The role of endogenous NO on cell survival was investigated in human
melanoma cells and melanocytes. Inducible
NO synthase (iNOS) was always expressed in a panel of
melanoma cell lines from metastatic lesions and in normal adult melanocytes. iNOS was also detected by immunohistochemistry in
melanoma cells from
metastases. Release of NO by
tumor cells and melanocytes was inhibited by a specific iNOS inhibitor,
aminoguanidine (AMG). Inhibition of endogenous NO synthesis did not affect cell cycle progression of
melanoma cells but led to cell death by apoptosis, as indicated by
Annexin V/
propidium iodide and terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling assays. By contrast, iNOS inhibition by AMG did not promote apoptosis in normal adult melanocytes. A mitochondrial pathway was involved in
melanoma apop tosis, as indicated by altered mitochondrial membrane potential (delta psi(m)) and down-regulation of Bcl-2
protein level after iNOS inhibition. AMG treatment triggered release of caspase-1, enzymatic activation of
caspase-3, and degradation of
poly(ADP-ribose) polymerase, one of the main
caspase-3 substrates.
Melanoma cell apoptosis induced by iNOS inhibition was completely blocked by
peptide inhibitors of caspase-1 and
caspase-3 (
Ac-DEVD-CHO and
AC-YVAD-CHO) or by an exogenous NO donor,
sodium nitroprusside, or by addition of serum. Finally, comparison of control and AMG-treated
melanoma cells by pathway-specific gene array analysis indicated that inhibition of NO synthesis led, before induction of apoptosis, to up-regulation of
mRNA levels of genes involved in the apoptosis pathway such as Bax, caspase-1,
caspase-3,
caspase-6, gadd45beta, mdm2, and TRAIL. Taken together, these results indicate that
melanoma cell survival is regulated by endogenous NO resulting from iNOS activity.