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.

Expression and Clinical Significance of the Autophagy Proteins BECLIN 1 and LC3 in Ovarian Cancer

“…Given the role of mitochondria in the apoptotic response to chemotherapeutics [32], we suspect that in the latter case apoptosis ensues because of the exaggerated mitophagy. Thus, to improve the chance to cure ovarian carcinomas, one should carefully consider whether to employ autophagy inhibitors or autophagy-enhancer drugs in the chemotherapy cocktail depending on the ratio of BECLIN 1 and BCL-2 expression and the actual level of autophagy in the cancer cells.…”

miR-483-5p determines mitochondrial fission and cisplatin sensitivity in tongue squamous cell carcinoma by targeting FIS1

“…Recent studies have demonstrated that cisplatin treatment activates mitochondrial fission and induces apoptosis [18,19]. However, the underlying mechanism of this effect remains poorly understood.…”

The Role of Secondary Metabolites on Gynecologic Cancer Therapy: Some Pathways and Mechanisms

“…Piperlongumine also enhanced cisplatin-induced apoptosis via increased levels of Drp 1-dependent mitochondrial fission. 13,110,142,143 …”