Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Anticancer Drug-Induced Epithelial-Mesenchymal Transition via p53/miR-34a axis in A549/ABCA3 Cells

Anticancer Drug-Induced Epithelial-Mesenchymal Transition via p53/miR-34a axis in A549/ABCA3 Cells J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 Anticancer Drug-Induced Epithelial-Mesenchymal Transition via p53/miR-34a axis in A549/ABCA3 Cells Ayano Yamamoto, Masashi Kawami, Takashi Konaka, Shinnosuke Takenaka, Ryoko Yumoto, Mikihisa Takano Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Received, July 25, 2019; Revised, September 30, 2019; Accepted, October 4, 2019; Published, October 7, 2019. ABSTRACT - PURPOSE. Several anticancer drugs including bleomycin (BLM) and methotrexate (MTX) cause serious lung diseases such as pulmonary fibrosis. Although evidences showing the association of epithelial-mesenchymal transition (EMT) with pulmonary fibrosis are increasing, the mechanism underlying anticancer drug-induced EMT has been poorly understood. On the other hand, miR-34a, a non-coding small RNA, has been highlighted as a key factor to regulate EMT in lung. In this study, we elucidated the role of miR-34a in anticancer drug-induced EMT using A549/ABCA3 cells as a novel type II alveolar epithelium model. METHODS. Expression levels of α-smooth muscle actin (α-SMA) mRNA, miR-34a, and p53 were evaluated by real-time PCR and western blot analysis, respectively. RESULTS. BLM and MTX induced EMT-like morphological changes and increase in mRNA expression level of α-SMA, an EMT marker. Also, both drugs increased the expression level of miR-34a. Furthermore, mRNA expression level of α-SMA was enhanced by introduction of miR-34a mimic into A549/ABCA3 cells. To examine the mechanism underlying drug-induced enhancement of miR-34a expression, we focused on p53/miR-34a axis. Both drugs upregulated protein expression of p53, an inducer of miR-34a, as well as phosphorylation of Ser15 in p53. CONCLUSIONS. These findings indicated that p53/miR-34a axis may contribute to anticancer drug-induced EMT in type II alveolar epithelial cells. _______________________________________________________________________________________ INTRODUCTION MicroRNAs (miRNA) are small single-stranded non-coding RNA of approximately 22 nucleotides There are increasing drugs with capacity of inducing and regulate post-transcriptional activity of their serious cytotoxic effects on lung such as pulmonary targeted genes by binding complementarily to the 3’ fibrosis as their adverse effects. In particular, untranslated regions (9). Current studies have anticancer drugs including bleomycin (BLM) (1) demonstrated that a number of miRNAs have and methotrexate (MTX) (2) have been reported to positive or negative effects on IPF via complicated induce severe lung diseases such as acute interstitial transcriptional regulations (10). In particular, miR- pneumonia and progressive pulmonary fibrosis. 200 family such as miR-200b and miR-200c inhibits Although these serious pulmonary diseases carry EMT in RLE-6TN cells, by downregulating the high mortality rates, development of the effective expression of several EMT inducing factors treatment has not been achieved, partly due to poor including zinc finger E-box-binding homeobox understanding of the drug-induced lung injury. (ZEB) 1/2 (11). On the other hand, miR-199a Recently, there are several suggested schemes stimulates fibroblasts in response to transforming concerning pathogenesis of serious pulmonary growth factor (TGF)-β1 via inhibitory effect on diseases such as idiopathic pulmonary fibrosis (IPF) caveolin 1, a negative regulator of TGF-β signaling (3). In these schemes, excessive extracellular matrix pathway (12). Thus, certain miRNAs would be (ECM) plays a crucial role in development of considerable biomarkers or therapeutic targets for pathogenic lung form. ECM is mainly produced by EMT-related diseases. myofibroblasts, which are derived from fibroblasts In the lung, EMT occurs in type II alveolar showing profibrotic phenotype (4, 5). When lung epithelial cells, as evidenced by several reports using injury occurs, on the other hand, myofibroblasts are human primary type II alveolar epithelial cells and also generated from injured type II alveolar lungs of patients with IPF (6, 13). __________________________________________________ epithelial cells via epithelial-mesenchymal transition (EMT) process (6). Therefore, EMT contributes to Corresponding Author: Prof. Mikihisa Takano, Department of development of pulmonary fibrosis (7, 8), and may Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 be considered to be a possible therapeutic target to Kasumi, Minami-ku, Hiroshima 734-8553, Japan; E-mail: prevent the drug-induced lung injury. takanom@hiroshima-u.ac.jp 516 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 So far, we established RLE/Abca3 cells by (TOYOBO Co. Ltd., Osaka, Japan). The PCR introducing Abca3 gene into RLE-6TN cells (rat- conditions were as follows: initial denaturation in derived alveolar epithelial cells), and mRNA one cycle of 5 min at 95℃, 30 cycles with 30 s at expression levels of several type II alveolar 95℃ (denaturation), 30 s at 65℃ (annealing), and 3 epithelial cell markers such as peptide transporter 2 min at 72℃ (extension). The purified fragment A (PEPT2) and formation of lamellar bodies were and B were cloned into pIRESpuro2 Vector and higher in RLE/Abca3 cells than those in the wild- pGEM-T Easy Vector, respectively. These plasmids type cells (14). In addition, we found that BLM and were digested by Psh AI and Not I, and isolated MTX could induce EMT, and miR-34a upregulation fragment B was ligated into fragment A-containing was observed in parallel with drug-induced EMT (14, vector by T4 DNA ligase, followed by pIRESpuro2 15). In this study, we attempted to establish a novel Vector containing full-length human ABCA3 gene. type II alveolar epithelial cell line, A549/ABCA3 Finally, the plasmid was digested by EcoRI and cells, by introducing ABCA3 gene into A549 cell ABCA3 gene isolated was cloned into EcoRI site of line like the case of RLE/Abca3 cells. Using this cell pMXs-Puro Retroviral Vector. Retrovirus infection line, we aimed to clarify the mechanism underlying into A549 cells was performed as reported drug-induced EMT focusing on miR-34a. On the previously (19). A549/ABCA3 cells were cultured in other hand, it has been reported that p53 plays a DMEM containing 10 % FBS, 100 IU/mL penicillin, crucial role in regulation of miR-34a (16). In and 100 mg/mL streptomycin in an atmosphere of addition, anticancer drugs such as BLM and MTX 5 % CO /95 % air at 37 ℃, and subcultured every 7 are well-known to stimulate p53 activity (17, 18). days using 0.25 % trypsin and 1 mM EDTA. Fresh However, these findings were independently medium was replaced every 2–3 days. reported, and evidence showing the relationship between drug-induced EMT and p53/miR-34a axis Evaluation of mRNA expression is still lacking. Therefore, we also aimed to clarify A549/ABCA3 cells grown on 12-well plate were the role of p53/miR-34a axis in drug-induced EMT treated with BLM (60 µM) and MTX (0.3 µM) for using A549/ABCA3 cells. 144 h. Extraction of total RNA from the treated cells, the reverse transcription, and the amplification of the MATERIALS and METHODS obtained cDNA were performed as reported previously (20). The primers were: sense 5'- Materials CGGGAAGACCACGACTTT-3', and antisense, 5'- BLM was purchased from TOKYO CHEMICAL GCTGCCGCACCTTTC-3' for ABCA3; sense 5'- INDUSTRY CO., LTD. (Tokyo, Japan). MTX and AGATCTACCTGTACACCTTGAATGACA-3', and PTX were purchased from Wako Pure Chemical Ind. antisense, 5'-CCATGATACCAGCAAGGAATTG- (Osaka, Japan). LysoTracker Red as a selective 3' for fibronectin; sense 5'- fluorescent probe for intracellular lipid droplets in AGGAAAATGGCTGTTGGTATGATC-3', and lamellar bodies was purchased from Life antisense 5'-CGCAACTGCAAATGCCAG-3' for Technologies (Carlsbad, CA, USA). All the other PEPT2. The primers for collagen type Ia1 chemicals used for the experiments were of the (COL1A1), α-smooth muscle actin (α-SMA), and highest purity that was commercially available. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were prepared as reported previously (20, Establishment of A549/ABCA3 cells and the cell 21). The expression level of mRNA was normalized culture as to that of GAPDH, a housekeeping gene. Full-length human ABCA3 gene (accession No. NM_001089) was constructed from two separated Confocal Laser Scanning Microscopy fragments, A and B. The amplification of these Staining of lamellar body and actin filaments were fragments were performed by using following performed as reported previously (19, 20). Each primers; ABCA3-fragment A-F663 (5’- florescence intensity in the cells was visualized by TCAGACCACCTACTTCTCTAGCAGC-3’), confocal laser scanning microscopy (CLSM) ABCA3-fragment A-R3660 (5’- (OLYPUS FV1000D). GCAGTGCGTCTTTCAGATGCTCTG-3’), ABCA3-fragment B-F3446 (5’- Evaluation of miRNA expression TTCCTGAAGAAGGCCGCATACAGC-3’), and A549/ABCA3 cells grown on 12-well plate were ABCA3-fragment B-R5962 (5’- treated with BLM (60 µM) and MTX (0.3 µM) for CACTCGTCCATTCTGTGCATACTGC-3’). Each 144 h. Quantitative evaluation of miR-34a fragment was amplified using ReverTra Dash expression level by real-time PCR was performed 517 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 using miScript Reverse Transcription Kit and performed using one-way ANOVA followed by miScript SYBR Green PCR Kit (QIAGEN, Hilden, Tukey’s test for multiple comparisons. A p value < Germany), as reported previously (14). 0.05 indicated significance. Transfection with the miR-34a Mimic RESULTS A549/ABCA3 cells grown on 12-well plates were transfected with 21 nM of miRIDIAN microRNA Establishment of A549/ABCA3 cells miR-34a-5p-mimic or miRIDIAN microRNA At first, we tried to establish the A549/ABCA3 cells Mimic Negative control (GE Healthcare Japan) as a novel type II alveolar epithelial cell line. So far, using Lipofectamine™2000 (Thermo Fisher we established RLE/Abca3 cells by introducing rat Scientific Inc., Waltham, MA) for 24 hr. After 72 hr, abca3 gene into RLE-6TN cells and found that the total RNA was obtained from the treated cells. increases in type II-like phenotypes of the cells were observed compared with those in the RLE/Vector Preparation of cytoplasmic and nuclear protein cells (19). Therefore, A549/ABCA3 cells were extracts prepared by introducing human ABCA3 gene into A549/ABCA3 cells grown on 100-mm culture dish A549 cells with the same strategy to RLE/Abca3 were treated with BLM (60 µM) and MTX (0.3 µM) cells as described in Materials and Methods. As for 12 h. After that, preparation of cytoplasmic and presented in Figure 1A, the expression of both type nuclear protein extracts was performed as reported II alveolar epithelium markers, ABCA3 and PEPT2, previously (22). Briefly, the treated cells were were markedly higher in A549/ABCA3 than in washed two times with PBS buffer, scraped, and A549/Vector cells. Furthermore, LysoTracker Red collected by centrifugation at 500 × g for 5 min at staining revealed that formation of lamellar body- 4℃. Cell lysis was performed in ice-cold hypotonic like structures was enhanced in A549/ABCA3 lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, compared with A549/Vector cells (Figure 1B). 1 mM EDTA, 1 mM EGTA, 0.5 mM Na VO , 0.5% These results suggested that type II-like phenotypes 3 4 v/v NP-40, 10% v/v glycerol, 1 mM dithiothreitol, 1 of A549/ABCA3 cells were enhanced compared mM phenylmethanesulfonyl fluoride (PMSF)). with the wild type cells. Therefore, further Nuclear and cytoplasmic fractions were separated by examination was proceeded using newly established centrifugation at 1000 × g for 5 min at 4℃. The A549/ABCA3 cells. resulting supernatant (cytoplasmic fraction) was stored at -80℃ until further analysis. The membrane EMT induction by various anticancer drugs in pellet was then resuspended in ice-cold hypertonic A549/ABCA3 cells lysis buffer (20 mM HEPES (pH 7.9), 420 mM NaCl, EMT-inducing effect of BLM and MTX, anticancer 1 mM EDTA, 1 mM EGTA, 500 µM Na VO , 20% drugs having pulmonary toxic effect, on 3 4 v/v glycerol, 1 mM dithiothreitol, 1 mM PMSF), and A549/ABCA3 cells was examined. As shown in incubated with shaking for 15 min at 4℃. Soluble Figure 2A, after treatment with these drugs for 144 compounds were then isolated by centrifugation at h, the morphology was drastically changed, and 12000 × g for 5 min at 4℃. The resulting supernatant actin filaments assembled into contractile stress (nuclear fraction) was stored at -80℃ until further fibers were observed compared to the control. In analysis. addition, both drugs significantly enhanced mRNA expression level of α-SMA, an EMT marker, while Western Blotting decreased that of ABCA3 (Figure 2B). Paclitaxel Western blot analysis was performed as reported (PTX) and gefitinib (GEF) are also well-known to previously (21). Phospho-p53 (Ser15), p53, lamin induce lung injury. Therefore, the effects of PTX and A/C, and α-tubulin were detected using antibodies GEF on mRNA expression level of α-SMA were against rabbit anti-phospho-p53 (Ser15) (1:250 examined. Notably, both drugs led to increase in dilution, Cell Signaling Technology, Inc), mouse mRNA expression of α-SMA in A549/ABCA3 cells anti-p53 (1:2000 dilution, Merck), Lamin A+C (Figure 2C). (1:3000 dilution, Gene Tex), and alpha Tubulin 4a (1:5000 dilution, GeneTex), respectively. Role of miR-34a in drug-induced EMT process in A549/ABCA3 cells STATISTICAL ANALYSIS We already showed that miR-34a increased α-SMA mRNA expression in RLE/Abca3 cells (14). Data are expressed as the mean ± standard error of Therefore, role of miR-34a in drug-induced EMT the mean (S.E.M.). Statistical analysis was was also examined in A549/ABCA3 cells. BLM and 518 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 MTX clearly upregulated the miR-34a expression in p53 regulates miR-34a expression by binding to the A549/ABCA3 cells (Figure 3A). In addition, promotor region (16). Considering those facts, we expression level of miR-34a was also enhanced by hypothesized that drug-induced upregulation of PTX and GEF (Figure 3B). As miR-34a expression miR-34a is associated with p53. At first, the effects was closely associated with α-SMA in drug-treated of BLM and MTX on phosphorylation of p53 were A549/ABCA3 cells, the contribution of miR-34a to examined using Phos-tag SDS-PAGE system. α-SMA expression was examined. As shown in Fig. Several phosphorylated p53 bands at different 3C, overexpression of miR-34a by introducing degree were observed, and all obtained bands tended mimic of miR-34a into the cells enhanced the to be high in nucleus than in cytoplasmic fraction. In mRNA expression level of α-SMA, indicating that both fractions, all bands were upregulated by the miR-34a may induce EMT in A549/ABCA3 cells. treatment of A549/ABCA3 cells with BLM and Furthermore, miR-34a mimic introduction led to MTX (Figure 4A), indicating that both anticancer upregulation of mRNA expression levels of drugs may lead to upregulation of p53. Furthermore, extracellular matrix components such as COL1A1 using monoclonal antibody against phosphorylated and fibronectin (Figure 3D), indicating that miR-34a p53 at Ser15, phosphorylation of Ser15 in nucleus may play an important role in production of fraction was upregulated by both drugs (Figure 4B), extracellular matrix through the EMT process. indicating that these anticancer drugs may lead to activation of p53 via phosphorylation of Ser15. Effects of BLM and MTX on phosphorylation of p53 at Ser15 Generally, BLM and MTX cause DNA damage, followed by upregulation of p53. On the other hand, 519 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 DISCUSSION considered to be one of the sources for active myofibroblasts in the lung (7, 14). Therefore, Several reports have indicated that myofibroblast- suitable type II alveolar epithelium model has an induced ECM production would be one of critical advantage in appropriate understanding on EMT in factors in the development of pulmonary fibrosis (5, clinical situation. In the present study, 6). During normal wound healing, resident A549/ABCA3 cells were newly established as a type fibroblasts are converted into active myofibroblasts, II alveolar epithelium model. So far, we experienced while EMT contributes to abnormal recruitment of that Abca3 gene introduction conferred type II myofibroblasts, followed by excessive ECM alveolar epithelium properties in RLE-6TN cells, production in pathological conditions (11). In with increases in mRNA expression level of PEPT2 particular, type II alveolar epithelial cells are and lamellar body formation (19), which were 520 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 comparable to current results in A549/ABCA3 cells. BLM and MTX. Therefore, ABCA3 may be On the other hand, we previously demonstrated that considered to be a key factor associated with EMT albumin transport capacity was higher in type II than and use of A549/ABCA3 cells may have advantage in type I-like alveolar epithelial cells using primary in terms of investigations on drug-induced lung cultured cells isolated from rat lung (23, 24). injury. Although albumin transport in RLE/Abca3 cells was BLM and MTX increased miR-34a expression higher than that in the wild type cells in previous level in A549/ABCA3 cells, indicating that common studies (19), there were no differences in function of factors may be associated with drug-induced albumin transport between A549/ABCA3 and the increase in expression level of miR-34a. According wild type cells (data not shown). Other factors may to the reports showing that both drugs enhanced p53 be needed to make A549/ABCA3 cells more activity in A549 cells (26, 27), the upregulation of validated model for type II alveolar epithelial cells. phosphorylated p53 by BLM and MTX was In A549/ABCA3 cells, BLM, MTX, PTX, and observed, which also corresponded to information GEF clearly induced EMT with increase in mRNA on miR-34a upregulation mediated by p53 (16, 28). expression level of α-SMA. However, differences in In addition, both drugs-induced increase in effects of EMT-inducing drugs on A549 and phosphorylation of Ser15 in p53 was observed. N- A549/ABCA3 cells have not been well examined. terminal phosphorylation at Ser15 has been Actually, we used A549 cells for investigations on generally known to stabilize p53 by inhibiting the drug-induced EMT (20, 21), and observed similar interaction between p53 and MDM2, which is well- EMT-like changes to the present study. On the other known to be a negative regulator of p53 as an E3 hand, ABCA3 plays an important role in regulation ubiquitin ligase (29). Therefore, BLM and MTX of pulmonary surfactant homeostasis in the alveoli may increase miR-34a expression by stabilizing p53 (25). In A549/ABCA3 cells, mRNA expression level in A549/ABCA3 cells. of ABCA3 was suppressed by the treatment with 521 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 Taken together, using A549/ABCA3 cells, we one hand, it has been reported that miR-34a clarified for the first time the significant relationship enhanced alveolar epithelial cell apoptosis and between drug-induced EMT and upregulation of p53 promoted BLM-induced pulmonary fibrosis in mice activity and miR-34a expression. (33). Furthermore, we found that miR-34a mimic As miR-34a mimic introduction led to introduction increased mRNA expression level of enhancement of α-SMA mRNA expression level in COL1A1 and fibronectin in A549/ABCA3 cells, A549/ABCA3 cells, miR-34a may have a capacity which are critical extracellular matrix components in to promote EMT. However, underlying mechanism pulmonary fibrosis, indicating that miR-34a may of miR-34a-induced EMT remains unclear at this contribute to the development of pulmonary fibrosis. moment. Several reports indicated that miR-34a To clarify the net contribution of miR-34a to EMT inhibited EMT via direct suppression of Snail in process and pulmonary fibrosis, further studies colorectal and ovarian cancer cell lines (30, 31), and should be required. miR-34a is considered as a promising therapeutic agent for malignant tumors (32), which is the CONCLUSION opposite information to our findings. Thus, the present study would provide new findings We established A549/ABCA3 cells as a novel type concerning the relationship between drug-induced II alveolar epithelial cell model, observing increase EMT and miR-34a in alveolar epithelial cells. On in type II features such as mRNA expression level of 522 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 PEPT2, a type II marker, and formation of lamellar fibrosis: prevailing and evolving hypotheses about its bodies compared to wild-type A549 cells. In this pathogenesis and implications for therapy. Ann model, the drugs having pulmonary toxicity Intern Med, 2001;134:136–151. doi:10.7326/0003- including BLM, MTX, GEF, and PTX induced the 4819-134-2-200101160-00015 remodeling of actin-filaments and increase in 4. Wynn TA, Integrating mechanisms of pulmonary mRNA expression of α-SMA, indicating that fibrosis. J Exp Med, 2011;208:1339–1350. A549/ABCA3 cells would be useful for EMT doi:10.1084/jem.20110551. studies. In addition, these drugs increased expression 5. Giannandrea M, Parks WC, Diverse functions of level of miR-34a, and introduction of miR-34a matrix metalloproteinases during fibrosis. Dis Model mimic into the cells led to induction of EMT, Mech, 2014;7:193–203. doi:10.1242/dmm.012062. suggesting that drug-induced EMT in A549/ABCA3 6. Goldmann T, Zissel G, Watz H, Drömann D, Reck M, cells may be associated with miR-34a. Furthermore, Kugler C, Rabe KF, Marwitz S, Human alveolar p53 was upregulated by BLM and MTX via increase epithelial cells type II are capable of TGFβ- in phosphorylation of p53 at Ser15. Taken together, dependent epithelial-mesenchymal-transition and A549/ABCA3 cells may be a useful cell model for collagen-synthesis. Respir Res, 2018;19:138. studying EMT process in type II alveolar epithelia, doi:10.1186/s12931-018-0841-9. and p53/miR-34a axis would be one of the 7. Thiery JP, Acloque H, Huang RYJ, Nieto MA, therapeutic target for drug-induced lung injury. Epithelial-mesenchymal transitions in development and disease. Cell, 2009;139:871–890. ACKNOWLEDGEMENT doi:10.1016/j.cell.2009.11.007. 8. Willis BC, Borok Z, TGF- beta -induced EMT : We appreciate Professor Tahara-H (Hiroshima mechanisms and implications for fibrotic lung University) and Professor Shimamoto-A (Sanyo- disease. Am J Physiol Lung Cell Mol Physiol, Onoda City University) for providing kind advises 2007;293:L525-34. doi:10.1152/ajplung.00163.2007. concerning establishment of A549/ABCA3 cells, 9. Bartel DP, MicroRNAs: target recognition and and Professor Koike-T (Hiroshima University) for regulatory functions. Cell, 2009;136:215–233. providing Phos-tag Acrylamide. Also, we thank doi:10.1016/j.cell.2009.01.002. Ms. Yamamoto-C for her assistance in the 10. Rajasekaran S, Rajaguru P, Sudhakar Gandhi PS, establishment of A549/ABCA3 cells. This study was MicroRNAs as potential targets for progressive supported in part by the Grants-in-Aid for Scientific pulmonary fibrosis. Front Pharmacol, 2015;6:254. Research from the Japan Society for the Promotion doi:10.3389/fphar.2015.00254. of Science (JP18H02586, JP18K06749, and 11. Yang S, Banerjee S, de Freitas A, Sanders YY, Ding JP16K18945). Q, Matalon S, Thannickal VJ, Abraham E, Liu G, Participation of miR-200 in pulmonary fibrosis. Am CONFLICT OF INTEREST STATEMENT J Pathol, 2012;180:484–493. doi:10.1016/j.ajpath.2011.10.005. The authors declare that there are no conflicts of 12. Lino Cardenas CL, Henaoui IS, Courcot E, interest. Roderburg C, Cauffiez C, Aubert S, Copin MC, Wallaert B, Glowacki F, Dewaeles E, Milosevic J, REFERENCES Maurizio J, Tedrow J, Marcet B, Lo-Guidice JM, Kaminski N, Barbry P, Luedde T, Perrais M, Mari B, 1. Froudarakis M, Hatzimichael E, Kyriazopoulou L, Pottier N, miR-199a-5p is upregulated during Lagos K, Pappas P, Tzakos AG, Karavasilis V, fibrogenic response to tissue injury and mediates Daliani D, Papandreou C, Briasoulis E, Revisiting TGFbeta-induced lung fibroblast activation by bleomycin from pathophysiology to safe clinical use. targeting caveolin-1. PLoS Genet, 2019;9:e1003291. Crit Rev Oncol Hematol, 2013;87:90–100. doi:10.1371/journal.pgen.1003291. doi:10.1016/j.critrevonc.2012.12.003. 13. Marmai C, Sutherland RE, Kim KK, Dolganov GM, 2. Barrera P, Laan RF, van Riel PL, Dekhuijzen PN, Fang X, Kim SS, Jiang S, Golden JA, Hoopes CW, Boerbooms AM, van de Putte LB, Methotrexate- Matthay MA, Chapman HA, Wolters PJ, Alveolar related pulmonary complications in rheumatoid epithelial cells express mesenchymal proteins in arthritis. Ann Rheum Dis, 1994;53:434–439. patients with idiopathic pulmonary fibrosis. Am J doi:10.1136/ard.53.7.434. Physiol Cell Mol Physiol, 2011;301:L71–L78. 3. Selman M, King TE, Pardo A, American Thoracic doi:10.1152/ajplung.00212.2010. Society, European Respiratory Society, American 14. Takano M, Nekomoto C, Kawami M, Yumoto R, College of Chest Physicians, Idiopathic pulmonary Role of miR-34a in TGF-β1- and drug-induced 523 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 epithelial-mesenchymal transition in alveolar type II epithelial cells. Expert Opin Drug Deliv, epithelial cells. J Pharm Sci, 2017;106:2868–2872. 2015;12:813-825. doi:10.1016/j.xphs.2017.04.002. doi:10.1517/17425247.2015.992778. 15. Takano M, Yamamoto C, Yamaguchi K, Kawami M, 25. Besnard V, Matsuzaki Y, Clark J, Xu Y, Wert SE, Yumoto R, Analysis of TGF-β1- and drug-induced Ikegami M, Stahlman MT, Weaver TE, Hunt AN, epithelial-mesenchymal transition in cultured Postle AD, Whitsett JA, Conditional deletion of alveolar epithelial cell line RLE/Abca3. Drug Metab Abca3 in alveolar type II cells alters surfactant Pharmacokinet, 2015;30:111-118. homeostasis in newborn and adult mice. Am J doi:10.1016/j.dmpk.2014.10.007. Physiol Lung Cell Mol Physiol, 2010;298:L646-659. 16. He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang doi:10.1152/ajplung.00409.2009. Y, Xue W, Zender L, Magnus J, Ridzon D, Jackson 26. Linge A, Weinhold K, Bläsche R, Kasper M, Barth AL, Linsley PS, Chen C, Lowe SW, Cleary MA, K, Downregulation of caveolin-1 affects bleomycin- Hannon GJ, A microRNA component of the p53 induced growth arrest and cellular senescence in tumour suppressor network. Nature, 2007;447:1130– A549 cells. Int J Biochem Cell Biol, 2007;39:1964– 1134. doi:10.1038/nature05939. 1974. doi:10.1016/j.biocel.2007.05.018. 17. Lee YS, Yoon S, Park MS, Kim JH, Lee JH, Song 27. Huang WY, Yang PM, Chang YF, Marquez VE, CW, Influence of p53 expression on sensitivity of Chen CC, Methotrexate induces apoptosis through cancer cells to bleomycin. J Biochem Mol Toxicol, p53/p21-dependent pathway and increases E- 2010;24:260-269. doi:10.1002/jbt.20334. cadherin expression through downregulation of 18. Fekry B, Esmaeilniakooshkghazi A, Krupenko SA, HDAC/EZH2. Biochem Pharmacol, 2011;81:510– Krupenko NI, Ceramide synthase 6 is a novel target 517. doi:10.1016/j.bcp.2010.11.014. of methotrexate mediating its antiproliferative effect 28. Wang B, Li D, Kovalchuk O, p53 Ser15 in a p53-dependent manner. PLoS One, phosphorylation and histone modifications contribute 2016;11:e0146618. to IR-induced miR-34a transcription in mammary doi:10.1371/journal.pone.0146618. epithelial cells. Cell Cycle, 2013;12:2073–2083. 19. Takano M, Yamamoto C, Sambuichi K, Oda K, doi:10.4161/cc.25135. Nagai J, Shimamoto A, Tahara H, Yumoto R, 29. Kruse J-P, Gu W, Modes of p53 Regulation. Cell, Introduction of a single transporter gene ABCA3 2009;137:609–622. doi:10.1016/j.cell.2009.04.050. directs RLE-6TN to more type II-like alveolar 30. Siemens H, Jackstadt R, Hünten S, Kaller M, epithelial cells. Membrane, 2013;38:246–253. Menssen A, Götz U, Hermeking H, miR-34 and 20. Kawami M, Harabayashi R, Harada R, Yamagami Y, SNAIL form a double-negative feedback loop to Yumoto R, Takano M, Folic acid prevents regulate epithelial-mesenchymal transitions, Cell methotrexate-induced epithelial-mesenchymal Cycle, 2011;10:4256–4271. transition via suppression of secreted factors from the doi:10.4161/cc.10.24.18552. human alveolar epithelial cell line A549. Biochem 31. Dong P, Xiong Y, Watari H, Hanley SJB, Konno Y, Biophys Res Commun, 2018;497:457-463. Ihira K, Yamada T, Kudo M, Yue J, Sakuragi N, doi:10.1016/j.bbrc.2018.02.111. MiR-137 and miR-34a directly target Snail and 21. Kawami M, Harabayashi R, Miyamoto M, Harada R, inhibit EMT, invasion and sphere-forming ability of Yumoto R, Takano M, Methotrexate-induced ovarian cancer cells. J Exp Clin Cancer Res, epithelial–mesenchymal transition in the alveolar 2016;35:132. doi:10.1186/s13046-016-0415-y. epithelial cell line A549. Lung, 2016;194:923–930. 32. Nie D, Fu J, Chen H, Cheng J, Fu J, Nie D, Fu J, Chen doi:10.1007/s00408-016-9935-7. H, Cheng J, Fu J, Roles of microRNA-34a in 22. Milosevic J, Bulau P, Mortz E, Eickelberg O, epithelial to mesenchymal transition, competing Subcellular fractionation of TGF-β1-stimulated lung endogenous RNA sponging and its therapeutic epithelial cells: A novel proteomic approach for potential. Int J Mol Sci, 2019;20:861. identifying signaling intermediates. Proteomics, doi:10.3390/ijms20040861. 2009;9:1230–1240. doi:10.1002/pmic.200700604. 33. Shetty SK, Tiwari N, Marudamuthu AS, Puthusseri 23. Ikehata M, Yumoto R, Nakamura K, Nagai J, Takano B, Bhandary YP, Fu J, Levin J, Idell S, Shetty S, p53 M, Comparison of albumin uptake in rat alveolar type and miR-34a feedback promotes lung epithelial II and type I-like epithelial cells in primary culture. injury and pulmonary fibrosis. Am J Pathol, Pharm Res, 2008;25:913–922. doi:10.1007/s11095- 2017;187:1016–1034. 007-9426-x. doi:10.1016/j.ajpath.2016.12.020. 24. Takano M, Kawami M, Aoki A, Yumoto R, Receptor-mediated endocytosis of macromolecules and strategy to enhance their transport in alveolar http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Pharmacy & Pharmaceutical Sciences Unpaywall

Anticancer Drug-Induced Epithelial-Mesenchymal Transition via p53/miR-34a axis in A549/ABCA3 Cells

Journal of Pharmacy & Pharmaceutical SciencesOct 10, 2019

Loading next page...
 
/lp/unpaywall/anticancer-drug-induced-epithelial-mesenchymal-transition-via-p53-mir-0E4eKvfyCg

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
1482-1826
DOI
10.18433/jpps30660
Publisher site
See Article on Publisher Site

Abstract

J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 Anticancer Drug-Induced Epithelial-Mesenchymal Transition via p53/miR-34a axis in A549/ABCA3 Cells Ayano Yamamoto, Masashi Kawami, Takashi Konaka, Shinnosuke Takenaka, Ryoko Yumoto, Mikihisa Takano Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Received, July 25, 2019; Revised, September 30, 2019; Accepted, October 4, 2019; Published, October 7, 2019. ABSTRACT - PURPOSE. Several anticancer drugs including bleomycin (BLM) and methotrexate (MTX) cause serious lung diseases such as pulmonary fibrosis. Although evidences showing the association of epithelial-mesenchymal transition (EMT) with pulmonary fibrosis are increasing, the mechanism underlying anticancer drug-induced EMT has been poorly understood. On the other hand, miR-34a, a non-coding small RNA, has been highlighted as a key factor to regulate EMT in lung. In this study, we elucidated the role of miR-34a in anticancer drug-induced EMT using A549/ABCA3 cells as a novel type II alveolar epithelium model. METHODS. Expression levels of α-smooth muscle actin (α-SMA) mRNA, miR-34a, and p53 were evaluated by real-time PCR and western blot analysis, respectively. RESULTS. BLM and MTX induced EMT-like morphological changes and increase in mRNA expression level of α-SMA, an EMT marker. Also, both drugs increased the expression level of miR-34a. Furthermore, mRNA expression level of α-SMA was enhanced by introduction of miR-34a mimic into A549/ABCA3 cells. To examine the mechanism underlying drug-induced enhancement of miR-34a expression, we focused on p53/miR-34a axis. Both drugs upregulated protein expression of p53, an inducer of miR-34a, as well as phosphorylation of Ser15 in p53. CONCLUSIONS. These findings indicated that p53/miR-34a axis may contribute to anticancer drug-induced EMT in type II alveolar epithelial cells. _______________________________________________________________________________________ INTRODUCTION MicroRNAs (miRNA) are small single-stranded non-coding RNA of approximately 22 nucleotides There are increasing drugs with capacity of inducing and regulate post-transcriptional activity of their serious cytotoxic effects on lung such as pulmonary targeted genes by binding complementarily to the 3’ fibrosis as their adverse effects. In particular, untranslated regions (9). Current studies have anticancer drugs including bleomycin (BLM) (1) demonstrated that a number of miRNAs have and methotrexate (MTX) (2) have been reported to positive or negative effects on IPF via complicated induce severe lung diseases such as acute interstitial transcriptional regulations (10). In particular, miR- pneumonia and progressive pulmonary fibrosis. 200 family such as miR-200b and miR-200c inhibits Although these serious pulmonary diseases carry EMT in RLE-6TN cells, by downregulating the high mortality rates, development of the effective expression of several EMT inducing factors treatment has not been achieved, partly due to poor including zinc finger E-box-binding homeobox understanding of the drug-induced lung injury. (ZEB) 1/2 (11). On the other hand, miR-199a Recently, there are several suggested schemes stimulates fibroblasts in response to transforming concerning pathogenesis of serious pulmonary growth factor (TGF)-β1 via inhibitory effect on diseases such as idiopathic pulmonary fibrosis (IPF) caveolin 1, a negative regulator of TGF-β signaling (3). In these schemes, excessive extracellular matrix pathway (12). Thus, certain miRNAs would be (ECM) plays a crucial role in development of considerable biomarkers or therapeutic targets for pathogenic lung form. ECM is mainly produced by EMT-related diseases. myofibroblasts, which are derived from fibroblasts In the lung, EMT occurs in type II alveolar showing profibrotic phenotype (4, 5). When lung epithelial cells, as evidenced by several reports using injury occurs, on the other hand, myofibroblasts are human primary type II alveolar epithelial cells and also generated from injured type II alveolar lungs of patients with IPF (6, 13). __________________________________________________ epithelial cells via epithelial-mesenchymal transition (EMT) process (6). Therefore, EMT contributes to Corresponding Author: Prof. Mikihisa Takano, Department of development of pulmonary fibrosis (7, 8), and may Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 be considered to be a possible therapeutic target to Kasumi, Minami-ku, Hiroshima 734-8553, Japan; E-mail: prevent the drug-induced lung injury. takanom@hiroshima-u.ac.jp 516 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 So far, we established RLE/Abca3 cells by (TOYOBO Co. Ltd., Osaka, Japan). The PCR introducing Abca3 gene into RLE-6TN cells (rat- conditions were as follows: initial denaturation in derived alveolar epithelial cells), and mRNA one cycle of 5 min at 95℃, 30 cycles with 30 s at expression levels of several type II alveolar 95℃ (denaturation), 30 s at 65℃ (annealing), and 3 epithelial cell markers such as peptide transporter 2 min at 72℃ (extension). The purified fragment A (PEPT2) and formation of lamellar bodies were and B were cloned into pIRESpuro2 Vector and higher in RLE/Abca3 cells than those in the wild- pGEM-T Easy Vector, respectively. These plasmids type cells (14). In addition, we found that BLM and were digested by Psh AI and Not I, and isolated MTX could induce EMT, and miR-34a upregulation fragment B was ligated into fragment A-containing was observed in parallel with drug-induced EMT (14, vector by T4 DNA ligase, followed by pIRESpuro2 15). In this study, we attempted to establish a novel Vector containing full-length human ABCA3 gene. type II alveolar epithelial cell line, A549/ABCA3 Finally, the plasmid was digested by EcoRI and cells, by introducing ABCA3 gene into A549 cell ABCA3 gene isolated was cloned into EcoRI site of line like the case of RLE/Abca3 cells. Using this cell pMXs-Puro Retroviral Vector. Retrovirus infection line, we aimed to clarify the mechanism underlying into A549 cells was performed as reported drug-induced EMT focusing on miR-34a. On the previously (19). A549/ABCA3 cells were cultured in other hand, it has been reported that p53 plays a DMEM containing 10 % FBS, 100 IU/mL penicillin, crucial role in regulation of miR-34a (16). In and 100 mg/mL streptomycin in an atmosphere of addition, anticancer drugs such as BLM and MTX 5 % CO /95 % air at 37 ℃, and subcultured every 7 are well-known to stimulate p53 activity (17, 18). days using 0.25 % trypsin and 1 mM EDTA. Fresh However, these findings were independently medium was replaced every 2–3 days. reported, and evidence showing the relationship between drug-induced EMT and p53/miR-34a axis Evaluation of mRNA expression is still lacking. Therefore, we also aimed to clarify A549/ABCA3 cells grown on 12-well plate were the role of p53/miR-34a axis in drug-induced EMT treated with BLM (60 µM) and MTX (0.3 µM) for using A549/ABCA3 cells. 144 h. Extraction of total RNA from the treated cells, the reverse transcription, and the amplification of the MATERIALS and METHODS obtained cDNA were performed as reported previously (20). The primers were: sense 5'- Materials CGGGAAGACCACGACTTT-3', and antisense, 5'- BLM was purchased from TOKYO CHEMICAL GCTGCCGCACCTTTC-3' for ABCA3; sense 5'- INDUSTRY CO., LTD. (Tokyo, Japan). MTX and AGATCTACCTGTACACCTTGAATGACA-3', and PTX were purchased from Wako Pure Chemical Ind. antisense, 5'-CCATGATACCAGCAAGGAATTG- (Osaka, Japan). LysoTracker Red as a selective 3' for fibronectin; sense 5'- fluorescent probe for intracellular lipid droplets in AGGAAAATGGCTGTTGGTATGATC-3', and lamellar bodies was purchased from Life antisense 5'-CGCAACTGCAAATGCCAG-3' for Technologies (Carlsbad, CA, USA). All the other PEPT2. The primers for collagen type Ia1 chemicals used for the experiments were of the (COL1A1), α-smooth muscle actin (α-SMA), and highest purity that was commercially available. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were prepared as reported previously (20, Establishment of A549/ABCA3 cells and the cell 21). The expression level of mRNA was normalized culture as to that of GAPDH, a housekeeping gene. Full-length human ABCA3 gene (accession No. NM_001089) was constructed from two separated Confocal Laser Scanning Microscopy fragments, A and B. The amplification of these Staining of lamellar body and actin filaments were fragments were performed by using following performed as reported previously (19, 20). Each primers; ABCA3-fragment A-F663 (5’- florescence intensity in the cells was visualized by TCAGACCACCTACTTCTCTAGCAGC-3’), confocal laser scanning microscopy (CLSM) ABCA3-fragment A-R3660 (5’- (OLYPUS FV1000D). GCAGTGCGTCTTTCAGATGCTCTG-3’), ABCA3-fragment B-F3446 (5’- Evaluation of miRNA expression TTCCTGAAGAAGGCCGCATACAGC-3’), and A549/ABCA3 cells grown on 12-well plate were ABCA3-fragment B-R5962 (5’- treated with BLM (60 µM) and MTX (0.3 µM) for CACTCGTCCATTCTGTGCATACTGC-3’). Each 144 h. Quantitative evaluation of miR-34a fragment was amplified using ReverTra Dash expression level by real-time PCR was performed 517 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 using miScript Reverse Transcription Kit and performed using one-way ANOVA followed by miScript SYBR Green PCR Kit (QIAGEN, Hilden, Tukey’s test for multiple comparisons. A p value < Germany), as reported previously (14). 0.05 indicated significance. Transfection with the miR-34a Mimic RESULTS A549/ABCA3 cells grown on 12-well plates were transfected with 21 nM of miRIDIAN microRNA Establishment of A549/ABCA3 cells miR-34a-5p-mimic or miRIDIAN microRNA At first, we tried to establish the A549/ABCA3 cells Mimic Negative control (GE Healthcare Japan) as a novel type II alveolar epithelial cell line. So far, using Lipofectamine™2000 (Thermo Fisher we established RLE/Abca3 cells by introducing rat Scientific Inc., Waltham, MA) for 24 hr. After 72 hr, abca3 gene into RLE-6TN cells and found that the total RNA was obtained from the treated cells. increases in type II-like phenotypes of the cells were observed compared with those in the RLE/Vector Preparation of cytoplasmic and nuclear protein cells (19). Therefore, A549/ABCA3 cells were extracts prepared by introducing human ABCA3 gene into A549/ABCA3 cells grown on 100-mm culture dish A549 cells with the same strategy to RLE/Abca3 were treated with BLM (60 µM) and MTX (0.3 µM) cells as described in Materials and Methods. As for 12 h. After that, preparation of cytoplasmic and presented in Figure 1A, the expression of both type nuclear protein extracts was performed as reported II alveolar epithelium markers, ABCA3 and PEPT2, previously (22). Briefly, the treated cells were were markedly higher in A549/ABCA3 than in washed two times with PBS buffer, scraped, and A549/Vector cells. Furthermore, LysoTracker Red collected by centrifugation at 500 × g for 5 min at staining revealed that formation of lamellar body- 4℃. Cell lysis was performed in ice-cold hypotonic like structures was enhanced in A549/ABCA3 lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, compared with A549/Vector cells (Figure 1B). 1 mM EDTA, 1 mM EGTA, 0.5 mM Na VO , 0.5% These results suggested that type II-like phenotypes 3 4 v/v NP-40, 10% v/v glycerol, 1 mM dithiothreitol, 1 of A549/ABCA3 cells were enhanced compared mM phenylmethanesulfonyl fluoride (PMSF)). with the wild type cells. Therefore, further Nuclear and cytoplasmic fractions were separated by examination was proceeded using newly established centrifugation at 1000 × g for 5 min at 4℃. The A549/ABCA3 cells. resulting supernatant (cytoplasmic fraction) was stored at -80℃ until further analysis. The membrane EMT induction by various anticancer drugs in pellet was then resuspended in ice-cold hypertonic A549/ABCA3 cells lysis buffer (20 mM HEPES (pH 7.9), 420 mM NaCl, EMT-inducing effect of BLM and MTX, anticancer 1 mM EDTA, 1 mM EGTA, 500 µM Na VO , 20% drugs having pulmonary toxic effect, on 3 4 v/v glycerol, 1 mM dithiothreitol, 1 mM PMSF), and A549/ABCA3 cells was examined. As shown in incubated with shaking for 15 min at 4℃. Soluble Figure 2A, after treatment with these drugs for 144 compounds were then isolated by centrifugation at h, the morphology was drastically changed, and 12000 × g for 5 min at 4℃. The resulting supernatant actin filaments assembled into contractile stress (nuclear fraction) was stored at -80℃ until further fibers were observed compared to the control. In analysis. addition, both drugs significantly enhanced mRNA expression level of α-SMA, an EMT marker, while Western Blotting decreased that of ABCA3 (Figure 2B). Paclitaxel Western blot analysis was performed as reported (PTX) and gefitinib (GEF) are also well-known to previously (21). Phospho-p53 (Ser15), p53, lamin induce lung injury. Therefore, the effects of PTX and A/C, and α-tubulin were detected using antibodies GEF on mRNA expression level of α-SMA were against rabbit anti-phospho-p53 (Ser15) (1:250 examined. Notably, both drugs led to increase in dilution, Cell Signaling Technology, Inc), mouse mRNA expression of α-SMA in A549/ABCA3 cells anti-p53 (1:2000 dilution, Merck), Lamin A+C (Figure 2C). (1:3000 dilution, Gene Tex), and alpha Tubulin 4a (1:5000 dilution, GeneTex), respectively. Role of miR-34a in drug-induced EMT process in A549/ABCA3 cells STATISTICAL ANALYSIS We already showed that miR-34a increased α-SMA mRNA expression in RLE/Abca3 cells (14). Data are expressed as the mean ± standard error of Therefore, role of miR-34a in drug-induced EMT the mean (S.E.M.). Statistical analysis was was also examined in A549/ABCA3 cells. BLM and 518 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 MTX clearly upregulated the miR-34a expression in p53 regulates miR-34a expression by binding to the A549/ABCA3 cells (Figure 3A). In addition, promotor region (16). Considering those facts, we expression level of miR-34a was also enhanced by hypothesized that drug-induced upregulation of PTX and GEF (Figure 3B). As miR-34a expression miR-34a is associated with p53. At first, the effects was closely associated with α-SMA in drug-treated of BLM and MTX on phosphorylation of p53 were A549/ABCA3 cells, the contribution of miR-34a to examined using Phos-tag SDS-PAGE system. α-SMA expression was examined. As shown in Fig. Several phosphorylated p53 bands at different 3C, overexpression of miR-34a by introducing degree were observed, and all obtained bands tended mimic of miR-34a into the cells enhanced the to be high in nucleus than in cytoplasmic fraction. In mRNA expression level of α-SMA, indicating that both fractions, all bands were upregulated by the miR-34a may induce EMT in A549/ABCA3 cells. treatment of A549/ABCA3 cells with BLM and Furthermore, miR-34a mimic introduction led to MTX (Figure 4A), indicating that both anticancer upregulation of mRNA expression levels of drugs may lead to upregulation of p53. Furthermore, extracellular matrix components such as COL1A1 using monoclonal antibody against phosphorylated and fibronectin (Figure 3D), indicating that miR-34a p53 at Ser15, phosphorylation of Ser15 in nucleus may play an important role in production of fraction was upregulated by both drugs (Figure 4B), extracellular matrix through the EMT process. indicating that these anticancer drugs may lead to activation of p53 via phosphorylation of Ser15. Effects of BLM and MTX on phosphorylation of p53 at Ser15 Generally, BLM and MTX cause DNA damage, followed by upregulation of p53. On the other hand, 519 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 DISCUSSION considered to be one of the sources for active myofibroblasts in the lung (7, 14). Therefore, Several reports have indicated that myofibroblast- suitable type II alveolar epithelium model has an induced ECM production would be one of critical advantage in appropriate understanding on EMT in factors in the development of pulmonary fibrosis (5, clinical situation. In the present study, 6). During normal wound healing, resident A549/ABCA3 cells were newly established as a type fibroblasts are converted into active myofibroblasts, II alveolar epithelium model. So far, we experienced while EMT contributes to abnormal recruitment of that Abca3 gene introduction conferred type II myofibroblasts, followed by excessive ECM alveolar epithelium properties in RLE-6TN cells, production in pathological conditions (11). In with increases in mRNA expression level of PEPT2 particular, type II alveolar epithelial cells are and lamellar body formation (19), which were 520 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 comparable to current results in A549/ABCA3 cells. BLM and MTX. Therefore, ABCA3 may be On the other hand, we previously demonstrated that considered to be a key factor associated with EMT albumin transport capacity was higher in type II than and use of A549/ABCA3 cells may have advantage in type I-like alveolar epithelial cells using primary in terms of investigations on drug-induced lung cultured cells isolated from rat lung (23, 24). injury. Although albumin transport in RLE/Abca3 cells was BLM and MTX increased miR-34a expression higher than that in the wild type cells in previous level in A549/ABCA3 cells, indicating that common studies (19), there were no differences in function of factors may be associated with drug-induced albumin transport between A549/ABCA3 and the increase in expression level of miR-34a. According wild type cells (data not shown). Other factors may to the reports showing that both drugs enhanced p53 be needed to make A549/ABCA3 cells more activity in A549 cells (26, 27), the upregulation of validated model for type II alveolar epithelial cells. phosphorylated p53 by BLM and MTX was In A549/ABCA3 cells, BLM, MTX, PTX, and observed, which also corresponded to information GEF clearly induced EMT with increase in mRNA on miR-34a upregulation mediated by p53 (16, 28). expression level of α-SMA. However, differences in In addition, both drugs-induced increase in effects of EMT-inducing drugs on A549 and phosphorylation of Ser15 in p53 was observed. N- A549/ABCA3 cells have not been well examined. terminal phosphorylation at Ser15 has been Actually, we used A549 cells for investigations on generally known to stabilize p53 by inhibiting the drug-induced EMT (20, 21), and observed similar interaction between p53 and MDM2, which is well- EMT-like changes to the present study. On the other known to be a negative regulator of p53 as an E3 hand, ABCA3 plays an important role in regulation ubiquitin ligase (29). Therefore, BLM and MTX of pulmonary surfactant homeostasis in the alveoli may increase miR-34a expression by stabilizing p53 (25). In A549/ABCA3 cells, mRNA expression level in A549/ABCA3 cells. of ABCA3 was suppressed by the treatment with 521 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 Taken together, using A549/ABCA3 cells, we one hand, it has been reported that miR-34a clarified for the first time the significant relationship enhanced alveolar epithelial cell apoptosis and between drug-induced EMT and upregulation of p53 promoted BLM-induced pulmonary fibrosis in mice activity and miR-34a expression. (33). Furthermore, we found that miR-34a mimic As miR-34a mimic introduction led to introduction increased mRNA expression level of enhancement of α-SMA mRNA expression level in COL1A1 and fibronectin in A549/ABCA3 cells, A549/ABCA3 cells, miR-34a may have a capacity which are critical extracellular matrix components in to promote EMT. However, underlying mechanism pulmonary fibrosis, indicating that miR-34a may of miR-34a-induced EMT remains unclear at this contribute to the development of pulmonary fibrosis. moment. Several reports indicated that miR-34a To clarify the net contribution of miR-34a to EMT inhibited EMT via direct suppression of Snail in process and pulmonary fibrosis, further studies colorectal and ovarian cancer cell lines (30, 31), and should be required. miR-34a is considered as a promising therapeutic agent for malignant tumors (32), which is the CONCLUSION opposite information to our findings. Thus, the present study would provide new findings We established A549/ABCA3 cells as a novel type concerning the relationship between drug-induced II alveolar epithelial cell model, observing increase EMT and miR-34a in alveolar epithelial cells. On in type II features such as mRNA expression level of 522 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 PEPT2, a type II marker, and formation of lamellar fibrosis: prevailing and evolving hypotheses about its bodies compared to wild-type A549 cells. In this pathogenesis and implications for therapy. Ann model, the drugs having pulmonary toxicity Intern Med, 2001;134:136–151. doi:10.7326/0003- including BLM, MTX, GEF, and PTX induced the 4819-134-2-200101160-00015 remodeling of actin-filaments and increase in 4. Wynn TA, Integrating mechanisms of pulmonary mRNA expression of α-SMA, indicating that fibrosis. J Exp Med, 2011;208:1339–1350. A549/ABCA3 cells would be useful for EMT doi:10.1084/jem.20110551. studies. In addition, these drugs increased expression 5. Giannandrea M, Parks WC, Diverse functions of level of miR-34a, and introduction of miR-34a matrix metalloproteinases during fibrosis. Dis Model mimic into the cells led to induction of EMT, Mech, 2014;7:193–203. doi:10.1242/dmm.012062. suggesting that drug-induced EMT in A549/ABCA3 6. Goldmann T, Zissel G, Watz H, Drömann D, Reck M, cells may be associated with miR-34a. Furthermore, Kugler C, Rabe KF, Marwitz S, Human alveolar p53 was upregulated by BLM and MTX via increase epithelial cells type II are capable of TGFβ- in phosphorylation of p53 at Ser15. Taken together, dependent epithelial-mesenchymal-transition and A549/ABCA3 cells may be a useful cell model for collagen-synthesis. Respir Res, 2018;19:138. studying EMT process in type II alveolar epithelia, doi:10.1186/s12931-018-0841-9. and p53/miR-34a axis would be one of the 7. Thiery JP, Acloque H, Huang RYJ, Nieto MA, therapeutic target for drug-induced lung injury. Epithelial-mesenchymal transitions in development and disease. Cell, 2009;139:871–890. ACKNOWLEDGEMENT doi:10.1016/j.cell.2009.11.007. 8. Willis BC, Borok Z, TGF- beta -induced EMT : We appreciate Professor Tahara-H (Hiroshima mechanisms and implications for fibrotic lung University) and Professor Shimamoto-A (Sanyo- disease. Am J Physiol Lung Cell Mol Physiol, Onoda City University) for providing kind advises 2007;293:L525-34. doi:10.1152/ajplung.00163.2007. concerning establishment of A549/ABCA3 cells, 9. Bartel DP, MicroRNAs: target recognition and and Professor Koike-T (Hiroshima University) for regulatory functions. Cell, 2009;136:215–233. providing Phos-tag Acrylamide. Also, we thank doi:10.1016/j.cell.2009.01.002. Ms. Yamamoto-C for her assistance in the 10. Rajasekaran S, Rajaguru P, Sudhakar Gandhi PS, establishment of A549/ABCA3 cells. This study was MicroRNAs as potential targets for progressive supported in part by the Grants-in-Aid for Scientific pulmonary fibrosis. Front Pharmacol, 2015;6:254. Research from the Japan Society for the Promotion doi:10.3389/fphar.2015.00254. of Science (JP18H02586, JP18K06749, and 11. Yang S, Banerjee S, de Freitas A, Sanders YY, Ding JP16K18945). Q, Matalon S, Thannickal VJ, Abraham E, Liu G, Participation of miR-200 in pulmonary fibrosis. Am CONFLICT OF INTEREST STATEMENT J Pathol, 2012;180:484–493. doi:10.1016/j.ajpath.2011.10.005. The authors declare that there are no conflicts of 12. Lino Cardenas CL, Henaoui IS, Courcot E, interest. Roderburg C, Cauffiez C, Aubert S, Copin MC, Wallaert B, Glowacki F, Dewaeles E, Milosevic J, REFERENCES Maurizio J, Tedrow J, Marcet B, Lo-Guidice JM, Kaminski N, Barbry P, Luedde T, Perrais M, Mari B, 1. Froudarakis M, Hatzimichael E, Kyriazopoulou L, Pottier N, miR-199a-5p is upregulated during Lagos K, Pappas P, Tzakos AG, Karavasilis V, fibrogenic response to tissue injury and mediates Daliani D, Papandreou C, Briasoulis E, Revisiting TGFbeta-induced lung fibroblast activation by bleomycin from pathophysiology to safe clinical use. targeting caveolin-1. PLoS Genet, 2019;9:e1003291. Crit Rev Oncol Hematol, 2013;87:90–100. doi:10.1371/journal.pgen.1003291. doi:10.1016/j.critrevonc.2012.12.003. 13. Marmai C, Sutherland RE, Kim KK, Dolganov GM, 2. Barrera P, Laan RF, van Riel PL, Dekhuijzen PN, Fang X, Kim SS, Jiang S, Golden JA, Hoopes CW, Boerbooms AM, van de Putte LB, Methotrexate- Matthay MA, Chapman HA, Wolters PJ, Alveolar related pulmonary complications in rheumatoid epithelial cells express mesenchymal proteins in arthritis. Ann Rheum Dis, 1994;53:434–439. patients with idiopathic pulmonary fibrosis. Am J doi:10.1136/ard.53.7.434. Physiol Cell Mol Physiol, 2011;301:L71–L78. 3. Selman M, King TE, Pardo A, American Thoracic doi:10.1152/ajplung.00212.2010. Society, European Respiratory Society, American 14. Takano M, Nekomoto C, Kawami M, Yumoto R, College of Chest Physicians, Idiopathic pulmonary Role of miR-34a in TGF-β1- and drug-induced 523 J Pharm Pharm Sci (www.cspsCanada.org) 22, 516 - 524, 2019 epithelial-mesenchymal transition in alveolar type II epithelial cells. Expert Opin Drug Deliv, epithelial cells. J Pharm Sci, 2017;106:2868–2872. 2015;12:813-825. doi:10.1016/j.xphs.2017.04.002. doi:10.1517/17425247.2015.992778. 15. Takano M, Yamamoto C, Yamaguchi K, Kawami M, 25. Besnard V, Matsuzaki Y, Clark J, Xu Y, Wert SE, Yumoto R, Analysis of TGF-β1- and drug-induced Ikegami M, Stahlman MT, Weaver TE, Hunt AN, epithelial-mesenchymal transition in cultured Postle AD, Whitsett JA, Conditional deletion of alveolar epithelial cell line RLE/Abca3. Drug Metab Abca3 in alveolar type II cells alters surfactant Pharmacokinet, 2015;30:111-118. homeostasis in newborn and adult mice. Am J doi:10.1016/j.dmpk.2014.10.007. Physiol Lung Cell Mol Physiol, 2010;298:L646-659. 16. He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang doi:10.1152/ajplung.00409.2009. Y, Xue W, Zender L, Magnus J, Ridzon D, Jackson 26. Linge A, Weinhold K, Bläsche R, Kasper M, Barth AL, Linsley PS, Chen C, Lowe SW, Cleary MA, K, Downregulation of caveolin-1 affects bleomycin- Hannon GJ, A microRNA component of the p53 induced growth arrest and cellular senescence in tumour suppressor network. Nature, 2007;447:1130– A549 cells. Int J Biochem Cell Biol, 2007;39:1964– 1134. doi:10.1038/nature05939. 1974. doi:10.1016/j.biocel.2007.05.018. 17. Lee YS, Yoon S, Park MS, Kim JH, Lee JH, Song 27. Huang WY, Yang PM, Chang YF, Marquez VE, CW, Influence of p53 expression on sensitivity of Chen CC, Methotrexate induces apoptosis through cancer cells to bleomycin. J Biochem Mol Toxicol, p53/p21-dependent pathway and increases E- 2010;24:260-269. doi:10.1002/jbt.20334. cadherin expression through downregulation of 18. Fekry B, Esmaeilniakooshkghazi A, Krupenko SA, HDAC/EZH2. Biochem Pharmacol, 2011;81:510– Krupenko NI, Ceramide synthase 6 is a novel target 517. doi:10.1016/j.bcp.2010.11.014. of methotrexate mediating its antiproliferative effect 28. Wang B, Li D, Kovalchuk O, p53 Ser15 in a p53-dependent manner. PLoS One, phosphorylation and histone modifications contribute 2016;11:e0146618. to IR-induced miR-34a transcription in mammary doi:10.1371/journal.pone.0146618. epithelial cells. Cell Cycle, 2013;12:2073–2083. 19. Takano M, Yamamoto C, Sambuichi K, Oda K, doi:10.4161/cc.25135. Nagai J, Shimamoto A, Tahara H, Yumoto R, 29. Kruse J-P, Gu W, Modes of p53 Regulation. Cell, Introduction of a single transporter gene ABCA3 2009;137:609–622. doi:10.1016/j.cell.2009.04.050. directs RLE-6TN to more type II-like alveolar 30. Siemens H, Jackstadt R, Hünten S, Kaller M, epithelial cells. Membrane, 2013;38:246–253. Menssen A, Götz U, Hermeking H, miR-34 and 20. Kawami M, Harabayashi R, Harada R, Yamagami Y, SNAIL form a double-negative feedback loop to Yumoto R, Takano M, Folic acid prevents regulate epithelial-mesenchymal transitions, Cell methotrexate-induced epithelial-mesenchymal Cycle, 2011;10:4256–4271. transition via suppression of secreted factors from the doi:10.4161/cc.10.24.18552. human alveolar epithelial cell line A549. Biochem 31. Dong P, Xiong Y, Watari H, Hanley SJB, Konno Y, Biophys Res Commun, 2018;497:457-463. Ihira K, Yamada T, Kudo M, Yue J, Sakuragi N, doi:10.1016/j.bbrc.2018.02.111. MiR-137 and miR-34a directly target Snail and 21. Kawami M, Harabayashi R, Miyamoto M, Harada R, inhibit EMT, invasion and sphere-forming ability of Yumoto R, Takano M, Methotrexate-induced ovarian cancer cells. J Exp Clin Cancer Res, epithelial–mesenchymal transition in the alveolar 2016;35:132. doi:10.1186/s13046-016-0415-y. epithelial cell line A549. Lung, 2016;194:923–930. 32. Nie D, Fu J, Chen H, Cheng J, Fu J, Nie D, Fu J, Chen doi:10.1007/s00408-016-9935-7. H, Cheng J, Fu J, Roles of microRNA-34a in 22. Milosevic J, Bulau P, Mortz E, Eickelberg O, epithelial to mesenchymal transition, competing Subcellular fractionation of TGF-β1-stimulated lung endogenous RNA sponging and its therapeutic epithelial cells: A novel proteomic approach for potential. Int J Mol Sci, 2019;20:861. identifying signaling intermediates. Proteomics, doi:10.3390/ijms20040861. 2009;9:1230–1240. doi:10.1002/pmic.200700604. 33. Shetty SK, Tiwari N, Marudamuthu AS, Puthusseri 23. Ikehata M, Yumoto R, Nakamura K, Nagai J, Takano B, Bhandary YP, Fu J, Levin J, Idell S, Shetty S, p53 M, Comparison of albumin uptake in rat alveolar type and miR-34a feedback promotes lung epithelial II and type I-like epithelial cells in primary culture. injury and pulmonary fibrosis. Am J Pathol, Pharm Res, 2008;25:913–922. doi:10.1007/s11095- 2017;187:1016–1034. 007-9426-x. doi:10.1016/j.ajpath.2016.12.020. 24. Takano M, Kawami M, Aoki A, Yumoto R, Receptor-mediated endocytosis of macromolecules and strategy to enhance their transport in alveolar

Journal

Journal of Pharmacy & Pharmaceutical SciencesUnpaywall

Published: Oct 10, 2019

There are no references for this article.