Antitumor effects of inhibitors of nitric oxide synthase or cyclooxygenase-2 on human KB carcinoma cells overexpressing COX-2

Abstract

Abstract. Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 are major inflammatory mediators. Nitric oxide (NO) produced by iNOS has been shown to have an important role in carcinogenesis. Recent studies have suggested that COX-2 expression also contributes to carcinogenesis, as well as tumor growth, invasion, and metastasis. COX-2 inhibitors such as celecoxib are widely recognized to have antitumor activity, but can cause adverse effects. We investigated possible relations between COX-2 and NO with the use of a human epidermoid carcinoma cell line, designated KB, in which overexpression of COX-2 protein was induced by gene transfer. We also assessed the possibility of using NOS inhibitor as an antitumor drug. We isolated a COX-2 transfected clone (KB/COX-2) and used a neomycin-transfected clone (KB/neo) as control. N G -nitro-Larginine-methyl ester (L-NAME) was used as a NOS inhibitor, dihydrochloride (1400W) as an iNOS inhibitor, and celecoxib as a selective COX-2 inhibitor. All agents inhibited the cell growth of both clones to similar extents in a dosedependent manner. Prostaglandin E 2 (PGE 2 ) production and COX-2 expression in KB/COX-2 were inhibited not only by celecoxib, but also by L-NAME and 1400W. The decreases in PGE 2 production and COX-2 expression were most prominent with celecoxib and L-NAME. In vivo, L-NAME and celecoxib significantly inhibited the proliferation of KB/COX-2-xenografted tumors. Tumor weight was reduced by L-NAME (60.6% decrease), 1400W (38.0% decrease), and celecoxib (74.5% decrease) as compared with the control after 21 days of treatment. Immunohistochemically, xenografted tumors expressed COX-2, iNOS, and eNOS. Such expression was suppressed by treatment with L-NAME and celecoxib. These results suggest that L-NAME and celecoxib significantly inhibit the proliferation of murine squamous cell carcinoma in vivo. L-NAME as well as celecoxib might thus be useful for the design and development of new antitumor drugs. IntroductionThe biosynthesis and release of nitric oxide (NO) and prostaglandins (PGs) share a number of similarities. Two major forms of nitric oxide synthase (NOS) and cyclooxygenase (COX) have been identified to date. NO is a multifunctional gaseous molecule synthesized from L-arginine by NOS. There are three isoforms of NOS: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible isoform of NOS (iNOS). nNOS and eNOS are constitutively expressed and are also referred to as constitutive NOS (cNOS). In contrast, iNOS is transcriptionally regulated and induced by inflammatory cytokines, endotoxins, hypoxia, and oxidative stress (1,2). iNOS produces high, sustained concentrations of NO, whereas the other two isoforms produce low, transient concentrations of NO (3).Previous studies have shown positive correlations between iNOS and poor outcomes in patients with breast cancer and melanoma (4,5). These observations suggest that NO generated by iNOS has multiple physiologic and pathologic effects. It has been reported that eNOS can modulate cancer-related ...