Vital Staining With Iodine Solution: Research of Glycogen Metabolism in Oral Squamous Cell Carcinoma
Vital staining with iodine solution has been used to distinguish dysplastic/malignant oral epithelium from normal mucosa in the case of an operation and a clinical diagnosis. However, little was known about its detailed mechanism a little while ago. By our study until the present, it is thought that iodine reacts with glycogen. Oral squamous cell carcinoma (OSCC) cells lacks glycogen and showed no-stain reaction with iodine. Therefore, the purpose of this study is to analyze glycogen metabolism (glycogen synthesis and breakdown) in iodine non-stained (oral dysplastic/malignant) epithelium.
Materials and Methods
Fifteen frozen tissue samples of iodine-stained and -unstained mucosa were obtained from 15 cases of OSCC. Serial frozen sections were cut and examined with hematoxylin-eosin (HE), periodic acid Schiff (PAS) methods and immunohistochemical staining for the enzyme glycogen synthesis (glycogen synthase, GS and phospho-glycogen synthase, PGS) and the enzyme glycogen phosphorylase (glycogen phosphorylase isoenzyme BB, GPBB and Glucagon-like peptide-1, GLP-1).
In addition, we carried out western blot analysis and fluorescence confocal microscopy to compare the oral cancer cell with the normal oral mucosa cell. Oral cancer cell was gotten from cell bank (SAS, HSC2, HSC3, SquuA, SquuB, Ca9-22)
Method of data analysis
Immunohistochemical staining: the percentages of positive cells in each layer for GS, PGS, and GPBB, GLP1 were calculated on a 400× field (five fields per area), irrespective of the intensity of the stain.
Western blot analysis: we compared the expression of the protein with the cancer cell between normal cells.
Fluorescence confocal microscopy: we compared the light quantity of the normal cell and cancer cell with two antibodies of GS and GPBB.
Results
There was no significant difference in immunoreaction for GS and PGS between iodine stained and non-stained epithelium. While, iodine non-stained epithelium presented significantly higher immunoreactions for GPBB in basal and parabasal layers compared to iodine stained epithelium. The immunoreaction of GLP-1 was not seen in all the epithelium layers.
There was significant difference in western blotting band intensity of GPBB between cancer cell and normal cells. However, there was no significant difference band of GS and PGS.
Significant difference was provided between a cancer cell and normal cells by the fluorescence dyeing using 2 antibodies GS and GPBB. Light quantity was significantly higher for GPBB in cancer call.
Conclusions
The results of this study suggested that dysplastic/malignant oral epithelium had activation of glycogen breakdown, and consequently lacked glycogen contents that resulted in negative stain reaction with iodine solution.
References
Kurita H, Kurashina K. Vital staining with iodine solution in delineating the border of oral dysplastic lesions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996;81(3):275-80.
Tiepeng Xiao , Kurita H. Vital staining with iodine solution in oral cancer: iodine infiltration, cell proliferation, and glucose transporter 1. Int J Clin Oncol 2012;DOI 10.1007/s10147-012-0450-4