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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.

SUBMITTER: Farrand L

PROVIDER: S-EPMC3767598 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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An improved quantitative approach for the assessment of mitochondrial fragmentation in chemoresistant ovarian cancer cells.

Farrand Lee L Kim Ji Young JY Im-Aram Akechai A Suh Jeong-Yong JY Lee Hyong Joo HJ Tsang Benjamin K BK

PloS one 20130909 9

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 ac ...[more]

PMID: 24040144

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Project description:Ovarian cancer ranks eighth in cancer incidence and mortality among women worldwide. Cisplatin-based chemotherapy is commonly used for patients with ovarian cancer. However, the clinical efficacy of cisplatin is limited due to the occurrence of adverse side effects and development of cancer chemoresistance during treatment. Trans-(±)-kusunokinin has been previously reported to inhibit cell proliferation and induce cell apoptosis in various cancer cell types, including breast, colon and cholangiocarcinoma. However, the potential effects of (±)-kusunokinin on ovarian cancer remains unknown. In the present study, chemosensitive ovarian cancer cell line A2780 and chemoresistant ovarian cancer cell lines A2780cis, SKOV-3 and OVCAR-3 were treated with trans-(±)-kusunokinin to investigate its potential effects. MTT, colony formation, apoptosis and multi-caspase assays were used to determine cytotoxicity, the ability of single cells to form colonies, induction of apoptosis and multi-caspase activity, respectively. Moreover, western blot analysis was performed to determine the proteins level of topoisomerase II, cyclin D1, CDK1, Bax and p53-upregulated modulator of apoptosis (PUMA). The results demonstrated that trans-(±)-kusunokinin exhibited the strongest cytotoxicity against A2780cis cells with an IC50 value of 3.4 µM whilst also reducing the colony formation of A2780 and A2780cis cells. Trans-(±)-kusunokinin also induced the cells to undergo apoptosis and increased multi-caspase activity in A2780 and A2780cis cells. This compound significantly downregulated topoisomerase II, cyclin D1 and CDK1 expression, but upregulated Bax and PUMA expression in both A2780 and A2780cis cells. In conclusion, trans-(±)-kusunokinin suppressed ovarian cancer cells through the inhibition of colony formation, cell proliferation and the induction of apoptosis. This pure compound could be a potential targeted therapy for ovarian cancer treatment in the future. However, studies in an animal model and clinical trial need to be performed to support the efficacy and safety of this new treatment.

Project description:BACKGROUND:How exosomal microRNAs (miRNAs) derived from macrophages contribute to the development of drug resistance in the context of the hypoxic tumor microenvironment in epithelial ovarian cancer (EOC) remains poorly understood. METHODS:The miRNA levels were detected by qRT-PCR. Protein levels of HIF-1α, CD163 and PTEN-PI3K/AKT pathway were assessed by Western blot (WB) and Immunohistochemistry (IHC). Exosomes were isolated, and then confirmed by Transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and WB. Internalization of macrophages-secreted exosomes in EOC cells was detected by Confocal microscope. Subsequently, Dual-luciferase reporter assay verified PTEN was the target of miR-223. Gain- and loss-of-function experiments, rescue experiments, and SKOV3 xenograft models were performed to uncover the underlying mechanisms of miR-223 and PTEN-PI3K/AKT pathway, as well as the exosomal miR-223 in inducing multidrug resistance in vitro and in vivo. RESULTS:Here, we showed hypoxic EOC cells triggered macrophages recruitment and induced macrophages into a tumor-associated macrophage (TAM)-like phenotype; exosomes derived from hypoxic macrophages enhanced the malignant phenotype of EOC cells, miR-223 was enriched in exosomes released from macrophages under hypoxia, which could be transferred to the co-cultivated EOC cells, accompanied by enhanced drug resistant of EOC cells. Besides, results from a functional assay revealed that exosomal miR-223 derived from macrophages promoted the drug resistance of EOC cells via the PTEN-PI3K/AKT pathway both in vivo and in vitro. Furthermore, patients with high HIF-1a expression had statistically higher CD163+ cell infiltration and intertumoral levels of miR-223. Finally, circulating exosomal miR-223 levels were closely related to the recurrence of EOC. CONCLUSIONS:These data indicate a unique role of exosomal miR-223 in the cross-talk between macrophages and EOC cells in chemotherapy resistance, through a novel exosomal miR-223/PTEN-PI3K/AKT signaling pathway.

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An improved quantitative approach for the assessment of mitochondrial fragmentation in chemoresistant ovarian cancer cells.

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An improved quantitative approach for the assessment of mitochondrial fragmentation in chemoresistant ovarian cancer cells.

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