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 different human neoplastic lymphoid cells through caspase activation. Here we studied the NO-mediated apoptosis in human breast cancer cell lines derived from primary tumor (BT-20) or from metastasis (MCF-7). NO donor glycerol trinitrate (GTN) induced apoptosis in both cell lines which was completely abrogated after pretreatment with the broad spectrum caspase inhibitor zVAD-fmk. NO triggered also a time-dependent activation of caspase-1, caspase-3, and 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 with low mitochondrial transmembrane potential and with high level of reactive oxygen species production. However, NO did not induce mRNA expression of CD95 (APO-1/Fas) ligand. FAS-associated phosphatase-1 (FAP-1) molecule was constitutively expressed at the mRNA level and did not show any changes upon NO treatment in both breast cancer cell lines. The expression of the pro-apoptotic protein Bax and of the anti-apoptotic protein Bcl-2 remained unchanged in MCF-7 and BT-20 cells upon GTN treatment. We suggest that the mechanism of NO-mediated activation of the caspase cascade and subsequent apoptosis in human breast cancer cells required mitochondrial damage (in particular, cytochrome c release, disruption of mitochondrial transmembrane potential and generation of reactive oxygen species) but not the activation of the CD95/CD95L pathway.
Umansky
V
V
Deutsches Krebsforschungszentrum, Abteilung Zellulare Immunologie G0100, FSP Tumorimmunologie, D-69120 Heidelberg, Germany.
Ushmorov
A
A
Ratter
F
F
Chlichlia
K
K
Bucur
M
M
Lichtenauer
A
A
Rocha
M
M
D017209:Apoptosis / Q000502:physiology*; D001943:Breast Neoplasms; D002352:Carrier Proteins / Q000096:biosynthesis; D020169:Caspases / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D053222:Fas Ligand Protein; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D009569:Nitric Oxide / Q000494:pharmacology / Q000502:physiology*; D054645:Protein Phosphatase 1; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000502:physiology*
C494826:BAX protein, human; D002352:Carrier Proteins; D003574:Cytochrome c Group; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D009569:Nitric Oxide; D054645:Protein Phosphatase 1; C516742:PTPN13 protein, human; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases; D020169:Caspases