Abstract
Taxanes have a broad spectrum of activity against various human cancers, including melanoma. In this study, we have examined the molecular mechanism of docetaxel-induced apoptosis of human melanoma. We report that docetaxel induced varying degrees of apoptosis in a panel of melanoma cell lines but not in normal fibroblasts. Induction of apoptosis was caspase dependent and associated with changes in mitochondrial membrane potential that could be inhibited by overexpression of Bcl-2. Docetaxel induced changes in Bax that correlated with sensitivity to docetaxel-induced apoptosis. These changes in Bax were not inhibited by overexpression of Bcl-2. Kinetic studies of caspase-2 activation by Western blotting and fluorogenic assays revealed that activation of caspase-2 seemed to be the initiating event. Inhibition of caspase-2 with z-VDVAD-fmk or by small interfering RNA knockdown inhibited changes in Bax and mitochondrial membrane potential and events downstream of mitochondria. Activation of caspase-8 and Bid seemed to be a late event, and docetaxel was able to induce apoptosis in cells deficient in caspase-8 and Bid. p53 did not seem to be involved as a p53 null cell line was sensitive to docetaxel and an inhibitor of p53 did not inhibit apoptosis. Small interfering RNA knockdown of PUMA and Noxa also did not inhibit apoptosis. These results suggest that docetaxel induces apoptosis in melanoma cells by pathways that are dependent on activation of caspase-2, which initiates mitochondrial dependent apoptosis by direct or indirect activation of Bax.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Chloromethyl Ketones / pharmacology
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Antineoplastic Agents / pharmacology*
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Apoptosis / drug effects*
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Apoptosis Regulatory Proteins / antagonists & inhibitors
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Apoptosis Regulatory Proteins / genetics
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Apoptosis Regulatory Proteins / metabolism
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Blotting, Western
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Caspase 2 / genetics
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Caspase 2 / metabolism*
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Caspase Inhibitors
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Cell Nucleus / drug effects
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Cell Nucleus / metabolism
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Cysteine Endopeptidases / genetics
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Cysteine Endopeptidases / metabolism*
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Cytochromes c / metabolism
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Docetaxel
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Enzyme Activation / drug effects*
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Enzyme Inhibitors / pharmacology
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Flow Cytometry
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Humans
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Melanoma / drug therapy*
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Melanoma / enzymology
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Melanoma / pathology
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Membrane Potential, Mitochondrial / drug effects
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Proto-Oncogene Proteins / antagonists & inhibitors
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-bcl-2 / antagonists & inhibitors
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Proto-Oncogene Proteins c-bcl-2 / genetics
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Proto-Oncogene Proteins c-bcl-2 / metabolism
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RNA, Small Interfering / pharmacology
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Radiation-Sensitizing Agents / pharmacology
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Taxoids / pharmacology*
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Transfection
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Tumor Cells, Cultured
Substances
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Amino Acid Chloromethyl Ketones
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Antineoplastic Agents
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Apoptosis Regulatory Proteins
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BBC3 protein, human
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Caspase Inhibitors
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Enzyme Inhibitors
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PMAIP1 protein, human
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-bcl-2
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RNA, Small Interfering
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Radiation-Sensitizing Agents
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Taxoids
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benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
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Docetaxel
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Cytochromes c
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CASP2 protein, human
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Caspase 2
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Cysteine Endopeptidases