Parasitology Research

, Volume 113, Issue 8, pp 3063–3071 | Cite as

Proteomic identification of potential Clonorchis sinensis excretory/secretory products capable of binding and activating human hepatic stellate cells

  • Xiaoyun Wang
  • Fengyu Hu
  • Xuchu Hu
  • Wenjun Chen
  • Yan Huang
  • Xinbing Yu
Original Paper

Abstract

Epidemiological and experimental evidence demonstrated that Clonorchis sinensis is an important risk factor of hepatic fibrosis and cholangiocarcinoma. C. sinensis excretory/secretory products (CsESPs) are protein complex including proteases, antioxidant enzymes, and metabolic enzymes, which may contribute to pathogenesis of liver fluke-associated hepatobiliary diseases. However, potential CsESP candidates involved into hepatic fibrosis and cholangiocarcinoma still remain to be elucidated. In the present study, we performed proteomic identification of CsESP candidates capable of binding and activating human hepatic stellate cell line LX-2. Immunofluorescence analysis confirmed the interaction of CsESPs with LX-2 cell membrane. LX-2 cells could be stimulated by CsESPs from 24 h post incubation (p < 0.05). Specifically, 50 μg/ml of CsESPs showed the strongest effect on cell proliferation in methyl thiazolyl tetrazolium (MTT) assay which could also be demonstrated by flow cytometry analysis (p < 0.01). Furthermore, expression level of human type III collagen in LX-2 cells treated with CsESPs was significantly higher than that in control cells measured by molecular beacon and semiquantitative reverse transcription (RT)-PCR approaches (p < 0.01). Finally, CsESPs before and after incubation with LX-2 cells were subjected to two-dimensional gel electrophoresis (2-DE) analysis and matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Nine proteins with abundance change above threefold were Rho GTPase-activating protein, mitochondrial cytochrome c oxidase subunit Va, α-enolase, phospholipase C, interleukin-15, insect-derived growth factor, cytochrome c oxidase subunit VI, DNAH1 protein, and kinesin light chain. Taken together, we identified potential CsESP candidates capable of binding and activating human hepatic stellate cells, providing more direct evidences that are previously unknown to accelerate strategies for C. sinensis prevention.

Keywords

Clonorchis sinensis Excretory/secretory products Hepatic stellate cells 

Notes

Acknowledgments

This work was supported by the National Key Basic Research and Development Project (973 project; No. 2010CB530000), National Natural Science Foundation of China (No. 81171602), National S&T Major Program (No. 2012ZX10004-220), and Fundamental Research Funds for the Central Universities (2013) to XBY and YH. The work was supported in part by Key Subject Programs of Guangzhou Municipal Health Bureau (No. 2009-Zdi-06) to FYH and National Natural Science Foundation of China (No. 81101270) to YH.

References

  1. Anthony B, Allen JT, Li YS, McManus DP (2010) Hepatic stellate cells and parasite-induced liver fibrosis. Parasit Vectors 3:60PubMedCentralPubMedCrossRefGoogle Scholar
  2. Boutorine AS, Novopashina DS, Krasheninina OA, Nozeret K, Venyaminova AG (2013) Fluorescent probes for nucleic acid visualization in fixed and live cells. Molecules 18:15357–15397PubMedCrossRefGoogle Scholar
  3. Cantacessi C, Mulvenna J, Young ND, Kasny M, Horak P, Aziz A, Hofmann A, Loukas A, Gasser RB (2012) A deep exploration of the transcriptome and “excretory/secretory” proteome of adult Fascioloides magna. Mol Cell Proteomics 11:1340–1353PubMedCentralPubMedCrossRefGoogle Scholar
  4. Carloni V, Romanelli RG, Pinzani M, Laffi G, Gentilini P (1997) Focal adhesion kinase and phospholipase C gamma involvement in adhesion and migration of human hepatic stellate cells. Gastroenterology 112:522–531PubMedCrossRefGoogle Scholar
  5. Chang D, Ramalho LN, Ramalho FS, Martinelli AL, Zucoloto S (2006) Hepatic stellate cells in human Schistosomiasis mansoni: a comparative immunohistochemical study with liver cirrhosis. Acta Trop 97:318–323PubMedCrossRefGoogle Scholar
  6. Chen X, Li S, He L, Wang X, Liang P, Chen W, Bian M, Ren M, Lin J, Liang C, Xu J, Wu Z, Li X, Huang Y, Yu X (2013) Molecular characterization of severin from Clonorchis sinensis excretory/secretory products and its potential anti-apoptotic role in hepatocarcinoma PLC cells. PLoS Negl Trop Dis 7:e2606PubMedCentralPubMedCrossRefGoogle Scholar
  7. Chuang SC, La Vecchia C, Boffetta P (2009) Liver cancer: descriptive epidemiology and risk factors other than HBV and HCV infection. Cancer Lett 286:9–14PubMedCrossRefGoogle Scholar
  8. Fang X, Mi Y, Li JJ, Beck T, Schuster S, Tan W (2002) Molecular beacons: fluorogenic probes for living cell study. Cell Biochem Biophys 37:71–81PubMedCrossRefGoogle Scholar
  9. Fried B, Reddy A, Mayer D (2011) Helminths in human carcinogenesis. Cancer Lett 305:239–49PubMedCrossRefGoogle Scholar
  10. Friedman SL (2003) Liver fibrosis—from bench to bedside. J Hepatol 38:S38–53PubMedCrossRefGoogle Scholar
  11. Hong ST, Fang Y (2012) Clonorchis sinensis and clonorchiasis, an update. Parasitol Int 61:17–24PubMedCrossRefGoogle Scholar
  12. Hu F, Hu X, Ma C, Zhao J, Xu J, Yu X (2009) Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A(2) and investigation of its potential contribution to hepatic fibrosis. Mol Biochem Parasitol 167:127–134PubMedCrossRefGoogle Scholar
  13. Ishihara K, Miyazaki A, Nabe T, Fushimi H, Iriyama N, Kanai S, Sato T, Uozumi N, Shimizu T, Akiba S (2012) Group IVA phospholipase A2 participates in the progression of hepatic fibrosis. FASEB J 26:4111–4121PubMedCrossRefGoogle Scholar
  14. Jex AR, Young ND, Sripa J, Hall RS, Scheerlinck JP, Laha T, Sripa B, Gasser RB (2012) Molecular changes in Opisthorchis viverrini (Southeast Asian liver fluke) during the transition from the juvenile to the adult stage. PLoS Negl Trop Dis 6:e1916PubMedCentralPubMedCrossRefGoogle Scholar
  15. Kim YJ, Choi MH, Hong ST, Bae YM (2008) Proliferative effects of excretory/secretory products from Clonorchis sinensis on the human epithelial cell line HEK293 via regulation of the transcription factor E2F1. Parasitol Res 102:411–417PubMedCrossRefGoogle Scholar
  16. Kim TI, Na BK, Hong SJ (2009) Functional genes and proteins of Clonorchis sinensis. Korean J Parasitol 47:S59–68PubMedCentralPubMedCrossRefGoogle Scholar
  17. Lee JH, Rim HJ, Sell S (1997) Heterogeneity of the “oval-cell” response in the hamster liver during cholangiocarcinogenesis following Clonorchis sinensis infection and dimethylnitrosamine treatment. J Hepatol 26:1313–1323PubMedCrossRefGoogle Scholar
  18. Li N, Long Y, Fan X, Liu H, Li C, Chen L, Wang Z (2009) Proteomic analysis of differentially expressed proteins in hepatitis B virus-related hepatocellular carcinoma tissues. J Exp Clin Cancer Res 28:122PubMedCentralPubMedCrossRefGoogle Scholar
  19. Liang P, Sun J, Huang Y, Zhang F, Zhou J, Hu Y, Wang X, Liang C, Zheng M, Xu Y, Mao Q, Hu X, Li X, Xu J, Lu G, Yu X (2013) Biochemical characterization and functional analysis of fructose-1,6-bisphosphatase from Clonorchis sinensis. Mol Biol Rep 40:4371–4382PubMedCrossRefGoogle Scholar
  20. Lun ZR, Gasser RB, Lai DH, Li AX, Zhu XQ, Yu XB, Fang YY (2005) Clonorchiasis: a key foodborne zoonosis in China. Lancet Infect Dis 5:31–41PubMedCrossRefGoogle Scholar
  21. Moon SY, Zheng Y (2003) Rho GTPase-activating proteins in cell regulation. Trends Cell Biol 13(1):13–22PubMedCrossRefGoogle Scholar
  22. Mulvenna J, Sripa B, Brindley PJ, Gorman J, Jones MK, Colgrave ML, Jones A, Nawaratna S, Laha T, Suttiprapa S, Smout MJ, Loukas A (2010) The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini. Proteomics 10:1063–1078PubMedCentralPubMedGoogle Scholar
  23. Pak JH, Moon JH, Hwang SJ, Cho SH, Seo SB, Kim TS (2009a) Proteomic analysis of differentially expressed proteins in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products. J Cell Biochem 108:1376–1388PubMedCrossRefGoogle Scholar
  24. Pak JH, Kim DW, Moon JH, Nam JH, Kim JH, Ju JW, Kim TS, Seo SB (2009b) Differential gene expression profiling in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products. Parasitol Res 104:1035–1046PubMedCrossRefGoogle Scholar
  25. Qian MB, Chen YD, Fang YY, Xu LQ, Zhu TJ, Tan T, Zhou CH, Wang GF, Jia TW, Yang GJ, Zhou XN (2011) Disability weight of Clonorchis sinensis infection: captured from community study and model simulation. PLoS Negl Trop Dis 5:e1377PubMedCentralPubMedCrossRefGoogle Scholar
  26. Qian MB, Chen YD, Liang S, Yang GJ, Zhou XN (2012) The global epidemiology of clonorchiasis and its relation with cholangiocarcinoma. Infect Dis Poverty 1:4PubMedCentralPubMedCrossRefGoogle Scholar
  27. Robinson MW, Menon R, Donnelly SM, Dalton JP, Ranganathan S (2009) An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host. Mol Cell Proteomics 8:1891–1907PubMedCentralPubMedCrossRefGoogle Scholar
  28. Santangelo PJ, Nix B, Tsourkas A, Bao G (2004) Dual FRET molecular beacons for mRNA detection in living cells. Nucleic Acids Res 32:e57PubMedCentralPubMedCrossRefGoogle Scholar
  29. Shi YF, Zhang Q, Cheung PY, Shi L, Fong CC, Zhang Y, Tzang CH, Chan BP, Fong WF, Chun J, Kung HF, Yang M (2006) Effects of rhDecorin on TGF-beta1 induced human hepatic stellate cells LX-2 activation. Biochim Biophys Acta 1760:1587–1595PubMedCrossRefGoogle Scholar
  30. Shin HR, Oh JK, Masuyer E, Curado MP, Bouvard V, Fang YY, Wiangnon S, Sripa B, Hong ST (2010) Epidemiology of cholangiocarcinoma: an update focusing on risk factors. Cancer Sci 101:579–585PubMedCrossRefGoogle Scholar
  31. Smout MJ, Laha T, Mulvenna J, Sripa B, Suttiprapa S, Jones A, Brindley PJ, Loukas A (2009) A granulin-like growth factor secreted by the carcinogenic liver fluke, Opisthorchis viverrini, promotes proliferation of host cells. PLoS Pathog 5:e1000611PubMedCentralPubMedCrossRefGoogle Scholar
  32. Wang X, Liang C, Chen W, Fan Y, Hu X, Xu J, Yu X (2009) Experimental model in rats for study on transmission dynamics and evaluation of Clonorchis sinensis infection immunologically, morphologically, and pathologically. Parasitol Res 106:15–21PubMedCrossRefGoogle Scholar
  33. Wang X, Chen W, Huang Y, Sun J, Men J, Liu H, Luo F, Guo L, Lv X, Deng C, Zhou C, Fan Y, Li X, Huang L, Hu Y, Liang C, Hu X, Xu J, Yu X (2011a) The draft genome of the carcinogenic human liver fluke Clonorchis sinensis. Genome Biol 12:R107PubMedCentralPubMedCrossRefGoogle Scholar
  34. Wang X, Chen W, Hu F, Deng C, Zhou C, Lv X, Fan Y, Men J, Huang Y, Sun J, Hu D, Chen J, Yang Y, Liang C, Zheng H, Hu X, Xu J, Wu Z, Yu X (2011b) Clonorchis sinensis enolase: identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products. Mol Biochem Parasitol 177:135–142PubMedCrossRefGoogle Scholar
  35. Wang X, Chen W, Tian Y, Huang Y, Li X, Yu X (2014a) RNAi-mediated silencing of enolase confirms its biological importance in Clonorchis sinensis. Parasitol Res 113:1451–1458PubMedCrossRefGoogle Scholar
  36. Wang X, Chen W, Tian Y, Mao Q, Lv X, Shang M, Li X, Yu X, Huang Y (2014b) Surface display of Clonorchis sinensis enolase on Bacillus subtilis spores potentializes an oral vaccine candidate. Vaccine 32:1338–1345PubMedCrossRefGoogle Scholar
  37. Xu L, Hui AY, Albanis E, Arthur MJ, O’Byrne SM, Blaner WS, Mukherjee P, Friedman SL, Eng FJ (2005) Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut 54:142–151PubMedCentralPubMedCrossRefGoogle Scholar
  38. Young ND, Campbell BE, Hall RS, Jex AR, Cantacessi C, Laha T, Sohn WM, Sripa B, Loukas A, Brindley PJ, Gasser RB (2010a) Unlocking the transcriptomes of two carcinogenic parasites, Clonorchis sinensis and Opisthorchis viverrini. PLoS Negl Trop Dis 4:e719PubMedCentralPubMedCrossRefGoogle Scholar
  39. Young ND, Jex AR, Cantacessi C, Campbell BE, Laha T, Sohn WM, Sripa B, Loukas A, Brindley PJ, Gasser RB (2010b) Progress on the transcriptomics of carcinogenic liver flukes of humans–unique biological and biotechnological prospects. Biotechnol Adv 28:859–870PubMedCrossRefGoogle Scholar
  40. Zhang F, Liang P, Chen W, Wang X, Hu Y, Liang C, Sun J, Huang Y, Li R, Li X, Xu J, Yu X (2013) Stage-specific expression, immunolocalization of Clonorchis sinensis lysophospholipase and its potential role in hepatic fibrosis. Parasitol Res 112:737–749PubMedCrossRefGoogle Scholar
  41. Zheng M, Hu K, Liu W, Hu X, Hu F, Huang L, Wang P, Hu Y, Huang Y, Li W, Liang C, Yin X, He Q, Yu X (2011) Proteomic analysis of excretory secretory products from Clonorchis sinensis adult worms: molecular characterization and serological reactivity of a excretory-secretory antigen-fructose-1,6-bisphosphatase. Parasitol Res 109:737–744PubMedCrossRefGoogle Scholar
  42. Zheng M, Hu K, Liu W, Li H, Chen J, Yu X (2013) Proteomic analysis of different period excretory secretory products from Clonorchis sinensis adult worms: molecular characterization, immunolocalization, and serological reactivity of two excretory secretory antigens-methionine aminopeptidase 2 and acid phosphatase. Parasitol Res 112:1287–1297PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Xiaoyun Wang
    • 1
  • Fengyu Hu
    • 2
  • Xuchu Hu
    • 1
  • Wenjun Chen
    • 1
  • Yan Huang
    • 1
  • Xinbing Yu
    • 1
  1. 1.Department of Parasitology, Zhongshan School of MedicineSun Yat-sen UniversityGuangzhouPeople’s Republic of China
  2. 2.Department of Infectious DiseasesGuangzhou No. 8 People’s HospitalGuangzhouPeople’s Republic of China

Personalised recommendations