Induction of apoptosis in MCF-7:WS8 breast cancer cells by beta-lapachone.
Beta-lapachone (beta-lap) affects a number of enzymes in vitro, including type I topoisomerase (Topo I); however, its exact intracellular target(s) and mechanism of cell killing remain unknown. We compared the cytotoxic responses of MCF-7:WS8 (MCF-7) human breast cancer cells after 4-h pulses of beta-lap or camptothecin (CPT), a known Topo I poison. A direct correlation between loss of survival and apoptosis was seen after beta-lap treatment (LD50 = 2.5 microM). A concentration-dependent, transient sub-2 N preapoptotic cell population was observed at 4-8 h. Estrogen deprivation-induced synchronization and bromodeoxyuridine-labeling studies revealed an apoptotic exit point near the G1-S border. Apoptosis activated by beta-lap was closely correlated with cleavage of lamin B but not with increases in p53/p21 or decreases in bcl-2. Loss of hyperphosphorylated forms of the retinoblastoma protein was observed within 5 h, but cyclins A, B1, and E levels were unaltered for up to 72 h after 5 microM beta-lap. Topo I and Topo IIalpha levels decreased at > 24 h. Logarithmic-phase MCF-7 cells were not affected by < or = 1 microM beta-lap. In contrast, dramatic and irreversible G2-M arrest with no apoptosis was observed in MCF-7 cells treated with 1 microM CPT, monitored for 6-10 days posttreatment. MCF-7 cells treated with supralethal doses of CPT (5 microM) resulted in only approximately 20% apoptosis. No correlation between apoptosis and loss of survival was observed. MCF-7 cells exposed to > 5 microM CPT arrested at key cell cycle checkpoints (i.e., G1, S, and G2-M), with little or no movement for 6 days. Ten-fold increases in p53/p21 and 2-5-fold decreases in bcl-2, Topo I, Topo IIalpha, and cyclins A and B1, with no change in cyclin E, were observed. Temporal decreases in bcl-2 and cleavage of lamin B corresponded to the minimal apoptotic response observed. Beta-lap activated apoptosis without inducing p53/p21 or cell cycle arrest responses and killed MCF-7 cells solely by apoptosis. In contrast, concentration-dependent increases in nuclear p53/p21 and various cell cycle checkpoint arrests were seen in MCF-7 cells after CPT. Despite dramatic p53/p21 protein induction responses, CPT-treated MCF-7 cells showed low levels of apoptosis, possibly due to protective cell cycle checkpoints or the lack of specific CPT-activated apoptotic pathways in MCF-7 cells.
Wuerzberger
S M
SM
Department of Human Oncology and the University of Wisconsin Comprehensive Cancer Center, University of Wisconsin-Madison, 53792, USA.
Pink
J J
JJ
Planchon
S M
SM
Byers
K L
KL
Bornmann
W G
WG
Boothman
D A
DA
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D002166:Camptothecin / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D007928:Lethal Dose 50; D009285:Naphthoquinones / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000972:Antineoplastic Agents, Phytogenic; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D009285:Naphthoquinones; D019253:Proto-Oncogene Proteins c-bcl-2; D018894:Reverse Transcriptase Inhibitors; D016159:Tumor Suppressor Protein p53; C014638:beta-lapachone; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin