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European Journal of Pharmacology 856 (2019) 172400
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Simvastatin inhibits the proliferation of HL-60 clone 15- derived eosinophils by inducing the arrest of the cell cycle in the G1/S phase
T
Chia-Hsiang Fua,b, Ta-Jen Leeb, Chi-Che Huanga,b, Po-Hung Changa,b, Jung-Wei Tsaia, Li-Pang Chuanga,c,*, Jong-Hwei Su Panga,d,∗ a
Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Tao-Yuan City, 333-02, Taiwan Department of Otolaryngology- Head and Neck Surgery, Chang Gung Memorial Hospital, Tao-Yuan City, 333-02, Taiwan c Department of Thoracic Medicine, Chang Gung Memorial Hospital, Tao-Yuan City, 333-02, Taiwan d Department of Physical Medicine and Rehabilitation, Chang Gung Memorial Hospital, Taoyuan City, 333-02, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Eosinophil HL-60 Eosinophil cationic protein ECP Simvastatin Cell cycle
Eosinophils and their granular proteins are crucial for combating allergic airway diseases. Eosinophils derived from HL-60 clone 15 (HC15) cells have been established as a feasible alternative cell model for human primary eosinophils. Simvastatin, a cholesterol-lowering agent, has been shown to exhibit anti-inflammatory and antiallergic effects. Among the granular eosinophil proteins, eosinophil cationic protein (ECP) is the one best recognised in allergic airway diseases. The aim of our study is to investigate the effect and regulatory mechanisms of simvastatin on ECP levels derived from eosinophils. Both HC15 cell counts and ECP levels decreased after simvastatin treatment in the animal and cell models; however, after a cell count adjustment, simvastatin was not observed to exert a significantly inhibitory effect on ECP expression. Real-time polymerase chain reaction and Western blotting analyses demonstrated that simvastatin did not inhibit the intracellular formation or release of ECP. Cell cycle analysis showed that the percentage of HC15 cells in the G1 and S phases significantly increased and decreased, respectively, after simvastatin treatment. Simvastatin inhibited the proliferation of HC15-derived eosinophils by inducing G1/S cell cycle arrest in a dose-dependent manner. Its effect on the cell cycle involved the downregulation of cyclin A but without the presence of mevalonate; therefore, total ECP expression from eosinophils decreased, not by suppressing the actual formation or release of ECP but by arresting the G1/S cell cycle phase and inhibiting subsequent cell proliferation through the mevalonate pathway.
1. Introduction Atopic airway diseases, such as allergic rhinitis and asthma, have critical impacts on the global health system, and their prevalence continues to increase (Chong and Chew, 2018; Lundbäck et al., 2016). Eosinophils play key roles in their pathogenesis after activation, migration into tissues and release of granular proteins. Among the granular proteins, eosinophil cationic protein (ECP) is the best characterised and most often used as a biomarker for the severity of allergic airway diseases. High ECP levels are highly predictive of an upcoming allergic attack, later development and poor asthma treatment response (Mogensen et al., 2016; Nielsen et al., 2009; Topic and Dodig, 2011). ECP can also serve as a prognostic marker for chronic rhinosinusitis, which would indicate that higher ECP levels result in more severe symptoms and higher rates of postoperative recurrence (Kim et al., 2013). The ability of eosinophils to produce ECPs is modified during
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secretion, most likely through post-translational glycosylation (Salazar et al., 2014). Eosinophils in sputum are associated with asthma control (Demarche et al., 2017; Wagener et al., 2015), and blood eosinophil counts significantly correlate with the stability and therapeutic effects of asthma (Krug et al., 2017; Zhang et al., 2014). Counts of blood eosinophils and their infiltration into the nasal tissue can predict symptom severity and post-surgery improvements in eosinophilic chronic rhinosinusitis or nasal polyps (Soler et al., 2010; Sreeparvathi et al., 2017). Treatments to attenuate their proliferation and survival would be effective for atopic diseases. Statins, which are cholesterol-lowering drugs, have additional immunomodulatory and anti-proliferative effects by involving the cell cycle (Mascitelli and Goldstein, 2017). For example, the arrest of tendon cell proliferation after statin therapy might cause tendinopathies (Tsai et al., 2016). Several statins involve the cell cycle and have anti-tumorigenic effects on diverse cancer cell lines (Henslee and
Corresponding authors. Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Tao-Yuan City, 333-02, Taiwan. E-mail addresses: [email protected] (L.-P. Chuang), [email protected] (J.-H. Su Pang).
https://doi.org/10.1016/j.ejphar.2019.05.029 Received 22 November 2018; Received in revised form 8 May 2019; Accepted 13 May 2019 Available online 16 May 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
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2.3. Immunohistochemical examination and scoring
Steele, 2018; Kany et al., 2018). Abdelrahman et al. (2015) showed that by inducing cell cycle arrest, statin treatment increased cell death and radiosensitivity in a myeloma cell line. The proliferative response of T cells is inhibited by statins owing to cell cycle arrest at the G0/G1 phase in patients with chronic idiopathic urticaria (Azor et al., 2011). Statin treatment reduces asthmatic airway inflammation in in vivo murine models (Gu et al., 2017; Han et al., 2017) and can supplement inhaled corticosteroids for asthmatics, with the additional effect of inhibiting eosinophils in sputum (Maneechotesuwan et al., 2010). We have shown that statins attenuate chemokine receptors on eosinophils and subsequent chemotactic abilities through the mevalonate pathway in an in vitro HL-60 clone 15 (HC15) cell model, an in vivo allergic rat model and human primary eosinophils (Fu et al., 2016); consequently, adequate doses and administration could play a therapeutic role in eosinophil-related allergic airway diseases. To our knowledge, the effect of statins on granular protein production and eosinophil proliferation has never been reported. This study aimed to determine the effect of simvastatin on ECP production and eosinophil cell proliferation to help advance our therapeutic principles related to atopic airway diseases.
Nasal turbinate tissue, which has been documented as the location of greater cellular infiltration of eosinophils (Fortin et al., 2010), was harvested from the lateral nasal wall of each allergic rat. After washing with PBS, the tissue was immersed in a fixative with 10% neutralbuffered formalin for 24 h at room temperature (RT) and embedded in paraffin for histological examination. Turbinate sections 5 μm thick were created, transferred to slides using an adherent coating and deparaffinised with xylene. The slides were soaked in a solution of 3% H2O2 for 15 min at RT to block endogenous peroxidases. After the slides were washed three times with PBS, primary ECP antibody (MyBioSource) was applied to each section and incubated for 30 min at RT. The slides were again washed three times with PBS, biotinylated goat antirabbit secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) was applied, and the tissues were incubated for 15 min at RT. Incubation was terminated by washing three times with PBS, after which the slides were conjugated with streptavidin–peroxidase (Thermo Fisher Scientific) for 10 min. After washing again three times with PBS, the sections were incubated in freshly prepared 3,3′-diaminobenzidine (DAB) substrate (Dako, Santa Clara, CA, USA) for 10 min. After rinsing and washing three times with PBS, the slides were counterstained with modified Harris hematoxylin solution (Sigma-Aldrich) for 2 min. After washing again with PBS, the slides were rinsed with ddH2O to avoid salt precipitation, after which the tissues were dehydrated, cleared and mounted. As the representative for ECP expression, a combined immunohistochemical (IHC) scoring system was applied with assessment of staining intensity (0–3) multiplied by the percentage of stained area (0–100%) for each intensity to obtain a final IHC score of 0–300 (Kuo et al., 2017; Alì et al., 2013). Strong, moderate, weak and negative staining intensities were assigned a score of 3, 2, 1 and 0, respectively. For each of the intensity scores, the corresponding percentage of stained area that stained at such level was estimated visually. IHC staining overview was performed by two independent physicians (C.C. Huang and P.H. Chang) who were blinded to the groups of the allergic rats.
2. Materials and methods 2.1. Reagents Simvastatin, RPMI-1640 medium, butyric acid (BA), mevalonate, ovalbumin (OVA), Triton X-100 and propidium iodide (PI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). ECP and the antibodies used to analyse ECP were purchased from MyBioSource (San Diego, CA, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Proteintech (Rosemont, IL, USA). The following primary antibodies were used in Western blotting to analyse cell cycle-associated proteins: mouse monoclonal anti-GAPDH (Proteintech, Rosemont, IL, USA), rabbit polyclonal anti-cdk 1, anti-cdk 2, and anti-cyclin B1 (Abclonal, Cambridge, MA, USA), and rabbit polyclonal anti-cyclin A and anti-cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The applied secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse IgG (Leinco Technologies Inc., St. Louis, MO, USA) and anti-rabbit IgG (Cell signaling Technology, Danvers, MA, USA).
2.4. Cell cultures HL-60 clone 15 (HC15) cells, derived from a leukaemia cell line, can be induced to differentiate into eosinophils after treatment with butyric acid in mildly alkaline conditions for 5–7 days (Fischkoff, 1988). The characteristics of these cells to respond to eosinophilic chemoattractants and produce eosinophil granular proteins and enzymes make them suitable for the study of eosinophilic functions (Fu et al., 2016). Briefly, HC15 cells obtained from Bioresource Collection and Research Center (Taiwan, ROC) were grown in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum purchased from Thermo Fisher Scientific and maintained under alkaline conditions (pH 7.6–7.8) to ensure eosinophil differentiation. Cells at 1 × 106 cells/ml were subcultured in a 1:5 dilution in fresh growth medium and maintained at 37 °C in an atmosphere of 95% air/5% CO2. After treating with 0.5 mM BA for 5 d, eosinophilic differentiation of the HC15 cells was induced, and the activated eosinophil simulation was used in the following experiments, as per our previous study (Fu et al., 2016).
2.2. Allergy rat model Pathogen-free male Sprague Dawley® rats (BioLASCO Taiwan Co., Ltd., Taiwan) 10–12 weeks old and weighing 150–250 g each were used in this study with approval by the Institutional Animal Care and Use Committee of Chang Gung University, Taoyuan City, Taiwan (No. CGU14-143). The rats were maintained in a temperature- and lightcontrolled room with free access to food and water. OVA sensitisation and challenge were conducted as previously reported (Fu et al., 2016). Briefly, the rats were divided into three groups as follows: control, sensitised and treatment. The rats in the sensitised and treatment groups were sensitised by subcutaneously injecting 1 ml saline containing 1 mg OVA (2 × 10 μg/0.1 ml) and 3.5 mg aluminium hydroxide gel (2%) on day 1; the control group received an injection of only phosphate buffered saline (PBS). The rats in the treatment group were administered 40 mg/kg simvastatin intragastrically 1 d before the aerosolised allergen challenge. Fourteen days after sensitisation, the allergen challenge was administered to all groups. The rats were administered aerosolised 0.5% (w/v) OVA through the PARI BOY® nebuliser (PARI GmbH, Starnberg, Germany) for 30 min/d for 3 consecutive days in a 40 × 50 × 60 cm exposure chamber, with an airflow rate of 4.41 L/min and mean air particle diameter of 3.7 μm. All rats were killed the day after the 3-d challenge was completed.
2.5. RNA extraction and real-time polymerase chain reaction The following RNA extraction and polymerase chain reaction (PCR) procedures were conducted as previously described (Fu et al., 2016). Briefly, the cells were lysed in 0.5 ml TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 100 μl chloroform–isoamyl alcohol (49:1 v:v) was added to the homogenate. After vortexing for 1 min, the solution was centrifuged at 13,362×g for 20 min at 4 °C. The RNA was precipitated by adding 0.5 ml isopropanol and stored for 1 h at −80 °C. After centrifuging the solution at 13,362×g for 20 min at 4 °C, the RNA 2
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procedures were conducted as described by Tsai et al. (2016). Briefly, after washing twice with PBS and centrifuging twice at 300×g for 5 min each, the cells were resuspended in 1 ml 0.5% (v/v) Triton X-100 (Sigma-Aldrich), 0.05% DNAse-free RNase A (Sigma-Aldrich) and 1 ml 50 μg/ml PI staining solution in PBS. Following incubation at 37 °C for 1 h, the stained cells were stored at 4 °C overnight before being analysed by flow cytometry using FACScan (Becton Dickinson, San Francisco, CA, USA).
pellet was rinsed in ice-cold 75% ethanol, air-dried and dissolved in diethyl pyrocarbonate-treated ddH2O. The cDNA was synthesised from total RNA using Moloney murine leukaemia virus reverse transcriptase (USB Corporation, Cleveland, OH, USA). Real-time PCR was conducted under programmable cycling conditions (15 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 1 min at 55 °C and 30 s at 72 °C). Cell cycle threshold values were determined by automated threshold analysis using an Mx-Pro Mx3005P v 4.00 real-time PCR detection system (Agilent Technologies, Santa Clara, CA, USA) with IQTM SYBR Green Supermix (Bio-Rad Labs, Hercules, CA, USA) according to the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was applied as an internal control. Oligonucleotide sequences for primers were as follows: GAPDH (forward: 5′-CACCTGACCTGCCG TCTA-3′; reverse: 5′-AGGAGTGGGTGTCGCTGT-3′); ECP (forward: 5′-AGATTCCGGGTGCCTTTACT-3′; reverse: 5′-AGGTGAACTGGAACCA CAGG-3′).
2.9. Statistical analyses The Mann–Whitney test was used to compare the presence of cdk, percentage of cells in each cell cycle and percentage of apoptotic cells among the groups. All values are expressed as the mean ± standard error of mean (S.E.M.). Statistical significance was set at P < 0.05 and was determined using Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA).
2.6. Enzyme-linked immunosorbent assay 3. Results ECP was measured in the culture supernatant using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (human RNASE3/ECP ELISA kit, MyBioSource) following the manufacturer's instructions. Briefly, the first dilution was 1:100, and the samples were further diluted over various calibration ranges. The plate was washed twice before adding the standard and sample wells. To each well, 100 μl of either standard or sample was added and kept at 37 °C for 90 min, after which 100 μl biotin-detection antibody was added as the working solution to each well and kept at 37 °C for 1 h. The plate was then washed three times with wash buffer, and 100 μl streptavidin conjugate working solution was applied to each well and incubated at 37 °C for 30 min. The plate was washed again five times and incubated with 90 μl 3,3′,5,5′-tetramethylbenzidine substrate at 37 °C for 15 min. Finally, 50 μl stop solution was added and mixed thoroughly, the optical density at 450 nm was read immediately using a microplate reader, and the concentration of ECP was calculated.
3.1. ECP in an allergic animal model After 3 consecutive days of OVA challenge, compared with the rats in the control group, nose rubbing and scratching events increased in the sensitised group but almost diminished in the simvastatin treatment group. To understand the mechanism by which simvastatin inhibits an allergic reaction, the expression of ECP in the in vivo model was investigated. We demonstrated the histological changes in sensitised nasal turbinate tissue during the allergen challenge using an allergic rat model (Fu et al., 2016). Greater submucosal cellular infiltration in the sensitised group and attenuated enhancement in the simvastatin treatment group were observed. We then determined the presence and levels of ECP, a powerful cytotoxic product of eosinophils created during an allergic reaction, in nasal turbinate tissue. Compared with the control group, immunohistochemical staining of ECP revealed a strongly enhanced expression in the sensitised group and a significantly inhibited expression in the simvastatin treatment group (Fig. 1). The overall expression of ECP in the allergic rat turbinate tissue markedly increased after the ovalbumin (OVA) challenge in the sensitised group and was suppressed after pre-treatment with simvastatin.
2.7. Western blotting Western blotting was conducted as previously described (Fu et al., 2016). Briefly, total proteins from the cell extracts were prepared in lysis buffer containing Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 2 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride and 1% Triton X-100. Protein concentration from the cell extracts was determined by Bradford assay (Bio-Rad Laboratories). Samples containing identical quantities of protein were separated using 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membrane was incubated at RT in blocking solution (1% bovine serum albumin and 1% goat serum in PBS) for 1 h, followed by incubation for 2 h in blocking solution containing an appropriate dilution of primary antibodies. After washing, the membrane was incubated for 1 h in PBS containing secondary antibodies conjugated with horseradish peroxidase (Sigma-Aldrich). After washing three times, the positive signals on the membranes were developed with enhanced chemiluminescence reagent (Amershan Pharmacia Biotech, Little Chalfont Buckinghamshire, UK). To quantify the band density of each protein normalised to GAPDH, the semi-quantitative measurement was calculated using digital analysis software (Kodak Digital Science TM, Eastman Kodak, Rochester, NY, USA).
3.2. Simvastatin effect on ECP To determine the significant correlation between ECP levels and the clinical severity of atopic airway diseases, as well as the inhibitory effects of statins on airway inflammation (Mogensen et al., 2016; Kim et al., 2013), the results of simvastatin treatment on ECP expression were investigated. Differentiation of HC15 cells toward eosinophils after treatment with BA for 5 d was documented in our previous study (Fu et al., 2016). We then examined the effect of simvastatin on the intracellular formation and extracellular release of ECP from the HC15 cells. After treating with different concentrations of simvastatin for 24 h, the expression level of ECP from the HC15 cells was analysed using real-time PCR (Fig. 2A). The results demonstrated that the expression of the ECP mRNA gene in the HC15 cells was not affected by simvastatin treatment. The cell culture supernatant from the HC15 cells with or without simvastatin treatment was collected, and a commercial ECP ELISA kit was used to determine the levels of ECP. Both the number of HC15 cells and ECP levels decreased after simvastatin treatment. Although the difference did not reach a significant level (P = 0.095), the number of HC15 cells tended to decrease at 25 μM simvastatin treatment (Fig. 2B). After adjusting the cell count, simvastatin was not observed to significantly suppress actual ECP expression (Fig. 2C). In addition, the conditioned medium of the HC15 cells with or without simvastatin treatment was collected, and ECP levels were analysed using Western
2.8. Cell cycle analysis HC15 cells were washed with PBS and fixed with 1 ml 70% ethanol in ice-cold PBS for 1 h. Statins can suppress the formation of mevalonate by inhibiting HMA-CoA reductase. To reverse this effect, mevalonate was supplemented along with simvastatin treatment. The 3
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Fig. 1. Eosinophil cationic protein (ECP) expression in nasal tissue in the allergic animal model. Rats in the control group were not sensitised by subcutaneous ovalbumin (OVA) injection on day 1. The treatment group was treated with simvastatin orally on day 14, 1 d before the OVA challenge. Rats in all groups received the aerosolised OVA after 15 d and were killed after 3 consecutive days of the challenge to examine ECP expression in nasal turbinate mucosa using immunohistochemical staining and a light microscope (400 × ) in (A) the control group, (B) the sensitised group and (C) the simvastatin treatment group. (D) The immunohistochemical stain (IHC) score of ECP expression significantly increased in the sensitised group and decreased in the simvastatin treatment group. Data are representative results from five independent replicates. *P < 0.05.
blotting. The ECP protein levels did not differ between the HC15 cells with and those without simvastatin treatment (Fig. 2D), which indicated that simvastatin had no effect on the release and production of ECP from the HC15 cells. At this point, we observed that simvastatin treatment could decrease the number of HC15 cells, but not its production or release from HC15-derived eosinophils. 3.3. Simvastatin induced cell cycle arrest in the G1/S phase of HC15 cells We treated HC15 cells with different concentrations of simvastatin (0, 5, 10 or 25 μM) for 24 h before cell cycle analysis. We found an increased percentage of cells in the G1 phase and a significant decreased percentage in the S phase after simvastatin treatment (Fig. 3; supplementary material, Table S1). Simvastatin was observed to arrest the cell cycle in a dose-dependent manner, which reached a statistical significance for both G1 and S phases at a concentration of 25 μM (both P = 0.008). The cell count percentages in the G2/M phase did not change with or without simvastatin treatment.
Fig. 3. The cell cycle was altered after simvastatin treatment. HC15 cells were treated with 0, 5, 10 or 25 μM simvastatin and subjected to cell cycle analysis using flow cytometry. The percentage of HC15 cells increased in the G1 phase and decreased in the S phase after co-treatment with simvastatin for 24 h. No statistical significance was found in the G2/M phase before and after treating with different concentrations of simvastatin. Data are the mean ± S.E.M. calculated from five independent experiments. *P < 0.05.
3.4. Simvastatin altered the expression of cyclins and cdks in HC15 cells To determine how simvastatin modulated the HC15 cell cycle, we investigated the expression of cell cycle-related cyclins and cdks. HC15
Fig. 2. Eosinophil cationic protein (ECP) expression did not differ after simvastatin treatment. (A) Analysis of ECP mRNA expression in HC15 cells after treatment with 0, 5, 10 or 25 μM simvastatin for 24 h using real-time polymerase chain reaction. The mRNA expression of ECP in HC15 cells did not differ with different concentrations of simvastatin treatment (P > 0.05); (B) HC15 cells counts after treatment with different concentrations of simvastatin; although not reach a statistical significance, cell counts tended to decrease at 25 μM simvastatin; (C) after a cell count adjustment, the expression of ECP levels in HC15 cells with different concentrations of simvastatin was analysed using enzyme-linked immunosorbent assay; there were no significant differences in ECP expression among the different concentrations of simvastatin (P > 0.05); (D) ECP protein levels in HC15 cells after treating with different concentrations of simvastatin were analysed using Western blotting; the levels did not differ before and after treatment (P > 0.05). Data are the mean ± S.E.M. calculated from four independent experiments. 4
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Fig. 4. Simvastatin inhibited the expression of cyclin A. (A) HC15 cells were treated with 0, 5, 10 or 25 μM simvastatin, and cell cycle-related cyclins and cdks were analysed using Western blotting; (B) relative band intensity revealed that cyclin A expression was inhibited by simvastatin treatment at a concentration of 25 μM. Data are representative results from experiments repeated at least four times. *P < 0.05.
combined with the pretreatment of 0.5 μM mevalonate for 1 h. The expression of proteins was examined by Western blotting, and the results showed that mevalonate supplement significantly reversed the inhibitory effect of simvastatin on the expression of cyclin A (Fig. 5B and C; supplementary material, Table S3). Collectively, the inhibitory effects of simvastatin on the cell cycle and its associated protein expression were mediated by blocking the mevalonate pathway.
cells were co-treated with 25 μM simvastatin for 24 h. The expression of associated cyclins and cdks was shown in Fig. 4. The inhibitory effect of simvastatin on cyclin A expression reached a statistical significance at 25 μM simvastatin (P = 0.029). No inhibitory effects on the protein levels of cyclin B1, cyclin E, cdk 1 or cdk 2 were observed after treatment with different concentrations of simvastatin. 3.5. Mevalonate reversed the effect of simvastatin on the cell cycle and the associated cyclin expression
3.6. Simvastatin induced cell apoptosis in HC15 cells in a dose-dependent manner
Statins inhibit HMA-CoA reductase by blocking mevalonate production. We examined the restorative effect of mevalonate to determine whether the effect of simvastatin on the cell cycle was mediated by the mevalonate pathway. HC15 cells were treated with either 25 μM simvastatin for 24 h or combined with the pretreatment of 0.5 μM mevalonate for 1 h. The results of flow cytometry demonstrated that mevalonate could reverse the inhibition of cell proliferation (Fig. 5A; supplementary material, Table S2). The effect of simvastatin on cell counts in the G1 and S phases recovered if HC15 cells were co-treated with mevalonate. To examine whether simvastatin-mediated inhibition of cell cyclerelated protein expression could also be reversed by mevalonate pretreatment, HC15 cells were treated with simvastatin alone for 24 h or
The effect of simvastatin treatment on cell apoptosis was also investigated. We treated HC15 cells with 0, 5, 10 or 25 μM simvastatin for 24 h and assessed the percentage of apoptotic cells using flow cytometry. The results revealed an increased percentage of apoptotic cells in a dose-dependent manner, and with significance observed at 10 and 25 μM simvastatin (P = 0.016 and 0.008, respectively; Fig. 6). Compared with the control group, apoptotic cells increased by an average of 3.42% after 25 μM simvastatin treatment, and the replacement of mevalonate could reverse the apoptosis induced by simvastatin treatment (Fig. 6; supplementary material, Table S4).
Fig. 5. Mevalonate supplementation reversed the effect of simvastatin. HC15 cells were treated with 25 μM simvastatin in either the presence or absence of 0.5 μM mevalonate. (A) The percentage of HC15 cells increased in the G1 phase and decreased in the S phase with simvastatin treatment, but the effects of simvastatin were reversed by supplementing with mevalonate; (B) Western blotting showed that the inhibitory effect of simvastatin on the expression of cell cyclerelated cyclin A was reversed in the presence of mevalonate; (C) relative band intensity revealed that the inhibitory effect of 25 μM simvastatin on the expression of cyclin A was reversed in the presence of mevalonate. Data are representative results from individual experiments repeated four times. *P < 0.05. 5
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in anaplastic thyroid cancers (Chen et al., 2017a). Further, other statins have been documented to present similar effects in HL-60 cells. Mevastatin has been reported to induce cell apoptosis of HL-60 cells by activating the caspase cascade through the modulation of mitochondrial functions (Kanno et al., 2002). Another lipophilic statin, lovastatin, has been documented to induce G1 arrest and apoptosis of HL60 cells in association with p27 production and Bcl-2 down-regulation, respectively (Park et al., 1999). The involved cell cycle–associated proteins included the decrease of cdk2/4/6 and cyclin E, which were not exactly the same as those found in this study. In the present investigation, the percentage of cells increased in G1 and significantly decreased in S phase after simvastatin treatment in a dose-dependent manner, leading to the arrest of HC15 cells at the G1/S transition. This effect could be reversed by supplementing with mevalonate, which is the downstream product of the mevalonate pathway. To our knowledge, this is the first study to demonstrate the ability of simvastatin to arrest the cell cycle at the G1/S phase and inhibit the proliferation of HC15-derived eosinophils through the mevalonate pathway. Despite the enhanced apoptosis of HC15 cells after simvastatin treatment in a dose-dependent manner, the percentage of apoptotic cells increased by only ∼3.4%, even at a simvastatin concentration of 25 μM. We might conclude that the significant decrease in HC15 cells after simvastatin treatment depended mainly on the inhibition of cell proliferation instead of on the enhancement of apoptosis. HMG-CoA reductase has been reported to be indispensable in T cell proliferation (Lacher et al., 2017). Through mevalonate replacement instead of the addition of cholesterol, cell death of HMG-CoA reductasedeficient lymphocytes could be prevented. Inhibition of HMG-CoA reductase by different statins would result in cell growth limitation, cell proliferation inhibition or apoptosis in various cell types through involvement of Akt signaling or ERK pathway (Beckwitt et al., 2018; Yeh et al., 2018; Chen et al., 2017a, b; Liu et al., 2015). Our previous investigation demonstrated that the inhibition effect of simvastatin for eosinophil chemotaxis was associated with the suppression of phosphorylation activity of p38 MAPK and ERK1/2 (Fu et al., 2016). Several cell cycle-related cyclins and cdks might be involved in the simvastatin effect on the cell cycle. Cyclin E functions at the G1/S boundary, while cyclin A is induced at a later phase, is required for progression through the G1/S transition and has the ability to activate cdk2 and cdk1 (Canavese et al., 2012; Jayapal et al., 2016). Cdk2 activity, which is restricted to the G1/S phase, is required for DNA replication, and its binding with cyclin E and A is essential for G1/S transition and the progression through the S phase, respectively (Sánchez and Dynlacht, 2005). Cdk1 governs the G2/M transition and drives cells into mitosis (Prevo et al., 2018). The simvastatin that affects cell cycle arrest and the associated cyclins has been reported in other cell types (Stine et al., 2016; Tsai et al., 2016). In this investigation, the downregulation of cyclin A in simvastatin-treated HC15-derived eosinophils further demonstrated the molecular mechanism of the arrest of the G1/S phase and subsequently provided the mechanism by which cell proliferation and eosinophilic inflammation are inhibited. In our previous study, we demonstrated that simvastatin significantly inhibits chemotaxis and CC chemokine receptor type 3 (CCR3) expression of activated HC15-derived eosinophils through the mevalonate pathway (Fu et al., 2016). This 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor in the current study could induce cell cycle G1/S arrest and inhibit cell proliferation of HC15derived eosinophil through the same pathway. Thus, this cholesterollowering agent has the ability to induce cell cycle arrest, cell proliferation and the CCR3-related chemotactic activity of eosinophils. Statins could most likely provide both mechanisms, if applied to the adequate route and at the correct dosage, to have favourable anti-eosinophilic inflammation effects and could be another effective therapeutic choice for treatment of allergic airway diseases.
Fig. 6. Mevalonate replacement reversed simvastatin-induced cell apoptosis. HC15 cells were treated with 0, 5, 10 or 25 μM simvastatin, and the percentage of apoptotic cells was analysed using flow cytometry. The percentage of apoptotic cells significantly increased with 10 and 25 μM simvastatin. Data are representative results from five independent replicates. *P < 0.05 compared with the control (without simvastatin); **P < 0.05 compared with 25 μM simvastatin treatment without mevalonate replacement.
4. Discussion Previous studies have documented that HC15-derived eosinophils can produce typical granular proteins and migrate across transwell filters, as observed in human primary eosinophils (Fischkoff et al., 1986; Fu et al., 2016). For eosinophils that make up only a small percentage in human peripheral blood, a suitable cell model can serve as an alternative to human primary eosinophils and help us understand more about the cellular features of the major components in allergic diseases. Phenotypes of atopic airway diseases could be distinct based on their inflammatory patterns and specific cytokine profiles, and various new therapeutic approaches have been developed for eosinophilic airway inflammation that is resistant to standard treatments (Van Crombruggen et al., 2011). ECP, an eosinophil-derived granular protein, has been recognised as an indicator for helper T cell type 2 (Th2)-related airway inflammation (Bystrom et al., 2011). Eosinophils release ECP after leaving the circulation system, which causes toxicity to epithelial cells, resulting in airway epithelial damage, hyper-reactivity and airway remodelling (Acharya and Ackerman, 2014; de Oliveira et al., 2012). ECP has been confirmed to exhibit post-translational processing during release, with its cytotoxicity enhanced by N-deglycosylation (Salazar et al., 2014; Woschnagg et al., 2009). Thus, the presentation of this eosinophilic granular protein involves its intracellular production and release from eosinophils. The relationship between the effect of statins on eosinophil-derived granular proteins and the reduction in airway inflammation has not been established. After a cell count adjustment in this investigation, the total ECP expression from HC15 cell-derived eosinophils decreased after simvastatin treatment only because the cell count decreased and not because simvastatin inhibited the actual production of ECP. The intracellular formation and extracellular release of ECP from HC15 cells were not attenuated by simvastatin treatment. Eosinophil proliferation has been reported to be associated with the severity (Ban et al., 2017; Poznanovic and Kingdom, 2007) and prognosis (Matsuwaki et al., 2008) of allergic airway diseases. Because statin treatment can attenuate airway eosinophilic inflammation, we investigated the details of the effects of simvastatin on cell proliferation and cell apoptosis of HC15-derived eosinophils. The main effect of a decrease in cell count after simvastatin treatment appeared to result from suppression of cell proliferation. Simvastatin has been report to have similar effects in different cell types, such as G1/S arrest in tendon cells (Tsai et al., 2016), G0/G1 arrest in hepatocellular carcinomas (Wang et al., 2017), and G1/S arrest 6
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5. Conclusion
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Impact of mucosal eosinophilia and nasal polyposis on quality-of-life outcomes after sinus surgery. Otolaryngol. Head Neck Surg. 142, 64–71. Sreeparvathi, A., Kalyanikuttyamma, L.K., Kumar, M., Sreekumar, N., Veerasigamani, N., 2017. Significance of blood eosinophil count in patients with chronic rhinosinusitis
The attenuated expression of ECP, a powerful indicator of allergic airway diseases, in HC15-derived eosinophils after simvastatin treatment did not result from the inhibition of ECP formation or its release, but from a decrease in cell counts. Simvastatin arrested the G1/S cell cycle phase to inhibit subsequent HC15 cell proliferation through the mevalonate pathway. This effect involved the downregulation of cyclin A and could be reversed by adding mevalonate, a downstream product of the mevalonate pathway. The present study could provide another treatment option for those whose allergic airway diseases are refractory to standard therapies. Conflicts of interest The authors declared no conflict of interest. Author contributions Conceived and designed the experiments: C.H.F. and J.H.S.P.; Performed the experiments: C.H.F., J.W.T and L.P.C.; Analysed the data: C.H.F., T.J.L. and J.H.S.P.; Contributed materials/analysis tools: C.C.H. and P.H.C.; Wrote the manuscript: C.H.F. and J.H.S.P. Acknowledgments This study was supported by grants from Chang Gung Memorial Hospital (CMRPG3E1671). We are grateful for the assistance provided by the faculty laboratory of Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejphar.2019.05.029. References Abdelrahman, I.Y., Helwa, R., Elkashef, H., Hassan, N.H., 2015. Induction of P3NS1 myeloma cell death and cell cycle arrest by simvastatin and/or γ-radiation. Asian Pac. J. Cancer Prev. APJCP 16, 7103–7110. Acharya, K.R., Ackerman, S.J., 2014. Eosinophil granule proteins: form and function. J. Biol. Chem. 289, 17406–17415. Alì, G., Borrelli, N., Riccardo, G., Proietti, A., Pelliccioni, S., Niccoli, C., Boldrini, L., Lucchi, M., Mussi, A., Fontanini, G., 2013. Differential expression of extracellular matrix constituents and cell adhesion molecules between malignant pleural mesothelioma and mesothelial hyperplasia. J. Thorac. Oncol. 8, 1389–1395. Azor, M.H., dos Santos, J.C., Futata, E.A., de Brito, C.A., Maruta, C.W., Rivitti, E.A., da Silva Duarte, A.J., Sato, M.N., 2011. Statin effects on regulatory and proinflammatory factors in chronic idiopathic urticaria. Clin. Exp. Immunol. 166, 291–298. Beckwitt, C.H., Shiraha, K., Wells, A., 2018. Lipophilic statins limit cancer cell growth and survival, via involvement of Akt signaling. PLoS One 13, e0197422. Ban, G.Y., Ye, Y.M., Kim, S.H., Hur, G.Y., Kim, J.H., Shim, J.J., Cho, K., Cho, J.Y., Park, H.S., PRANA group, 2017. Plasma LTE4/PGF2α ratio and blood eosinophil count are increased in elderly asthmatics with previous asthma exacerbation. Allergy Asthma Immunol. Res. 9, 378–382. Bystrom, J., Amin, K., Bishop-Bailey, D., 2011. Analysing the eosinophil cationic proteina clue to the function of the eosinophil granulocyte. Respir. Res. 12, 10. Canavese, M., Santo, L., Raje, N., 2012. Cyclin dependent kinases in cancer: potential for therapeutic intervention. Cancer Biol. Ther. 13, 451–457. Chen, M.C., Tsai, Y.C., Tseng, J.H., Liou, J.J., Horng, S., Wen, H.C., Fan, Y.C., Zhong, W.B., Hsu, S.P., 2017a. Simvastatin inhibits cell proliferation and migration in human anaplastic thyroid cancer. Int. J. Mol. Sci. 18, E2690. Chen, S., Dong, S., Li, Z., Guo, X., Zhang, N., Yu, B., Sun, Y., 2017b. Atorvastatin calcium inhibits PDGF-ββ-induced proliferation and migration of VSMCs through the G0/G1 cell cycle arrest and suppression of activated PDGFRβ-PI3K-Akt signaling cascade. Cell. Physiol. Biochem. 44, 215–228. Chong, S.N., Chew, F.T., 2018. Epidemiology of allergic rhinitis and associated risk factors in Asia. World Allergy Organ J 11, 17. de Oliveira, P.C., de Lima, P.O., Oliveira, D.T., Pereira, M.C., 2012. Eosinophil cationic protein: overview of biological and genetic features. DNA Cell Biol. 31, 1442–1446. Demarche, S.F., Schleich, F.N., Paulus, V.A., Henket, M.A., Van Hees, T.J., Louis, R.E., 2017. Asthma control and sputum eosinophils: a longitudinal study in daily practice. J. Allergy Clin. Immunol. Pract. 5, 1334–1343.
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Wagener, A.H., de Nijs, S.B., Lutter, R., Sousa, A.R., Weersink, E.J., Bel, E.H., Sterk, P.J., 2015. External validation of blood eosinophils, FE(NO) and serum periostin as surrogates for sputum eosinophils in asthma. Thorax 70, 115–120. Woschnagg, C., Rubin, J., Venge, P., 2009. Eosinophil cationic protein (ECP) is processed during secretion. J. Immunol. 183, 3949–3954. Yeh, Y.S., Jheng, H.F., Iwase, M., Kim, M., Mohri, S., Kwon, J., Kawarasaki, S., Li, Y., Takahashi, H., Ara, T., Nomura, W., Kawada, T., Goto, T., 2018. The mevalonate pathway is indispensable for adipocyte survival. iScience 9, 175–191. Zhang, X.Y., Simpson, J.L., Powell, H., Yang, I.A., Upham, J.W., Reynolds, P.N., Hodge, S., James, A.L., Jenkins, C., Peters, M.J., Lin, J.T., Gibson, P.G., 2014. Full blood count parameters for the detection of asthma inflammatory phenotypes. Clin. Exp. Allergy 44, 1137–1145.
with nasal polyposis. J. Clin. Diagn. Res. 11 MC08–MC11. Topic, R.Z., Dodig, S., 2011. Eosinophil cationic protein- current concepts and controversies. Biochem. Med. 21, 111–121. Tsai, W.C., Yu, T.Y., Lin, L.P., Cheng, M.L., Chen, C.L., Pang, J.H., 2016. Prevention of Simvastatin-induced inhibition of tendon cell proliferation and cell cycle progression by geranylgeranyl pyrophosphate. Toxicol. Sci. 149, 326–334. Van Crombruggen, K., Zhang, N., Gevaert, P., Tomassen, P., Bachert, C., 2011. Pathogenesis of chronic rhinosinusitis: inflammation. J. Allergy Clin. Immunol. 128, 728–732. Wang, S.T., Ho, H.J., Lin, J.T., Shieh, J.J., Wu, C.Y., 2017. Simvastatin-induced cell cycle arrest through inhibition of STAT3/SKP2 axis and activation of AMPK to promote p27 and p21 accumulation in hepatocellular carcinoma cells. Cell Death Dis. 8, e2626.
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Full length article
Simvastatin inhibits the proliferation of HL-60 clone 15- derived eosinophils by inducing the arrest of the cell cycle in the G1/S phase
T
Chia-Hsiang Fua,b, Ta-Jen Leeb, Chi-Che Huanga,b, Po-Hung Changa,b, Jung-Wei Tsaia, Li-Pang Chuanga,c,*, Jong-Hwei Su Panga,d,∗ a
Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Tao-Yuan City, 333-02, Taiwan Department of Otolaryngology- Head and Neck Surgery, Chang Gung Memorial Hospital, Tao-Yuan City, 333-02, Taiwan c Department of Thoracic Medicine, Chang Gung Memorial Hospital, Tao-Yuan City, 333-02, Taiwan d Department of Physical Medicine and Rehabilitation, Chang Gung Memorial Hospital, Taoyuan City, 333-02, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Eosinophil HL-60 Eosinophil cationic protein ECP Simvastatin Cell cycle
Eosinophils and their granular proteins are crucial for combating allergic airway diseases. Eosinophils derived from HL-60 clone 15 (HC15) cells have been established as a feasible alternative cell model for human primary eosinophils. Simvastatin, a cholesterol-lowering agent, has been shown to exhibit anti-inflammatory and antiallergic effects. Among the granular eosinophil proteins, eosinophil cationic protein (ECP) is the one best recognised in allergic airway diseases. The aim of our study is to investigate the effect and regulatory mechanisms of simvastatin on ECP levels derived from eosinophils. Both HC15 cell counts and ECP levels decreased after simvastatin treatment in the animal and cell models; however, after a cell count adjustment, simvastatin was not observed to exert a significantly inhibitory effect on ECP expression. Real-time polymerase chain reaction and Western blotting analyses demonstrated that simvastatin did not inhibit the intracellular formation or release of ECP. Cell cycle analysis showed that the percentage of HC15 cells in the G1 and S phases significantly increased and decreased, respectively, after simvastatin treatment. Simvastatin inhibited the proliferation of HC15-derived eosinophils by inducing G1/S cell cycle arrest in a dose-dependent manner. Its effect on the cell cycle involved the downregulation of cyclin A but without the presence of mevalonate; therefore, total ECP expression from eosinophils decreased, not by suppressing the actual formation or release of ECP but by arresting the G1/S cell cycle phase and inhibiting subsequent cell proliferation through the mevalonate pathway.
1. Introduction Atopic airway diseases, such as allergic rhinitis and asthma, have critical impacts on the global health system, and their prevalence continues to increase (Chong and Chew, 2018; Lundbäck et al., 2016). Eosinophils play key roles in their pathogenesis after activation, migration into tissues and release of granular proteins. Among the granular proteins, eosinophil cationic protein (ECP) is the best characterised and most often used as a biomarker for the severity of allergic airway diseases. High ECP levels are highly predictive of an upcoming allergic attack, later development and poor asthma treatment response (Mogensen et al., 2016; Nielsen et al., 2009; Topic and Dodig, 2011). ECP can also serve as a prognostic marker for chronic rhinosinusitis, which would indicate that higher ECP levels result in more severe symptoms and higher rates of postoperative recurrence (Kim et al., 2013). The ability of eosinophils to produce ECPs is modified during
∗
secretion, most likely through post-translational glycosylation (Salazar et al., 2014). Eosinophils in sputum are associated with asthma control (Demarche et al., 2017; Wagener et al., 2015), and blood eosinophil counts significantly correlate with the stability and therapeutic effects of asthma (Krug et al., 2017; Zhang et al., 2014). Counts of blood eosinophils and their infiltration into the nasal tissue can predict symptom severity and post-surgery improvements in eosinophilic chronic rhinosinusitis or nasal polyps (Soler et al., 2010; Sreeparvathi et al., 2017). Treatments to attenuate their proliferation and survival would be effective for atopic diseases. Statins, which are cholesterol-lowering drugs, have additional immunomodulatory and anti-proliferative effects by involving the cell cycle (Mascitelli and Goldstein, 2017). For example, the arrest of tendon cell proliferation after statin therapy might cause tendinopathies (Tsai et al., 2016). Several statins involve the cell cycle and have anti-tumorigenic effects on diverse cancer cell lines (Henslee and
Corresponding authors. Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Tao-Yuan City, 333-02, Taiwan. E-mail addresses: [email protected] (L.-P. Chuang), [email protected] (J.-H. Su Pang).
https://doi.org/10.1016/j.ejphar.2019.05.029 Received 22 November 2018; Received in revised form 8 May 2019; Accepted 13 May 2019 Available online 16 May 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
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2.3. Immunohistochemical examination and scoring
Steele, 2018; Kany et al., 2018). Abdelrahman et al. (2015) showed that by inducing cell cycle arrest, statin treatment increased cell death and radiosensitivity in a myeloma cell line. The proliferative response of T cells is inhibited by statins owing to cell cycle arrest at the G0/G1 phase in patients with chronic idiopathic urticaria (Azor et al., 2011). Statin treatment reduces asthmatic airway inflammation in in vivo murine models (Gu et al., 2017; Han et al., 2017) and can supplement inhaled corticosteroids for asthmatics, with the additional effect of inhibiting eosinophils in sputum (Maneechotesuwan et al., 2010). We have shown that statins attenuate chemokine receptors on eosinophils and subsequent chemotactic abilities through the mevalonate pathway in an in vitro HL-60 clone 15 (HC15) cell model, an in vivo allergic rat model and human primary eosinophils (Fu et al., 2016); consequently, adequate doses and administration could play a therapeutic role in eosinophil-related allergic airway diseases. To our knowledge, the effect of statins on granular protein production and eosinophil proliferation has never been reported. This study aimed to determine the effect of simvastatin on ECP production and eosinophil cell proliferation to help advance our therapeutic principles related to atopic airway diseases.
Nasal turbinate tissue, which has been documented as the location of greater cellular infiltration of eosinophils (Fortin et al., 2010), was harvested from the lateral nasal wall of each allergic rat. After washing with PBS, the tissue was immersed in a fixative with 10% neutralbuffered formalin for 24 h at room temperature (RT) and embedded in paraffin for histological examination. Turbinate sections 5 μm thick were created, transferred to slides using an adherent coating and deparaffinised with xylene. The slides were soaked in a solution of 3% H2O2 for 15 min at RT to block endogenous peroxidases. After the slides were washed three times with PBS, primary ECP antibody (MyBioSource) was applied to each section and incubated for 30 min at RT. The slides were again washed three times with PBS, biotinylated goat antirabbit secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) was applied, and the tissues were incubated for 15 min at RT. Incubation was terminated by washing three times with PBS, after which the slides were conjugated with streptavidin–peroxidase (Thermo Fisher Scientific) for 10 min. After washing again three times with PBS, the sections were incubated in freshly prepared 3,3′-diaminobenzidine (DAB) substrate (Dako, Santa Clara, CA, USA) for 10 min. After rinsing and washing three times with PBS, the slides were counterstained with modified Harris hematoxylin solution (Sigma-Aldrich) for 2 min. After washing again with PBS, the slides were rinsed with ddH2O to avoid salt precipitation, after which the tissues were dehydrated, cleared and mounted. As the representative for ECP expression, a combined immunohistochemical (IHC) scoring system was applied with assessment of staining intensity (0–3) multiplied by the percentage of stained area (0–100%) for each intensity to obtain a final IHC score of 0–300 (Kuo et al., 2017; Alì et al., 2013). Strong, moderate, weak and negative staining intensities were assigned a score of 3, 2, 1 and 0, respectively. For each of the intensity scores, the corresponding percentage of stained area that stained at such level was estimated visually. IHC staining overview was performed by two independent physicians (C.C. Huang and P.H. Chang) who were blinded to the groups of the allergic rats.
2. Materials and methods 2.1. Reagents Simvastatin, RPMI-1640 medium, butyric acid (BA), mevalonate, ovalbumin (OVA), Triton X-100 and propidium iodide (PI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). ECP and the antibodies used to analyse ECP were purchased from MyBioSource (San Diego, CA, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Proteintech (Rosemont, IL, USA). The following primary antibodies were used in Western blotting to analyse cell cycle-associated proteins: mouse monoclonal anti-GAPDH (Proteintech, Rosemont, IL, USA), rabbit polyclonal anti-cdk 1, anti-cdk 2, and anti-cyclin B1 (Abclonal, Cambridge, MA, USA), and rabbit polyclonal anti-cyclin A and anti-cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The applied secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse IgG (Leinco Technologies Inc., St. Louis, MO, USA) and anti-rabbit IgG (Cell signaling Technology, Danvers, MA, USA).
2.4. Cell cultures HL-60 clone 15 (HC15) cells, derived from a leukaemia cell line, can be induced to differentiate into eosinophils after treatment with butyric acid in mildly alkaline conditions for 5–7 days (Fischkoff, 1988). The characteristics of these cells to respond to eosinophilic chemoattractants and produce eosinophil granular proteins and enzymes make them suitable for the study of eosinophilic functions (Fu et al., 2016). Briefly, HC15 cells obtained from Bioresource Collection and Research Center (Taiwan, ROC) were grown in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum purchased from Thermo Fisher Scientific and maintained under alkaline conditions (pH 7.6–7.8) to ensure eosinophil differentiation. Cells at 1 × 106 cells/ml were subcultured in a 1:5 dilution in fresh growth medium and maintained at 37 °C in an atmosphere of 95% air/5% CO2. After treating with 0.5 mM BA for 5 d, eosinophilic differentiation of the HC15 cells was induced, and the activated eosinophil simulation was used in the following experiments, as per our previous study (Fu et al., 2016).
2.2. Allergy rat model Pathogen-free male Sprague Dawley® rats (BioLASCO Taiwan Co., Ltd., Taiwan) 10–12 weeks old and weighing 150–250 g each were used in this study with approval by the Institutional Animal Care and Use Committee of Chang Gung University, Taoyuan City, Taiwan (No. CGU14-143). The rats were maintained in a temperature- and lightcontrolled room with free access to food and water. OVA sensitisation and challenge were conducted as previously reported (Fu et al., 2016). Briefly, the rats were divided into three groups as follows: control, sensitised and treatment. The rats in the sensitised and treatment groups were sensitised by subcutaneously injecting 1 ml saline containing 1 mg OVA (2 × 10 μg/0.1 ml) and 3.5 mg aluminium hydroxide gel (2%) on day 1; the control group received an injection of only phosphate buffered saline (PBS). The rats in the treatment group were administered 40 mg/kg simvastatin intragastrically 1 d before the aerosolised allergen challenge. Fourteen days after sensitisation, the allergen challenge was administered to all groups. The rats were administered aerosolised 0.5% (w/v) OVA through the PARI BOY® nebuliser (PARI GmbH, Starnberg, Germany) for 30 min/d for 3 consecutive days in a 40 × 50 × 60 cm exposure chamber, with an airflow rate of 4.41 L/min and mean air particle diameter of 3.7 μm. All rats were killed the day after the 3-d challenge was completed.
2.5. RNA extraction and real-time polymerase chain reaction The following RNA extraction and polymerase chain reaction (PCR) procedures were conducted as previously described (Fu et al., 2016). Briefly, the cells were lysed in 0.5 ml TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 100 μl chloroform–isoamyl alcohol (49:1 v:v) was added to the homogenate. After vortexing for 1 min, the solution was centrifuged at 13,362×g for 20 min at 4 °C. The RNA was precipitated by adding 0.5 ml isopropanol and stored for 1 h at −80 °C. After centrifuging the solution at 13,362×g for 20 min at 4 °C, the RNA 2
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procedures were conducted as described by Tsai et al. (2016). Briefly, after washing twice with PBS and centrifuging twice at 300×g for 5 min each, the cells were resuspended in 1 ml 0.5% (v/v) Triton X-100 (Sigma-Aldrich), 0.05% DNAse-free RNase A (Sigma-Aldrich) and 1 ml 50 μg/ml PI staining solution in PBS. Following incubation at 37 °C for 1 h, the stained cells were stored at 4 °C overnight before being analysed by flow cytometry using FACScan (Becton Dickinson, San Francisco, CA, USA).
pellet was rinsed in ice-cold 75% ethanol, air-dried and dissolved in diethyl pyrocarbonate-treated ddH2O. The cDNA was synthesised from total RNA using Moloney murine leukaemia virus reverse transcriptase (USB Corporation, Cleveland, OH, USA). Real-time PCR was conducted under programmable cycling conditions (15 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 1 min at 55 °C and 30 s at 72 °C). Cell cycle threshold values were determined by automated threshold analysis using an Mx-Pro Mx3005P v 4.00 real-time PCR detection system (Agilent Technologies, Santa Clara, CA, USA) with IQTM SYBR Green Supermix (Bio-Rad Labs, Hercules, CA, USA) according to the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was applied as an internal control. Oligonucleotide sequences for primers were as follows: GAPDH (forward: 5′-CACCTGACCTGCCG TCTA-3′; reverse: 5′-AGGAGTGGGTGTCGCTGT-3′); ECP (forward: 5′-AGATTCCGGGTGCCTTTACT-3′; reverse: 5′-AGGTGAACTGGAACCA CAGG-3′).
2.9. Statistical analyses The Mann–Whitney test was used to compare the presence of cdk, percentage of cells in each cell cycle and percentage of apoptotic cells among the groups. All values are expressed as the mean ± standard error of mean (S.E.M.). Statistical significance was set at P < 0.05 and was determined using Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA).
2.6. Enzyme-linked immunosorbent assay 3. Results ECP was measured in the culture supernatant using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (human RNASE3/ECP ELISA kit, MyBioSource) following the manufacturer's instructions. Briefly, the first dilution was 1:100, and the samples were further diluted over various calibration ranges. The plate was washed twice before adding the standard and sample wells. To each well, 100 μl of either standard or sample was added and kept at 37 °C for 90 min, after which 100 μl biotin-detection antibody was added as the working solution to each well and kept at 37 °C for 1 h. The plate was then washed three times with wash buffer, and 100 μl streptavidin conjugate working solution was applied to each well and incubated at 37 °C for 30 min. The plate was washed again five times and incubated with 90 μl 3,3′,5,5′-tetramethylbenzidine substrate at 37 °C for 15 min. Finally, 50 μl stop solution was added and mixed thoroughly, the optical density at 450 nm was read immediately using a microplate reader, and the concentration of ECP was calculated.
3.1. ECP in an allergic animal model After 3 consecutive days of OVA challenge, compared with the rats in the control group, nose rubbing and scratching events increased in the sensitised group but almost diminished in the simvastatin treatment group. To understand the mechanism by which simvastatin inhibits an allergic reaction, the expression of ECP in the in vivo model was investigated. We demonstrated the histological changes in sensitised nasal turbinate tissue during the allergen challenge using an allergic rat model (Fu et al., 2016). Greater submucosal cellular infiltration in the sensitised group and attenuated enhancement in the simvastatin treatment group were observed. We then determined the presence and levels of ECP, a powerful cytotoxic product of eosinophils created during an allergic reaction, in nasal turbinate tissue. Compared with the control group, immunohistochemical staining of ECP revealed a strongly enhanced expression in the sensitised group and a significantly inhibited expression in the simvastatin treatment group (Fig. 1). The overall expression of ECP in the allergic rat turbinate tissue markedly increased after the ovalbumin (OVA) challenge in the sensitised group and was suppressed after pre-treatment with simvastatin.
2.7. Western blotting Western blotting was conducted as previously described (Fu et al., 2016). Briefly, total proteins from the cell extracts were prepared in lysis buffer containing Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 2 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride and 1% Triton X-100. Protein concentration from the cell extracts was determined by Bradford assay (Bio-Rad Laboratories). Samples containing identical quantities of protein were separated using 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membrane was incubated at RT in blocking solution (1% bovine serum albumin and 1% goat serum in PBS) for 1 h, followed by incubation for 2 h in blocking solution containing an appropriate dilution of primary antibodies. After washing, the membrane was incubated for 1 h in PBS containing secondary antibodies conjugated with horseradish peroxidase (Sigma-Aldrich). After washing three times, the positive signals on the membranes were developed with enhanced chemiluminescence reagent (Amershan Pharmacia Biotech, Little Chalfont Buckinghamshire, UK). To quantify the band density of each protein normalised to GAPDH, the semi-quantitative measurement was calculated using digital analysis software (Kodak Digital Science TM, Eastman Kodak, Rochester, NY, USA).
3.2. Simvastatin effect on ECP To determine the significant correlation between ECP levels and the clinical severity of atopic airway diseases, as well as the inhibitory effects of statins on airway inflammation (Mogensen et al., 2016; Kim et al., 2013), the results of simvastatin treatment on ECP expression were investigated. Differentiation of HC15 cells toward eosinophils after treatment with BA for 5 d was documented in our previous study (Fu et al., 2016). We then examined the effect of simvastatin on the intracellular formation and extracellular release of ECP from the HC15 cells. After treating with different concentrations of simvastatin for 24 h, the expression level of ECP from the HC15 cells was analysed using real-time PCR (Fig. 2A). The results demonstrated that the expression of the ECP mRNA gene in the HC15 cells was not affected by simvastatin treatment. The cell culture supernatant from the HC15 cells with or without simvastatin treatment was collected, and a commercial ECP ELISA kit was used to determine the levels of ECP. Both the number of HC15 cells and ECP levels decreased after simvastatin treatment. Although the difference did not reach a significant level (P = 0.095), the number of HC15 cells tended to decrease at 25 μM simvastatin treatment (Fig. 2B). After adjusting the cell count, simvastatin was not observed to significantly suppress actual ECP expression (Fig. 2C). In addition, the conditioned medium of the HC15 cells with or without simvastatin treatment was collected, and ECP levels were analysed using Western
2.8. Cell cycle analysis HC15 cells were washed with PBS and fixed with 1 ml 70% ethanol in ice-cold PBS for 1 h. Statins can suppress the formation of mevalonate by inhibiting HMA-CoA reductase. To reverse this effect, mevalonate was supplemented along with simvastatin treatment. The 3
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Fig. 1. Eosinophil cationic protein (ECP) expression in nasal tissue in the allergic animal model. Rats in the control group were not sensitised by subcutaneous ovalbumin (OVA) injection on day 1. The treatment group was treated with simvastatin orally on day 14, 1 d before the OVA challenge. Rats in all groups received the aerosolised OVA after 15 d and were killed after 3 consecutive days of the challenge to examine ECP expression in nasal turbinate mucosa using immunohistochemical staining and a light microscope (400 × ) in (A) the control group, (B) the sensitised group and (C) the simvastatin treatment group. (D) The immunohistochemical stain (IHC) score of ECP expression significantly increased in the sensitised group and decreased in the simvastatin treatment group. Data are representative results from five independent replicates. *P < 0.05.
blotting. The ECP protein levels did not differ between the HC15 cells with and those without simvastatin treatment (Fig. 2D), which indicated that simvastatin had no effect on the release and production of ECP from the HC15 cells. At this point, we observed that simvastatin treatment could decrease the number of HC15 cells, but not its production or release from HC15-derived eosinophils. 3.3. Simvastatin induced cell cycle arrest in the G1/S phase of HC15 cells We treated HC15 cells with different concentrations of simvastatin (0, 5, 10 or 25 μM) for 24 h before cell cycle analysis. We found an increased percentage of cells in the G1 phase and a significant decreased percentage in the S phase after simvastatin treatment (Fig. 3; supplementary material, Table S1). Simvastatin was observed to arrest the cell cycle in a dose-dependent manner, which reached a statistical significance for both G1 and S phases at a concentration of 25 μM (both P = 0.008). The cell count percentages in the G2/M phase did not change with or without simvastatin treatment.
Fig. 3. The cell cycle was altered after simvastatin treatment. HC15 cells were treated with 0, 5, 10 or 25 μM simvastatin and subjected to cell cycle analysis using flow cytometry. The percentage of HC15 cells increased in the G1 phase and decreased in the S phase after co-treatment with simvastatin for 24 h. No statistical significance was found in the G2/M phase before and after treating with different concentrations of simvastatin. Data are the mean ± S.E.M. calculated from five independent experiments. *P < 0.05.
3.4. Simvastatin altered the expression of cyclins and cdks in HC15 cells To determine how simvastatin modulated the HC15 cell cycle, we investigated the expression of cell cycle-related cyclins and cdks. HC15
Fig. 2. Eosinophil cationic protein (ECP) expression did not differ after simvastatin treatment. (A) Analysis of ECP mRNA expression in HC15 cells after treatment with 0, 5, 10 or 25 μM simvastatin for 24 h using real-time polymerase chain reaction. The mRNA expression of ECP in HC15 cells did not differ with different concentrations of simvastatin treatment (P > 0.05); (B) HC15 cells counts after treatment with different concentrations of simvastatin; although not reach a statistical significance, cell counts tended to decrease at 25 μM simvastatin; (C) after a cell count adjustment, the expression of ECP levels in HC15 cells with different concentrations of simvastatin was analysed using enzyme-linked immunosorbent assay; there were no significant differences in ECP expression among the different concentrations of simvastatin (P > 0.05); (D) ECP protein levels in HC15 cells after treating with different concentrations of simvastatin were analysed using Western blotting; the levels did not differ before and after treatment (P > 0.05). Data are the mean ± S.E.M. calculated from four independent experiments. 4
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Fig. 4. Simvastatin inhibited the expression of cyclin A. (A) HC15 cells were treated with 0, 5, 10 or 25 μM simvastatin, and cell cycle-related cyclins and cdks were analysed using Western blotting; (B) relative band intensity revealed that cyclin A expression was inhibited by simvastatin treatment at a concentration of 25 μM. Data are representative results from experiments repeated at least four times. *P < 0.05.
combined with the pretreatment of 0.5 μM mevalonate for 1 h. The expression of proteins was examined by Western blotting, and the results showed that mevalonate supplement significantly reversed the inhibitory effect of simvastatin on the expression of cyclin A (Fig. 5B and C; supplementary material, Table S3). Collectively, the inhibitory effects of simvastatin on the cell cycle and its associated protein expression were mediated by blocking the mevalonate pathway.
cells were co-treated with 25 μM simvastatin for 24 h. The expression of associated cyclins and cdks was shown in Fig. 4. The inhibitory effect of simvastatin on cyclin A expression reached a statistical significance at 25 μM simvastatin (P = 0.029). No inhibitory effects on the protein levels of cyclin B1, cyclin E, cdk 1 or cdk 2 were observed after treatment with different concentrations of simvastatin. 3.5. Mevalonate reversed the effect of simvastatin on the cell cycle and the associated cyclin expression
3.6. Simvastatin induced cell apoptosis in HC15 cells in a dose-dependent manner
Statins inhibit HMA-CoA reductase by blocking mevalonate production. We examined the restorative effect of mevalonate to determine whether the effect of simvastatin on the cell cycle was mediated by the mevalonate pathway. HC15 cells were treated with either 25 μM simvastatin for 24 h or combined with the pretreatment of 0.5 μM mevalonate for 1 h. The results of flow cytometry demonstrated that mevalonate could reverse the inhibition of cell proliferation (Fig. 5A; supplementary material, Table S2). The effect of simvastatin on cell counts in the G1 and S phases recovered if HC15 cells were co-treated with mevalonate. To examine whether simvastatin-mediated inhibition of cell cyclerelated protein expression could also be reversed by mevalonate pretreatment, HC15 cells were treated with simvastatin alone for 24 h or
The effect of simvastatin treatment on cell apoptosis was also investigated. We treated HC15 cells with 0, 5, 10 or 25 μM simvastatin for 24 h and assessed the percentage of apoptotic cells using flow cytometry. The results revealed an increased percentage of apoptotic cells in a dose-dependent manner, and with significance observed at 10 and 25 μM simvastatin (P = 0.016 and 0.008, respectively; Fig. 6). Compared with the control group, apoptotic cells increased by an average of 3.42% after 25 μM simvastatin treatment, and the replacement of mevalonate could reverse the apoptosis induced by simvastatin treatment (Fig. 6; supplementary material, Table S4).
Fig. 5. Mevalonate supplementation reversed the effect of simvastatin. HC15 cells were treated with 25 μM simvastatin in either the presence or absence of 0.5 μM mevalonate. (A) The percentage of HC15 cells increased in the G1 phase and decreased in the S phase with simvastatin treatment, but the effects of simvastatin were reversed by supplementing with mevalonate; (B) Western blotting showed that the inhibitory effect of simvastatin on the expression of cell cyclerelated cyclin A was reversed in the presence of mevalonate; (C) relative band intensity revealed that the inhibitory effect of 25 μM simvastatin on the expression of cyclin A was reversed in the presence of mevalonate. Data are representative results from individual experiments repeated four times. *P < 0.05. 5
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in anaplastic thyroid cancers (Chen et al., 2017a). Further, other statins have been documented to present similar effects in HL-60 cells. Mevastatin has been reported to induce cell apoptosis of HL-60 cells by activating the caspase cascade through the modulation of mitochondrial functions (Kanno et al., 2002). Another lipophilic statin, lovastatin, has been documented to induce G1 arrest and apoptosis of HL60 cells in association with p27 production and Bcl-2 down-regulation, respectively (Park et al., 1999). The involved cell cycle–associated proteins included the decrease of cdk2/4/6 and cyclin E, which were not exactly the same as those found in this study. In the present investigation, the percentage of cells increased in G1 and significantly decreased in S phase after simvastatin treatment in a dose-dependent manner, leading to the arrest of HC15 cells at the G1/S transition. This effect could be reversed by supplementing with mevalonate, which is the downstream product of the mevalonate pathway. To our knowledge, this is the first study to demonstrate the ability of simvastatin to arrest the cell cycle at the G1/S phase and inhibit the proliferation of HC15-derived eosinophils through the mevalonate pathway. Despite the enhanced apoptosis of HC15 cells after simvastatin treatment in a dose-dependent manner, the percentage of apoptotic cells increased by only ∼3.4%, even at a simvastatin concentration of 25 μM. We might conclude that the significant decrease in HC15 cells after simvastatin treatment depended mainly on the inhibition of cell proliferation instead of on the enhancement of apoptosis. HMG-CoA reductase has been reported to be indispensable in T cell proliferation (Lacher et al., 2017). Through mevalonate replacement instead of the addition of cholesterol, cell death of HMG-CoA reductasedeficient lymphocytes could be prevented. Inhibition of HMG-CoA reductase by different statins would result in cell growth limitation, cell proliferation inhibition or apoptosis in various cell types through involvement of Akt signaling or ERK pathway (Beckwitt et al., 2018; Yeh et al., 2018; Chen et al., 2017a, b; Liu et al., 2015). Our previous investigation demonstrated that the inhibition effect of simvastatin for eosinophil chemotaxis was associated with the suppression of phosphorylation activity of p38 MAPK and ERK1/2 (Fu et al., 2016). Several cell cycle-related cyclins and cdks might be involved in the simvastatin effect on the cell cycle. Cyclin E functions at the G1/S boundary, while cyclin A is induced at a later phase, is required for progression through the G1/S transition and has the ability to activate cdk2 and cdk1 (Canavese et al., 2012; Jayapal et al., 2016). Cdk2 activity, which is restricted to the G1/S phase, is required for DNA replication, and its binding with cyclin E and A is essential for G1/S transition and the progression through the S phase, respectively (Sánchez and Dynlacht, 2005). Cdk1 governs the G2/M transition and drives cells into mitosis (Prevo et al., 2018). The simvastatin that affects cell cycle arrest and the associated cyclins has been reported in other cell types (Stine et al., 2016; Tsai et al., 2016). In this investigation, the downregulation of cyclin A in simvastatin-treated HC15-derived eosinophils further demonstrated the molecular mechanism of the arrest of the G1/S phase and subsequently provided the mechanism by which cell proliferation and eosinophilic inflammation are inhibited. In our previous study, we demonstrated that simvastatin significantly inhibits chemotaxis and CC chemokine receptor type 3 (CCR3) expression of activated HC15-derived eosinophils through the mevalonate pathway (Fu et al., 2016). This 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor in the current study could induce cell cycle G1/S arrest and inhibit cell proliferation of HC15derived eosinophil through the same pathway. Thus, this cholesterollowering agent has the ability to induce cell cycle arrest, cell proliferation and the CCR3-related chemotactic activity of eosinophils. Statins could most likely provide both mechanisms, if applied to the adequate route and at the correct dosage, to have favourable anti-eosinophilic inflammation effects and could be another effective therapeutic choice for treatment of allergic airway diseases.
Fig. 6. Mevalonate replacement reversed simvastatin-induced cell apoptosis. HC15 cells were treated with 0, 5, 10 or 25 μM simvastatin, and the percentage of apoptotic cells was analysed using flow cytometry. The percentage of apoptotic cells significantly increased with 10 and 25 μM simvastatin. Data are representative results from five independent replicates. *P < 0.05 compared with the control (without simvastatin); **P < 0.05 compared with 25 μM simvastatin treatment without mevalonate replacement.
4. Discussion Previous studies have documented that HC15-derived eosinophils can produce typical granular proteins and migrate across transwell filters, as observed in human primary eosinophils (Fischkoff et al., 1986; Fu et al., 2016). For eosinophils that make up only a small percentage in human peripheral blood, a suitable cell model can serve as an alternative to human primary eosinophils and help us understand more about the cellular features of the major components in allergic diseases. Phenotypes of atopic airway diseases could be distinct based on their inflammatory patterns and specific cytokine profiles, and various new therapeutic approaches have been developed for eosinophilic airway inflammation that is resistant to standard treatments (Van Crombruggen et al., 2011). ECP, an eosinophil-derived granular protein, has been recognised as an indicator for helper T cell type 2 (Th2)-related airway inflammation (Bystrom et al., 2011). Eosinophils release ECP after leaving the circulation system, which causes toxicity to epithelial cells, resulting in airway epithelial damage, hyper-reactivity and airway remodelling (Acharya and Ackerman, 2014; de Oliveira et al., 2012). ECP has been confirmed to exhibit post-translational processing during release, with its cytotoxicity enhanced by N-deglycosylation (Salazar et al., 2014; Woschnagg et al., 2009). Thus, the presentation of this eosinophilic granular protein involves its intracellular production and release from eosinophils. The relationship between the effect of statins on eosinophil-derived granular proteins and the reduction in airway inflammation has not been established. After a cell count adjustment in this investigation, the total ECP expression from HC15 cell-derived eosinophils decreased after simvastatin treatment only because the cell count decreased and not because simvastatin inhibited the actual production of ECP. The intracellular formation and extracellular release of ECP from HC15 cells were not attenuated by simvastatin treatment. Eosinophil proliferation has been reported to be associated with the severity (Ban et al., 2017; Poznanovic and Kingdom, 2007) and prognosis (Matsuwaki et al., 2008) of allergic airway diseases. Because statin treatment can attenuate airway eosinophilic inflammation, we investigated the details of the effects of simvastatin on cell proliferation and cell apoptosis of HC15-derived eosinophils. The main effect of a decrease in cell count after simvastatin treatment appeared to result from suppression of cell proliferation. Simvastatin has been report to have similar effects in different cell types, such as G1/S arrest in tendon cells (Tsai et al., 2016), G0/G1 arrest in hepatocellular carcinomas (Wang et al., 2017), and G1/S arrest 6
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5. Conclusion
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The attenuated expression of ECP, a powerful indicator of allergic airway diseases, in HC15-derived eosinophils after simvastatin treatment did not result from the inhibition of ECP formation or its release, but from a decrease in cell counts. Simvastatin arrested the G1/S cell cycle phase to inhibit subsequent HC15 cell proliferation through the mevalonate pathway. This effect involved the downregulation of cyclin A and could be reversed by adding mevalonate, a downstream product of the mevalonate pathway. The present study could provide another treatment option for those whose allergic airway diseases are refractory to standard therapies. Conflicts of interest The authors declared no conflict of interest. Author contributions Conceived and designed the experiments: C.H.F. and J.H.S.P.; Performed the experiments: C.H.F., J.W.T and L.P.C.; Analysed the data: C.H.F., T.J.L. and J.H.S.P.; Contributed materials/analysis tools: C.C.H. and P.H.C.; Wrote the manuscript: C.H.F. and J.H.S.P. Acknowledgments This study was supported by grants from Chang Gung Memorial Hospital (CMRPG3E1671). We are grateful for the assistance provided by the faculty laboratory of Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejphar.2019.05.029. References Abdelrahman, I.Y., Helwa, R., Elkashef, H., Hassan, N.H., 2015. Induction of P3NS1 myeloma cell death and cell cycle arrest by simvastatin and/or γ-radiation. Asian Pac. J. Cancer Prev. APJCP 16, 7103–7110. Acharya, K.R., Ackerman, S.J., 2014. Eosinophil granule proteins: form and function. J. Biol. Chem. 289, 17406–17415. Alì, G., Borrelli, N., Riccardo, G., Proietti, A., Pelliccioni, S., Niccoli, C., Boldrini, L., Lucchi, M., Mussi, A., Fontanini, G., 2013. Differential expression of extracellular matrix constituents and cell adhesion molecules between malignant pleural mesothelioma and mesothelial hyperplasia. J. Thorac. Oncol. 8, 1389–1395. Azor, M.H., dos Santos, J.C., Futata, E.A., de Brito, C.A., Maruta, C.W., Rivitti, E.A., da Silva Duarte, A.J., Sato, M.N., 2011. Statin effects on regulatory and proinflammatory factors in chronic idiopathic urticaria. Clin. Exp. Immunol. 166, 291–298. Beckwitt, C.H., Shiraha, K., Wells, A., 2018. Lipophilic statins limit cancer cell growth and survival, via involvement of Akt signaling. PLoS One 13, e0197422. Ban, G.Y., Ye, Y.M., Kim, S.H., Hur, G.Y., Kim, J.H., Shim, J.J., Cho, K., Cho, J.Y., Park, H.S., PRANA group, 2017. Plasma LTE4/PGF2α ratio and blood eosinophil count are increased in elderly asthmatics with previous asthma exacerbation. Allergy Asthma Immunol. Res. 9, 378–382. Bystrom, J., Amin, K., Bishop-Bailey, D., 2011. Analysing the eosinophil cationic proteina clue to the function of the eosinophil granulocyte. Respir. Res. 12, 10. Canavese, M., Santo, L., Raje, N., 2012. Cyclin dependent kinases in cancer: potential for therapeutic intervention. Cancer Biol. Ther. 13, 451–457. Chen, M.C., Tsai, Y.C., Tseng, J.H., Liou, J.J., Horng, S., Wen, H.C., Fan, Y.C., Zhong, W.B., Hsu, S.P., 2017a. Simvastatin inhibits cell proliferation and migration in human anaplastic thyroid cancer. Int. J. Mol. Sci. 18, E2690. Chen, S., Dong, S., Li, Z., Guo, X., Zhang, N., Yu, B., Sun, Y., 2017b. Atorvastatin calcium inhibits PDGF-ββ-induced proliferation and migration of VSMCs through the G0/G1 cell cycle arrest and suppression of activated PDGFRβ-PI3K-Akt signaling cascade. Cell. Physiol. Biochem. 44, 215–228. Chong, S.N., Chew, F.T., 2018. Epidemiology of allergic rhinitis and associated risk factors in Asia. World Allergy Organ J 11, 17. de Oliveira, P.C., de Lima, P.O., Oliveira, D.T., Pereira, M.C., 2012. Eosinophil cationic protein: overview of biological and genetic features. DNA Cell Biol. 31, 1442–1446. Demarche, S.F., Schleich, F.N., Paulus, V.A., Henket, M.A., Van Hees, T.J., Louis, R.E., 2017. Asthma control and sputum eosinophils: a longitudinal study in daily practice. J. Allergy Clin. Immunol. Pract. 5, 1334–1343.
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