World Class University Major in Biomodulation, Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Republic of Korea.
PLoS One. 2013 Sep 9;8(9):e74008. doi: 10.1371/journal.pone.0074008. eCollection 2013.
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.
线粒体裂变是一个将线粒体分裂成较小片段的过程，由 GTPase 相关蛋白 1（Drp1）调节。较高水平的线粒体裂变与癌细胞凋亡的诱导有关。然而，目前用于准确量化线粒体裂变以比较针对该过程的治疗方法的方法往往不明确或依赖于主观评估。线粒体也容易聚集，使得准确分析变得困难。在这里，我们描述了一种改进的线粒体碎片化定量方法，与目前存在的方法有几个不同之处。首先，细胞经历细胞离心，这降低了细胞的 z 轴高度并分散了单个线粒体，以便于观察。然后应用三种商业上可用的荧光分析工具来消除需要进一步检查的剩余线粒体簇的歧义。最后，应用截止评分，可根据个别细胞类型进行调整。该方法可用于高效和客观地评估治疗引起的线粒体碎片化。我们将该技术应用于一个涉及化疗敏感和化疗耐药卵巢癌细胞（OVCA）的实验问题。顺铂和植物化学物质胡椒碱都能诱导化疗敏感细胞的线粒体裂变和细胞凋亡，而只有胡椒碱能够在化疗耐药细胞中引发这些细胞反应。胡椒碱诱导的细胞凋亡似乎是由 Drp1 依赖性线粒体裂变介导的，因为 Drp1 抑制剂 mDivi-1 的存在减弱了细胞凋亡反应。我们的研究为更客观地量化线粒体裂变提供了基础，并进一步阐明了胡椒碱在治疗化疗耐药 OVCA 中的潜在作用机制。
