Short CommunicationCell proliferation and apoptosis culminate in early stages of oral oncogenesis
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.
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2009, Oral OncologyCitation Excerpt :Both human and hamster OSCCs were characterized by increased cell proliferation and reduced apoptosis. Overexpression of PCNA, GST-P, and NF-κB involved in cell proliferation and antiapoptotic reactions with downregulation of IκB, p53 and p21 that play sentinel roles in cell-cycle arrest, DNA repair, and apoptosis, may be key factors that confer a survival advantage to both human and hamster OSCCs.14–16 Upregulation of Bcl-2 associated with downregulation of Bax, Fas, Apaf-1, cytochrome c, caspases, and PARP in human and hamster OSCCs indicates apoptosis avoidance.