Changes of Nitric Oxide and Cell Proliferation after Bacillus Calmette-Guerin and Nitric Oxide Synthase Inhibitor Treatment on Murine Bladder Tumor-2 Cell Line / 대한비뇨기과학회지

PURPOSE: We hypothesize that the cytolytic activity of Bacillus Calmette-Guerin (BCG) may act through nitric oxide (NO) production, and that cell death is correlated with apoptosis, changes of NO and cell proliferation following BCG and/or nitric oxide synthase (NOS) inhibition treatment of a murine bladder tumor-2 (MBT-2) cell line. If these cell lines show cell death due to apoptosis was also determined. MATERIALS AND METHODS: NO production and proliferation activity of the MBT-2 cell line were measured after stimulation, with BCG and/or L-NAME, using an enzyme- linked immunosorbent assay (ELISA). After incubation of the MBT-2 cells with BCG and/or L-NAME, the cell cycle was analysis was performed by immunocytometry. RESULTS: The production of NO in the MBT-2 cells was significantly increased by the BCG. The BCG had direct dose-dependent cytotoxic effects on the MBT-2 cell line. After the L-NAME treatment, both the NO production and cytotoxicity were decreased (p<0.05). When the MBT-2 cells were cultured with BCG, the apoptotic cell ratio increased compared to that of the MBT-2 cells treated with L-NAME (p<0.05). CONCLUSIONS: The up-regulation of the production of NO following BCG treatment of the MBT-2 cell line may be due, in part, to the cytolytic action of the BCG. The cell death, when BCG was instilled, correlated with apoptosis.

Effect of Nitric Oxide and Peroxynitrite ( ONOO- ) on the Apoptosis of Murine Bladder Tumor-2 Cell Line / 대한비뇨기과학회지

Nitric oxide (NO) has been emerged as an important intracellular and intercellular regulatory molecule having functions as diverse vasodilatation, neural communication, and host defense. In the immune system, NO produced by activated macrophage or neutrophil is known to kill tumor cells as a defense molecule. In addition, recent reports demonstrated that NO could interact with superoxide to generate peroxynitrite (ONOO-), an anion and a potent oxidant, in macrophages or other cellular systems. The production of peroxynitrite has been recognized to be associated with the activation and expression of inducible NO synthase (iNOS). In this study, to evaluate the role of NO and peroxynitrite in murine bladder tumor cells, the author investigate the effect of NO and peroxynitrite on the viability, cytotoxicity, and DNA fragmentation of MBT-2 cells. The results are as followings: 1. Activated macrophages treated with INF-r, LPS, or INF-r+ LPS showed increment of nitrite (NO2) production and cytotoxicity against MBT-2 cells in a dose dependent manner. However, treatment with NGMMA, a NOS inhibitor, decreased NO2- production and cytotoxicity. 2. Treatment with SNP, a nitric oxide donor, increased NO2 production and DNA fragmentation (%), but decreased viability (%) of MBT-2 cells in a concentration dependent manner. 3. Treatment with peroxynitrite increased cytotoxicity and DNA fragmentation, but decreased viability of MBT-2 cells in a concentration dependent manner. 4. NO- and peroxynitrite-mediated increment of cytotoxicity in MBT-2 cells was corresponded to the programmed cell death, apoptosis. Taken together, these data indicate that NO and peroxynitrite elaborated from macrophages or other cellular systems may increase the cytotoxicity of MBT-2 cells via the mechanism of apoptosis.

Effects of Nitrogen Oxidation Metabolism of Murine Macrophages on the Growth of MBT-2 Cell Line / 대한비뇨기과학회지

Recently it is known that nitric oxide(NO) generated by an activatedmacrophage plays an important role in tumoricidal or bactericidal activity. Thisstudy was done to know the effects of NO produced by the activated macrophages onthe growth of the murine bladder tumor cell line (MBT-2). For the activation ofmacrophages, RAW 264.7 cell line ( macrophage-derived cell line) and peritonealmacrophages from Balb/c mouse were treated with interferon-r (INF-r),lipopolysaccharide ( LPS) or INF-r plus LPS and for the evaluation of growthinhibition of MBT -2 cell line, tritiated thymidine (3[H] -thymidine)incorporation was measured after coculturing of MBT-2 cell line with macrophageswhich had been activated to produce NO. The results are as follows : l.Peritoneal macrophages from Balb/c mouse could be activated to produce NO onlywhen they are stimulated by the combination of INF-r and LPS (35+/-1.0uM/L). Theycould produce a slight increase amount of NO when they had been stimulated byINF-r (11+/-2.5uM/ L) or LPS (14+/-3.5uM/L) compared to control macrophages(8+/-2.5uM/L). The induction of NO production by INF-r plus LPS could be abrogatedby the use of NG-monomethyl-L- arginine (NGMMA), a competitive inhibitor of NOsynthase (5+/-1.5 M/L). 2. RAW 264.7 cell lines could be activated to produce NOby the treatment of INF-r, LPS, and INF-r plus LPS (40+/-2.5uM/L, 37+/-3.0uM/Land 51+/-2.6uM/L respectively). When they are treated with NGMMA, they produced nomore NO even though they had been stimulated with INF-r plus LPS (14+/-4.0uM/L),and they could produce small .amount of NO(19+/-1.0uM/L) in the absence of thestimulation. 3. The incorporation of 3[H] -thymidine of the MBT-2 and peritonealmacrophages was reduced when the cells had been treated with INF-r plus LPScompared to the control (14,519+/-1,087cpm, 20,716+/-1,474cpm respectively).There was no reduction in the incorporation of 3[H] -thymidine when the cellshad been treated with INF-r or LPS alone. The incorporation of 3[H]-thymidineincreased when the cells had been treated with INF-r plus LPS in the presence ofNGMMA(19,622+/-1,341cpm). 4. The incorporation of 3[H] -thymidine of the MBT-2 and RAW 264.7 cell lines were reduced when the cells had been treated with INF-r, LPS and INF-r plus LPS (19.068+/-144cpm, 15,070+/-122cpm and 7,543+/-85cpm respectively) compared to control( 20,708+/-142cpm). When the cells had been pretreated with NGMMA, the incorporation of 3[H]- thymidine recovered to same degree (12,605+/-108cpm) compared to the cells stimulated with INF-r plus LPS. The above correlation of the NO production from macrophages which had been stimulated by INF-r and/or LPS and the inhibition of growth of the tumor cells suggests that NO produced by stimulated macrophages might be the responsible molecule in the defense system of the body against tumors.