C-Myc Down-Regulation Increases Susceptibility to Cisplatin through Reactive Oxygen Species-Mediated Apoptosis in M14 Human Melanoma Cells
- Annamaria Biroccio,
- Barbara Benassi,
- Sarah Amodei,
- Chiara Gabellini,
- Donatella Del Bufalo and
- Gabriella Zupi
- Experimental Chemotherapy Laboratory, Experimental Research Center, Regina Elena Cancer Institute, Rome, Italy
- Dr. G. Zupi, Experimental Chemotherapy Laboratory, Experimental Research Center, Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy. E-mail: zupi{at}ifo.it
Abstract
Our aim in this work was to define the role of c-Myc in the susceptibility to cisplatin [cis-diamminedichloroplatinum(II) (CDDP)] in human melanoma cells. Two M14 melanoma cell clones obtained by transfection and expressing six to ten times lower c-Myc protein levels than the parental cells and the control clone were employed. Analysis of survival curves demonstrates an increase in CDDP sensitivity in c-Myc low-expressing clones if compared with the control clone and the parental line. The enhanced sensitivity is unrelated to the impairment in enzymatic DNA repair activity. Cell cycle analysis demonstrates that although the control clone is able to completely recover from the CDDP-induced S-G2/M block, this arrest is prolonged in c-Myc low-expressing clones and a fraction of cells undergoes apoptosis. Although no changes in P53, Bax, Bcl-2, and Bcl-xL/S protein levels are observed, apoptosis is associated with the formation of reactive oxygen species (ROS), activation of caspase-1, caspase-3 and cleavage of the specific caspase substrate poly-ADP-ribose polymerase. The use of the antioxidantN-acetyl cysteine and caspase inhibitors prevents CDDP-induced apoptosis in c-Myc low-expressing clones, demonstrating that ROS, caspase-1, and caspase-3 are required for apoptotic cell death. Moreover, ROS generation depends on caspase-1-like activation because the Ac-YVAD-cho inhibitor abrogates CDDP-induced ROS in the c-Myc low-expressing clones.
Footnotes
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This work was supported by grants from A.I.R.C. and Ministero della Sanità, and CNR-MURST. B.B. and S.A. are recipients of a fellowship from Italian Foundation for Cancer Research (FIRC).
- Abbreviations:
- CPP32
- caspase-3
- CDDP
- cisplatin [cis-diamminedichloroplatinum(II)]
- ODN
- oligodeoxynucleotide
- ICE
- caspase-1
- ROS
- reactive oxygen species
- ADR
- doxorubicin (Adriamycin)
- CPT
- camptothecin
- Z-VAD-fmk
- Z-Val-Ala-Asp (OMe)-fluoromethylketone
- Ac-YVAD-cho
- N-acetyl-Tyr-Val-Ala-Asp-CHO
- Ac-DEVD-cho
- N-acetyl-Asp-Glu-Val-Asp-CHO
- β-gal
- β-galactosidase
- BrdU
- bromodeoxyuridine
- NAC
- N-acetyl-l-cysteine
- LUC
- luciferase
- PARP
- poly-ADP-ribose polymerase
- PI
- propidium iodide
- CHO
- Chinese hamster ovary
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- Received November 28, 2000.
- Accepted March 30, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
