoxic-induced PASMCs proliferation and migration. A: Cultured PASMCs were exposed to dacomitinib treatment, Ki67 labeled fluorescence images were obtained through immunocytochemistry (n = 3). B: The protein expression of PCNA in PASMCs treated with hypoxia in the absence or presence of dacomitinib (* P < 0.05; ** P < 0.01; n = 6). C: Scratch-wounding cell migration assay. The migration induced by hypoxia was blocked by dacomitinib in PASMCs of human (** P < 0.01; n = 6). D: Boyden chamber migration assays were performed, and the number of migratory cells under normoxic and hypoxic conditions was assessed by crystal violet staining (** P < 0.01; n = 6). All values are presented as the mean ± S.E.M. Nor, normoxia; Hyp, hypoxia; Dac, Dacomitinib.
induce a protective autophagy to resist proliferation and cell cycle progression effects (Li et al., 2013). So, we speculated dacomitinib effecting in autophagy of PAH. First, we evaluated the expression of BECN-1, LC3B-Ⅱand SQSTM-1 in pulmonary arteries from animal models of experimental PAH (hypoxia-induced PAH or MCT-induced PAH) treated with dacomitinib. Hypoxia-induced or MCT-induced
highly expressed in BECN-1 and LC3B-Ⅱ was attenuated by dacomitinib and degradation of SQSTM-1 was markedly increased by dacomitinib (Fig. 6A–B). In PASMCs, hypoxia-induced elevated expressed of BECN-1 and LC3B-Ⅱ which was attenuated by dacomitinib, and degradation of SQSTM-1 was markedly increased by dacomitinib in PASMCs (Fig. 6C). To elucidate the inductive role of dacomitinib in autophagy, expression 103
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Fig. 5. Dacomitinib play an important role in PASMCs cell cycle progression. A: The number of cells in each phase of the cell cycle was examined by FACS analysis (n = 3). The Result showed that dacomitinib reduced the percentage of cells phase accompanied by a concomitant increase of cells at G0/G1 phase. B: The expression of cyclin A and cyclin D1 of PASMCs was inhibited by dacomitinib (** P < 0.01; n = 6). C: hypoxia-induced the increased the expression of CDK1 and CDK2 of PASMCs was inhibited by dacomitinib (** P < 0.01; n = 6).
cell cycle progression and promoted autophagy. (3) The analysis of dacomitinib affects the proliferation and migration, the cell cycle progression and autophagy of PASMCs are closely related to the activation of PI3K-AKT-mTOR signaling. Chronic hypoxia and MCT-induced PAH were the mainly models for studying human PAH (Pugliese et al., 2015). Both chronic hypoxia and MCT-induced PAH can promote pulmonary vascular remodeling, causing PAH and even heart faiure (Pugliese et al., 2015; Sakao and Tatsumi, 2011). MCT induced PAH involving inflammatory response, and closer to the pathogenesis of PAH associated with connective tissue disease (Gomez-Arroyo et al., 2012). PAH and cancer share growth factor and protein kinase signaling pathways that result in smooth muscle cell proliferation and vasculopathy((Alkhatib et al., 2016). Therefore, Our research with other laboratories have apply anti-tumor drugs (kinase inhibors) to experimental treatment of PAH models (Chhina et al., 2010; Dahal et al., 2010; Liang et al., 2017; Yu et al., 2018, 2015). Kinase inhibitors inhibited the proliferation of PAECs or PASMCs by blocking the signaling pathway of growth factors (Chhina et al., 2010; Dahal et al., 2010; Liang et al., 2017; Nielsen et al., 2017; Yu et al., 2018, 2015). However, apparent lack of translation of these two models for the human disease as kinase inhibitors are concerned. An accumulating of evidence has revealed that the cell differentiation, proliferation, survival, and migration mediated by EGFR/ErbB signaling are crucially involved in the pathobiology of PAH and thus this signaling is a major target for PAH therapy (Normanno et al., 2006; Wieduwilt and Moasser, 2008). Research has confirmed that gefitinib (Iressa) and erlotinib (Tarceva) reduced the RVSP, right heart hypertrophy and medial wall thickness and degree of muscularization of small peripheral pulmonary arteries revealed that the pulmonary vascular remodeling was improved in MCT-induced PAH, but none of the two inhibitors provided significant therapeutic benefit, either in hemodynamic or in pulmonary vascular remodeling in chronic hypoxic PAH (Dahal et al., 2010). The therapeutic effect of lapatinib (Tykerb) in the field of pulmonary hypertension is controversial (Dahal et al., 2010). Studies have shown that in two experimental model of PAH (hypoxia- or MCT-induced PAH), lapatinib did not reverse PAH and pulmonary vascular remodeling (Dahal et al., 2010). Some studies have shown experiments on animal models has shown an effect of lapatinib
of EGFR was knocked down with its inhibitor, dacomitinib. Further experiments confirmed the altered autophagy markers and the appearance of the eGFP-mRFP-LC3 adenoviruses under hypoxia were inhibited by dacomitinib (Fig. 6D). These results confirmed that dacomitinib played an important role in autophagy during hypoxia. 3.6. Dacomitinib attenuated pulmonary vascular remodeling and PASMCs proliferation involved PI3K-AKT-mTOR signaling The PI3K-AKT-mTOR pathway signaling pathway is critical in the regulation of cell growth, metabolism and survival, angiogenesis, tumor invasion, cell cycle regulation, and DNA repair (Courtney et al., 2010; Shen et al., 2013). Previous studies have confirmed that the EGF/EGFR regulation of autophagy through PI3K involves Akt and mTOR (Sobolewska et al., 2009). Interesting is that EGFR inhibitors, dacomitinib can affect autophagy (Brana et al., 2017). So, we investigated the role of dacomitinib affect PI3K-AKT-mTOR signaling pathway in animal models of experimental PAH and PASMCs. We evaluated the expression of PI3K-AKT-mTOR in pulmonary arteries from animal models of experimental PAH (hypoxia-induced PAH or MCT-induced PAH) treated with dacomitinib. The results showed that dacomitinib decreased the expression of p-Akt (Ser 473) and p-mTOR (Ser 248), without altering the total protein levels of Akt and mTOR (Fig. 7A–B). We observed similar results of p-Akt (Ser 473) and p-mTOR (Ser 248) in PASMCs (Fig. 7C–D). The above results indicated that dacomitinib attenuated pulmonary vascular remodeling and PASMCs proliferation involved PI3K-AKT-mTOR signaling. Fig. 8 4. Discussion and conclusions The major findings of this study are (1) the EGFR antagonists dacomitinib exhibit therapeutic efficacy in animal models of experimental PAH (including chronic hypoxia and MCT-induced PAH). Dacomitinib has a significant role in attenuating pulmonary artery pressure and RVH, and dacomitinib has a significant inhibitory effect on vascular remodeling. (2) Dacomitinib showed no significant difference from gefitinib, erlotinib, and lapatinib in inhibit of PASMCs proliferation, however, dacomitinib inhibited hypoxia-induced PASMCs migration, 104
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Fig. 6. Dacomitinib promotes protective autophagy in PASMCs. A-B: Autophagy-associated alteration of LC3B-, BECN-1, SQSTM1 were analyzed in the pulmonary vascular from hypoxia-induced PAH rats and MCT-induced PAH rats (* P < 0.05; ** P < 0.01; n = 6). C: Autophagy-associated alteration of LC3B-, BECN-1, SQSTM1 were analyzed in PASMCs were treated with dacomitinib (* P < 0.05; ** P < 0.01; n = 6). D: PASMCs was treated with the eGFP-mGFP-LC3B plasmid. Yellow and red dots refer to autolysosomes and autophagosomes, respectively (** P < 0.01; n = 4). All values are presented as the mean ± S.E.M. Nor, normoxia; Hyp, hypoxia; Dac, Dacomitinib.
in attenuating induced PAH in rats (Alkhatib et al., 2016). Lapatinib chronic use might be associated with the development of PAH (Alkhatib et al., 2016). Stopping treatment in cases where no other reasons were identified might be associated with reversibility of the elevated pulmonary artery pressure (Dahal et al., 2010). Our research first time showed that dacomitinib exhibit therapeutic efficacy in animal models of experimental PAH (including chronic hypoxia PAH and MCT-induced PAH). Dacomitinib has a significant role in attenuating pulmonary artery pressure and RVH, and dacomitinib has a significant inhibitory effect on vascular remodeling. Our study
provides a new indication of dacomitinib and provides a new idea for the treatment of PAH. However, the study has some limitations. Dacomitinib is still in clinical trials, the study of patients with PAH is currently unable to carry out, and could not determine whether dacomitinib has other adverse reactions, which we need in the follow-up experiments in the exploration. PASMCs proliferation and migration constitutes an important incentive for pulmonary vascular remodeling. Our results have confirmed that effect of dacomitinib could significantly alter the protein and mRNA level of EGFR and through blocking EGFR activation, attenuated 105
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Fig. 7. Dacomitinib attenuated pulmonary vascular remodeling and PASMCs proliferation involved PI3K-AKT-mTOR signaling. A–B: The expression of p-Akt (Ser 473) and p-mTOR (Ser 248) were analyzed by western-blot in pulmonary vascular from hypoxia-induced PAH rats and MCT-induced PAH rats (* P < 0.05; ** P < 0.01; n = 6). C-D: The expression of p-Akt (Ser 473) and p-mTOR (Ser 248) were analyzed by western-blot in PASMCs (* P < 0.05; ** P < 0.01; n = 6). All values are presented as the mean ± S.E.M. Nor, normoxia; Hyp, hypoxia; Dac, Dacomitinib.
the pulmonary vascular remodeling also reduced the positive rate of EGFR expression in PASMCs. Previous studies have shown that EGFR signaling contributes to vascular SMC proliferation and migration (Chan et al., 2003; Kalmes et al., 2000; Zhou et al., 2007). In addition, EGFR signaling has also been implicated in the survival of PASMCs (Merklinger et al., 2005). There are results showed that dacomitinib has an effect on cell viability, self-renewal, and proliferation both in EGFRamplified ± EGFRvIII GBM cells and H1975 human NSCLC line (Zahonero et al., 2015). Our results proved the solid evidence that dacomitinib through blocking EGFR activating mediated in hypoxiainduced proliferation and migration of PASMCs, resulting in reduced pulmonary vascular remodeling. Disorders of cell cycle components may lead to cell proliferation. The percentage of cancer cells in the S and G2 phases of the cell cycle significantly decreased, and Cdk1 and Cdk2 protein levels declined after dacomitinib treatment in SKOV3 and OV4 cells (Xu et al., 2016).
Dacomitinib can significantly regulate the cell cycle of cancer cells (Xu et al., 2016). In our research, the percentage of PASMCs in the S and G2 phases of the cell cycle significantly decreased, and cyclin A; cyclin D; Cdk1 and Cdk2 protein levels declined after dacomitinib treatment in PASMCs. This is the first time confirmed that dacomitinib regulates the cell cycle in PASMCs. Autophagy is a major intracellular degradation and recycling process that maintains cellular homeostasis, which is involved in structural and functional abnormalities of PAH (Mao et al., 2017). Previous studies suggested that EGFR tyrosine kinases inhibitors including erlotinib and gefitinib, the first generation EGFR tyrosine kinases inhibitors, induce a protective autophagy to resist theirs in proliferation and cell cycle progression effects (Li et al., 2013). Results showed that a novel autophagic inhibitor cepharanthine enhanced the anti-cancer property of dacomitinib in non-small cell lung cancer (Tang et al., 2018). Furthermore, some studies have confirmed that dacomitinib in phase III
Fig. 8. A schematic model showing the proposed mechanism for dacomitinib attenuated pulmonary vascular remodeling and pulmonary hypertension. 106
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clinical trials for NSCLC treatment induced a protective autophagy to decrease its anti-cancer effect (Tang et al., 2018). Combined treatment with cepharanthine increased the anti-proliferative and apoptotic effects of dacomitinib in vitro and enhanced the anti-cancer effect of dacomitinib in NCI-H1975 x-enograft mice (Tang et al., 2018). In view of the above studies, we explored the effect of dacomitinib on autophagy in PASMCs. Our study found that dacomitinib induced a protective autophagy in two experimental model of PAH (hypoxia or MCT induced PAH) and PASMCs, which effect may be an important mechanism that causes dacomitinib to attenuate pulmonary vascular remodeling. PI3K-AKT-mTOR signaling pathway is critical in the regulation of cell growth, metabolism and survival, angiogenesis, tumor invasion, cell cycle regulation and DNA repair (Courtney et al., 2010; Shen et al., 2013). Dacomitinib has been reported to be involved regulated the PI3K-AKT-mTOR signaling pathway in PTEN-deficient patient-derived tumor xenografts (Brana et al., 2017). Here, we found that PI3K/AKT/ mTOR signaling pathway involved two experimental model of PAH (hypoxia or MCT induced PAH) and PASMCs treated with dacomitinib. This is the first time confirmed that dacomitinib inhibited PI3K-AKTmTOR signaling pathway in PASMCs and animal models of hypoxiainduced PAH and MCT- induced PAH. In conclusion, this study indicates that dacomitinib inhibits hypoxia-induced proliferation, migration, autophagy and Cell cycle progression involved PI3K-AKT-mTOR signaling pathway. Moreover, dacomitinib has a significant inhibitory effect on the thickening of the media; fibrosis of the adventitia. Dacomitinib has a significant role in attenuating pulmonary artery pressure and right ventricular hypertrophy andmay serve as new potential therapeutic for the treatment of PAH.
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Acknowledgments This work was supported by National Natural Science Foundation of China (contract grant numbers: 31820103007 and 31771276 to D.Z.; 81800047 to X.Y.) University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (contract grant numbers: UNPYSCT-2018067 to X.Y.); Youth Science Foundation of Heilongjiang Province (contract grant number: QC2016111 to X.Y.); Postdoctoral Foundation of Heilongjiang Province (contract grant number: LBHZ16241 to X.Y.); Wu Liande Young Scientific Research Foundation of Harbin Medical University Daqing (contract grant number: DQWLD20 to X.Y.); China Postdoctoral Science Foundation Funded Project (contract grant number: 2016M591557 to X.Y.); and Academician Mr. Yu Weihan Foundation for Distinguished Young Scholars (contract grant numbers: DQYWH201601 to L.Q.). Author contributions Daling Zhu, Lihui Qu, Xiufeng Yu created this rearch design. Xiufeng Yu and Xijuan Zhao analysed data and drafeted manuscript. Junting Zhang performed animal experiments and histological experiments. Yiying Li, Ping Sheng, Cui Ma, Xuewei Hao and Qiao Hui performed the cell culture and molecular biology experiments. Lixin Zhang performed Echocardiography and cell cycle DNA analysis experiments. Xiaodong Zheng and Yan Xing performed The Hemodynamic Evaluation of PAH–model. All authors read and approved the manuscript. Conflict of interests There is no conflict of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ejphar.2019.02.008. 107
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