Nitric oxide-mediated apoptosis in human breast cancer cells requires changes in mitochondrial functions and is independent of CD95 (APO-1/Fas)

We have previously shown that nitric oxide (NO) induces apoptosis in differ ent human neoplastic lymphoid cells through caspase activation. Here we stu died the NO-mediated apoptosis in human breast cancer cell lines derived fr om primary tumor (BT-20) or from metastasis (MCF-7). NO donor glycerol trin itrate (GTN) induced apoptosis in both cell lines which was completely abro gated after pretreatment with the broad spectrum caspase inhibitor zVAD-fmk , NO triggered also a time-dependent activation of caspase-1, caspase-3, an d caspase-6 in these cells. Moreover, NO caused a release of mitochondrial protein cytochrome c into the cytosol, an increase in the number of cells w ith low mitochondrial transmembrane potential and with high level of reacti ve oxygen species production. However, NO did not induce mRNA expression of CD95 (APO-1/Fas) ligand. FAS-associated phosphatase-l (FAP-1) molecule was constitutively expressed at the mRNA level and did not show any changes up on NO treatment in both breast cancer cell lines. The expression of the pro -apoptotic protein Pax and of the anti-apoptotic protein Bcl-2 remained unc hanged in MCF-7 and BT-20 cells upon GTN treatment. We suggest that the mec hanism of NO-mediated activation of the caspase cascade and subsequent apop tosis in human breast cancer cells required mitochondrial damage (in partic ular, cytochrome c release, disruption of mitochondrial transmembrane poten tial and generation of reactive oxygen species) but not the activation of t he CD95/CD95L pathway.