Effect of Acriflavine-Guanosine Compound (AG60) Treatment on the Gastrointestinal Endocrine Cells of Mouse Inoculated with Ehrlich Carcinoma Cells

Park, Kyung Ho; Song, Hyoung Gon; Ko, Jeong Sik; Ahn, E Tay; Kim, Jin Gook

Abstract

To study the tumor-suppression effect of a newly developed anti-tumor agent AG60 [ acriflavine (1) : guanosine (1) composition, Taerim Pharm. Co., Seoul, Korea], each Ehrlich carcinoma (107 cells)-inoculated mouse received the subcutaneous injection of 0.2 ml of saline, 5mg/kg of AG60, and 30 mg/kg of AG60, every other day for two weeks. Animals were sacrificed, and stomach, duodenum, appendix vermiformis and rectal tissues were resected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut in 6 microgram-thick sections. For immunocytochemistry, the streptavidine-biotin-peroxidse method was used with a InnoGenex (San Ramon, Calif., USA) staining kit. The tissues were incubated with rabbit antisera against somatostatin (Biogenesis, Poole, England, UK) diluted 1 : 300, secretin (Biogenesis, Poole, England, UK) diluted 1 : 2,400, neurotensin (Biogenesis, Poole, England, UK) diluted 1 : 2,600, or motilin (Biogenesis, Poole, England, UK) diluted 1 : 1,000 for 24 hour at 4dreeges C, followed by incubation in biotinylated antirabbit IgG and horseradish peroxidase-streptavidin conjugate for 1 hour at room temperature. The antigen-antibody reaction sites were visualized by incubating the sections with diaminobezidine tetrahydrochloride (DAB) for 5~15 minutes at room temperature. After mounting in canada balsam, they were examined in a Leica DM RB microscope. The number of the immunoreactive cells in the area of gastrointestinal mucosae (mean number of immunoreactive cells per 0.25mm2) were observed and calculated. The results are as follows : 1. In the fundic gland of normal mouse, somatostatin immunoreactive cells were detected (18.5+/-0.71), but neurotensin, secretin, or motilin immunoreactive cells were not found. In the duodenal mucosa of normal mouse, somatostatin immunoreactive cells were detected (7.0+/-0.10), but neurotensin, secretin or motilin immunoreactive cells were rarely found. 2. Immunoreactivity of somatostatin, secretin, neurotensin or motilin cells was not found in appendix vermiformis and rectum of normal mouse. 3. On immunocytochemical study, somatostatin immunoreactive cells in the fundic glands of normal, experimental control, AG60 (5mg/kg)-treated, AG60 (30 mg/kg)-treated and 5-fluorouracil (60 mg/kg)-treated groups were 18.5+/-0.71, 10.0+/-4.20, 11.5+/-0.71, 13.5+/-2.10, 11.5+/-2.71, respectively. 4. On immunocytochemical study, somatostatin immunoreactive cells in the duodenal mucosae of normal, experimental control, AG60 (5 mg/kg)-treated, AG60 (30 mg/kg)-treated and 5-fluorouracil (60 mg/kg)-treated groups were 7.0+/-2.10, 0.5+/-2.71, 3.0+/-1.41, 0.5+/-0.71, 2.50+/-0.71, respectively. 5. On immunocytochemical study, secretin immunoreactive cells in the duodenal mucosae of normal, experimental control, AG60 (5 mg/kg)-treated, AG60 (30 mg/kg)-treated and 5-fluorouracil (60 mg/kg)-treated groups were rarely found. 6. On immunocytochemical study, neurotensin and motilin immunoreactive cells in the duodenal mucosae of normal groups were detected, but immunoreactivies were not detected in experimental control, AG60 (5 mg/kg)-treated, AG60 (30 mg/kg)-treated or 5-fluorouracil (60 mg/kg)-treated groups.