Advertisement

Journal of Zhejiang University-SCIENCE B

, Volume 19, Issue 12, pp 895–909 | Cite as

Comparative transcriptomic analysis reveals adriamycin-induced apoptosis via p53 signaling pathway in retinal pigment epithelial cells

  • Yu-chen Lin
  • Ze-ren Shen
  • Xiao-hui Song
  • Xin Liu
  • Ke Yao
Article
  • 7 Downloads

Abstract

Objective

This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin (ADR) in treating proliferative vitreoretinopathy (PVR) using ARPE-19 cells.

Methods

The growth inhibitory effects of ADR on ARPE-19 cells were assessed by sulforhodamine B (SRB) assay and propidium iodide (PI) staining using flow cytometry. The differentially expressed genes between ADR-treated ARPE-19 cells and normal ARPE-19 cells and the signaling pathways involved were investigated by microarray analysis. Mitochondrial function was detected by JC-1 staining using flow cytometry and the Bcl-2/Bax protein family. The phosphorylated histone H2AX (γ-H2AX), phosphorylated checkpoint kinase 1 (p-CHK1), and phosphorylated checkpoint kinase 2 (p-CHK2) were assessed to detect DNA damage and repair.

Results

ADR could significantly inhibit ARPE-19 cell proliferation and induce caspasedependent apoptosis in vitro. In total, 4479 differentially expressed genes were found, and gene ontology items and the p53 signaling pathway were enriched. A protein–protein interaction analysis indicated that the TP53 protein molecules regulated by ADR were related to DNA damage and oxidative stress. ADR reduced mitochondrial membrane potential and the Bcl-2/Bax ratio. p53-knockdown restored the activation of c-caspase-3 activity induced by ADR by regulating Bax expression, and it inhibited ADR-induced ARPE-19 cell apoptosis. Finally, the levels of the γ-H2AX, p-CHK1, and p-CHK2 proteins were up-regulated after ADR exposure.

Conclusions

The mechanism of ARPE-19 cell death induced by ADR may be caspase-dependent apoptosis, and it may be regulated by the p53-dependent mitochondrial dysfunction, activating the p53 signaling pathway through DNA damage.

Key words

Adriamycin Proliferative vitreoretinopathy Retinal pigment epithelial p53 Apoptosis 

比较转录组学分析揭示阿霉素经p53信号通路诱导视网膜色素上皮细胞凋亡

摘要

目的

通过比较阿霉素(ADR)作用的视网膜色素上皮细胞(ARPE-19)和正常ARPE-19 间的差异表达基因和信号通路,探究ADR 治疗玻璃体视网膜增殖性疾病的潜在机制。

创新点

首次运用基因芯片技术通过比较转录组学分析探究ADR促进ARPE-19 细胞凋亡的机制。

方法

采用磺酰罗丹明B(sulforhodamine B,SRB)比色法和碘化丙啶(PI)单染结合流式细胞术检测ADR 对ARPE-19 细胞的增殖抑制作用;通过基因芯片技术筛选ADR 作用的ARPE-19 细胞(实 验组)和正常ARPE-19 细胞(对照组)间的差异表达基因和相关信号通路;用JC-1 染色结合流式细胞术和Bcl2/Bax 蛋白表达比率检测线粒体功 能;通过检测γ-H2AX、p-CHK1、 p-CHK2 等蛋 白表达量分析DNA 的损伤和修复。

结论

ADR 通过启动DNA 损伤反应,引起p53 信号通路依赖的线粒体功能失调并激活caspase 依赖的凋亡,最终导致ARPE-19 细胞死亡。

关键词

阿霉素 玻璃体视网膜增殖性疾病 视网膜色素上皮层 p53 凋亡 

CLC number

R3 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

11585_2018_408_MOESM1_ESM.pdf (323 kb)
Comparative transcriptomic analysis reveals adriamycin-induced apoptosis via p53 signaling pathway in retinal pigment epithelial cells

References

  1. Amarnani D, Machuca-Parra AI, Wong LL, et al., 2017. Effect of methotrexate on an in vitro patient-derived model of proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci, 58(10):3940–3949. https://doi.org/10.1167/iovs.16-20912 Google Scholar
  2. Asaria RHY, Kon CH, Bunce C, et al., 2001. Adjuvant 5-fluorouracil and heparin prevents proliferative vitreoretinopathy: results from a randomized, double-blind, controlled clinical trial. Ophthalmology, 108(7):1179–1183. https://doi.org/10.1016/S0161-6420(01)00589-9 Google Scholar
  3. Brazma A, Hingamp P, Quackenbush J, et al., 2001. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet, 29(4): 365–371. https://doi.org/10.1038/ng1201-365 Google Scholar
  4. Charteris DG, Sethi CS, Lewis GP, et al., 2002. Proliferative vitreoretinopathy-developments in adjunctive treatment and retinal pathology. Eye (Lond), 16(4):369–374. https://doi.org/10.1038/sj.eye.6700194 Google Scholar
  5. Charteris DG, Aylward GW, Wong D, et al., 2004. A randomized controlled trial of combined 5-fluorouracil and low-molecular-weight heparin in management of established proliferative vitreoretinopathy. Ophthalmology, 111(12):2240–2245. https://doi.org/10.1016/j.ophtha.2004.05.036 Google Scholar
  6. Christmann M, Kaina B, 2013. Transcriptional regulation of human DNA repair genes following genotoxic stress: trigger mechanisms, inducible responses and genotoxic adaptation. Nucleic Acids Res, 41(18):8403–8420. https://doi.org/10.1093/nar/gkt635 Google Scholar
  7. Claes C, Lafetá AP, 2014. Proliferative vitreoretinopathy. Dev Ophthalmol, 54:188–195. https://doi.org/10.1159/000360466 Google Scholar
  8. Consortium M, Shi L, Reid LH, et al., 2006. The microarray quality control (MAQC) project shows inter-and intraplatform reproducibility of gene expression measurements. Nat Biotechnol, 24(9):1151–1161. https://doi.org/10.1038/nbt1239 Google Scholar
  9. Daniels SA, Coonley KG, Yoshizumi MO, 1990. Taxol treatment of experimental proliferative vitreoretinopathy. Graefes Arch Clin Exp Ophthalmol, 228(6):513–516. https://doi.org/10.1007/BF00918482 Google Scholar
  10. Dash SK, Chattopadhyay S, Ghosh T, et al., 2015. Self-assembled betulinic acid protects doxorubicin induced apoptosis followed by reduction of ROS-TNF-a-caspase-3 activity. Biomed Pharmacother, 72:144–157. https://doi.org/10.1016/j.biopha.2015.04.017 Google Scholar
  11. Deus CM, Zehowski C, Nordgren K, et al., 2015. Stimulating basal mitochondrial respiration decreases doxorubicin apoptotic signaling in H9c2 cardiomyoblasts. Toxicology, 334:1–11. https://doi.org/10.1016/j.tox.2015.05.001 Google Scholar
  12. di Marco A, Gaetani M, Scarpinato B, 1969. Adriamycin (NSC-123,127): a new antibiotic with antitumor activity. Cancer Chemother Rep, 53(1):33–37.Google Scholar
  13. Eibl KH, Fisher SK, Lewis GP, 2009. Alkylphosphocholines: a new approach to inhibit cell proliferation in proliferative vitreoretinopathy. Dev Ophthalmol, 44:46–55. https://doi.org/10.1159/000223945 Google Scholar
  14. Flores ER, Tsai KY, Crowley D, et al., 2002. p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature, 416(6880):560–564. https://doi.org/10.1038/416560a Google Scholar
  15. Heimann H, Bartz-Schmidt KU, Bornfeld N, et al., 2007. Scleral buckling versus primary vitrectomy in rhegmatogenous retinal detachment: a prospective randomized multicenter clinical study. Ophthalmology, 114(12):2142–2154. https://doi.org/10.1016/j.ophtha.2007.09.013 Google Scholar
  16. Kumar A, Nainiwal S, Choudhary I, et al., 2002. Role of daunorubicin in inhibiting proliferative vitreoretinopathy after retinal detachment surgery. Clin Exp Ophthalmol, 30(5):348–351. https://doi.org/10.1046/j.1442-9071.2002.00554.x Google Scholar
  17. Kuo HK, Wu PC, Yang PM, et al., 2007. Effects of topoisomerase II inhibitors on retinal pigment epithelium and experimental proliferative vitreoretinopathy. J Ocul Pharmacol Ther, 23(1):14–20. https://doi.org/10.1089/jop.2006.0059 Google Scholar
  18. Kuo HK, Chen YH, Wu PC, et al., 2012. Attenuated glial reaction in experimental proliferative vitreoretinopathy treated with liposomal doxorubicin. Invest Ophthalmol Vis Sci, 53(6):3167–3174. https://doi.org/10.1167/iovs.11-7972 Google Scholar
  19. Leaver PK, 1995. Proliferative vitreoretinopathy. Br J Ophthalmol, 79(10):871–872. https://doi.org/10.1136/bjo.79.10.871 Google Scholar
  20. Leiderman YI, Miller JW, 2009. Proliferative vitreoretinopathy: pathobiology and therapeutic targets. Semin Ophthalmol, 24(2):62–69. https://doi.org/10.1080/08820530902800082 Google Scholar
  21. Lisenko K, Dingeldein G, Cremer M, et al., 2017. Addition of rituximab to CHOP-like chemotherapy in first line treatment of primary mediastinal B-cell lymphoma. BMC Cancer, 17:359. https://doi.org/10.1186/s12885-017-3332-3 Google Scholar
  22. Machemer R, van Horn D, Aaberg TM, 1978. Pigment epithelial proliferation in human retinal detachment with massive periretinal proliferation. Am J Ophthalmol, 85(2): 181–191. https://doi.org/10.1016/S0002-9394(14)75946-X Google Scholar
  23. Marchenko ND, Zaika A, Moll UM, 2000. Death signalinduced localization of p53 protein to mitochondria. A potential role in apoptotic signaling. J Biol Chem, 275(21): 16202–16212.Google Scholar
  24. Martins-Neves SR, Paiva-Oliveira DI, Wijers-Koster PM, et al., 2016. Chemotherapy induces stemness in osteosarcoma cells through activation of Wnt/β-catenin signaling. Cancer Lett, 370(2):286–295. https://doi.org/10.1016/j.canlet.2015.11.013 Google Scholar
  25. Matt S, Hofmann TG, 2016. The DNA damage-induced cell death response: a roadmap to kill cancer cells. Cell Mol Life Sci, 73(15):2829–2850. https://doi.org/10.1007/s00018-016-2130-4 Google Scholar
  26. Minotti G, Menna P, Salvatorelli E, et al., 2004. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev, 56(2):185–229. https://doi.org/10.1124/pr.56.2.6 Google Scholar
  27. Moritera T, Ogura Y, Yoshimura N, et al., 1992. Biodegradable microspheres containing adriamycin in the treatment of proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci, 33(11):3125–3130.Google Scholar
  28. Patel N, Garikapati KR, Pandita RK, et al., 2017. miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin. Sci Rep, 7(1):4263. https://doi.org/10.1038/s41598-017-02800-2 Google Scholar
  29. Polo SE, Jackson SP, 2011. Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev, 25(5):409–433. https://doi.org/10.1101/gad.2021311 Google Scholar
  30. Rong A, Li J, 2002. Intravitreal injection of doxorobicin and dexamethason in treatment of proliferative vitreoretinopathy. Natl Med J China, 82(8):546–548 (in Chinese). https://doi.org/10.3760/j:issn:0376-2491.2002.08.012 Google Scholar
  31. Savitskaya MA, Onishchenko GE, 2015. Mechanisms of apoptosis. Biochemistry (Mosc), 80(11):1393–1405. https://doi.org/10.1134/S0006297915110012 Google Scholar
  32. Steinhorst UH, Chen EP, Machemer R, et al., 1993. N,Ndimethyladriamycin for treatment of experimental proliferative vitreoretinopathy: efficacy and toxicity on the rabbit retina. Exp Eye Res, 56(4):489–495. https://doi.org/10.1006/exer.1993.1062 Google Scholar
  33. Sunalp M, Wiedemann P, Sorgente N, et al., 1984. Effects of cytotoxic drugs on proliferative vitreoretinopathy in the rabbit cell injection model. Curr Eye Res, 3(4):619–623. https://doi.org/10.3109/02713688409003063 Google Scholar
  34. Sunalp MA, Wiedemann P, Sorgente N, et al., 1985. Effect of adriamycin on experimental proliferative vitreoretinopathy in the rabbit. Exp Eye Res, 41(1):105–115. https://doi.org/10.1016/0014-4835(85)90099-5 Google Scholar
  35. Tanaka H, Arakawa H, Yamaguchi T, et al., 2000. A ribonucleotide reductase gene involved in a p53-dependent cellcycle checkpoint for DNA damage. Nature, 404(6773): 42–49. https://doi.org/10.1038/35003506 Google Scholar
  36. Tewey KM, Rowe TC, Yang L, et al., 1984. Adriamycininduced DNA damage mediated by mammalian DNA topoisomerase II. Science, 226(4673):466–468. https://doi.org/10.1126/science.6093249 Google Scholar
  37. van Bockxmeer FM, Martin CE, Thompson DE, et al., 1985. Taxol for the treatment of proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci, 26(8):1140–1147.Google Scholar
  38. Vaseva AV, Marchenko ND, Ji K, et al., 2012. p53 opens the mitochondrial permeability transition pore to trigger necrosis. Cell, 149(7):1536–1548. https://doi.org/10.1016/j.cell.2012.05.014 Google Scholar
  39. Wang J, Nachtigal MW, Kardami E, et al., 2013. FGF-2 protects cardiomyocytes from doxorubicin damage via protein kinase C-dependent effects on efflux transporters. Cardiovasc Res, 98(1):56–63. https://doi.org/10.1093/cvr/cvt011 Google Scholar
  40. Wang SW, Konorev EA, Kotamraju S, et al., 2004. Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms: intermediacy of H2O2-and p53-dependent pathways. J Biol Chem, 279(24): 25535–25543. https://doi.org/10.1074/jbc.M400944200 Google Scholar
  41. Wang ZC, Wang J, Xie RF, et al., 2015. Mitochondria-derived reactive oxygen species play an important role in doxorubicin-induced platelet apoptosis. Int J Mol Sci, 16(5):11087–11100. https://doi.org/10.3390/ijms160511087 Google Scholar
  42. Wickham L, Bunce C, Wong D, et al., 2007. Randomized controlled trial of combined 5-fluorouracil and lowmolecular-weight heparin in the management of unselected rhegmatogenous retinal detachments undergoing primary vitrectomy. Ophthalmology, 114(4):698–704. https://doi.org/10.1016/j.ophtha.2006.08.042 Google Scholar
  43. Wiedemann P, Hilgers RD, Bauer P, et al., 1998. Adjunctive daunorubicin in the treatment of proliferative vitreoretinopathy: results of a multicenter clinical trial. Am J Ophthalmol, 126(4):550–559. https://doi.org/10.1016/S0002-9394(98)00115-9 Google Scholar
  44. Xu XD, Zhao Y, Zhang M, et al., 2017. Inhibition of autophagy by deguelin sensitizes pancreatic cancer cells to doxorubicin. Int J Mol Sci, 18(2):370. https://doi.org/10.3390/ijms18020370 Google Scholar
  45. Zhao LF, Shan YJ, Liu B, et al., 2017. Functional screen analysis reveals miR-3142 as central regulator in chemoresistance and proliferation through activation of the PTEN-AKT pathway in CML. Cell Death Dis, 8(5): e2830. https://doi.org/10.1038/cddis.2017.223 Google Scholar

Copyright information

© Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Eye Center, the Second Affiliated HospitalZhejiang University School of MedicineHangzhouChina
  2. 2.Key Laboratory of Ophthalmology of Zhejiang ProvinceHangzhouChina

Personalised recommendations