Nitric-oxide production by murine mammary adenocarcinoma cells promotes tumor-cell invasiveness

The role of nitric oxide (NO) in tumor biology remains controversial and po orly understood. While a few reports indicate that the presence of NO in tu mor cells or their micro-environment is detrimental for tumor-cell survival , and consequently their metastatic ability, a large body of data suggests that NO promotes tumor progression, The purpose of this study was to identi fy the source of NO in the spontaneously metastasizing C3-L5 murine mammary -adenocarcinoma model, the role of tumor-derived NO in tumor-cell invasiven ess, and the mechanisms underlying the invasion-stimulating effects of tumo r-derived NO. The source of NO was established by immunocytochemical locali zation of NO synthase (NOS) enzymes in C3-L5 cells in vitro and transplante d tumors in vivo. An in vitro transwell Matrigel invasion assay was used to test the invasiveness of C3-L5 cells in the presence or the absence of NO blocking agents or iNOS inducers (IFN-gamma and LPS), The mechanisms underl ying the invasion-stimulating effects of tumor-derived NO were examined by measuring mRNA expression of matrix metalloproteinases (MMP)-2 and -9, and tissue inhibitors of metalloproteinases (TIMP) 1, 2 and 3 in C3-L5 cells in various experimental conditions. Results showed that C3-L5 cells expressed high level of eNOS protein in vitro, and in vivo, both in primary and in m etastatic tumors. C3-L5 cells also expressed iNOS mRNA and protein when cul tured in the presence of IFN-gamma and LPS, Constitutively produced NO prom oted tumor-cell invasiveness in vitro by down-regulating TIMP 2 and TIMP 3, In addition, there was up-regulation of MMP-2, when extra NO was induced b y IFN-gamma and LPS, In conclusion, NO produced by C3-L5 cells promoted tum or-cell invasiveness by altering the balance between MMP-2 and its inhibito rs TIMP-2 and 3. Thus, our earlier observations of anti-tumor and anti-meta static effects of NO inhibitors in vivo in this tumor model can be explaine d, at least in part, by reduced tumor cell invasiveness. (C) 1999 Wiley-Lis s, Inc.