Tacrolimus reduces cell viability in rheumatoid arthritis fibroblast like synoviocytes through inhibition of autophagic flux

Background We aimed to investigate the effect of tacrolimus on human fibroblast-like synoviocyte-rheumatoid arthritis (HFLS-RA) cells and determine whether it induces apoptosis. Methods Cell viability and cell cycle progression were analyzed using the CCK-8 assay and flow cytometry, respectively, after treating HFLS-RA cells with tacrolimus. Western blot analysis was performed by pretreating the cells with the autophagy inhibitors 3-Methyladenine (3-MA) and Bafilomycin A1 (BafA1) to determine the effect on the expression of LC-3 and p62. To analyze the effect of tacrolimus on lysosomal function of tacrolimus, flow cytometry analysis was performed using LysoTracker, and confocal microscopy was conducted via double-staining for lysosome-associated membrane protein 1 (LAMP-1) and LC-3. Results Treatment of HFLS-RA cells with tacrolimus at concentrations of 0–80 µM resulted in a concentration-dependent decrease in cell viability to 20%, 60%, and 93%, respectively, and in a time-dependent manner to 23%, 53%, and 65%, respectively, at 8–24 h. Regarding cell cycle progression, the number of cells in the sub-G1 stage increased in a concentration-dependent manner (control: 6.05%, 40 µM: 11.5%, 60 µM: 33.45%, and 80 µM: 61.5%). Increased LC-3 II protein expression and decreased p62 protein expression were offset when cells were treated with the autophagy inhibitor 3-MA, but not when pretreated with BafA1. Confocal microscopy revealed an increase in the formation of LAMP-1 puncta in HFLS-RA treated with tacrolimus; however, LC-3 was localized within the nucleus, and Lamp-1 and LC-3 did not co-localize. Conclusion Our findings revealed that tacrolimus induces lysosomal accumulation in cells through autophagy, indicating potential lysosomal dysfunction and subsequent apoptosis.