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 AG60 against Ehrlich Tumor

BACKGROUND: AG60 is a complex of acriflavine and guanosine. Our previous study revealed that AG60 had not only in vitro antitumor activities in several human cancer cell lines, but also strong antitumor effects in animal experiments using p388 or S180 cells-implanted mice. METHODS: Antitumor effects of AG60 were compared with those of Adriamycin, acriflavine, guanosine or control group. Body weight, tumor weight change, and survival time were measured in Ehrlich carcinoma cells implanted ICR mice. RESULTS: Body weights in AG60, acriflavine, or Adriamycin treated groups were significantly lower than those in control group during 30 day observation period(p<0.05). The percent tumor growth inhibition of AG60, Adriamycin, acriflavine, or guanosine two weeks after last treatment was respectively 86% (T/C%=14), 83% (T/C%=17), 68%(T/C%=32), 41% (T/C%=59). According to above data, tumor growth inhibition in AG60 treated group was significantly stronger than that in control, acriflavine or guanosine treated group(p<0.01), but there was no significant difference between AG60 and Adriamycin treated group. Mean survival time in control, AG60, Adriamycin, acriflavine, or guanosine treated group was respectively 33+/-3.9 days, 68+/-4.2 days, 54+/-5.8 days, 36+/-3.8 days, 50+/-8.1 days. CONCLUSIONS: The anti-tumor effect of AG60 against Ehrlich tumor was significantly stronger than that of control, acriflavine or guanosine, and comparable with Adriamycin. Mean survival time in AG60 treated group was significantly longer than that in control, acrifavine, guanosine or Adriamysin treated group.

Effects of BCG Treatment on the Mouse Thymic Cortex : An Electron Microscopic Study / 대한해부학회지

This experiment was performed to study the morphological responses of the thymic cortex of the mice after administration of BCG. Healthy adult mice weighing 25gm each were divided into normal and experimental groups. BCG[0.03X108-0.32X108 CFU] were injected subcutaneously to the animals every other day, and animals were sacrificed at 4 days, 1 week, 2 weeks and 8 weeks following the first injection. Thymus were removed immediately after sacrifice and transferred to cold phosphate buffered 2.5% glutaraldehyde-1.5% paraformaldehyde solution[pH 7.3], and cut into small pieces. Tissue samples were fixed for 2-3 hours in the same fixative, postfixed with phosphate buffered 1% osmium tetroxide solution[pH 7.3], dehydrated in a graded series of alcohol, and embedded in araldite mixture. Ultrathin sections stained with uranyl acetate and lead citrate were observed with a JEM 100CX-II electron microscope. The observed results were as follow : 1. In the early BCG treated groups, a few eosinophile leucocytes were observed, but more eosinophils were observed in later groups. Some elongated and bar-shaped lysosomes with eletron lucent gap were often obserced in the macrophages. 2. Cortical population of thymocytes in the thymus were reduced, whereas territoris of the epithelial reticular cells were expanded especially in 2 weeks and 8 weeks groups. Some portion of the thymic cortex exhibited large intercellular spaces, and a few nuclear bodies filled with materials of medium density were observed in the epithelial reticular cells. 3. In the 8 weeks groups, macrophages, plasma cells and eosinopile leucocytes and developing eosinophile leucocytes were often observed in the thymic cortex. Distended cisternae of granular endoplasmic reticula and newly forming prosecretory granulses in the Golgi complex were ovserved in som plasma cells. From the above results, it was suggested that repeated treatment with BCG could induce disturb the maturation and differentiation of the T lymphocytes. In turn, BCG, if repeatedly injected, may disturb the immunological medchanism.

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.

Contrast Sensitivity Changes in Patients with Diabetic Retinopathy

Changes in contrast sensitivity have been demonstrated in patients with normal Snellen acuity. In an attempt to elucidate more sensitively the visual dysfunction before developement of either overt retinopathy or a reduction in Snellen acuity in patients with retinal disorders, contrast sensitivity test was performed in diabetic patients with normal Snellen acuity and control subjects matched for age and sex. The results were as follows. 1) Throughout all spatial frequencies(1.5 - 3.0 - 6.0 - 12.0 - 18.0 cpd), contrast sensitivity was significantly lower(P-value<0.01) in the diabetic eyes with retinopathy(30.7 - 49.3 - 52.5 - 16.1 - 7.8) than in the normal controls(42.5 - 84.3 - 103.0 - 60.5 - 25.1) or the diabetic eyes without retinopathy(43.1 - 92.2 - 95.8 - 43.4 - 16.4 ). 2) In high spatial frequencies(12.0 - 18.0 cpd) contrast sensitivity in the diabetic eyes without retinopathy group(43.4 - 16.4) was significantly decreased(P-value<0.01) in comparison with the normal controls(60.5 - 25.1). So, contrast sensitivity test is more sensitive test for central visual function than Snellen acuity.