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Abstract
Background: Granulosa cell tumors (GCT) are a rare ovarian neoplasm but prognosis is poor following recurrence. Keratin intermediate filaments expressed in these tumors are a diagnostic marker, yet paradoxically, may also constitute a target for therapeutic intervention. In the current study, we evaluated keratin 8/18 (K8/18) filament expression as a mechanism of resistance to apoptosis in GCT, specifically focusing on regulation of the cell surface death receptor, Fas (FAS).
Methods: The GCT cell line, KGN, was transiently transfected with siRNA to KRT8 and KRT18 to reduce K8/18 filament expression. Expression of K8/18, FAS, and apoptotic proteins (PARP, cleaved PARP) were evaluated by fluorescence microscopy, flow cytometric analysis, and immunoblotting, respectively. The incidence of FAS-mediated apoptosis in KGN cells was measured by caspase 3/7 activity. All experiments were performed independently three to six times, using a fresh aliquot of KGN cells for each experiment. Quantitative data were analyzed by one- or two-way analysis of variance (ANOVA), followed by a Tukey’s post-test for multiple comparisons; differences among means were considered statistically significant at P < 0.05.
Results: Control cultures of KGN cells exhibited abundant K8/18 filament expression (~90 % of cells), and minimal expression of FAS (<25 % of cells). These cells were resistant to FAS-activating antibody (FasAb)-induced apoptosis, as determined by detection of cleaved PARP and measurement of caspase 3/7 activity. Conversely, siRNA-mediated knock-down of K8/18 filament expression enhanced FAS expression (> 70 % of cells) and facilitated FasAb-induced apoptosis, evident by increased caspase 3/7 activity (P < 0.05). Additional experiments revealed that inhibition of protein synthesis, but not MEK1/2 or PI3K signaling, also prompted FasAb-induced apoptosis.
Conclusions: The results demonstrated that K8/18 filaments provide resistance to apoptosis in GCT by impairing FAS expression. The abundance of keratin filaments in these cells and their role in apoptotic resistance provides a greater mechanistic understanding of ovarian tumorgenicity, specifically GCT, as well as a clinically-relevant target for potential therapeutic intervention.
Publication Date
2-24-2016
Journal Title
Journal of Ovarian Research
Publisher
BioMed Central (BMC)
Digital Object Identifier (DOI)
Scientific Contribution Number
2644
Document Type
Article
Rights
© Trisdale et al. 2016
Recommended Citation
Sarah K. Trisdale, Nicolette M. Schwab, Xiaoying Hou, John S. Davis and David H. Townson. Molecular manipulation of keratin 8/18 intermediate filaments: modulators of FAS-mediated death signaling in human ovarian granulosa tumor cells. J Ovarian Res. 2016; 9: 8. https://dx.doi.org/10.1186/s13048-016-0217-z
Comments
This is an article published by BioMed Central (BMC) in Journal of Ovarian Research in Z, available online: https://dx.doi.org/10.1186/s13048-016-0217-z