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Reactive oxygen species‐mediated regulation of eNOS and iNOS expression in multicellular prostate tumor spheroids

Nitric oxide (NO) generated by either endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS) may be involved in prostate tumorigenesis through the inhibition of reactive oxygen species (ROS)‐induced apoptosis. Multicellular DU‐145 prostate tumor spheroids endogenously generated NO that paralleled the production of ROS. With increasing spheroid size, eNOS expression was downregulated, whereas an upregulation of iNOS expression was observed. In parallel, NO generation declined, as evaluated by the NO indicator diaminofluorescein‐2 diacetate (DAF‐2DA), suggesting that NO generation in DU‐145 tumor spheroids is mainly mediated by eNOS. Elevation of ROS by treatment of tumor spheroids with either buthionine sulfoximine (BSO) or hydrogen peroxide resulted in upregulation of eNOS, whereas iNOS was downregulated. Furthermore, eNOS expression was increased by epidermal growth factor (EGF) in a redox‐sensitive manner. Upregulation of eNOS after treatment with hydrogen peroxide was apparently transduced through receptor tyrosine kinase signaling pathways since it was abolished by the protein kinase C (PKC) inhibitor bisindolylmaleimide‐1 (BIM‐1), the p21ras inhibitor S‐trans‐trans‐farnesylthiosalicylic acid (FTS), the c‐Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1/2 activation. Endogenous NO may serve to escape from oxidative stress‐induced apoptosis since treatment of tumor spheroids with the NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethyl imidazoline‐1‐oxyl 3‐oxide (carboxy‐PTIO) as well as the NO synthase inhibitor N‐ω‐amino‐L‐arginine (L‐NAA) increased cleaved caspase‐3. Consequently, lowering intracellular NO levels with either L‐NAA or PTIO significantly raised ROS levels, indicating that endogenously generated NO may play a role as a ROS scavenger, thereby protecting exponentially growing tumor spheroids from ROS‐induced apoptosis. © 2003 Wiley‐Liss, Inc.