Identification of protein changes in the blood plasma of lung cancer patients subjected to chemotherapy using a 2D-DIGE approach

Autoři: Andrzej Ciereszko aff001;  Mariola A. Dietrich aff001;  Mariola Słowińska aff001;  Joanna Nynca aff001;  Michał Ciborowski aff002;  Joanna Kisluk aff003;  Anna Michalska-Falkowska aff003;  Joanna Reszec aff004;  Ewa Sierko aff005;  Jacek Nikliński aff003
Působiště autorů: Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland aff001;  Clinical Research Centre, Medical University of Białystok, Białystok, Poland aff002;  Department of Clinical Molecular Biology, Medical University of Bialystok, Bialystok, Poland aff003;  Department of Medical Pathomorphology, Medical University of Bialystok, Bialystok, Poland aff004;  Department of Oncology, Medical University of Bialystok, Bialystok, Poland aff005
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223840


A comparative analysis of blood samples (depleted of albumin and IgG) obtained from lung cancer patients before chemotherapy versus after a second cycle of chemotherapy was performed using two-dimensional difference gel electrophoresis (2D-DIGE). The control group consisted of eight patients with non-cancerous lung diseases, and the experimental group consisted of four adenocarcinoma (ADC) and four squamous cell carcinoma (SCC) patients. Analyses of gels revealed significant changes in proteins and/or their proteoforms between control patients and lung cancer patients, both before and after a second cycle of chemotherapy. Most of these proteins were related to inflammation, including acute phase proteins (APPs) such as forms of haptoglobin and transferrin, complement component C3, and clusterin. The variable expression of APPs can potentially be used for profiling lung cancer. The greatest changes observed after chemotherapy were in transferrin and serotransferrin, which likely reflect disturbances in iron turnover after chemotherapy-induced anaemia. Significant changes in plasma proteins between ADC and SCC patients were also revealed, suggesting use of plasma vitronectin as a potential marker of SCC.

Klíčová slova:

Biomarkers – Blood plasma – Cancer chemotherapy – Fibrinogen – Haptoglobins – Lung and intrathoracic tumors – Protein expression – Acute phase proteins


1. Hoseok I, Cho J-Y. Lung cancer biomarkers. Adv Clin Chem. 2015;72: 107–170. doi: 10.1016/bs.acc.2015.07.003 26471082

2. Niklinski J, Kretowski A, Moniuszko M, Reszec J, Michalska-Falkowska A, Niemira M, et al. Systematic biobanking, novel imaging techniques, and advanced molecular analysis for precise tumor diagnosis and therapy: The Polish MOBIT project. Adv Med Sci. 2017;62: 405–413. doi: 10.1016/j.advms.2017.05.002 28646744

3. Pamungkas AD, Park C, Lee S, Jee SH, Park YH. High resolution metabolomics to discriminate compounds in serum of male lung cancer patients in south Korea. Resp Res. 2016;17. doi: 10.1186/s12931-016-0419-3 27506545

4. Villalobos P, Wistuba II. Lung cancer biomarkers. Hematol Oncol Clin North Am. 2017;31: 13–29. doi: 10.1016/j.hoc.2016.08.006 27912828

5. Hanash SM, Ostrin EJ, Fahrmann JF. Blood based biomarkers beyond genomics for lung cancer screening. Transl Lung Cancer Res. 2018;7: 327–333. doi: 10.21037/tlcr.2018.05.13 30050770

6. Boeri M, Verri C, Conte D, Roz L, Modena P, Facchinetti F, et al. MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Nat Acad Sci USA. 2011;108: 3713–3718. doi: 10.1073/pnas.1100048108 21300873

7. Ma L, Sun XZ, Kuai WX, Hu J, Yuan YF, Feng WJ, et al. Long noncoding RNA SOX2OT accelerates the carcinogenesis of Wilms' tumor through ceRNA through miR-363/FOXP4 axis. DNA Cell Biol. 2018;37: 1031–1038. doi: 10.1089/dna.2018.4420

8. Nolen BM, Langmead CJ, Choi S, Lomakin A, Marrangoni A, Bigbee WL, et al. Serum biomarker profiles as diagnostic tools in lung cancer. Canc Biomark. 2011;10: 3–12. doi: 10.3233/CBM-2012-0229 22297547

9. Hoseok I, Cho JY. Chapter Three—Lung Cancer Biomarkers. Ad Clin Chem. 2015;72: 107–170. doi: 10.1016/bs.acc.2015.07.003

10. Birse CE, Lagier RJ, FitzHugh W, Pass HI, Rom WN, Edell ES, et al. Blood-based lung cancer biomarkers identified through proteomic discovery in cancer tissues, cell lines and conditioned medium. Clin Proteom. 2015;12: 8. doi: 10.1186/s12014-015-9090-9 26279647

11. Huang Z, Ma L, Huang C, Li Q, Nice EC. Proteomic profiling of human plasma for cancer biomarker discovery. Proteomics 2017; 17. doi: 10.1002/pmic.201600240 27550791

12. Jung YJ, Katilius E, Ostroff RM, Kim Y, Seok M, Lee S, et al. Development of a protein biomarker panel to detect non–small-cell lung cancer in Korea. Clin Lung Canc. 2017;18: e99–e107. doi: 10.1016/j.cllc.2016.09.012 27836219

13. Pastor MD, Nogal A, Molina-Pinelo S, Carnero A, Paz-Ares L. Proteomic biomarkers in lung cancer. Clin Transl Oncol. 2013;15: 671–682. doi: 10.1007/s12094-013-1034-0 23606351

14. Kondo T. Cancer biomarker development and two-dimensional difference gel electrophoresis (2D-DIGE). Biochim Biophys Acta Proteins Proteom. 2019;1867: 2–8. doi: 10.1016/j.bbapap.2018.07.002 30392560

15. Meleady P. Two-dimensional gel electrophoresis and 2D-DIGE. Methods Mol Biol 2018;1664: 3–14. doi: 10.1007/978-1-4939-7268-5_1 29019120

16. Arnold GJ, Frohlich T. Dynamic proteome signatures in gametes, embryos and their maternal environment. Reprod Fertil Dev 2011;23: 81–93. doi: 10.1071/RD10223 21366984

17. Kondo T, Seike M, Mori Y, Fujii K, Yamada T, Hirohashi S. Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool. Proteomics 2003;3: 1758–66. doi: 10.1002/pmic.200300531 12973736

18. Ohlendieck K. Comparative DIGE proteomics. Methods Mol Biol. 2018;1664: 17–24. doi: 10.1007/978-1-4939-7268-5_2 29019121

19. Strohkamp S, Gemoll T, Habermann JK. Possibilities and limitations of 2DE-based analyses for identifying low-abundant tumor markers in human serum and plasma. Proteomics 2016;16: 2519–2532. doi: 10.1002/pmic.201600154 27377442

20. Fujii K, Kondo T, Yokoo H, Yamada T, Iwatsuki K, Hirohashi S. Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye. Proteomics 2005;5: 1411–22. doi: 10.1002/pmic.200401004 15751005

21. Seike M, Kondo T, Fujii K, Okano T, Yamada T, Matsuno Y, et al. Proteomic signatures for histological types of lung cancer. Proteomics 2005;5: 2939–48. doi: 10.1002/pmic.200401166 15996008

22. Okano T, Seike M, Kuribayashi H, Soeno C, Ishii T, Kida K et al. Identification of haptoglobin peptide as a novel serum biomarker for lung squamous cell carcinoma by serum proteome and peptidome profiling. Int J Oncol. 2016;48: 945–952. doi: 10.3892/ijo.2016.3330 26783151

23. Ünlü M, Morgan ME, Minden JS. Difference gel electrophoresis: A single gel method for detecting changes in protein extracts. Electrophoresis 1997;18: 2071–2077. doi: 10.1002/elps.1150181133 9420172

24. Beckett P. The basics of 2D DIGE. Methods Mol Biol. 2012;854: 9–19. doi: 10.1007/978-1-61779-573-2_2 22311750

25. Zakharchenko O, Greenwood C, Lewandowska A, Hellman U, Alldridge L, Souchelnytskyi S. Meta-data analysis as a strategy to evaluate individual and common features of proteomic changes in breast cancer. Canc Genom Proteom. 2011;8: 1–14.

26. Posch A, Kohn J, Oh K, Hammond M, Liu N. V3 stain-free workflow for a practical, convenient, and reliable total protein loading control in western blotting. J Visualized Exp 2013;82: e50948.

27. Repetto O, Mussolin L, Elia C, Martina L, Bianchi M, Buffardi S, et al. Proteomic identification of plasma biomarkers in children and adolescents with recurrent hodgkin lymphoma. J Cancer 2018;9: 4650–8. doi: 10.7150/jca.27560 30588249

28. Mallikaratchy P, Tang Z, Kwame S, Meng L, Shangguan D, Tan W. Aptamer directly evolved from live cells recognizes membrane bound immunoglobin heavy mu chain in burkitt's lymphoma cells. Mol Cell Proteom. 2007;6: 2230–2238. doi: 10.1074/mcp.M700026-MCP200 17875608

29. Gianazza E, Miller I, Palazzolo L, Parravicini C, Eberini I. With or without you—proteomics with or without major plasma/serum proteins. J Proteom. 2016;140: 62–80. doi: 10.1016/j.jprot.2016.04.002 27072114

30. Wen C, Chen K, Chen C, Chuang J, Yang P, Chow L. Development of an AlphaLISA assay to quantify serum core-fucosylated E-cadherin as a metastatic lung adenocarcinoma biomarker. J Proteom. 2012;75: 3963–3976. doi: 10.1016/j.jprot.2012.05.015 22634079

31. Dowling P, O'Driscoll L, Meleady P, Henry M, Roy S, Ballot J, et al. 2-D difference gel electrophoresis of the lung squamous cell carcinoma versus normal sera demonstrates consistent alterations in the levels of ten specific proteins. Electrophoresis 2007;28: 4302–4310. doi: 10.1002/elps.200700246 18041032

32. Rodríguez-Piñeiro AM, Blanco-Prieto S, Sánchez-Otero N, Rodríguez-Berrocal FJ, Páez de la Cadena M. On the identification of biomarkers for non-small cell lung cancer in serum and pleural effusion. J Proteom. 2010;73: 1511–1522. doi: 10.1016/j.jprot.2010.03.005 20230924

33. Kisluk J, Ciborowski M, Niemira M, Kretowski A, Niklinski J. Proteomics biomarkers for non-small cell lung cancer. J Pharm Biomed Anal. 2014;101: 40–49. doi: 10.1016/j.jpba.2014.07.038 25175018

34. Okano T, Kondo T, Kakisaka T, Fujii K, Yamada M, Kato H, et al. Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis. Proteomics 2006;6: 3938–3948. doi: 10.1002/pmic.200500883 16767791

35. Hoagland IV LFM, Campa MJ, Gottlin EB, Herndon II JE, Patz Jr. EF. Haptoglobin and posttranslational glycan-modified derivatives as serum biomarkers for the diagnosis of nonsmall cell lung cancer. Cancer 2007;110: 2260–2268. doi: 10.1002/cncr.23049 17918261

36. Guergova-Kuras M, Kurucz I, Hempel W, Tardieu N, Kádas J, Malderez-Bloes C et al. Discovery of lung cancer biomarkers by profiling the plasma proteome with monoclonal antibody libraries. Mol Cell Proteom. 2011;10. doi: 10.1074/mcp.M111.010298 21947365

37. Dowling P, Clarke C, Hennessy K, Torralbo-Lopez B, Ballot J, Crown J et al. Analysis of acute-phase proteins, AHSG, C3, CLI, HP and SAA, reveals distinctive expression patterns associated with breast, colorectal and lung cancer. Int J Canc. 2012;131: 911–923. doi: 10.1002/ijc.26462 21953030

38. Bhatnagar S, Katare DP, Jain SK. Serum-based protein biomarkers for detection of lung cancer. Cent Eur J Biol. 2014;9: 341–358. doi: 10.2478/s11535-013-0271-0

39. Sideras K, Kwekkeboom J. Cancer inflammation and inflammatory biomarkers: Can neutrophil, lymphocyte, and platelet counts represent the complexity of the immune system? Transpl Int. 2014;27: 28–31. doi: 10.1111/tri.12229 24350721

40. Mantovani A. Cancer: Inflaming metastasis. Nature 2009;457: 36–37. doi: 10.1038/457036b 19122629

41. Partridge M, Fallon M, Bray C, McMillan D, Brown D, Laird B. Prognostication in advanced cancer: A study examining an inflammation-based score. J Pain Sympt Manag. 2012;44: 161–167. doi: 10.1016/j.jpainsymman.2011.08.013 22732417

42. Shiels MS, Katki HA, Hildesheim A, Pfeiffer RM, Engels EA, Williams M, et al. Circulating inflammation markers, risk of lung cancer, and utility for risk stratification. J Nat Canc Inst, 2015;107. doi: 10.1093/jnci/djv199 26220734

43. Shiels MS, Shu X, Chaturvedi AK, Gao Y, Xiang Y, Cai Q, et al. A prospective study of immune and inflammation markers and risk of lung cancer among female never smokers in shanghai. Carcinogenesis 2017;38: 1004–1010. doi: 10.1093/carcin/bgx075 28981818

44. Wang Y, Song G, Wang Y, Qiu L, Qin X, Liu H, et al. Elevated serum levels of circulating immunoinflammation-related protein complexes are associated with cancer. J Proteom Res. 2014;13: 710–719. doi: 10.1021/pr4008255 24295561

45. Smeets MB, Fontijn J, Kavelaars A, Pasterkamp G, De Kleijn DPV. The acute phase protein haptoglobin is locally expressed in arthritic and oncological tissues. International J Exp Pathol. 2003;84: 69–74. doi: 10.1046/j.1365-2613.2003.00336.x 12801280

46. Hanash S, Taguchi A, Wang H, Ostrin EJ. Deciphering the complexity of the cancer proteome for diagnostic applications. Exp Rev Mol Diagn. 2016;16: 399–405. doi: 10.1586/14737159.2016.1135738 26694525

47. Guerin M, Gonçalves A, Toiron Y, Baudelet E, Audebert S, Boyer J, et al. How may targeted proteomics complement genomic data in breast cancer? Exp Rev Proteom. 2017;14: 43–54. doi: 10.1080/14789450.2017.1256776 27813428

48. Gu H, Ren JM, Jia X, Levy T, Rikova K, Yang V, et al. Quantitative profiling of post-translational modifications by immunoaffinity enrichment and LC-MS/MS in cancer serum without immunodepletion. Mol Cell Proteom. 2016;15: 692–702. doi: 10.1074/mcp.O115.052266 26635363

49. Takahashi S, Sugiyama T, Shimomura M, Kamada Y, Fujita K, Nonomura N, et al. Site-specific and linkage analyses of fucosylated N-glycans on haptoglobin in sera of patients with various types of cancer: Possible implication for the differential diagnosis of cancer. Glycoconjugate J. 2016;33: 471–482. doi: 10.1007/s10719-016-9653-7 26869352

50. Schmidlin T, Garrigues L, Lane CS, Mulder TC, van Doorn S, Post H. Assessment of SRM, MRM3, and DIA for the targeted analysis of phosphorylation dynamics in non-small cell lung cancer. Proteomics 2016;16: 2193–2205. doi: 10.1002/pmic.201500453 27219855

51. Zavialova MG, Zgoda VG, Nikolaev EN. Analysis of the role of protein phosphorylation in the development of diseases. Biochemistry (Moscow) Supplement Series B: Biomed Chem. 2017;11: 203–218. doi: 10.1134/S1990750817030118

52. Li QQ, Hao JJ, Zhang Z, Krane LS, Hammerich KH, Sanford T, et al. Proteomic analysis of proteome and histone post-translational modifications in heat shock protein 90 inhibition-mediated bladder cancer therapeutics. Sci Rep. 2017;7: 201. doi: 10.1038/s41598-017-00143-6 28298630

53. Ludwig H, Müldür E, Endler G, Hübl W. Prevalence of iron deficiency across different tumors and its association with poor performance status, disease status and anemia. Ann Oncol. 2013;24: 1886–1892. doi: 10.1093/annonc/mdt118 23567147

54. Ludwig H, Aapro M, Bokemeyer C, Glaspy J, Hedenus M, Littlewood TJ, et al. A European patient record study on diagnosis and treatment of chemotherapy-induced anaemia. Supp Care Canc. 2014;22: 2197–2206. doi: 10.1007/s00520-014-2189-0 24659244

55. Nakamura T, Takahashi M, Niigata R, Yamashita K, Kume M, Hirai M, et al. Changes in blood concentrations of trace metals in cancer patients receiving cisplatin-based chemotherapy. Biomed Rep. 2016;5: 737–744. doi: 10.3892/br.2016.789 28105341

56. Michlmayr A, Bachleitner-Hofmann T, Baumann S, Marchetti-Deschmann M, Rech-Weichselbraun I, Burghuber C, et al. Modulation of plasma complement by the initial dose of epirubicin/docetaxel therapy in breast cancer and its predictive value. Brit J Canc. 2010;103: 1201–1208. doi: 10.1038/sj.bjc.6605909 20877360

57. Lin K, He S, He L, Chen J, Cheng X, Zhang G, et al. Complement component 3 is a prognostic factor of non-small cell lung cancer. Mol Med Rep. 2014;10: 811–817. doi: 10.3892/mmr.2014.2230 24819254

58. Kwak JW, Laskowski J, Li HY; McSharry MV, Sippel TR, Bullock BL, et al. Complement Activation via a C3a Receptor Pathway Alters CD4(+) T Lymphocytes and Mediates Lung Cancer Progression. Canc Res. 2018;78: 143–156. doi: 10.1158/0008-5472.CAN-17-0240 29118090

59. Schneider G, Bryndza E, Poniewierska-Baran A, Serwin K, Suszynska M, Sellers ZP, et al. Evidence that vitronectin is a potent migration-enhancing factor for cancer cells chaperoned by fibrinogen: A novel view of the metastasis of cancer cells to low-fibrinogen lymphatics and body cavities. Oncotarget 2016;7: 69829–69843. doi: 10.18632/oncotarget.12003 27634880

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2019 Číslo 10