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Induction of apoptosis in MCF-7:WS8 breast cancer cells by beta-lapachone
S M Wuerzberger1, J J Pink, S M Planchon
1Department of Human Oncology and the University of Wisconsin Comprehensive Cancer Center, University of Wisconsin-Madison, 53792, USA.
Abstract:
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.
Insights
Beta-lapachone (beta-lap) induces apoptosis in breast cancer cells by targeting enzymes like topoisomerase I, distinct from camptothecin (CPT) which causes cell cycle arrest. Beta-lapachone triggers apoptosis without increasing p53/p21 or arresting the cell cycle.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Beta-lapachone (beta-lap) is known to affect enzymes like topoisomerase I (Topo I) in vitro.
- The precise intracellular targets and cell-killing mechanisms of beta-lapachone remain unclear.
- Camptothecin (CPT) is a known Topo I poison used as a comparative agent.
Purpose of the Study:
- To compare the cytotoxic responses of MCF-7 human breast cancer cells to beta-lapachone and camptothecin.
- To elucidate the specific intracellular targets and apoptotic pathways activated by beta-lapachone.
- To investigate the role of cell cycle regulation and key proteins (p53, p21, bcl-2, retinoblastoma protein) in the differential responses to these agents.
Main Methods:
- Exposure of MCF-7 cells to 4-hour pulses of beta-lapachone or camptothecin.
- Analysis of cell survival, apoptosis, cell cycle progression (G1, S, G2-M), and protein levels (p53, p21, bcl-2, cyclins, Topo I, Topo IIalpha, retinoblastoma protein).
- Utilized estrogen deprivation-induced synchronization and bromodeoxyuridine-labeling studies.
Main Results:
- Beta-lapachone treatment showed a direct correlation between decreased survival and apoptosis (LD50 = 2.5 microM), inducing a preapoptotic cell population near the G1-S border and lamin B cleavage.
- Beta-lapachone-induced apoptosis occurred without significant increases in p53/p21, decreases in bcl-2, or alterations in cyclin levels, and without cell cycle arrest.
- Camptothecin induced dramatic G2-M arrest, with minimal apoptosis at higher doses, and was associated with increased p53/p21, decreased bcl-2, Topo I, Topo IIalpha, and cyclins A/B1, suggesting protective cell cycle checkpoints.
Conclusions:
- Beta-lapachone effectively kills MCF-7 cells solely through apoptosis, independent of p53/p21 induction or cell cycle arrest.
- Camptothecin primarily induces cell cycle arrest and limited apoptosis in MCF-7 cells, potentially due to protective mechanisms.
- The distinct mechanisms of cell death induced by beta-lapachone and camptothecin highlight differential drug responses in cancer cells.