泌尿器科領域の組織培養, 特に癌をめぐる線溶系とその臨床的応用 (第30回泌尿器科中部連合地方会)

Abstract

In this special lecture presented were the results of tissue culture studies of urological cancers in our department. The tissue cultures have been introduced into our experimental investigations of the fibrinolytic system in neoplasms, particularly bladder cancer, and of an in vitro sensitivity test of anticancer drugs. In addition, the results of our clinically significant studies related to the fibrinolytic system in the diagnostic and therapeutic aspects of bladder cancer were reported. Using a micro-single radial immunodiffusion method, serum protease inhibitors includingα1-antitrypsin, α1-antichymotrypsin and Cis-inhibitor were increased, whereas plasminogen was decreased in bladder cancer patients. These changes were remarkable in patients with an advanced bladder cancer and the decrease of α2-macroglobulin, a distinct condition in DIC, was also found. The depressed fibrinolytic activity indicated by these results would be a predisposing cause of fibrin depositions in cancerous tissues or blood vessels, and explain that patients with an advanced tumor are prone to develop vascular thrombosis and tend to occur a peculiar form of intravascular coagulation. During last 6 years, 75 out of In human urological malignant tumors subjected to tissue culture studies were successed in a primary culture, and 2 cell lines, KK-47 and KW-103, derived from transitional cell carcinomas of the bladder, and 2 cell lines, KN-41 and KH-39, from renal cell carcinomas, have been established and these cell lines utilized for the subsequent studies. Three human bladder carcinoma cell lines, T24, RT4 and MANO, a human bladder non-malignant epithelial cell line, HCV-29, and a human lung fibroblast line, 460 HI, were investigated for their ability to induce fibrinolytic, urokinase and plasmin inhibitory activities in cell culture, using serum-free medium, for up to 36 hrs. Generally, the non-malignant cell line and the fibroblast line had a greater ability to produce urokinase inhibitor than did the malignant cell lines. The low concentration of plasminogen activator, immunologically identical with urokinase, and its accumulation in culture supernatant were found with RT4 after 12-hr and 24-hr cultivations, whereas no plasminogen activator was detected in all other cell lines for periods up to 36 hrs. No plasmin, non-specific protease or plasmin inhibitory activities were detected in any of the supernatants from the cell lines. Moreover, [125]I-fibrin coated culture dishes were adopted for a meticulous detection of in vitro plasminogen activator production by cultured cells in Ham Fl2 medium supplemented with 20 per cent calf serum. In this assay system, KK-47, KW-103 and KN-41 cells resulted in an accumulation of plasminogen activator in the medium along with their logarithmic cell growth curves. However, in case of culture dishes not coated by fibrin, a temporary production of plasminogen activator occurred for the first 24 hrs of culture periods, and then a progressive lowering of the production was found in spite of their logarithmic cell proliferation. These in vitro results suggested that the activator production by cultured cells may be stimulated by the coexistence of fibrin and it may overcome the activity of fibrinolysis inhibitors. This suggestion would be able to explain a mechanism between the production and elimination of fibrin depositions in cancerous tissues and a rapid turnover of fibrinogen or fibrin in cancer patients. Clinically, the presence of this turnover would be manifested by an increased urinary FDP in bladder cancer patients, and urinary FDP would be considered as a biochemical marker in bladder cancer. Enhanced cell killing effect by thio-TEPA and urokinase on KK-47 cells was investigated using methods of assessing cell growth and 3H-thymidine uptake. A combined use of urokinase and thioTEPA resulted in a significant increase of the thio-TEPA toxicity with a 2-hr and 24-hr exposure times. A peak in 32P-thio-TEPA uptake in the cells was observed 2 hrs after administration of the agent, and about 2-time increase in the peak was obtained by adding urokinase. From the results, it was suggested that the increased cytotoxicity may be related with a gross change in cell permeability to thio-TEPA through plasminogen activation by urokinase. This conclusion was introduced into the topical prophylactic combination use of thio-TEPA, carboquone or cytosine arabinoside and urokinase in bladder cancer patients. The recurrence rates of the thio-TEPA and carboquone therapies for the postoperative 12 months were 10 and 14 per cent, and 22 and 33 per cent for the postoperative 24 months, respectively. While, combined instillation of cytosine arabinoside, a time-dependent anticancer agent, and urokinase resulted in high recurrence rates, 24.0 and 33.5 per cent for the postoperative 3 and 6 months, respectively. These data would recommend the topical use of dose-dependent anticancer agents such as alkylating agent and antibiotic rather than time-dependent anticancer agents. KK-47, KW-103 and KN-41 were used for studies on the cytocidal effect of 7 anticancer agents. The effect on in vitro cultured cells was determined by a colony-forming method. Based on 50 per cent and 90 per cent lethal dosages in 2-hr and 24-hr exposures, the most prominent cytocidal effect on the cells was obtained by carboquone, adriamycin and mitomycin C. A mild cell killing effect was obtained by cis-platinum, VP-16, bleomycin and thio-TEPA. There were slight differences of cell killing effect among the cell lines, and the survival curves characteristic in cell killing modality would provide a valuable reference for their clinical use. The in vitro cytotoxicity of anticancer drugs should be compared with their clinical benefits and the improvement of the test system including much more significant cell lines is necessary to establish its clinical usefulness. Enhanced cell killing effect by t-AMCHA, a potent antiplasmin agent, and carboquone or bleomycin on KK-47, KW-103 and KN-41 cells was investigated using the colony-forming method. The effect was observed in the combinations of 50 per cent lethal dose of the agents and 150 to 1500 µg/ml of t-AMCHA. Much more clearly enhanced cell killing effect was also obtained in combination with another most potent antiplasmin agent, DV-I006, and these anticancer agents. While, EACA, a relatively weak antiplasmin agent compared with t-AMCHA or DV-I006, possessed less combination effect. It is certain that an enhanced inhibitory action for the cell growth is achieved by combination of anticancer drugs and induced antifibrinolysis. However, further in vivo studies of the combination are necessary to clarify the role of antifibrinolysis in the combination effect. A future new promising plan for tissue culture studies with regard to the development of diagnosis and treatment of bladder cancer was discussed.