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

The role of nitric oxide (NO)in tumor biology remains controversial and poorly understood. While a few reports indicate that the presence of NO in tumor 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 identify the source of NO in the spontaneously metastasizing C3-L5 murine mammary-adenocarcinoma model, the role of tumor-derived NO in tumor-cell invasiveness, and the mechanisms underlying the invasion-stimulating effects of tumor-derived NO. The source of NO was established by immunocytochemical localization of NO synthase (NOS) enzymes in C3-L5 cells in vitro and transplanted 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-γ and LPS). The mechanisms underlying 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 metastatic tumors. C3-L5 cells also expressed iNOS mRNA and protein when cultured in the presence of IFN-γ and LPS. Constitutively produced NO promoted 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 by IFN-γ and LPS. In conclusion, NO produced by C3-L5 cells promoted tumor-cell invasiveness by altering the balance between MMP-2 and its inhibitors TIMP-2 and 3. Thus, our earlier observations of anti-tumor and anti-metastatic effects of NO inhibitors in vivo in this tumor model can be explained, at least in part, by reduced tumor-cell invasiveness.