Background Effective treatments for Osteoarthritis (OA) are not available. In aging-related diseases, including OA, failure of cellular homeostasis mechanisms, such as autophagy can cause extracellular matrix destruction and cell death (1). With aging, chondrocyte function is diminished, contributing to a cellular senescence phenotype often observed in OA chondrocytes. In addition, a defect in autophagy is observed in both aging and cartilage degeneration (2,3).
Objectives The objective of this study was to identify anti-senescence and pro-autophagy molecules by a cell-based high-throughput screening (HTS) in human chondrocytes.
Methods To induce cellular senescence or reduced autophagy, immortalized human chondrocytes (TC28a2) were seeded (2500 cells/well) in 384 well plates, and treated with IL-6 (20 ng/ml) for 72 or 18 hours, respectively. Then, chondrocytes were incubated with Prestwick Chemical Library (1120 approved drugs with chemical and pharmacological diversity, as well as bioavailability and safety in humans) at 10 μM for 72 hours. To identify anti-senescence hits, nuclei was stained with Hoechst 33342 (2,5μg/ml), while β–galactosidase subcellular structures was stained by using Imagene Green C12FDG substrate (30 μM). To evaluate autophagic flux, a reporter cell line was generated by retrovirus transfection of pBABE-mCherry-EGFP-LC3 plasmid in TC28a2 chondrocytes. Plates were imaged by using Operetta® High Content Screening (HCS) system in non-confocal mode using the 20x WD objective. For each well, 4 fields and 4 planes of bright field, Hoechst and fluorescein channels were obtained. Relative intensity of C12FDG in cytoplasm and number of autophagosomes/autolysosomes per area of cytoplasm were determined to quantitate β–galactosidase activity and autophagy flux respectively. Compound validation was performed in TC28a2 chondrocytes and in primary human chondrocytes by evaluating cell senescence, autophagy pathway and cell death by apoptosis.
Results A primary screening was performed to identify anti-senescence compounds by measurement of senescence-associated β-galactosidase activity. 283 compounds with anti-senescence effects were identified. The anti-senescence hits were analyzed by monitorizing autophagic flux. 29 compounds with both anti-senescence and pro-autophagy effects were selected. Then, one compound was selected for further validation. The compound reduced chondrocyte senescence, increased autophagy (p<0.0001) and protected against inflammation and cell death by apoptosis in human chondrocytes (p<0.05) in response to IL-6. Interestingly, this protective effect was partially mediated by mTOR inhibition, a proposed mechanism to prevent cartilage aging.
Conclusions Our screening methodology provides a unique opportunity to repurpose drugs and mechanisms to prevent cartilage aging. Autophagy activation and protection against senescence in chondrocytes may provide benefits for delaying cartilage degeneration.
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Acknowledgements This study was supported by Instituto de Salud Carlos III- Ministerio de Economía y Competitividad, Spain-PI14/01324 and Fondo Europeo de Desarrollo Regional (FEDER).
Disclosure of Interest None declared