Related Experiment Videos
Flow cytometric measurement of mitochondrial mass and function: a novel method for assessing chemoresistance
M Mancini1, M Sedghinasab, K Knowlton
1Department of Surgery, University of Washington, Seattle 98195, USA.
Background:
Chemotherapeutic agents induce apoptosis in cancer cells. Drugs failing to induce apoptosis are likely to have decreased clinical efficacy. We hypothesize that (1) chemotherapeutic agents induce mitochondrial changes and apoptosis through mechanisms associated with reactive oxidant species production; (2) the anti-apoptotic protein Bcl-2 prevents drug-induced mitochondrial changes, reactive oxygen species (ROS) production, and apoptosis; and (3) the assay of drug-induced mitochondrial changes can reflect drug-specific chemoresistance in a given cancer cell line.
Methods:
A stable Bcl-2 transfectant of the Bcl-2 negative breast cancer cell line SKBr3 was created (SKBr3/Bcl2-2). Both SKBr3 and SKBr3/Bcl2-2 cells were treated with Herbimycin A (300 ng/mL) or vehicle (1% DMSO). Cell cycle changes were assessed by BRDU staining. Apoptosis was determined by electron microscopy, TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining, and diphenylamine assay of DNA fragmentation. Changes in mitochondrial mass and transmembrane potential (deltapsi(m)) were assessed by flow cytometric assessment of JC-1 fluorescence. Reactive oxygen species production was measured by 2',7'-dichlorodihydrofluorescein diacetate (DCFH) fluorescence.
Results:
Both SKBr3 and SKBr3/Bcl2-2 cells show cell cycle arrest after Herbimycin treatment. However, SKBr3 cells, but not SKBr3/Bcl2-2 cells, undergo apoptosis. Herbimycin-treated SKBr3 cells show increased mitochondrial mass (JC-1 green fluorescence), with no corresponding increase in deltapsi(m) (JC-1 red fluorescence). By contrast, Herbimycin-treated SKBr3/Bcl2-2 cells show no change in mitochondrial mass or deltapsi(m). Similarly, drug-treated SKBr3 cells, but not SKBr3/Bcl2-2 cells, demonstrate increased reactive oxygen species (ROS) production concomitant with the development of apoptosis.
Conclusion:
SKBr3 cells undergoing apoptosis demonstrate mitochondrial changes associated with ROS production. Bcl-2 transfection prevents these changes because it prevents apoptosis and induces chemoresistance to Herbimycin in SKBr3. Flow cytometric measurement of drug induced mitochondrial changes and ROS production may facilitate in vitro assessment of chemosensitivity or chemoresistance in breast cancer.
Insights
Chemotherapy induces cancer cell apoptosis via mitochondrial changes and reactive oxygen species (ROS) production. The anti-apoptotic protein Bcl-2 blocks these effects, indicating chemoresistance and suggesting mitochondrial assays can predict treatment response.
Area of Science:
- Cell Biology
- Molecular Oncology
- Biochemistry
Background:
- Chemotherapeutic agents are designed to induce apoptosis (programmed cell death) in cancer cells.
- Failure of drugs to induce apoptosis correlates with diminished clinical efficacy.
- The anti-apoptotic protein Bcl-2 is a key regulator of cell death pathways.
Purpose of the Study:
- To investigate if chemotherapeutic agents induce mitochondrial alterations and apoptosis through reactive oxygen species (ROS) production.
- To determine if the anti-apoptotic protein Bcl-2 can inhibit drug-induced mitochondrial changes, ROS production, and apoptosis.
- To assess if measuring drug-induced mitochondrial changes can predict chemoresistance in cancer cells.
Main Methods:
- Utilized a breast cancer cell line (SKBr3) and its stable Bcl-2 transfectant (SKBr3/Bcl2-2).
- Treated cells with Herbimycin A and assessed cell cycle, apoptosis (via electron microscopy, TUNEL, DNA fragmentation), mitochondrial mass and potential (JC-1 fluorescence), and ROS production (DCFH fluorescence).
Main Results:
- Herbimycin A induced cell cycle arrest and apoptosis in SKBr3 cells, accompanied by increased mitochondrial mass and ROS production.
- SKBr3/Bcl2-2 cells, expressing Bcl-2, showed cell cycle arrest but not apoptosis, with no significant changes in mitochondrial parameters or ROS levels.
- Bcl-2 expression prevented Herbimycin A-induced mitochondrial alterations and ROS generation, correlating with resistance to apoptosis.
Conclusions:
- Apoptosis in SKBr3 cells involves mitochondrial changes linked to ROS production.
- Bcl-2 overexpression confers chemoresistance to Herbimycin A by preventing these drug-induced mitochondrial and ROS events.
- Flow cytometry assays measuring mitochondrial changes and ROS production show potential for predicting breast cancer chemosensitivity or chemoresistance in vitro.