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Della Porta et al. Comparison of p53 and prohibitin expression

hepatocytes as a consequence of chronic inflammation, potential differences in the pathogenesis of human
[5]
which occurs in viral hepatitis. However, little is known hepatic cancer.
about the interaction of NFκB activation and tumour
microenvironment in the sequence of hepatitis C virus METHODS
(HCV), cirrhosis (CIR), and HCC.
Tissue specimens
The inflammatory cytokine tumor necrosis factor-α Primary liver carcinoma samples were obtained
(TNFα) participates in the control of cellular from the Department of Pathology at the University
proliferation and differentiation and cell death. [6] Hospital of the Faculty of Medicine of Ribeirão Preto,
Although TNFα was initially identified as inducing University of São Paulo, Ribeirão Preto, São Paulo,
cell death in some tumours, an association between Brazil. Twenty samples of normal liver from autopsies
activation of the TNFα/NFκB pathway by inflammation with causes other than liver disease (NL), 30 cases
and hepatocarcinogenesis has also been reported. [7] of HCV, 30 cases of CIR associated with HCV and
Binding of TNFα to TNF receptor-1 (TNFR1) results in 60 cases of HCC related to HCV-CIR were selected
NFκB activation and may induce hepatocyte survival for this study [Table 1]. Patients with evidence of any
[8]
and proliferation. Additionally, activated NFκB can be other cause of liver disease were excluded. The study
considered as a protector of TNFα-induced apoptosis. [9] was approved by the local Ethics Committee (number
1611/2011).
TNF-related apoptosis-inducing ligand (TRAIL) has
been demonstrated as a proapoptotic mediator of Immunohistochemistry
various tumour cells. [10] Similarly, TRAIL caused Liver preparations were submitted for immuno-
cytotoxic effects in transformed hepatocytes of histochemical analysis. Sections were incubated
HCC, perhaps related to inhibition of NFκB survival with monoclonal primary antibodies specific for
signalling. [11] In contrast, participation of TRAIL TNFR1, TRAIL, RelA/p65, and p53 (Santa Cruz
in hepatocyte’s apoptosis during chronic hepatic Biotechnology, Santa Cruz, CA, USA, dilution 1:100)
diseases remains controversial. and PHB (Thermo Fisher Scientific Waltham, MA,
USA, dilution 1:100). Following this, a secondary
The p53 gene is a classical tumor suppressor gene. antibody (Vectastain Elite ABC Kit, Universal, Vector
p53 mutations occur in diverse human cancers, Laboratories Inc.) was applied. Next, the slides were
including HCC. p53 tumor suppressor function incubated with avidin-biotin-peroxidase complex
involves cell cycle control, transcriptional regulation, (Vectastain Elite ABC Kit) and developed with Vector
[2]
and apoptosis. In spite of the p53 mutation NovaRED kit (Vector Laboratories Inc.) for 5 min. The
being rare in HCC not induced by aflatoxin, the slides were counterstained with Harris haematoxylin
high incidence of HCV-related HCC justifies novel and mounted with Permount (Biomeda, Foster City,
studies even in this context. NFκB linked to HCV CA, USA). Percentages of nuclear RelA/p65 and
non-structural 5A (NS5A) protein inhibits the p53 p53, cytoplasmic TNFR1 and TRAIL, and nuclear/
tumor suppressor role and leads to cell survival and cytoplasmic PHB-positive cells were obtained blindly
hepatocarcinogenesis. [12] for each case at least 10 representative high-power
fields (40×) by two of the authors (LDP and LNR).
Human prohibitin (PHB), a pleiotropic protein, was For statistical purposes, the samples were scored
identified first as a potential tumour suppressor [13] as follows: -, no stained cells; +, weak or moderate
and has been implicated in cellular differentiation, staining in less than 25% positive cells; ++, moderate
antiproliferation, and morphogenesis. [14] Although a to strong staining in 25-50% positive cells; and +++,
liver-specific deletion of PHB has been observed in strong staining in more than 50% positive cells. [18]
a wide spectrum of liver injury types, fibrosis, and
hepatocarcinogenesis in mice, [15] PHB has also been Southwestern histochemistry analysis
[16]
reported to be overexpressed in cases of human HCV. Non-radioactive in situ detection of NFκB in paraffin-
On the other handit has been suggested that PHB may embedded liver tissue preparations was performed
have a pivotal role in cellular proliferation and malignant using the Southwestern histochemistry method,
transformation. Thus, the role of PHB in development with digoxigenin labelling and detection kits (Roche
[17]
of human hepatic cancer remains controversial. Applied Science, Indianapolis, USA). Briefly, synthetic
sense DNAs (Imprint Genetics Corporation, Hialeah,
Our study aimed to compare the importance of the USA), which contain sequences of NFκB, were used
p53 and PHB pathway through the NFκB signalling in as probes. After annealing with the complementary
the spectrum of HCV, CIR, and HCC, considering the sequence, the DNA probe was labelled with digoxigenin.
Hepatoma Research ¦ Volume 3 ¦ February 28, 2017 35