All Title Author
Keywords Abstract

PLOS ONE  2012 

Molecular Effects of Doxycycline Treatment on Pterygium as Revealed by Massive Transcriptome Sequencing

DOI: 10.1371/journal.pone.0039359

Full-Text   Cite this paper   Add to My Lib

Abstract:

Pterygium is a lesion of the eye surface which involves cell proliferation, migration, angiogenesis, fibrosis, and extracellular matrix remodelling. Surgery is the only approved method to treat this disorder, but high recurrence rates are common. Recently, it has been shown in a mouse model that treatment with doxycycline resulted in reduction of the pterygium lesions. Here we study the mechanism(s) of action by which doxycycline achieves these results, using massive sequencing techniques. Surgically removed pterygia from 10 consecutive patients were set in short term culture and exposed to 0 (control), 50, 200, and 500 μg/ml doxycycline for 24 h, their mRNA was purified, reverse transcribed and sequenced through Illumina’s massive sequencing protocols. Acquired data were subjected to quantile normalization and analyzed using cytoscape plugin software to explore the pathways involved. False discovery rate (FDR) methods were used to identify 332 genes which modified their expression in a dose-dependent manner upon exposure to doxycycline. The more represented cellular pathways included all mitochondrial genes, the endoplasmic reticulum stress response, integrins and extracellular matrix components, and growth factors. A high correlation was obtained when comparing ultrasequencing data with qRT-PCR and ELISA results. Doxycycline significantly modified the expression of important cellular pathways in pterygium cells, in a way which is consistent with the observed efficacy of this antibiotic to reduce pterygium lesions in a mouse model. Clinical trials are under way to demonstrate whether there is a benefit for human patients.

References

[1]  Coroneo M (2011) Ultraviolet radiation and the anterior eye. Eye Contact Lens 37: 214–224. 10.1097/ICL.0b013e318223394e [doi].
[2]  Bradley JC, Yang W, Bradley RH, Reid TW, Schwab IR (2010) The science of pterygia. Br J Ophthalmol 94: 815–820. bjo.2008.151852 [pii];10.1136/bjo.2008.151852 [doi].
[3]  Chui J, Di GN, Wakefield D, Coroneo MT (2008) The pathogenesis of pterygium: current concepts and their therapeutic implications. Ocul Surf 6: 24–43.
[4]  Dzunic B, Jovanovic P, Veselinovic D, Petrovic A, Stefanovic I, et al. (2010) Analysis of pathohistological characteristics of pterygium. Bosn J Basic Med Sci 10: 307–313.
[5]  Chui J, Coroneo MT, Tat LT, Crouch R, Wakefield D, et al. (2011) Ophthalmic pterygium: a stem cell disorder with premalignant features. Am J Pathol 178: 817–827. S0002-9440(10)00133-1 [pii];10.1016/j.ajpath.2010.10.037 [doi].
[6]  Di GN, Chui J, Coroneo MT, Wakefield D (2004) Pathogenesis of pterygia: role of cytokines, growth factors, and matrix metalloproteinases. Prog Retin Eye Res 23: 195–228. 10.1016/j.preteyeres.2004.02.002 [doi];S1350946204000126 [pii].
[7]  Marcovich AL, Bahar I, Srinivasan S, Slomovic AR (2010) Surgical management of pterygium. Int Ophthalmol Clin 50: 47–61. 10.1097/IIO.0b013e3181e218f7 [doi];00004397-201005030-00006 [pii].
[8]  Kandavel R, Kang JJ, Memarzadeh F, Chuck RS (2010) Comparison of pterygium recurrence rates in Hispanic and white patients after primary excision and conjunctival autograft. Cornea 29: 141–145. 10.1097/ICO.0b013e3181b11630 [doi].
[9]  Mauro J, Foster CS (2009) Pterygia: pathogenesis and the role of subconjunctival bevacizumab in treatment. Semin Ophthalmol 24: 130–134. 911113760 [pii];10.1080/08820530902801106 [doi].
[10]  Besharati MR, Manaviat MR, Souzani A (2011) Subconjunctival bevacizumab injection in treatment of pterygium. Acta Med Iran 49: 179–183. 18268 [pii].
[11]  Cox CA, Amaral J, Salloum R, Guedez L, Reid TW, et al. (2010) Doxycycline’s effect on ocular angiogenesis: an in vivo analysis. Ophthalmology 117: 1782–1791. .S0161-6420(10)00110-7 [pii];10.1016/j.ophtha.2010.01.037 [doi].
[12]  Dan L, Shi-long Y, Miao-li L, Yong-ping L, Hong-jie M, et al. (2008) Inhibitory effect of oral doxycycline on neovascularization in a rat corneal alkali burn model of angiogenesis. Curr Eye Res 33: 653–660. 901525287 [pii];10.1080/02713680802245772 [doi].
[13]  Sapadin AN, Fleischmajer R (2006) Tetracyclines: nonantibiotic properties and their clinical implications. J Am Acad Dermatol 54: 258–265. S0190-9622(05)03231-7 [pii];10.1016/j.jaad.2005.10.004 [doi].
[14]  Golub LM, Lee HM, Ryan ME, Giannobile WV, Payne J, et al. (1998) Tetracyclines inhibit connective tissue breakdown by multiple non-antimicrobial mechanisms. Adv Dent Res 12: 12–26.
[15]  Lee CZ, Xu B, Hashimoto T, McCulloch CE, Yang GY, et al. (2004) Doxycycline suppresses cerebral matrix metalloproteinase-9 and angiogenesis induced by focal hyperstimulation of vascular endothelial growth factor in a mouse model. Stroke 35: 1715–1719. 10.1161/01.STR.0000129334.05181.b6 [doi];01.STR.0000129334.05181.b6 [pii].
[16]  Morin R, Bainbridge M, Fejes A, Hirst M, Krzywinski M, et al. (2008) Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing. Biotechniques 45: 81–94. 000112900 [pii];10.2144/000112900 [doi].
[17]  Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5: 621–628. nmeth.1226 [pii];10.1038/nmeth.1226 [doi].
[18]  Bullard JH, Purdom E, Hansen KD, Dudoit S (2010) Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments. BMC Bioinformatics 11: 94. 1471-2105-11-94 [pii];10.1186/1471-2105-11-94 [doi].
[19]  Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, et al. (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4: 249–264. 10.1093/biostatistics/4.2.249 [doi];4/2/249 [pii].
[20]  Tarazona S, Garcia-Alcalde F, Dopazo J, Ferrer A, Conesa A (2011) Differential expression in RNA-seq: a matter of depth. Genome Res 21: 2213–2223. gr.124321.111 [pii];10.1101/gr.124321.111 [doi].
[21]  John-Aryankalayil M, Dushku N, Jaworski CJ, Cox CA, Schultz G, et al. (2006) Microarray and protein analysis of human pterygium. Mol Vis 12: 55–64. v12/a6 [pii].
[22]  Tong L, Chew J, Yang H, Ang LP, Tan DT, et al. (2009) Distinct gene subsets in pterygia formation and recurrence: dissecting complex biological phenomenon using genome wide expression data. BMC Med Genomics 2: 14. 1755-8794-2-14 [pii];10.1186/1755-8794-2-14 [doi].
[23]  Jaworski CJ, Aryankalayil-John M, Campos MM, Fariss RN, Rowsey J, et al. (2009) Expression analysis of human pterygium shows a predominance of conjunctival and limbal markers and genes associated with cell migration. Mol Vis 15: 2421–2434.
[24]  Riau AK, Wong TT, Finger SN, Chaurasia SS, Hou AH, et al. (2011) Aberrant DNA methylation of matrix remodeling and cell adhesion related genes in pterygium. PLoS One 6: e14687. 10.1371/journal.pone.0014687 [doi].
[25]  Dushku N, Reid TW (1994) Immunohistochemical evidence that human pterygia originate from an invasion of vimentin-expressing altered limbal epithelial basal cells. Curr Eye Res 13: 473–481.
[26]  Ugalde C, Vogel R, Huijbens R, Van Den Heuvel B, Smeitink J, et al. (2004) Human mitochondrial complex I assembles through the combination of evolutionary conserved modules: a framework to interpret complex I deficiencies. Hum Mol Genet 13: 2461–2472. 10.1093/hmg/ddh262 [doi];ddh262 [pii].
[27]  Olgun A, Akman S (2007) Mitochondrial DNA-deficient models and aging. Ann N Y Acad Sci 1100: 241–245. 1100/1/241 [pii];10.1196/annals.1395.025 [doi].
[28]  Pello R, Martin MA, Carelli V, Nijtmans LG, Achilli A, et al. (2008) Mitochondrial DNA background modulates the assembly kinetics of OXPHOS complexes in a cellular model of mitochondrial disease. Hum Mol Genet 17: 4001–4011. ddn303 [pii];10.1093/hmg/ddn303 [doi].
[29]  Sourdeval M, Lemaire C, Brenner C, Boisvieux-Ulrich E, Marano F (2006) Mechanisms of doxycycline-induced cytotoxicity on human bronchial epithelial cells. Front Biosci 11: 3036–3048. 2031 [pii].
[30]  Wu J, Liu T, Xie J, Xin F, Guo L (2006) Mitochondria and calpains mediate caspase-dependent apoptosis induced by doxycycline in HeLa cells. Cell Mol Life Sci 63: 949–957. 10.1007/s00018-005-5565-6 [doi].
[31]  Sagar J, Sales K, Taanman JW, Dijk S, Winslet M (2010) Lowering the apoptotic threshold in colorectal cancer cells by targeting mitochondria. Cancer Cell Int 10: 31. 1475-2867-10-31 [pii];10.1186/1475-2867-10-31 [doi].
[32]  Onoda T, Ono T, Dhar DK, Yamanoi A, Nagasue N (2006) Tetracycline analogues (doxycycline and COL-3) induce caspase-dependent and -independent apoptosis in human colon cancer cells. Int J Cancer 118: 1309–1315. 10.1002/ijc.21447 [doi].
[33]  Rubins JB, Charboneau D, Alter MD, Bitterman PB, Kratzke RA (2001) Inhibition of mesothelioma cell growth in vitro by doxycycline. J Lab Clin Med 138: 101–106. S0022-2143(01)29869-2 [pii];10.1067/mlc.2001.116591 [doi].
[34]  Lai HC, Yeh YC, Ting CT, Lee WL, Lee HW, et al. (2010) Doxycycline suppresses doxorubicin-induced oxidative stress and cellular apoptosis in mouse hearts. Eur J Pharmacol 644: 176–187. S0014-2999(10)00707-7 [pii];10.1016/j.ejphar.2010.07.010 [doi].
[35]  Yeh YC, Lai HC, Ting CT, Lee WL, Wang LC, et al. (2007) Protection by doxycycline against doxorubicin-induced oxidative stress and apoptosis in mouse testes. Biochem Pharmacol 74: 969–980. S0006-2952(07)00407-8 [pii];10.1016/j.bcp.2007.06.031 [doi].
[36]  Lai E, Teodoro T, Volchuk A (2007) Endoplasmic reticulum stress: signaling the unfolded protein response. Physiology (Bethesda) 22: 193–201. 22/3/193 [pii];10.1152/physiol.00050.2006 [doi].
[37]  Xu C, Bailly-Maitre B, Reed JC (2005) Endoplasmic reticulum stress: cell life and death decisions. J Clin Invest 115: 2656–2664. 10.1172/JCI26373 [doi].
[38]  Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D (2000) Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol 2: 326–332. 10.1038/35014014 [doi].
[39]  Patil C, Walter P (2001) Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals. Curr Opin Cell Biol 13: 349–355. S0955-0674(00)00219-2 [pii].
[40]  Hetz C, Bernasconi P, Fisher J, Lee AH, Bassik MC, et al. (2006) Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1alpha. Science 312: 572–576. 312/5773/572 [pii];10.1126/science.1123480 [doi].
[41]  Yoshida H, Haze K, Yanagi H, Yura T, Mori K (1998) Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors. J Biol Chem 273: 33741–33749.
[42]  Wang XZ, Ron D (1996) Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP Kinase. Science 272: 1347–1349.
[43]  Borradori L, Sonnenberg A (1999) Structure and function of hemidesmosomes: more than simple adhesion complexes. J Invest Dermatol 112: 411–418. 10.1046/j.1523-1747.1999.00546.x [doi].
[44]  Lipscomb EA, Mercurio AM (2005) Mobilization and activation of a signaling competent alpha6beta4integrin underlies its contribution to carcinoma progression. Cancer Metastasis Rev 24: 413–423. 10.1007/s10555-005-5133-4 [doi].
[45]  Shaw LM, Rabinovitz I, Wang HH, Toker A, Mercurio AM (1997) Activation of phosphoinositide 3-OH kinase by the alpha6beta4 integrin promotes carcinoma invasion. Cell 91: 949–960. S0092-8674(00)80486-9 [pii].
[46]  Bon G, Folgiero V, Di CS, Sacchi A, Falcioni R (2007) Involvement of alpha6beta4 integrin in the mechanisms that regulate breast cancer progression. Breast Cancer Res 9: 203. bcr1651 [pii];10.1186/bcr1651 [doi].
[47]  Sithanandam G, Smith GT, Fields JR, Fornwald LW, Anderson LM (2005) Alternate paths from epidermal growth factor receptor to Akt in malignant versus nontransformed lung epithelial cells: ErbB3 versus Gab1. Am J Respir Cell Mol Biol 33: 490–499. 2005-0049OC [pii];10.1165/rcmb.2005-0049OC [doi].
[48]  Jin J, Guan M, Sima J, Gao G, Zhang M, et al. (2003) Decreased pigment epithelium-derived factor and increased vascular endothelial growth factor levels in pterygia. Cornea 22: 473–477.
[49]  Tsai YY, Chiang CC, Bau DT, Cheng YW, Lee H, et al. (2008) Vascular endothelial growth factor gene 460 polymorphism is associated with pterygium formation in female patients. Cornea 27: 476–479. 10.1097/ICO.0b013e3181644581 [doi];00003226-200805000-00015 [pii].
[50]  Parkash V, Lindholm P, Peranen J, Kalkkinen N, Oksanen E, et al. (2009) The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional. Protein Eng Des Sel 22: 233–241. gzn080 [pii];10.1093/protein/gzn080 [doi].
[51]  Di GN, Kumar RK, Coroneo MT, Wakefield D (2002) UVB-mediated induction of interleukin-6 and -8 in pterygia and cultured human pterygium epithelial cells. Invest Ophthalmol Vis Sci 43: 3430–3437.
[52]  Onoda T, Ono T, Dhar DK, Yamanoi A, Fujii T, et al. (2004) Doxycycline inhibits cell proliferation and invasive potential: combination therapy with cyclooxygenase-2 inhibitor in human colorectal cancer cells. J Lab Clin Med 143: 207–216. 10.1016/j.lab.2003.12.012 [doi]; S0022214304000083 [pii].
[53]  Lai PB, Chi TY, Chen GG (2007) Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line in vitro. Apoptosis 12: 387–393. 10.1007/s10495-006-0571-1 [doi].
[54]  Montojo J, Zuberi K, Rodriguez H, Kazi F, Wright G, et al. (2010) GeneMANIA Cytoscape plugin: fast gene function predictions on the desktop. Bioinformatics 26: 2927–2928. btq562 [pii];10.1093/bioinformatics/btq562 [doi].
[55]  Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3: 1101–1108.

Full-Text

comments powered by Disqus

Contact Us

service@oalib.com

QQ:3279437679

微信:OALib Journal