Upregulation of p21WAF1/CIP1 in human breast cancer cell lines MCF-7 and MDA-MB-468 undergoing apoptosis induced by natural product anticancer drugs 10-hydroxycamptothecin and camptothecin through p53-dependent and independent pathways.
Recently, natural product DNA topoisomerase I inhibitors 10-hydroxycamptothecin (HCPT) and camptothecin (CPT) have been shown to have therapeutic effects in both in vitro and in vivo models of human breast cancer. In the present study, we characterized the in vitro and in vivo apoptotic pathways induced by HCPT and CPT in the human breast cancer cell lines MCF-7 and MDA-MB-468. Using various DNA fragmentation analyses and the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, the apoptosis induced by HCPT and CPT was shown to be dose- and time-dependent. The MDA-MB-468 cells were more sensitive to both HCPT and CPT than MCF-7 cells. HCPT induced apoptosis in MDA-MB-468 cells more effectively than CPT; however, in MCF-7 cells, CPT was more effective than HCPT. The levels of p53 and p21WAF1/CIP1 protein increased in MCF-7 cells treated with HCPT or CPT in a dose- and time-dependent manner. The levels of p21WAF1/CIP1 protein also increased in a dose- and time-dependent manner in MDA-MB-468 cells treated with HCPT or CPT, whereas the mutated p53 protein levels had no significant change. The elevation of p53 protein levels in MCF-7 cells treated with CPT was significantly inhibited by preincubation with DNA breaks inhibitor aphidicolin, while the elevation of p21WAF1/CIP1 protein levels was not inhibited. The elevation of p21WAF1/CIP1 in MDA-MB-468 cells treated with CPT was not inhibited by aphidicolin. Using Northern blot analysis, the transcription of p21WAF1/CIP1 was shown to increase in a dose-dependent manner in MCF-7 and MDA-MB-468 cells treated with HCPT or CPT. These results suggest that treatment with HCPT and CPT results in increased levels of p21WAF1/CIP1 protein and mRNA, and that they induce apoptosis in human breast cancer cells through both p53-dependent and -independent pathways. These findings may be significant in further understanding the mechanisms of actions of camptothecins in the treatment of human cancers.
Liu
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Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Zhang
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D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis* / Q000235:genetics; D004249:DNA Damage; D005260:Female; D006801:Humans; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
D000972:Antineoplastic Agents, Phytogenic; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; C028098:10-hydroxycamptothecin; D002166:Camptothecin