Abstract
Camptothecin (CPT) and Nutlin-3 caused apoptosis by increasing p53 protein and its activation in intestinal epithelial cells (IEC-6). We studied the effectiveness of these inducers on apoptosis in human colon cancer cells (Caco2) lacking p53 expression. CPT failed to activate caspase-3 and cause apoptosis in these cells. The absence of p53 expression, higher basal Bcl-xL and lower Bax proteins prevented CPT-induced apoptosis. However, the Mdm2 antagonist Nutlin-3 induced apoptosis in a dose dependent manner by activating caspases-9 and -3. Nutlin-3 prevented the activation of AKT via PTEN-mediated inhibition of the PI3K pathway. Nutlin-3 increased the phosphorylation of retinoblastoma protein causing E2F1 release leading to induction of Siva-1. Nutlin-3-mediated degradation of Mdm2 caused the accumulation of p73, which induced the expression of p53 up-regulated modulator of apoptosis (PUMA). E2F1 and p73 knockdown decreased the expression of Siva and PUMA, respectively and abolished Nutlin-3-induced caspase-3 activation. Cycloheximide (CHX) inhibited Nutlin-3-induced Siva, Noxa, and PUMA expression and inhibited apoptosis in IEC-6 and Caco2 cells. These results indicate that translation of mRNAs induced by Nutlin-3 is critical for apoptosis. In summary, apoptosis in Caco2 cells lacking functional p53 occurred following the disruption of Mdm2 binding with p73 and Rb leading to the expression of pro-apoptotic proteins, PUMA, Noxa, and Siva-1.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Apoptosis / drug effects
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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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Caco-2 Cells
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Camptothecin / pharmacology
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Caspases / genetics
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Caspases / metabolism
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Cell Line, Transformed
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Colonic Neoplasms / genetics
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Colonic Neoplasms / metabolism*
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Colonic Neoplasms / pathology
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Colonic Neoplasms / therapy
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DNA Fragmentation / drug effects
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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E2F1 Transcription Factor / genetics
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E2F1 Transcription Factor / metabolism
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Gene Expression
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Humans
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Imidazoles / pharmacology
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Piperazines / pharmacology
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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 / genetics
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Proto-Oncogene Proteins c-bcl-2 / metabolism*
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Proto-Oncogene Proteins c-mdm2 / antagonists & inhibitors*
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Proto-Oncogene Proteins c-mdm2 / metabolism
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RNA, Small Interfering / pharmacology
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Tumor Protein p73
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Tumor Suppressor Protein p53 / genetics
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Tumor Suppressor Protein p53 / metabolism
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Tumor Suppressor Proteins / genetics
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Tumor Suppressor Proteins / metabolism
Substances
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Apoptosis Regulatory Proteins
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BBC3 protein, human
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DNA-Binding Proteins
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E2F1 Transcription Factor
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E2F1 protein, human
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Imidazoles
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Nuclear Proteins
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PMAIP1 protein, human
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Piperazines
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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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SIVA1 protein, human
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TP73 protein, human
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Tumor Protein p73
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Tumor Suppressor Protein p53
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Tumor Suppressor Proteins
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nutlin 3
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MDM2 protein, human
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Proto-Oncogene Proteins c-mdm2
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Caspases
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Camptothecin