Elsevier

Oral Oncology

Volume 42, Issue 5, May 2006, Pages 540-550
Oral Oncology

Short Communication
Cell proliferation and apoptosis culminate in early stages of oral oncogenesis

https://doi.org/10.1016/j.oraloncology.2005.10.008Get rights and content

Summary

Markers of cell proliferation (Ki-67 antigen) and apoptosis (Bax, Bcl-2) were studied in an experimental system of induced oral carcinogenesis in Syrian golden hamsters. Thirty-seven animals were divided into one control group and three experimental groups, which were treated with a carcinogen and sacrificed at 10, 14 and 19 weeks after treatment. The histological status of the lesions in the three experimental groups corresponded well with tumour advancement (from oral mucosal dysplasia to moderately differentiated squamous cell carcinoma). Tumour sections were studied using monoclonal antibodies against Bax, Bcl-2 and Ki-67 proteins. Pro-apoptotic Bax expression maintained high levels during all stages of oral carcinogenesis. Anti-apoptotic Bcl-2 expression decreased significantly in dysplastic and early invasion lesions and consequently increased almost to normal tissue level in consequent stages. Finally, Ki-67 expression increased sharply in initial stages of oral carcinogenesis, but significantly decreased in later stages.

Introduction

Oral squamous cell carcinoma (OSCC) is a major health problem, since more than 300 000 new cases annually are being diagnosed worldwide.1 Despite advances in treatment with surgery, radiation, and chemotherapy, prognosis of advanced stage oral cancer remains poor over the last four decades.1, 2 Advancement of our understanding of molecular mechanisms involved in oncogenesis may provide the means for better prevention and treatment of OSCC in initial stages.3

A series of genetic alterations such as the activation of oncogenes and the inactivation of tumour suppressor genes are involved in stages of carcinogenesis in the oral region.4 Triggering factors for initial mutational events may include traditional risk factors such as tobacco and alcohol as well as other risk factors, such as chronic immunodeficiency states, viral infection and diets low in fruit and vegetables.5, 6

Cell proliferation is a biological process of vital importance to all living organisms both in embryonic and in post-embryonic existence. Down regulation control on this important biological process is thought to be lost in cancer. Among the most common immunohistochemical markers used to study cell proliferation is the Ki-67 antigen.7 The mammal Ki-67 nuclear antigen is expressed during the G1, S, G2 and M phases in the cell cycle, but is absent in G0 phase.7 Ki-67 may be employed to measure the growth fraction of normal tissues and malignant tumours.8 High indices of Ki-67 have been observed in OSCC and correlated with disease progression and poor prognosis.9, 10

Recent studies have shown that the process of carcinogenesis may involve not only increased cell proliferation but also decreased cell death (apoptosis) or increased cell survival.11 Apoptosis plays an important role in morphogenesis, homeostasis, and cancer regression12 and is regulated by a number of genes of the Bcl-2 family, including anti-apoptotic (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1/Bfl-1, Boo/Diva and NR-13), pro-apoptotic (Bax, Bak, Bcl-xs and Bok/Mtd), and BH3-only members (Bad, Bid, Bim, Hrk, Blk, Bik, Nip3, Nix).13 The Bcl-2 protein family has up to four highly conserved sequence homology domains, Bcl-2 homologues 1–4 (BH1-4), which mediate protein interactions.14, 15 It has been shown that the proteins of the Bcl-2 family heterodimerize and homodimerize with each other, and the relative proportions of these dimers may determine whether or not a cell becomes apoptotic.16, 17

The Bcl-2 protein is heavily localized to the outer mitochondrial membrane.13 High levels of Bcl-2 in a cell will prevent the induction of many forms of apoptosis.18 The related protein Bax is localized to cytosol.13 Following a death signal, Bax undergoes conformational change that enables it to target and integrate into the outer mitochondrial membrane.13 Activation of Bax may be inhibited by Bcl-2 which forms complexes with Bax. The ratio of Bcl-2/Bax regulates the release of cytochrome c from the mitochondria.19 Cytochrome c triggers the death of the cell by activating the caspases, which initiate a proteolytic cascade leading to the dismantling of the cell.20, 21

A previous study of human OSCC found a decreased Bcl-2/Bax ratio and increased apoptosis in comparison to normal epithelium.22 Moreover, increased expression of Bax correlated with tumour histological grading; significantly more Bax positive cells were detected in well differentiated than in poor differentiated OSCC.22 Another study proposed that the ratio of Bcl-2/Bax expression seems to be the best variable in predicting disease specific survival in tongue SCC.23

In order to investigate the balance of cell proliferation and apoptosis in all stages of oral carcinogenesis, we simultaneously studied Ki-67, Bax and Bcl-2 in an experimental system of chemically induced oral cancer at different stages, in Syrian golden hamsters.

Section snippets

Experimental carcinogenesis

Forty male Syrian golden hamsters (Mesocricetus auratus) purchased from the Hellenic Pasteur Institute (Athens) at the age of five weeks and weighted approximately 100 g each, were used in this study. The hamsters were handled in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). The animals were randomly divided into four groups: one control group (n = 7) and three experimental groups

Results

The histological status of biopsies in the control group and in the three experimental groups is shown in Table 1. A progression towards OSCC formation in correlation to increased time of carcinogen application is evident. Therefore, as expected, this experimental model seems valid and further analysis of data was implemented.

The percentages of cells positively stained for apoptosis-related proteins Bax and Bcl-2 in the various categories of histological status are shown in Table 2, Table 3,

Discussion

In this study, an experimental system of chemically induced oral cancer was established and the expression of markers of apoptosis (Bax, Bcl-2) and cell proliferation (Ki-67) was investigated.

Bax expression was detected in high levels during the development of OSCC and it did not change significantly in any stage of histological status. In the contrary, Bcl-2 expression was detected in significantly decreased levels in dysplastic lesions as well as during early invasion, but in the latter

Acknowledgements

This work was supported in part by “Pythagoras” EPEAEK 70/3/7391 Grant of the Greek Secretariat of Research and Technology to E.V.

References (30)

  • B.B. Singh et al.

    Immunohistochemical evaluation of Bcl-2 oncoprotein in oral dysplasia and carcinoma

    Oral Surg Oral Med Oral Pathol Oral Radio Endod

    (1998)
  • R.T. Greenlee et al.

    Cancer statistics 2000

    CA Cancer J Clin

    (2000)
  • J. Mork

    Forty years of monitoring head and neck cancer in Norway—no good news

    Anticancer Res

    (1998)
  • J.J. Lee et al.

    Predicting cancer development in oral leukoplakia: ten years of translational research

    Clin Cancer Res

    (2000)
  • A. Iamaroon et al.

    Co-expression of p53 and Ki-67 and lack of EBV expression in oral squamous cell carcinoma

    J Oral Pathol Med

    (2004)
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