Mdm2 inhibition induces apoptosis in p53 deficient human colon cancer cells by activating p73- and E2F1-mediated expression of PUMA and Siva-1.
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.
Ray
Ramesh M
RM
Department of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163, USA. rray3@uthsc.edu
Bhattacharya
Sujoy
S
Johnson
Leonard R
LR
D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D018938:Caco-2 Cells; D002166:Camptothecin / Q000494:pharmacology; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D053938:DNA Fragmentation / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D004268:DNA-Binding Proteins; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D007093:Imidazoles; D009687:Nuclear Proteins; C498702:PMAIP1 protein, human; D010879:Piperazines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; C106549:SIVA1 protein, human; C000606460:TP73 protein, human; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D020169:Caspases; D002166:Camptothecin