title: An improved quantitative approach for the assessment of mitochondrial fragmentation in chemoresistant ovarian cancer cells.
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.
authors.affiliation: World Class University Major in Biomodulation, Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Republic of Korea.
authors.forename: Lee
authors.initials: L
authors.lastname: Farrand
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Kim
authors.forename: Akechai
authors.initials: A
authors.lastname: Im-Aram
authors.forename: Jeong-Yong
authors.initials: JY
authors.lastname: Suh
authors.forename: Hyong Joo
authors.initials: HJ
authors.lastname: Lee
authors.forename: Benjamin K
authors.initials: BK
authors.lastname: Tsang
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D004148:Dioxolanes / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D034281:Dynamins; D005260:Female; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D063154:Mitochondrial Dynamics / Q000187:drug effects; D024101:Mitochondrial Proteins / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000378:metabolism*; D010881:Piperidones
chemical_list: D000970:Antineoplastic Agents; D002945:Cisplatin; D004148:Dioxolanes; D034281:Dynamins; D020558:GTP Phosphohydrolases; D008869:Microtubule-Associated Proteins; D024101:Mitochondrial Proteins; C110768:DNM1L protein, human; C498077:piperlongumine; D010881:Piperidones
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