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-gamma 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-gamma 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-gamma 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.
Orucevic
A
A
Department of Surgery, University of Pittsburgh, PA, USA.
Bechberger
J
J
Green
A M
AM
Shapiro
R A
RA
Billiar
T R
TR
Lala
P K
PK
D000230:Adenocarcinoma / Q000201:enzymology / Q000473:pathology* / Q000503:physiopathology / Q000556:secondary; D000818:Animals; D004790:Enzyme Induction; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008175:Lung Neoplasms / Q000201:enzymology / Q000473:pathology / Q000503:physiopathology / Q000556:secondary*; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology / Q000473:pathology* / Q000503:physiopathology; D051379:Mice; D008809:Mice, Inbred C3H; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009361:Neoplasm Invasiveness; D009569:Nitric Oxide / Q000096:biosynthesis / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D011994:Recombinant Proteins; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics; D014407:Tumor Cells, Cultured; D019323:omega-N-Methylarginine / Q000494:pharmacology
D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D019717:Tissue Inhibitor of Metalloproteinase-3; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496317:Nos2 protein, mouse; C496315:Nos3 protein, mouse; D019331:NG-Nitroarginine Methyl Ester