iNOS expression and NO production contribute to the direct effects of BCG on urothelial carcinoma cell biology.
OBJECTIVE
Evidence suggests that oxidative stress occurring as a consequence of inducible nitric oxide synthase/nitric oxide (iNOS/NO) contributes to the biologic effects of bacille Calmette-Guérin (BCG). Objective of this study is to examine iNOS expression, NO production, and the biologic effect of NO on established intermediate end points for the human urothelial carcinoma cell response to BCG.


METHODS
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's 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.


RESULTS
Exposure of UC cells to BCG significantly increased both iNOS expression and NO production. Inhibition of iNOS activity with 1400W significantly inhibited BCG's direct biologic effect on UC cells for all of the end points evaluated.


CONCLUSIONS
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.
Department of Urology, Medical College of Wisconsin, Milwaukee, WI.
Gopitkumar
G
Shah
Department of Urology, Medical College of Wisconsin, Milwaukee, WI.
Guangjian
G
Zhang
Department of Urology, Medical College of Wisconsin, Milwaukee, WI.
Fanghong
F
Chen
Department of Urology, Medical College of Wisconsin, Milwaukee, WI.
Yanli
Y
Cao
Department of Biophysics and the Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI.
Balaraman
B
Kalyanaraman
Department of Urology, Medical College of Wisconsin, Milwaukee, WI. Electronic address: wsee@mcw.edu.
William A
WA
See
D000578:Amidines / Q000494:pharmacology; D001500:BCG Vaccine / Q000494:pharmacology*; D001596:Benzylamines / Q000494:pharmacology; D002295:Carcinoma, Transitional Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D054418:Chemokine CCL20 / Q000235:genetics; D054360:Chemokine CXCL1 / Q000235:genetics; D019743:Chemokines, CXC / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics; D016209:Interleukin-8 / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology
D000578:Amidines; D001500:BCG Vaccine; D001596:Benzylamines; C493956:CDKN1A protein, human; C499707:CXCL3 protein, human; D054418:Chemokine CCL20; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D050759:Cyclin-Dependent Kinase Inhibitor p21; D015850:Interleukin-6; D016209:Interleukin-8; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D016328:NF-kappa B; D018799:Intercellular Adhesion Molecule-1; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II
BCG; Bladder cancer; NO; iNOS