Expression of Apoptotic versus Anti-apoptotic protein in Middle Ear Cholesteatoma

Abstract

Cholesteatoma is an expanding growth of keratinizing squamous epithelium with the aggressive clinical characteristics of bony destruction in the middle ear and temporal bone. Although cholesteatoma is a pathologically benign lesion, its destructive and expansive characteristics might induce the serious problems of hearing loss, otorrhea and intracranial complications. Cholesteatoma is classified into congenital and acquired cholesteatoma according to the onset of disease. Various hypothesis had been suggested to explain the etiology of cholesteatoma including retraction pocket, proliferation, immigration, and metaplasia. Those theories share the issue of hyperproliferation and hyperkeratosis. Hyperproliferation might imply the breakdown of epithelial homeostasis, which is maintained by proliferation and apoptosis. Though researchers investigated the role of apoptosis in cholesteatoma, apoptosis is still a highly controversial topic. c-FLIP (Cellular FLICE inhibitory protein) is a close homologue of Caspase 8, inhibiting the initiation of apoptosis cascade of the extrinsic pathway. There was an up-regulation of c-FLIP in various types of malignancy and benign hyperplastic skin disease. p53 is a well-known tumor suppressor gene activating the intrinsic pathway of apoptosis via Caspase 3 and regulating the cell cycle preventing cancer development. Though various studies had been conducted regarding the role of p53 in cholesteatoma. The expression of p53 in cholesteatoma tissue is in debate. Ki-67 is a representative proliferation marker which is expressed only in the proliferative phase of the cell cycle. Up-regulation of Ki-67 was identified in many benign epithelial diseases including cholesteatoma. The author evaluated the expression of anti-apoptotic and proliferative proteins (c-FLIP, Ki-67) and the apoptosis marker of p53 in the cholesteatoma and compared the expression rates between retroauricular skin and cholesteatoma. In addition, subgroup analysis of congenital and acquired cholesteatoma was also performed. An immunohistochemistry was performed with 35 cholesteatoma specimens and 10 normal retroauricular skin tissues to evaluate the expression of c-FLIP, p53 and Ki-67. Thirty five cholesteatoma were composed of 14 congenital cholesteatoma and 21 acquired cholesteatoma. The expression rate of each marker was measured with a cell count plug-in software operated in an image processing program of ImageJ to determine the differences between retroauricular skin and cholesteatoma, as well as between congenital and acquired cholesteatoma. The results are as follows:

1. c-FLIP was expressed in basal, spinous and granular layer of cholesteatoma and retroauricular skin. The expression rate of c-FLIP in basal layer did not differ between cholesteatoma and retroauricular skin (p=0.946). In suprabasal and granular layer, a significantly higher expression rate was identified in cholesteatoma than in retroauricular skin (p<0.001, p<0.001). The results revealed that acquired cholesteatoma showed a higher expression rate of c-FLIP in granular layer than that of congenital cholesteatoma (p<0.001).

2. The expression of p53 was identified in the basal and suprabasal layer of cholesteatoma and retroauricular epithelium. The expression rate did not significantly differ between cholesteatoma and retroauricular skin (p<0.396). Congenital cholesteatoma showed no difference with acquired cholesteatoma regarding the expression rate of p53.

3. Ki-67 positive keratinocyte was identified in the basal and suprabasal layers of epithelium. The expression of Ki-67 in cholesteatoma was significantly increased than in retroauricular skin (p<0.001). In the suprabasal layer of acquired cholesteatoma, the expression rate of Ki-67 was significantly higher than that of congenital cholesteatoma.

4. In the suprabasal layer of epithelium, the expression rate of c-FLIP had a significant positive correlation with the expression rate of Ki-67 (r=0.47 p=0. 001). However, there was no significant correlation between the expression rate of c-FLIP and p53 (r= 0.152, p=0.319).

Based on these results, the up-regulation of c-FLIP correlated with the expression of Ki-67 in cholesteatoma, while the expression of p53 was not increased. Additionally, there was a prevalent expression of c-FLIP in acquired cholesteatoma than in congenital one. The author postulates that the anti-apoptotic process may play a significant role in the development of cholesteatoma leading to the inhibition of apoptosis and an increase in cell proliferation. Following studies regarding the molecular regulator of c-FLIP might provide further knowledge about the role of anti-apoptotic pathway in cholesteatoma