Study On Expression Of NF-κB,Cyclin D1 And CD105 In Oral Leukoplakia And Oral Squamous Cell Carcinoma

Oral Leukoplakia (OLK) assessed histopathologically as epithelial unusual hyperplasias is the most common premalignant lesion in oral cavity and is a white patch or plaque that cannot be characterized clinically as any other condition. There are 3%-5% patients of OLK will develop into oral squamous cell carcinoma (OSCC), and the average course of this disease reach to 8.2 years. The risk that OLK change into OSCC is more higher than that of the common oral mucosa. OSCC is the leading malignant tumor in oral carvity, amounting for more than 80% of the oral malignant tumor. The process of OLK to OSCC is multi-step, so it is generally accepted that we examine OLK and study the relation between OLK and OSCC.Neoplasia is a complicated process, which is controlled by gene expression and regulation, cell multiplication and apoptosis, angiogenesis and so on. Nuclear Factor-kappa B (NF-κB/Rel) is a transcription factor that induces the expression of various genes, leading to inflammatory reactions, embryonic morphogenesis, and antiapoptosis. NF-κB was first found in B cell binding withκB binding site by Sen in 1986, and the mammalian members of which include p50 (NF-κB₁), p52 (NF-κB₂), p65 (RelA), Rel-B, and c-Rel. Heterodimers of p65 and p50, also known as classical NF-kB, are present in the cytoplasm bound with their inhibitory proteins IκBs, of which IκB-a is the best characterized. In recent years, further studies suggested that NF-κB has been implicated in the regulation of cell proliferation, transformation, and tumor development. NF-κB was found to stimulate transcription of cyclin D1, a key regulator of G1 checkpoint control. NF-κB binding site in the human cyclin D1 promoter conferred activation by NF-κB as well as by growth factors. Both levels and kinetics of cyclin D1 expression during G1 phase were controlled by NF-κB. Recent advances in biological research have revealed that NF-κB can stimulate neovascularization. CD105 (endoglin) is a homodimeric membrane glycoprotein expressed on Endothelial Cells that can bind transforming growth factor-β. Microvessel density (MVD) was determined with an anti-CD 105 mAb (CD105/MVD). It has been recently reported that CD105/MVD preferentially reacts with "activated" endothelial cells in angiogenic tissues. At present, less is known about study on the expression of NF-κB/p65, cyclin D1 as well as their relationship with angiogenesis in OLK and OSCC.Objective The aim of study was to investigate the expression of NF-κB/p65 and cyclin D1 as well as their relationship with angiogenesis activity in OLK and OSCC, and to evaluate their functions in tumorigenesis and progession of OSCC, so as to provide a potential new way for early diagnosis, to prognosis assessment and indicating theraputics of OSCC.Materials and methods1. Eighth-five cases of formalin-fixed,paraffin-embedded specimens of oral mucosa lesions were obtained from Deparment of pathology, the First Affiliated Hospital of Zhengzhou University. The OLK specimens were assessed histopathologically as 12 cases of simple hyperplasis, 13 cases of mild dysplasia, 13 cases of moderate dysplasia, 10 cases of severe dysplasia. The OSCC specimens were divided into three groups: 13 cases of grade I, 13 cases of grade II, and 11 cases of grade III. 10 cases of specimens from oral normal mucosa (NM) were taken as control.2. Immunohistochemical staining technique was used to determine the expression NF-κB/p65, cyclin D1 and CD105 in all specimens. Immunoreactivity in the tissues was estimated by counting the number of positive cells per 1000 tumor cells for NF-κB/p65 protein and cyclin D1 protein. MVD as visualized by staining for CD105 was quantified by light microoscopy. The most vascular areas in a tumor wer located at low magnification, and vessels were counted at x400 magnification.3.The results were analyzed by SPSS11.0 software ,which using Chi-square, Fisher's exact test, analysis of variance (ANOVA) and t-test. Possible correlations between variables of the analyzed samples were examined by Spearman test. Statistically significant level was considered as "alpha equals 0.05".Results1. The positive staining for protein was mainly observed in the cytoplasm with yellow staining, with only few nucleus staining. NF-κB/p65 protein was negatively expressed in group of simple hyperplasia and group of NM, while it was positively expressed in dysplasia OLK and OSCC groups. The positive rates of NF-κB/p65 protein in dysplasia OLK and OSCC were 41.67% and 78.38% respectively. It has statistical differece significance in the four groups (P=0.000). The strength of NF-κB/p65 protein staining increased with cell proliferation and carcinogenesis, and there were significant differences between the four groups (P=0.001). There were significant differences in OSCC groups of NF-κB/p65 protein staining strength (P=0.039), while there were no significant differences in dysplasia OLK groups (P=0.065).2. The positive staining of cyclin D1 protein was mainly observed in the nucleus with yellow staining. In the process of oral carcinogenesis, there were expression of cyclin D1 in each stage with different level which showed a dynamic process. In NM, simple hyperplasia OLK, dysplasia OLK and OSCC were found that positive rates of cyclin D1 expression were 20.00%, 58.33%, 72.22%, and 81.08%respectively, and a statistical significance was observed among them (P=0.000). The strength of cyclin D1 protein staining increased with cell proliferation and carcinogenesis, and there were significant differences between the four groups (P=0.000). But there were no significant differences in dysplasia OLK and OSCC groups of cyclin D1 protein staining strength and positive rates (P>0.05).3. The MVD was assessed following immunohistochemical staining with anti-CD 105 antibody. The positive staining of CD105 was mainly observed in the cytoplasm of vascular endothelial cell.The mean CD105/MVD in NM, simple hyperplasia OLK, dysplasia OLK and OSCC were 0.00±0.000, 3.92±1.929, 10.44±1.858 and 15.92±3.427 respectively. The result showed a significant difference among the groups (P<0.05). In grade I, II, III OSCC groups,the mean CD105/MVD showed a statistical significance among them (P<0.05).4. In all samples, Spearman rank correlation analysis showed a significant correlation between expression of NF-κB/p65 and cyclin D1 protein (r_s=0.482, P<0.05).5. In all samples, the CD105/MVD in the NF-κB/p65 positive group and the NF-κB/p65 negative group were 13.71±4.742 and 7.90±5.539 respectively, and there was significant difference between them (P<0.05).Conclusion1. NF-κB/p65 protein has a close relationship with cell proliferation and carcinogenesis of malignant tumors. It may be useful for evaluating the malignant potential of OLK and early diagnosis of OSCC , and may be used as a biomarker for supervising oral malignancy.2. cyclin D1 is overexpression in OLK and OSCC. But expression of cyclin D1 has the down regulation in gradeIII OSCC which may be an early event during oral carcinogenesis.3. The increasing of CD105/MVD with carcinogenesis of OLK and the degree of differentiation in OSCC identified that the induction of tumor angiogenesis is an important event of carcinogenesis which may be a good marker in evaluating angiogenesis and the biological character of OLK and OSCC.4. There was a significant correlation between the expression of NF-κB/p65 and cyclin D1 in the canceriztion of OLK, which played a cooperative role in cell proliferation and took part in the carcinogenesis of OSCC. A significant correlation was observed between the expression of NF-κB/p65 and CD105/MVD, suggesting NF-κB/p65 played an important role in oral tumor angiogenesis.5. The combined examination of the expression of NF-κB/p65, cyclin D1 and CD105 by immunohistochemistry staining contributes to supervising oral malignancy.