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Ductular reaction, cytokeratin 7 positivity, and gamma-glutamyl transferase in multistage hepatocarcinogenesis in rats

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Abstract

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is characterized by multistage formation. The presence of ductular reaction, cytokeratin 7 positivity (PCK7), and increased levels of gamma glutamyltransferase (γGT) has been observed during liver carcinogenesis and contribute to tumor progression. Our goal was to evaluate the ductular reaction in multistage carcinogenesis and to correlate PCK7 and γGT levels with tumor incidence, histological characteristics, liver DNA damage index, and the expression of oxidative stress proteins. HCC was induced in 24 male Wistar rats weighing 145–150 g by chronic and intermittent exposure to 50 or 100 mg/kg diethylnitrosamine (DEN). Six control animals received only vehicle. Blood was collected to determine hepatic enzyme levels. Animals were divided into three groups: control (CO), precancerous lesions (PL), and advanced HCC. Liver samples were obtained for immunohistochemical analyses and the measurement of protein expression. Statistical analyses included Tukey’s test and Pearson’s correlation analyses. We observed an extensive ductular reaction in advanced HCC and a strong correlation between PCK7 and levels of γGT and the poor prognosis and aggressiveness of HCC. The extent of PCK7 and high γGT levels were associated with overexpression of inducible nitric oxide synthase (iNOS) and heat shock factor protein 1 (HSF-1). However, PCK7 and γGT levels were negatively correlated with protein expression of nuclear factor erythroid 2-related factor 2 (NRF2) and inducible heat shock protein 70 (iHSP70). These findings suggest that ductular reaction is involved in the progression of multistage hepatocarcinogenesis.

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Acknowledgments

We wish to thank Dr. Widlak and our group for the important contribution to the understanding of the role of HSF-1. This study was supported by grants from the following Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundo de Incentivo à Pesquisa e Eventos (FIPE) of the Hospital de Clínicas of Porto Alegre (HCPA), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), and Laboratório Experimental de Hepatologia e Gastroenterologia (HCPA/UFRGS).

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Authors and Affiliations

  1. Center of Experimental Research, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2400, Porto Alegre, RS, Brazil

    Andrea Janz Moreira, Silvia Bona, Leila Xavier Sinigaglia Fratta, Giovana Regina Weber, Jorge Luiz dos Santos & Norma Possa Marroni

  2. Department of Biological Sciences: Physiology, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500, Porto Alegre, RS, Brazil

    Andrea Janz Moreira & Norma Possa Marroni

  3. Department of Physical Therapy of Porto Alegre Institute, IPA, Rua Joaquim Pedro Salgado, 80, Porto Alegre, Brazil

    Andrea Janz Moreira

  4. Gene Therapy Center, Center of Experimental Research, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2400, Porto Alegre, Brazil

    Graziella Ramos Rodrigues

  5. Program in Cell and Molecular Biology Applied to Health, Universidade Luterana do Brasil, Av. Farroupilha, 8001, Canoas, Brazil

    Jaqueline Nascimento Picada & Norma Possa Marroni

  6. Pediatric Hepatology Unit, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2400, Porto Alegre, Brazil

    Jorge Luiz dos Santos

  7. Department of Pathology, School of Medicine, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

    Carlos Thadeu Cerski

  8. Program in Liver Diseases, Universidade Federal de Ciências da Saúde de Porto Alegre, Rua Sarmento Leite, 245, Porto Alegre, Brazil

    Claudio Augusto Marroni

  9. Rua José Kanan Aranha 102, 91760-470, Porto Alegre, RS, Brazil

    Norma Possa Marroni

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  1. Andrea Janz Moreira
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  2. Graziella Ramos Rodrigues
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  9. Claudio Augusto Marroni
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  10. Norma Possa Marroni
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Correspondence to Norma Possa Marroni.

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All experiments were performed in accordance with the Guiding Principles for Research Involving Animals (NAS) and were approved by the Ethics in Research Committee (protocol 120355 – Ethical Committee of Hospital de Clínicas de Porto Alegre). Animals were killed at the end of the experiment by exsanguination under deep anesthesia, as described in the American Veterinary Medical Association (AVMA) Guidelines on Euthanasia (AVMA 2013).

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Moreira, A.J., Rodrigues, G.R., Bona, S. et al. Ductular reaction, cytokeratin 7 positivity, and gamma-glutamyl transferase in multistage hepatocarcinogenesis in rats. Protoplasma 254, 911–920 (2017). https://doi.org/10.1007/s00709-016-1000-0

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