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Updated: May 7, 2026
Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis
Published on: November 18, 2019
An improved quantitative approach for the assessment of mitochondrial fragmentation in chemoresistant ovarian cancer
Lee Farrand1, Ji Young Kim, Akechai Im-Aram
1World Class University Major in Biomodulation, Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Republic of Korea.
Abstract:
Mitochondrial fission is a process that involves cleavage of mitochondria into smaller fragments and is regulated by the GTPase Dynamin-related protein 1 (Drp1). Higher levels of mitochondrial fission are associated with the induction of apoptosis in cancer cells. However, current methods to accurately quantify mitochondrial fission in order to compare therapeutics that target this process are often ambiguous or rely on subjective assessment. Mitochondria are also prone to aggregation, making accurate analysis difficult. Here we describe an improved approach for the quantification of mitochondrial fragmentation involving several differences from currently existing methods. Cells are first subjected to cytological centrifugation, which reduces cellular z-axis height and disperses individual mitochondria for easier observation. Three commercially available fluorescence analysis tools are then applied to disambiguate remaining mitochondrial clusters that require further inspection. Finally, cut-off scoring is applied, which can be tailored to individual cell type. The resultant approach allows for the efficient and objective assessment of mitochondrial fragmentation in response to treatment. We applied this technique to an experimental question involving chemosensitive and chemoresistant ovarian cancer (OVCA) cells. Cisplatin and the phytochemical piperlongumine were found to induce both mitochondrial fission and apoptosis in chemosensitive cells, while only piperlongumine was able to elicit these cellular responses in chemoresistant cells. Piperlongumine-induced apoptosis appeared to be mediated by Drp1-dependent mitochondrial fission since the apoptotic response was attenuated by the presence of the Drp1 inhibitor mDivi-1. Our study provides groundwork for a more objective approach to the quantification of mitochondrial fragmentation, and sheds further light on a potential mechanism of action for piperlongumine in the treatment of chemoresistant OVCA.
Insights
This study introduces a new objective method to quantify mitochondrial fission, a key process in cancer cell apoptosis. The method successfully identified piperlongumine as a potential therapeutic for chemoresistant ovarian cancer by inducing mitochondrial fission.
Area of Science:
- Cell Biology
- Cancer Research
- Biochemistry
Background:
- Mitochondrial fission, regulated by Dynamin-related protein 1 (Drp1), is linked to apoptosis in cancer cells.
- Current methods for quantifying mitochondrial fission are often subjective and ambiguous, hindering therapeutic development.
- Mitochondrial aggregation complicates accurate analysis.
Purpose of the Study:
- To develop an improved, objective approach for quantifying mitochondrial fragmentation.
- To assess the efficacy of novel therapeutics targeting mitochondrial fission in ovarian cancer.
- To elucidate the mechanism of action of piperlongumine in chemoresistant ovarian cancer.
Main Methods:
- Cells were subjected to cytological centrifugation to disperse mitochondria.
- Three fluorescence analysis tools were used to disambiguate mitochondrial clusters.
- Cut-off scoring, tailored to cell type, was applied for objective assessment.
- The technique was applied to chemosensitive and chemoresistant ovarian cancer cells treated with cisplatin and piperlongumine.
Main Results:
- The new method allows for efficient and objective assessment of mitochondrial fragmentation.
- Cisplatin and piperlongumine induced mitochondrial fission and apoptosis in chemosensitive ovarian cancer cells.
- Piperlongumine induced mitochondrial fission and apoptosis in chemoresistant ovarian cancer cells, unlike cisplatin.
- Piperlongumine's apoptotic effect in chemoresistant cells was mediated by Drp1-dependent mitochondrial fission, as shown by inhibition with mDivi-1.
Conclusions:
- The developed approach provides a more objective quantification of mitochondrial fragmentation.
- Piperlongumine demonstrates potential as a therapeutic agent for chemoresistant ovarian cancer by inducing Drp1-dependent mitochondrial fission and apoptosis.
- This study offers a mechanistic understanding of piperlongumine's action against resistant cancer cells.
