Inhibition of Angiogenesis by the First Type I Repeat Peptides of Thrombospondin-1 / 체질인류학회지

Angiogenesis is the fundamental biological phenomenon in the development of vertebrates and various pathophysiological process such as cancer, inflammation and wound healing. Thrombospondin-1 is a well-known anti-angiogenic molecule which is distributed in the extracellular matrix of various tissues. The second and third type I repeats of human TSP-1 have inhibitory effects on endothelial cell migration and induce angiogenesis inhibition. However the role of the first type I repeat was not elucidated. In addition, the first type I repeat of bovine TSP-1 has CSVTCG amino acid sequence which is known to have anti-angiogenic activity. In the present study, we compared the inhibition of angiogenesis to investigate the role of the first type I repeat of the human and bovine TSP-1. Matrigel was mixed with or without TSR-1 peptides and then injected into C57BL/6J mice. We compared angiogenesis inhibition activity by hemoglobin assay, microvessel density and optical density value after 7 days. Furthermore, inhibition of angiogenesis was confirmed on CAM assay by TSR-1 peptides. For in vitro angiogenesis assay, TSR-1 peptides were treated on the proliferation, migration, and tube formation assay of HUVEC. Apoptosis effect of TSR-1 peptides was confirmed by apoptosis assay kit and flow cytometry. Bovine and human TSR-1 peptides blocked neovascularization in in vivo Matrigel plug assay and CAM assay at 10 microM. Bovine TSR-1 peptides have shown stronger angiogenesis inhibition in bFGF-induced angiogenesis than human TSR-1 and CSVTCG peptides. However, all of TSR-1 peptides inhibit migration and tube formation of HUVEC in in vitro. Furthermore, these peptides also induced apoptosis of HUVEC. These results suggest that TSR-1 peptides of bovine and human TSP-1 have angiogenesis inhibition activity.

Effect of Antioxidant Vitamin Supplementation on Hyperbaric Oxygen Injury in Rats / 항공우주의학회지

BACKGROUND: Antioxidants vitamin C and vitamin E may protect against the toxic effect of oxygen free radicals that are preferentially produced after exposures to hyperbaric oxygen (HBO). This study investigated the effect of vitamin C and vitamin E on serum nitric oxide (NO) concentration and intercellular adhesion molecule-1 (ICAM-1) expression on lung after HBO exposure. METHODS: Male Sprague-Dawley rats weighing 200 to 250 g were exposed to HBO at 3 ATA of 100% O2 for 3 hours. The experimental groups were given vitamin C (125 mg/day per rat) and/or vitamin E (50 mg/day per rat) orally, from 5 days prior to the HBO exposure to the day of sacrifice. Serum NO concentrations were determined by measuring NO end product nitrite by non-enzymatic Griess assay. Expression of ICAM-1 on lung was observed by immunohistochemical analysis. RESULTS: The serum nitrite levels were significantly increased after HBO exposure and were higher at 24 hours after HBO exposure than at 0 h (P<0.05). The expression of ICAM-1 was weak immediately after HBO exposure and enhanced at 24 hours. There were no pronounced suppressive effects of vitamins on serum NO production and ICAM-1 expression induced by the 3 hours HBO exposure. CONCLUSION: The 3 hours HBO exposure induces the serum NO production and ICAM-1 expression on lung. The short-term supplementation of vitamin C or/and E do not suppress the NO production and ICAM-1 expression on lung.

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.

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.

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.

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.