Quantitative reverse transcriptase-polymerase chain reaction and real-time measurement of NO was used to assess iNOS and NO production, respectively, in 2 human urothelial carcinoma (UC) cell lines, in response to BCG. The effect of blocking NO production using the specific iNOS inhibitor 1400W was determined for multiple intermediate end points characterizing BCG鈥檚 direct effects on tumor cell biology. Activation of nuclear factor kappa B and nuclear factor (erythroid-derived 2)-like 2 signaling pathways, transactivation of genes, including p21, CD54, IL6, IL8, CXCL1, CXCL3, CCL20, and cytotoxicity, as measured by vital dye exclusion, lactate dehydrogenase release, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were measured in response to BCG with and without iNOS inhibition.
Exposure of UC cells to BCG significantly increased both iNOS expression and NO production. Inhibition of iNOS activity with 1400W significantly inhibited BCG鈥檚 direct biologic effect on UC cells for all of the end points evaluated.
iNOS expression, NO production, and the associated oxidative stress play a central role in the response of UC cells to BCG exposure. Manipulation of oxidative stress may afford an opportunity to enhance the antitumor effects of BCG.