Parasitology Research

, Volume 117, Issue 10, pp 3127–3136 | Cite as

Exploring tumourigenic potential of the parasite Anisakis: a pilot study

  • María Teresa Corcuera
  • Cruz Rodríguez-Bobada
  • Jaime Zuloaga
  • Fernando Gómez-Aguado
  • Rosa Rodríguez-Perez
  • Ángel Mendizabal
  • Pablo González
  • Javier Arias-Díaz
  • María Luisa CaballeroEmail author
Original Paper


Anisakiasis is a global disease caused by consumption of raw or lightly cooked fish parasitised with Anisakis spp. third-stage larvae. Cases in the literature show colocalised anisakiasis and colorectal cancer, and the incidental finding of Anisakis larvae at the tumour site was reported. Data from our group suggested an epidemiological link between previous infection and gastrointestinal cancer. Furthermore, it has recently been reported that Anisakis products lead to inflammation and DNA damage. Based on these facts, the aim was to investigate whether Anisakis antigens are able to induce changes in the proliferation of epithelial cells in vitro or in the expression of serum microRNA (miRNA) in Sprague-Dawley rats. Anisakis complete extract (CE) induced increases in cell proliferation and decreases in apoptosis compared with nontreated cells, which resulted in a significant increase in the absolute number of viable cells at 48 h of exposure (P < .05). Furthermore, the miRNAs mmu-miR-1b-5p and mmu-miR-10b-5p (a cancer-related miRNA) were significantly decreased (P < .05) in sera from the rats inoculated with Anisakis CE, compared with control rats inoculated with saline. Additionally, based on their relative quantification values, four other cancer-related miRNAs were considered to be differently expressed, rno-miR-218a-5p and mmu-miR-224-5p (decreased) and rno-miR-125a-3p and rno-miR-200c-3p (increased). Anisakis CE was able to induce changes both in epithelial cells in vitro and in an animal model. The results obtained with Anisakis CE, in terms of increasing cell proliferation, decreasing apoptosis and inducing changes in the expression of serum cancer-related miRNAs in rats, suggest that Anisakis could have tumourigenic potential.


Anisakiasis Anisakis Cell proliferation microRNA Gastrointestinal cancer 



We thank Drs. Noemí Fernandez and Ricardo Ramos for their expert assistance with the analysis and interpretation of the results of the miRNA expression study.

This research was co-financed by Fondo de Investigación Sanitario (FIS) from the Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FEDER) (grant No. FIS-PI12/00888).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.


  1. AAITO-IFIACI Anisakis Consortium (2011) Anisakis hypersensitivity in Italy: prevalence and clinical features: a multicenter study. Allergy 66:1563–1569Google Scholar
  2. Abdel-Ghaffar F, Badr AM, Morsy K, Ebead S, El Deeb S, Al Quraishy S, Mehlhorn H (2015) Cytokine signature and antibody-mediated response against fresh and attenuated Anisakis simplex (L3) administration into Wistar rats: implication for anti-allergic reaction. Parasitol Res 114:2975–2984CrossRefPubMedGoogle Scholar
  3. Abou-Rahma Y, Abdel-Gaber R, Kamal Ahmed A (2016) First record of Anisakis simplex third-stage larvae (Nematoda, Anisakidae) in European hake Merluccius merluccius lessepsianus in Egyptian water. J Parasitol Res 16:9609752Google Scholar
  4. Adamopoulos PG, Kontos CK, Rapti SM, Papadopoulos IN, Scorilas A (2015) miR-224 overexpression is a strong and independent prognosticator of short-term relapse and poor overall survival in colorectal adenocarcinoma. Int J Oncol 46:849–859CrossRefPubMedGoogle Scholar
  5. Ahmed M, Ayoob F, Kesavan M, Gumaste V, Khalil A (2016) Gastrointestinal anisakidosis—watch what you eat. Cureus 8:e860PubMedPubMedCentralGoogle Scholar
  6. Audicana MT, Fernandez de Corres L, Munoz D, Fernandez E, Navarro JA, del Pozo MD (1996) Recurrent anaphylaxis caused by Anisakis simplex parasitizing fish. J Allergy Clin Immunol 96:558–560CrossRefGoogle Scholar
  7. Audicana MT, Kennedy MW (2008) Anisakis simplex: from obscure infectious worm to inducer of immune hypersensitivity. Clin Microbiol Rev 21:360–379CrossRefPubMedPubMedCentralGoogle Scholar
  8. Ayremlou N, Mozdarani H, Mowla SJ, Delavari A (2015) Increased levels of serum and tissue miR-107 in human gastric cancer: correlation with tumor hypoxia. Cancer Biomark 15:851–860CrossRefPubMedPubMedCentralGoogle Scholar
  9. Balkwill F, Mantovani A (2001) Inflammation and cancer: back to Virchow? Lancet 357:539–545CrossRefGoogle Scholar
  10. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297CrossRefPubMedPubMedCentralGoogle Scholar
  11. Berridge MV, Tan AS (1993) Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys 303:474–482CrossRefPubMedGoogle Scholar
  12. Blick C, Ramachandran A, McCormick R, Wigfield S, Cranston D, Catto J, Harris AL (2015) Identification of a hypoxia-regulated miRNA signature in bladder cancer and a role for miR-145 in hypoxia-dependent apoptosis. Br J Cancer 113:634–644CrossRefPubMedPubMedCentralGoogle Scholar
  13. Botelho M, Ferreira AC, Oliveira MJ, Domingues A, Machado JC, da Costa JM (2009) Schistosoma haematobium total antigen induces increased proliferation, migration and invasion, and decreases apoptosis of normal epithelial cells. Int J Parasitol 39:1083–1091CrossRefPubMedGoogle Scholar
  14. Botelho MC, Oliveira PA, Lopes C, Correia da Costa JM, Machado JC (2011) Urothelial dysplasia and inflammation induced by Schistosoma haematobium total antigen instillation in mice normal urothelium. Urol Oncol 29:809–814CrossRefPubMedGoogle Scholar
  15. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedPubMedCentralGoogle Scholar
  16. Caballero ML, Moneo I, Gómez-Aguado F, Corcuera MT, Casado I, Rodríguez-Pérez R (2008) Isolation of Ani s 5, an excretory-secretory and highly heat-resistant allergen useful for the diagnosis of Anisakis larvae sensitization. Parasitol Res 103:1231–1233CrossRefPubMedGoogle Scholar
  17. Caballero ML, Umpierrez A, Moneo I, Rodriguez-Perez R (2011) Ani s 10 a new Anisakis simplex allergen: cloning and heterologous expression. Parasitol Int 60:209–212CrossRefPubMedGoogle Scholar
  18. Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, Guo J, Zhang Y, Chen J, Guo X, Li Q, Li X, Wang W, Zhang Y, Wang J, Jiang X, Xiang Y, Xu C, Zheng P, Zhang J, Li R, Zhang H, Shang X, Gong T, Ning G, Wang J, Zen K, Zhang J, Zhang CY (2008) Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18:997–1006CrossRefPubMedPubMedCentralGoogle Scholar
  19. Daschner A, Alonso-Gómez A, Cabañas R, Suarez-de-Parga JM, MC L-S (2000) Gastroallergic anisakiasis: borderline between food allergy and parasitic disease-clinical and allergologic evaluation of 20 patients with confirmed acute parasitism by Anisakis simplex. J Allergy Clin Immunol 105:176–181CrossRefPubMedGoogle Scholar
  20. Del Pozo MD, Audicana M, Diez JM, Munoz D, Ansotegui IJ, Fernandez E, García M, Etxenagusia M, Moneo I, Fernández de Corres L (1997) Anisakis simplex, a relevant etiologic factor in acute urticaria. Allergy 52:576–579CrossRefPubMedGoogle Scholar
  21. Duan F, Kaijuan W, Dai L, Zhao X, Feng Y, Song C, Cui S, Wang C (2016) Prognostic significance of low microRNA-218 expression in patients with different types of cancer: evidence from published studies. Medicine (Baltimore) 95:e4773CrossRefGoogle Scholar
  22. García-Mayoral MF, Treviño MA, Pérez-Piñar T, Caballero ML, Knaute T, Umpierrez A, Bruix M, Rodríguez-Pérez R (2014) Relationships between IgE/IgG4 epitopes, structure and function in Anisakis simplex Ani s 5, a member of the SXP/RAL-2 protein family. PLoS Negl Trop Dis 8:e2735CrossRefPubMedPubMedCentralGoogle Scholar
  23. Garcia-Perez JC, Rodríguez-Perez R, Ballestero A, Zuloaga J, Fernandez-Puntero B, Arias-Díaz J, Caballero ML (2015) Previous exposure to the fish parasite Anisakis as a potential risk factor for gastric or colon adenocarcinoma. Medicine (Baltimore) 94:e1699CrossRefGoogle Scholar
  24. Hashiguchi Y, Nishida N, Mimori K, Sudo T, Tanaka F, Shibata K, Ishii H, Mochizuki H, Hase K, Doki Y, Mori M (2012) Down-regulation of miR-125a-3p in human gastric cancer and its clinicopathological significance. Int J Oncol 40:1477–1482PubMedGoogle Scholar
  25. Hashimoto Y, Akiyama Y, Yuasa Y (2013) Multiple-to-multiple relationships between microRNAs and target genes in gastric cancer. PLoS One 8:e62589CrossRefPubMedPubMedCentralGoogle Scholar
  26. Heffler E, Sberna ME, Sichili S, Intravaia R, Nicolosi G, Porto M, Liuzzo MT, Picardi G, Fichera S, Crimi N (2016) High prevalence of Anisakis simplex hypersensitivity and allergy in Sicily, Italy. Ann Allergy Asthma Immunol 116:146–150CrossRefPubMedGoogle Scholar
  27. Hochberg NS, Hamer DH (2010) Anisakidosis: perils of the deep. Clin Infect Dis 51:806–812CrossRefPubMedGoogle Scholar
  28. Ishikura H, Kikuchi K, Nagasawa K, Ooiwa T, Takamiya H, Sato N, Sugane K (1993) Anisakidae and anisakidosis. Prog Clin Parasitol 3:43–102CrossRefPubMedGoogle Scholar
  29. Jansson MD, Lund AH (2012) MicroRNA and cancer. Mol Oncol 6:590–610CrossRefPubMedPubMedCentralGoogle Scholar
  30. Ke TW, Hsu HL, Wu YH, Chen WT, Cheng YW, Cheng CW (2014) MicroRNA-224 suppresses colorectal cancer cell migration by targeting Cdc42. Dis Markers 2014:617150, 1, 11CrossRefGoogle Scholar
  31. Kim K, Lee HC, Park JL, Kim M, Kim SY, Noh SM, Song KS, Kim JC, Kim YS (2011) Epigenetic regulation of microRNA-10b and targeting of oncogenic MAPRE1 in gastric cancer. Epigenetics 6:740–751CrossRefPubMedGoogle Scholar
  32. 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 102:411–417CrossRefPubMedGoogle Scholar
  33. Klimpel S, Palm HW, Rückert S, Piatkowski U (2004) The life cycle of Anisakis simplex in the Norwegian Deep (northern North Sea). Parasitol Res 94:1–9CrossRefPubMedGoogle Scholar
  34. Kuhn T, Garcıa-Marquez J, Klimpel S (2011) Adaptive radiation within marine anisakid nematodes: a zoogeographical modelling of cosmopolitan, zoonotic parasites. PLoS One 6:e28642CrossRefPubMedPubMedCentralGoogle Scholar
  35. Li Z, Lei H, Luo M, Wang Y, Dong L, Ma Y, Liu C, Song W, Wang F, Zhang J, Shen J, Yu J (2015) DNA methylation downregulated mir-10b acts as a tumor suppressor in gastric cancer. Gastric Cancer 18:43–54CrossRefPubMedGoogle Scholar
  36. Ling H, Pickard K, Ivan C, Isella C, Ikuo M, Mitter R, Spizzo R, Bullock MD, Braicu C, Pileczki V, Vincent K, Pichler M, Stiegelbauer V, Hoefler G, Almeida MI, Hsiao A, Zhang X, Primrose JN, Packham GK, Liu K, Bojja K, Gafà R, Xiao L, Rossi S, Song JH, Vannini I, Fanini F, Kopetz S, Zweidler-McKay P, Wang X, Ionescu C, Irimie A, Fabbri M, Lanza G, Hamilton SR, Berindan-Neagoe I, Medico E, Mirnezami AH, Calin GA, Nicoloso MS (2016) The clinical and biological significance of MIR-224 expression in colorectal cancer metastasis. Gut 65:977–989CrossRefPubMedGoogle Scholar
  37. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR (2005) MicroRNA expression profiles classify human cancers. Nature 435:834–838CrossRefPubMedGoogle Scholar
  38. Maggi P, Caputi-Iambrenghi O, Scardigno A, Scoppetta L, Saracino A, Valente M, Pastore G, Angarano G (2000) Gastrointestinal infection due to Anisakis simplex in southern Italy. Eur J Epidemiol 16:75–78CrossRefPubMedGoogle Scholar
  39. Messina CM, Pizzo F, Santulli A, Bušelić I, Boban M, Orhanović S, Mladineo I (2016) Anisakis pegreffii (Nematoda: Anisakidae) products modulate oxidative stress and apoptosis-related biomarkers in human cell lines. Parasit Vectors 9:607CrossRefPubMedPubMedCentralGoogle Scholar
  40. Mineta S, Shimanuki K, Sugiura A, Tsuchiya Y, Kaneko M, Sugiyama Y, Akimaru K, Tajiri T (2006) Chronic anisakiasis of the ascending colon associated with carcinoma. J Nippon Med Sch 73:169–174CrossRefPubMedGoogle Scholar
  41. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 105:10513–10518CrossRefPubMedPubMedCentralGoogle Scholar
  42. Moneo I, Caballero ML, Gómez F, Ortega E, Alonso MJ (2000) Isolation and characterization of a major allergen from the fish parasite Anisakis simplex. J Allergy Clin Immunol 06:177–182CrossRefGoogle Scholar
  43. Namiki N, Yazaki Y (1989) Endoscopic findings of gastric anisakiasis with acute symptoms. In: Ishikura H, Namiki M (ed) Gastric anisakiasis in Japan. Epidemiology, diagnosis, treatment. Springer-Verlag, Tokyo, pp 47–51CrossRefGoogle Scholar
  44. Pak JH, Kim IK, Kim SM, Maeng S, Song KJ, Na BK, Kim TS (2014) Induction of cancer-related microRNA expression profiling using excretory-secretory products of Clonorchis sinensis. Parasitol Res 113:4447–4455CrossRefPubMedGoogle Scholar
  45. Pampiglione S, Rivasi F, Criscuolo M, De Benedittis A, Gentile A, Russo S, Testini M, Villan M (2002) Human Anisakiasis in Italy: a report of eleven new cases. Pathol Res Pract 198:429–434CrossRefPubMedGoogle Scholar
  46. Plieskatt J, Rinaldi G, Feng Y, Peng J, Easley S, Jia X, Potriquet J, Pairojkul C, Bhudhisawasdi V, Sripa B, Brindley PJ, Bethony J, Mulvenna J (2015) A micro-RNA profile associated with Opisthorchis viverrini-induced cholangiocarcinoma in tissue and plasma. BMC Cancer 15:309CrossRefPubMedPubMedCentralGoogle Scholar
  47. Rodriguez-Perez R, Caballero ML, Gonzalez-Munoz M, Rodriguez-Mahillo A, Moneo I (2007) Cloning and expression of a biologically active Anisakis simplex allergen Ani s 1 in the yeast Pichia pastoris. Mol Biochem Parasitol 154:115–118CrossRefPubMedGoogle Scholar
  48. Rodriguez-Perez R, Moneo I, Rodriguez-Mahillo A, Caballero ML (2008) Cloning and expression of Ani s 9, a new Anisakis simplex allergen. Mol Biochem Parasitol 159:92–97CrossRefPubMedGoogle Scholar
  49. Speciale A, Trombetta D, Saija A, Panebianco A, Giarratana F, Ziino G, Minciullo PL, Cimino F, Gangemi S (2017) Exposure to Anisakis extracts can induce inflammation on in vitro cultured human colonic cells. Parasitol Res 116:2471–2477CrossRefPubMedGoogle Scholar
  50. Thuwajit C, Thuwajit P, Kaewkes S, Sripa B, Uchida K, Miwa M, Wongkham S (2004) Increased cell proliferation of mouse fibroblast NIH-3T3 in vitro induced by excretory/secretory product(s) from Opisthorchis viverrini. Parasitology 129:455–464CrossRefPubMedGoogle Scholar
  51. Toiyama Y, Hur K, Tanaka K, Inoue Y, Kusunoki M, Boland CR, Goel A (2014) Serum miR-200c is a novel prognostic and metastasis-predictive biomarker in patients with colorectal cancer. Ann Surg 259:735–743CrossRefPubMedPubMedCentralGoogle Scholar
  52. Tsutsumi Y, Fujimoto Y (1983) Early cancer superimposed on infestation of an Anisakis-like larva: a case report. Tokai J Exp Clin Med 8:265–273PubMedGoogle Scholar
  53. Valladares-Ayerbes M, Reboredo M, Medina-Villaamil V, Iglesias-Díaz P, Lorenzo-Patiño M, Haz M, Santamarina I, Blanco M, Fernández-Tajes J, Quindós M, Carral A, Figueroa A, Antón-Aparicio L, Calvo L (2012) Circulating miR-200c as a diagnostic and prognostic biomarker for gastric cancer. J Transl Med 10:186CrossRefPubMedPubMedCentralGoogle Scholar
  54. Wang XX, Ge SJ, Wang XL, Jiang1 LX, Sheng2 MF, Ma2 JJ (2016) miR-218 tissue expression level is associated with aggressive progression of gastric cancer. Genet Mol Res 5:2Google Scholar
  55. Wang YY, Ye ZY, Zhao ZS, Li L, Wang YX, Tao HQ, Wang HJ, He XJ (2013) Clinicopathologic significance of miR-10b expression in gastric carcinoma. Hum Pathol 44:1278–1285CrossRefPubMedGoogle Scholar
  56. Xin SY, Feng XS, Zhou LQ, Sun JJ, Gao XL, Yao GL (2014) Reduced expression of circulating microRNA-218 in gastric cancer and correlation with tumor invasion and prognosis. World J Gastroenterol 20:6906–6911CrossRefPubMedPubMedCentralGoogle Scholar
  57. Xu X, Jia R, Zhou Y, Song X, Wang J, Qian G, Ge S, Fan X (2011) Microarray-based analysis: identification of hypoxia-regulated microRNAs in retinoblastoma cells. Int J Oncol 38:1385–1393PubMedGoogle Scholar
  58. Yokogawa M, Yoshimura H (1967) Clinicopathologic studies on larval anisakiasis in Japan. Amer J Trop Med Hyg 16:723–728CrossRefGoogle Scholar
  59. Yuan K, Xie K, Fox J, Zeng H, Gao H, Huang C, Wu M (2013) Decreased levels of miR-224 and the passenger strand of miR-221 increase MBD2, suppressing Maspin and promoting colorectal tumor growth and metastasis in mice. Gastroenterology 145:853–64.e9, 864.e9CrossRefPubMedCentralGoogle Scholar
  60. Zhang JY, Wang YM, Song LB, Chen C, Wang YC, Song NH (2016) Prognostic significance of microRNA-200c in various types of cancer: an updated meta-analysis of 34 studies. Mol Clin Oncol 4:933–941CrossRefPubMedPubMedCentralGoogle Scholar
  61. Zuloaga J, Rodríguez-Bobada C, Corcuera MT, Gómez-Aguado F, González P, Rodríguez-Perez R, Arias-Díaz J, Caballero ML (2013) A rat model of intra-gastric infection with Anisakis spp. live larvae: histopathological study. Parasitol Res 112:2409–2411CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • María Teresa Corcuera
    • 1
  • Cruz Rodríguez-Bobada
    • 2
  • Jaime Zuloaga
    • 3
  • Fernando Gómez-Aguado
    • 4
  • Rosa Rodríguez-Perez
    • 5
  • Ángel Mendizabal
    • 6
  • Pablo González
    • 2
  • Javier Arias-Díaz
    • 3
  • María Luisa Caballero
    • 7
    Email author
  1. 1.Department of MicrobiologyLa Paz University HospitalMadridSpain
  2. 2.Experimental Medicine and Department of SurgeryHospital Clínico San CarlosMadridSpain
  3. 3.Department of Surgery, Hospital Clínico San CarlosComplutense UniversityMadridSpain
  4. 4.Department of HaematologyLa Paz University HospitalMadridSpain
  5. 5.La Paz University Hospital Institute for Health Research (IdiPAZ)MadridSpain
  6. 6.Department of Health Services, Quality and ConsumptionMadridSpain
  7. 7.Department of AllergyLa Paz University Hospital Institute for Health Research (IdiPAZ)MadridSpain

Personalised recommendations