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Thapsigargin enhances camptothecin-induced apoptosis in cardiomyocytes
1University of British Columbia, Vancouver, Canada.
Abstract:
Topoisomerase I inhibitors are promising new chemotherapeutic agents for the treatment of certain malignancies. The present study investigated the impact of the topoisomerase I inhibitor camptothecin on cell death in cardiomyocytes and sought to determine whether the sesquiterpene gamma-lactone--thapsigargin, that alter sarcoplasmic reticulum calcium flux, modulates the effect of camptothecin on the cardiomyocyte. Camptothecin-induced cell death was demonstrated in cardiomyocytes maintained in culture, from 7 day old embryonic chick hearts, by the trypan blue and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, two independent indicators of the loss of cell viability. The type of cell death was attributed to apoptosis based on cell structure, DNA fragmentation and flow cytometry studies. Camptothecin-treated cardiomyocytes were shrunken with membrane blebs and nuclear fragmentation. Camptothecin produced a dose-dependent increase in DNA fragments of 180 base pairs, or multiples thereof, which are characteristic of apoptosis. A two-fold increase in this type of DNA fragmentation was produced by camptothecin (10 microM) compared to control (diluent-treated) cells. Flow cytometry analysis of populations of 10,000 cardiomyocytes stained with propidium iodide demonstrated a significant increase in the proportion of the population with alterations of DNA content consistent with apoptosis. Pretreatment of cells with thapsigargin, which selectively inhibits sarcoplasmic reticulum and endoplasmic reticulum Ca+2-dependent ATPase, significantly augmented camptothecin-induced apoptosis. Exploring further the role of calcium in camptothecin-induced cell death, we found that the Ca+2 chelator EGTA decreased camptothecin-induced DNA fragmentation. These data indicate the potential for cardiotoxicity from camptothecin through the process of apoptosis and suggest that agents which affect cellular calcium regulation enhance camptothecin-induced apoptosis.
Insights
Camptothecin, a topoisomerase I inhibitor, induces apoptosis in cardiomyocytes. Thapsigargin enhances this cell death, suggesting calcium regulation influences camptothecin cardiotoxicity.
Area of Science:
- Cardiology
- Molecular Biology
- Pharmacology
Background:
- Topoisomerase I inhibitors are emerging chemotherapeutics for malignancies.
- Cardiomyocytes are susceptible to drug-induced cell death.
- Calcium flux plays a role in cellular homeostasis and apoptosis.
Purpose of the Study:
- Investigate camptothecin's effect on cardiomyocyte cell death.
- Determine if thapsigargin modulates camptothecin-induced apoptosis.
- Elucidate the role of calcium in camptothecin's cardiotoxicity.
Main Methods:
- Cardiomyocytes cultured from embryonic chick hearts.
- Cell viability assessed by trypan blue and MTT assays.
- Apoptosis confirmed by DNA fragmentation analysis and flow cytometry.
Main Results:
- Camptothecin induced dose-dependent apoptosis in cardiomyocytes.
- Thapsigargin pretreatment significantly augmented camptothecin-induced apoptosis.
- EGTA, a calcium chelator, reduced camptothecin-induced DNA fragmentation.
Conclusions:
- Camptothecin exhibits potential cardiotoxicity via apoptosis.
- Agents affecting calcium regulation can enhance camptothecin-induced apoptosis.
- Understanding calcium's role is crucial for managing camptothecin therapy.