Effects of AG60 on Gastric Chief Cells of the Mouse Implanted with Ehrlich Carcinoma Cells / 대한해부학회지

This experiment was performed to evaluate the morphological responses of the gastric epithelial cells and the gastric chief cells of the mouse inoculated with Ehrlich carcinoma cells in the inguinal area following administration of acriflavine-guanosine composition (AG60). Healthy adult ICR mice were divided into normal and experimental groups. In the experimental groups, each mouse was inoculated with 1x10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. The day following the 7th injection of saline or AG60, each mouse was injected with methyl-3H-thymidine through tail vein. Seventy minutes after the thymidine injection, gastric tissues were taken and fixed in 10% buffered neutral formalin. Deparaffinized sections were coated with autoradiographic emulsion EM-1 and dried, and then placed in a light-tight box. The number of labeled epithelial cells in the gastric mucosae were observed and calculated. And for electron microscopic observation, gastric tissues were prefixed with 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation with 1% osmium tetroxide solution. The ultrathin sections were stained with uranyl acetate and lead citrate. The size of zymogen granules and mitochondria in the gastric chief cells were observed and calculated. On the autoradiographic study, number of labeled cells in the area of 3.5 mm width (6 micrometer thickness) of mouse gastric mucosae of normal control, tumor control and AG60-treated groups were 319.7+/-66.46, 343.7+/-47.72 and 102.3+/-54.99 respectively. On the electron microscopic study, the size of zymogen granule in the gastric chief cells of normal control, tumor control and AG60-treated groups were 0.74+/-0.208 micrometer, 1.18+/-0.291 micrometer and 0.97+/-0.259 micrometer, respectively. And the mitochondrial size of the gastric chief cells of normal control, tumor control and AG60-treated groups were 0.86+/-0.364 micrometer, 1.02+/-0.466 micrometer and 0.92+/-0.390 micrometer, respectively. And in the AG60 treated group, most chief cells did not show any difference in ultrastructure, except that myelin figures were more frequently observed, in comparison with that of nornmal control group. From the above results, AG60 may suppress the DNA synthesis of the gastric epithelial cells, but does not results severe fine structural defect on the gastric chief cells. These results suggest that AG60 is expected as one of the most effective anticancer drugs.

Effects of 5-Fluorouracil, Mitomycin C or AG60 to the DNA Synthesis of Rectal Epithelium of Mice Implanted with Ehrlich Carcinoma Cells / 체질인류학회지

This experiment was performed to evaluate the morphological responses of the intestinal gland of the mouse, rectum inoculated with Ehrlich carcinoma cells, following administration of 5- fluorouracil, mitomycin C or AG60. Healthy adult ICR mice weighing 25 gm each were divided into normal and experimental groups (tumor control group, 5-fluorouracil, mitomycin C treated group, and AG60 treated group). In the experimental groups, each mouse was inoculated with 1*10 (7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 mL of saline, 5-fluorouracil (30 mg/kg), mitomycin C (400 microgram/kg) or AG60 (5 mg/kg) were injected subcutaneously to the animals every other day, respectively. The day following the 7th injection of anticancer drugs, each mouse was injected with a single dose of 0.7 microCi/gm of methyl-3H-thymidine through tail vein. Seventy minutes after the thymidine injection, animals were sacrificed. The number of the labeled epithelial cells of the rectal crypts (mean number of labeled epithelial cells per 3.5 mm length of mucosa) were observed and calculated. On histological study, in the rectum of mitomycin C treated groups, narrowed intestinal gland, a number of the nectotic changed epithelial nuclei and loosely arranged lamina propria were observed. But in the AG60 treated group, morphological changes of the rectum were not observed. On autoradiographic study, number of the labeled cells of normal control, tumor control, 5-fluorouracil (30 mg/kg) treated, mitomycin C (400 microgram/kg) treated and AG60 (5 mg/kg) treated groups were 246.3+/-42.30, 253.8+/-20.54, 172.7+/-19.02, 108.7+/-17.67 and 53.8+/-11.70, respectively. In the AG60 and mitomycin C treated group, poorly-labeled cells containing only a few silver grains of 3H-thymidine were observed more frequently than in those of the normal control group. From the above results, AG60 (5 mg/kg) and mitomycin C (400 microgram/kg) are more suppressed the DNA synthesis of the cells of the rectal crypts as compare with 5- fluorouracil (30 mg/kg). And AG60 does not result any histological defect on the rectal mucosa. These results suggest that AG60 is expected as one of most effective anticancer drugs.

Effects of Adriamycin or AG60 to the DNA Synthesis of Duodenal Epithelium of Mice Implanted with Ehrlich Carcinoma Cells: An Autoradiographic Study / 체질인류학회지

This experiment was performed to evaluate the morphological responses of the intestinal gland of the mouse, duodenum inoculated with Ehrlich carcinoma cells, following administration of adriamycin or acriflavine -guanosine composition (AG60, Taerim Pharm. Co. Seoul, Korea). Healthy adult ICR mice weighing 25 g each were divided into normal and experimental groups (experimental control group, adriamycin treated group, and AG60 treated group). In the experimental groups, each mouse was inoculated with 1 x10 7 Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 ml of saline, adriamycin (2 mg/ kg), AG60 (5 mg/kg) or AG60 (30 mg/kg) were injected subcutaneously to the animals every other day, respectively. The day following the 7th injection of anticancer drugs, each mouse was injected with a single dose of 0.7 microCi/gm of methyl -3 H -thymidine (25 Ci/mmol, Amersham Lab., England) through tail vein. Seventy minutes after the thymidine injection, animals were sacrificed. The number of the labeled epithelial cells of the duodenal crypts (mean number of labeled epithelial cells per 3.5 mm length of mucosa) were observed and calculated. On histological study, in the duodenum of adriamycin treated groups, vesiculated epithelial cells of the intestinal villi, expanded lumen of the intestinal gland (G) and loosely arranged lamina propria were observed. But in the AG60 treated group, morphological changes of the duodenum were not observed. On autoradiographic study, number of the labeled cells of normal control, experimental control, adriamycin -treated, AG60 (5 mg/kg)-, and AG60 (30 mg/kg)-treated groups were 595.3 +/-48.96, 715.+/-89.11, 96.0 +/-15.62, 632.0 +/-83.16 and 370.3 +/-49.65, respectively. In the adriamycin and AG60 30mg/kg -treated group, poorly -labeled cells containing only a few silver grains of 3 H -thymidine were observed more frequently than in those of the normal control group. But in the experimental control group, number of the heavy labeled cells were observed more frequently than in those of the normal control group. From the above results, adriamycin and AG60 (30 mg/kg) may suppress the DNA synthesis of the cells of the duodenal crypts. But AG60 does not result any histological defect on the duodenal mucosa. These results suggest that AG60 is expected as one of most effective anticancer drugs.

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

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.

Morphological Study on the Thymic Cortex of the Ehrlich Carcinoma-Inoculated Mice, Treated with 5-Fluorouracil and Acriflavine-Guanosine Complex (AG60) / 대한해부학회지

In cancer therapy, immunological disorder is one of most severe problem. Since thymic cortex is the home of T-cell proliferation and "education", thymic morphology following administration of certain drugs can be used as a parameter of immunological safety of the drug. In this study, morphology of thymic cortex, following administration of 5-fluorouracil or AG60, was studied. AG60 is a newly developed anti-cancer remedy, the compound of acriflavine and guanosine (1 : 1). ICR mice were subcutaneously inoculated with Ehrlich carcinoma cells (10(7) cells/mouse) in their inguinal areas. Each mouse in 5-fluorouracil group was injected subcutaneously with a single dose of 30 mg/kg of 5-fluorouracil every other day, and the mouse in AG60 group, with 30 mg/kg of AG60 (Taerim Pharm. Co., Seoul) every other day. The control mouse was injected with saline. The mice were sacrificed on the day after 7th injection. Tissues of thymic cortices were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution (0.1 M Millonig's phosphate buffer, pH 7.3), and refixed in 2% osmium tetroxide solution (0.1 M Millonig's phosphate buffer, pH 7.3). Tissue blocks were dehydrated, and were embedded in araldite mixture. For the overview-comparison, semithin sections stained with toluidine blue solution were photographed. And the typical portions were cut with ultratome, stained and observed with electron microscope. In light microscopy, thymic cortical morphology of AG60-injected mouse was similar with that of control mouse. But the cortical morphology of 5-fluorouracil-injected mouse was impressively different from those of the control or AG60 group mice. Thymocytes in the thymic cortex of 5-fluorouracil-injected mice were severely depleted. In electron microscopy, thymocytes in the thymic cortices of the control or AG60 group mice were crowded, and small groups of thymocytes were surrounded by the cytoplasmic processes of epithelial reticular cells. Mitotic figures were randomly seen. Thymocytes of 5-fluorouracil-injected mouse were naked out from the epithelial reticular cells, and were completely depleted out from the cortex composed mainly of enlarged epithelial reticular cells. Numerous microvilli were protruded from the naked thymocytes. The results were interpreted as that 5-fluorouracil induce leukopenia, and homing of lymphocytes to thymic cortex is severely depressed. 5-fluorouracil also disturb the normal protective and supportive function of epithelial reticular cells for thymocytes. Whereas the complex of acriflavine-guanosine compound (AG60) is immunologically safe, as seen in thymic cortical morphology.

Effects of BCG or AG60 on Gastric Parietal Cells of the Mouse Implanted with Ehrlich Carcinoma Cells / 대한해부학회지

This experiment was performed to evaluate the morphological responses of the gastric parietal cells of mouse inoculated with Ehrlich carcinoma cells, following administration of Bacillus Calmette -Guerin (BCG) or acriflavine -guanosine composition (AG60, Taerim Pharm. Co. Seoul, Korea). In the experimental groups, each mouse was inoculated with 1 X 10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 ml of saline, BCG (0.03 X 10(8) ~0.32 X 10(8) CFU) or AG60 (30 mg/kg) was injected subcutaneously to the animals every other day. Animals were sacrificed on the 14th day from the first injection. Pieces of the tissue were taken from the stomach, prefixed with 2.5% glutaraldehyde -1.5% paraformaldehyde, followed by post -fixation with 1% osmium tetroxide. The ultrathin sections stained with uranyl acetate and lead citrate were observed with a JEM 100CX -II electron microscope. In the experimental control, the BCG and the AG60 treated groups, most parietal cells showed reduced lumenal spaces of the intracellular canaliculi, since microvilli of intracellular canaliculi were very irregularly shaped and crowed with each other. And in the BCG and the AG60 treated mice, myelin figures, lysosomes and multivesicular bodies in the parietal cells were observed more frequently than in those of the experimental control ones. In the BCG treated rats, membranes of the tubulovesicles of the parietal cells were disintegrated, but the similar changes were not observed in the AG60 treated mice,. Above results suggest that the BCG treated animals inoculated with Ehrlich carcinoma cells might suffer from reduced acid secretion of the parietal cell, since the disintegration of the tubulovesicular membranes in the parietal cells are occurred following injections. Whereas AG60 dose not affect remakably defect on the parietal cells.

Effects of Antitumor Agents on the Spleen of Mouse Implanted with Ehrlich Carcinoma Cells: An Autoradiographic Study / 대한해부학회지

In this experiment, side effects of three anticancer drugs (5-fluorouracil, mitomycin C and AG60) on the structure of spleen were histologically studied. Each of ICR mice was inoculated with 1X10(7) Ehrlich carcinoma cells subcutaneously in the inguinal area. From next day, 0.2 ml of saline solution, 5-fluorouracil (30 mg/kg), mitomycin C (400 micorgram/kg), 5 mg/kg of AG60, or 30 mg/kg of AG60 (acriflavine (1) : guanosine (1) composition, Taerim Pharm. Co., Seoul, Korea) were injected subcutaneously every other day. The day following the 7th injection of anticancer drugs, each mouse was injected with a single dose of 0.7 microCi/gm of methyl-(3)H-thymidine (25 Ci/mmol, Amersham Lab., England) through tail vein. Seventy minutes after the thymidine injection, animals were sacrificed, and splenic tissues were collected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut into 6 micrometer-thick sections. Deparaffinized sections were coated with autoradiographic emulsion EM-1 (Amersham Lab., England) in the dark room and dried, and were kept in a light-tight box. The sections were exposured for 5 weeks in the dark room, and were deveoped in D-19 developer. The number of the labeled cells in the areas of the white pulp, the red pulp and the marginal zone (mean number of labeled cells per 0.21 mm2) were observed and calculated. The results were as follow: 1. On histological study, in the spleen of mitomycin C treated group, vacuoles containing pyknotic nuclei were observed more frequently than in those of 5-fluorouracil treated group. Whereas in the AG60 treated group, morphological changes of the spleen were not observed. 2. On autoradiographic study, most of the labeled cells were located near the marginal zone of the spleen. 3. On autoradiographic study, number of the labeled cells of normal control, experimental control, 5-fluorouracil-treated, mitomycin C-treated, AG60 (5 mg/kg)-, and AG60 (30 mg/kg)-treated groups were 214.0+/-56.87, 235.7+/-59.69, 331.0+/-67.20, 137.0+/-33.48, 124.6+/-34.28, and 64.9+/-16.26, respectively. 4. In the mitomycin C treated group and AG60 (5 mg/kg or 30 mg/kg) treated group, labeled cells containing a few silver grains of (3)H-thymidine were observed more frequently than in those of the normal and experimental control groups. But in the 5-fluorouracil treated group, number of the heavy labeled cells were observed more frequently than in those of the normal and experimental control groups. From the above results, DNA synthesis, in the cells of the marginal zone were more active than in the cells of the white pulp or the red pulp. And mitomycin C and AG60 may suppress the DNA synthesis of the splenic tissues. Especially, AG60 does not results any histological defect on the splenic tissues. These result suggest that AG60 is expected as one of most effective anticancer drugs.

Effects of Lower Dose-Injections of Acriflavine-Guanosine Compound (AG60) on the DNA Synthesis and Ultrastructure of the Ehrlich Carcinoma Cells Inoculated to the Mouse / 대한해부학회지

AG60, a recently introduced anti-cancer compound, was reported to show highly effective anti-cancer activities, when injected with doses from 30 mg/kg to 5 mg/kg.The purpose of this study was to know the lower effective doses of AG60, and to give the informations for preparing more advanced therapeutic tools for anti-cancer war. Ehrlich cancer cells were inoculated in the subcutaneous tissue of inguinal region of ICR mice, and saline (treated control groups) or AG60 (experimental groups) were injected daily. Animals of experimental groups were injected subcutaneously with doses of 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, or 2.0 mg/kg body weight, according to their subgroups. Five mice from each subgroup were sacrificed on 1 week, 2 weeks, 3 weeks following the first injection. Seventy minutes before sacrifice, each mouse was injected with 0.7 microCi/g body weight of 3H-thymidine (Amersham Lab.) through tail vein. After sacrifice, cancer masses were fixed in 10% formalin solution for autoradiography and light microscopy, and in 2.5% glutaraldehyde-1.5% paraformaldehyde solution, followed by post-fixation in 2% osmium tetroxide solution, for electron microscopy. The observed results were as follows: Autoradiographic observations show, 1. Labelled cancer cell indices of the experimental groups received AG60 were decreased around to 80% (0.2 mg/kg), to 74% (0.5 mg/kg), to 75~60% (1.0 mg/kg), and to 70~50% (2.0 mg/kg), as compared with those of the controls. 2. The contents of silver grains were dramatically decreased nearly to 35% (0.2 mg/kg), to 20% (0.5 mg/kg), to 21~16% (1.0 mg/kg), and to 20~15% (2.0 mg/kg), as compared with those of the controls. 3. Total granular content in 100 cancer cells on the third week of the experiment decreased nearly to 30% (0.2 mg/kg), to 15% (0.5 mg/kg), to 10% (1.0 mg/kg), and to 8% (2.0 mg/kg), as compared with those of the controls. Histological observations show, 1. AG60 induces large amount of apoptosis on Ehrlich cancer cells. 2. Following the treatment with AG60, multinuclear cells or giant cells were increased in number. Comparing by autoradiography and histology, multinuclear or giant cells were interpreted as those cells supplied by poor amounts of thymidine, or almost no new DNA content. Electronmicroscopic readings show, 1. AG60 induces numerous macroclefts and microclefts within the nuclei of Ehrlich cancer cells. 2. AG60 induces numerous apoptosis among Ehrlich cancer cells. 3. Apoptotic bodies are phagocytosed by adjacent cancer cells or by macrophages. From the above results, AG60 is expected to be a very successful anti-cancer candidate. And it is suggested that combined or cocktail therapy including AG60 may greatly improve the anti-cancer therapy on certain kind of cancer.

Autoradiographic Study on the Effect of AG60 to the DNA Synthesis of Gastric Epithelium of Mouse Inoculated with Ehrlich Carcinoma / 대한해부학회지

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 (10(7) cells)-inoculated mouse received the subcutaneous injection of 0.2 ml of saline, 5 mg/kg of AG60, and 30 mg/kg of AG60 per day for a week. The day following the last injection, each mouse was injected with a single dose of 0.7 microcurie/g of methyl-3H-thymidine (25Ci/mmol, Amersham Lab., England) through tail vein. Seventy minutes after the thymidine injection, animals were sacrificed, and gastric tissues were collected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut in 6 micrometer-thick sections. Deparaffinized sections were coated with autoradiographic emulsion EM 1 (Amersham Lab. England) in a dark room and dried and were placed in a light-tight box. The sections were exposured for 5 weeks in the dark room, and were then developed in D-19 developer. Labeled indices (mean number of labeled cells per 100 epithelial cells in the isthmus) were observed and calculated. The results are as follows; 1. On histological study, gastric mucosa had no morphological changes following the injection of AG60. 2. On autoradiographic study, labeled grains of 3H-thymidine were restricted on the isthmus portion of the gatric gland. 3. On autoradiographic study, labeling indicies of gastric epithelial cells of normal control, experimental control, AG60 (5 mg/kg)-treated and AG60 (30 mg/kg)-treated groups were 21.9+/-0.28%, 18.8+/-0.03%, 21.6+/-1.61% and 6.3+/-0.93%, respectively. These result suggest that AG60 is expected as one of most effective anticancer drugs, and the dosage under 5 mg/kg of AG60 does not result any defect on the DNA synthesis in gastric epithelial cells.

Fluorescent Study on the Tissue-Distribution of Acriflavine-Guanosine Compound (AG60) / 대한해부학회지

The study was carried out to evaluate the tissue-distribution of acriflavine or AG60 (acriflavine-guanosine compound, 1 : 1), the newly developed anticancer remedy. Successful access or distribution of a drug to specific tissue is important to attack the cancer cells in the same area. But it also means that the drug may disturb the activities of labelled tissues or cells. On the other hand, unlabelled elements may survive from massive treatment with the drug. In this study, distribution of acriflavine or AG60 in Yac-1 leukemic cells (0.25~25 microgram/ml) and in the tissues of rats or mice (5~50 mg/kg) were evaluated. Yac-1 cells showed prominent fluorescence on the heterochromatin and more or less prominent fluorescence on the nucleoplasm, cytoplasm and plasma membrane. Cytotoxicity of AG60 led to morphologic changes such as bleb- or baloon-formation on the surface, general swelling of the cell, and lysis of the cell. Following the subcutaneous administration of acriflavine or AG60 (5~50 mg/kg) to the Ehrlich carcinoma-inoculat-ed rats or mice, most tissues including cancer cells showed acriflavine-fluorescence with some exception. The nuclei of cells of tissues were labelled more strongly than those of cytoplasm. Fluorescence was especially strong over biliary tree, renal corpuscle, gastrointestinal mucous coat, and bronchial mucous coat. But parenchymal components of central nervous system did not show any fluorescence. As shown in Yac-1 cells treated with AG60, the drug strongly attached to nucleic acids, and it induced swelling and disintegration of cancer cells. Fast turn-over of AG60 was seen in the secretory passages of bile juice, urine, gastrointestinal mucin, and bronchial mucin. The results show that AG60 could reach most tissues except parenchymes of central nervous system.

Anticancer Effect of AG60 (Acriflavine-Guanosine Compound) on the Ehrlich Cancer Cells Light Microscopic, Autoradiographic and Electron Microscopic Study / 대한해부학회지

To evaluate the effect and working mechanism of a newly developed anti-cancer drug, AG60 (acriflavine-guanosine compound, Taerim Pharm. Co. Seoul, Korea), histotologic, autoradiographic and electron microscopic studies were carried out. For the histologic study, each Ehrlich carcinoma cells (10(7) cells)-inoculated mouse was subcutaneously injected with saline (0.2 ml), 10 mg/kg of AG60, or 30 mg/kg of AG60, every other day, respectively. Animals were sacrified on the 14th day from the first injection, and tumor masses were fixed in 10% formalin solution. Tissue sections of the tumor were stained with hematoxylin and eosin. For the electron microscopic study, Ehrlich carcinoma (10(7) cells)-inoculated mice were subcutaneously injected every other day with saline (0.2 ml) or 30 mg/kg of AG60, respectively. The day after 7th injection (14th day), animals were sacrified, small piece of tumor masses were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution followed by fixation in 2% osmium tetroxide solution. Ultrathin sections were counter stained with uranyl acetate-lead citrate solutions, and observed with JEM 100CX electron microscope. For the autoradiographic study, each Ehrlich carcinoma (10(7) cells)-inoculated mouse was injected every day with 0.2 ml of saline, 5 mg/kg of AG60, or 30 mg/kg of AG60, respectively. The day following the last injection, each animal was given a single dose of 0.7 micricurie/g of methyl-3H-thymidine (Amersham Lab., England) through the tail vein. Seventy minutes after the thymidine injection, animals were sacrified, tumor masses were collected and fixed in 10% neutral formalin. Tissue blocks were washed, dehydrated, embedded and cut in 6 micrometer-thick sections. Deparaffinzied sections were dipped in the autradiographic emulsion E1 (Amersham Lab., England) and dried and stocked in the dark room. Filmed sections were exposured five weeks in the dark room, and were developed in the developer. Labeled indices (mean number of labeled cells per 100 cancer cells) and labeled grain indices (mean number of labeled silver grains per one cancer cell, and total granule numbers per every 100 cancer cell) were observed and calculated. The results were as follows : 1. On histological study, massive apoptosis were occured following the injection of AG60. Only small number of live cancer cells were observed. 2. On electron microscopic study, massive apoptotic figures including fragmentation of nuclei and cytoplasms, multiple nucleoli, condensation of nucleus and cytoplasm, deep invaginations and microcleft formations of nuclei, margination of heterochromatin along the inner nuclear membrane and microcleft , etc. were noticed. Giant cells represent the "tumor cell-tumor cell emperipolesis", and many of them seem to be in process of "cytolytic emperipolesis". 3. On autoradiographic study, labeled grains of 3H-thymidine were suppressed to only 11%~5% of control cancer cells following AG60 administrations. Discussed on the above experiments, it is suggested that severe suppression of DNA, RNA and protein syntheses by AG60 induce massive apoptosis of cancer cells. AG60 is expected as one of most effective anticancer drugs for the cytostatic therapy, the disease stabilization, the improved quality of life, the prolongation of life, and possibly the chemoprevention.

Anti-tumor Effect of the Complex of Acriflavine and Guanosine (AG60) / 대한암학회지

PURPOSE: The anti-tumor effect of the complex of acriflavine and guanosine (AG60) was investigated. MATERIALS AND METHODS: In vitro cytotoxicity of AG60 was measured using SRB assay, and in vivo antitumor activity of AG60 was examined in CDF1 mice intraperitoneally inoculated with the P388 leukemic cells and in ICR mice inguinally implanted with S-180 cells. Tumor size and mean survival time were determined. RESULTS: AG60 and acriflavine showed strong anti-tumor effect in vitro on lung cancer (A549), renal cancer (UO-31) and colon cancer (COLO205) cells. However, AG60 did not show the cytotoxicity against normal cell line, 3T3. The range of the IC50 of AG60 to the various tumor cell lines was 0.09 microgram/ml through 1.94 microgram/ml. The treatment of ascitic tumor bearing CDF1 mice with AG60 resulted in over 160% increases in the mean survival time. The most effective dose of AG60 was 30 mg/kg body weight in tumor implanted mice. In solid tumor bearing ICR mice tumor growth and progression were suppressed in response to the different doses at 30 days; 69.8% suppression of tumor size in response to acriflavine, 16.0% to guanosine, 87.7% to AG60 and 78.5% to doxorubicin. In addition, 35% increases were observed in the means survival time of AG60 treated group compared with control group. CONCLUSION: The prominant anti-tumor effects of AG60 shown in this report would represent the possibility of the clinical trials.