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Mitomycin C induces apoptosis and caspase-8 and -9 processing through a caspase-3 and Fas-independent pathway
F Pirnia1, E Schneider, D C Betticher
1Institute of Medical Oncology, Department for Clinical Research, University of Bern, Inselspital, 3010 Bern, Switzerland.
Abstract:
Caspase-3 activity has been described to be essential for drug-induced apoptosis. Recent results suggest that in addition to its downstream executor function, caspase-3 is also involved in the processing of upstream caspase-8 and -9. To test the absolute requirement for caspase-3, we examined mitomycin C (MMC)-induced apoptosis in the caspase-3 deficient human breast cancer cell line MCF-7. MMC was used as anticancer drug since this agent was preferentially active compared to chemotherapeutic compounds with differing mechanisms of action such as cisplatin, docetaxel, or lovastatin. MMC treatment led to pronounced caspase-8, -9, and -7 processing and early morphological features of apoptosis within 48 h. This could be inhibited by the broad-spectrum caspase inhibitor z-VAD.fmk and to a lesser extent by z-IETD.fmk and z-LEHD.fmk, which have a certain preference for inhibiting caspase-8 and -9, respectively. MMC induced apoptosis in MCF-7 cells was not mediated by the death receptor pathway as demonstrated by experiments using the inhibiting anti-Fas antibody ZB4 and transfections with CrmA, a viral serpin inhibitor of caspase-8, and the dominant negative Fas-associated death domain (FADD-DN). Stable expression with Bcl-2 significantly prevented the processing of caspase-9 but also of caspase-8 and blocked the induction of apoptosis. Thus, we provide evidence that caspase-3 activity is dispensable for MMC-induced apoptosis and for caspase-8 and -9 processing in MCF-7 cells.
Insights
Caspase-3 is not essential for mitomycin C-induced apoptosis in breast cancer cells. This study shows caspase-3 deficiency does not prevent apoptosis or caspase-8 and -9 processing, challenging previous assumptions.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Caspase-3 is traditionally considered crucial for drug-induced apoptosis.
- Emerging evidence suggests caspase-3 may also process upstream caspases like caspase-8 and -9.
Purpose of the Study:
- To investigate the absolute requirement of caspase-3 for mitomycin C (MMC)-induced apoptosis.
- To determine if caspase-3 is necessary for the processing of caspase-8 and -9 during MMC-induced apoptosis in a caspase-3 deficient cell line.
Main Methods:
- Utilized the caspase-3 deficient human breast cancer cell line MCF-7.
- Treated cells with the anticancer drug mitomycin C (MMC).
- Assessed apoptosis induction, caspase processing (caspase-8, -9, -7), and utilized caspase inhibitors (z-VAD.fmk, z-IETD.fmk, z-LEHD.fmk), anti-Fas antibody (ZB4), CrmA, and FADD-DN transfections.
Main Results:
- MMC induced significant apoptosis and processing of caspase-8, -9, and -7 in MCF-7 cells.
- Apoptosis and caspase processing were inhibited by broad-spectrum and specific caspase inhibitors.
- The death receptor pathway was not involved in MMC-induced apoptosis.
- Stable Bcl-2 expression inhibited caspase processing and apoptosis.
Conclusions:
- Caspase-3 activity is dispensable for MMC-induced apoptosis in MCF-7 cells.
- Caspase-3 is not required for the processing of caspase-8 and -9 during MMC-induced apoptosis.
- These findings challenge the established role of caspase-3 as an essential executor in all drug-induced apoptotic pathways.