Autophagy Contributes to Celecoxib-Induced Cell Death in Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Abstract

Aggressive proliferation of the synovium is the hallmark of rheumatoid arthritis (RA). Recently, it has been proposed that autophagy regulates the apoptosis resistance and hyperplasia of fibroblast-like synoviocytes (FLS) in RA. This study was done to investigate the influence of celecoxib on the viability of RA-FLS and to reveal how celecoxib affects the viability of RA-FLS. RA synovial tissue was obtained from patients during total knee replacement surgery. Cell viability was measured by MTS assay. The expression of autophagy-related protein was analyzed by western blot, and apoptosis was measured by a caspase activity assay. Celecoxib dose-dependently decreased the cell viability (IC50 of 120 μM) of RA-FLS. Combination treatment of the caspase inhibitor and celecoxib increased cell viability considerably, compared to that observed with celecoxib treatment alone. Celecoxib also increased the expression of LC3, Atg5 and Beclin1 in RA-FLS. Inhibition of autophagy by 3-methyladenine, knockdown of Atg5 or Beclin1 were restored celecoxib-decreased cell viability in RA-FLS. Furthermore, celecoxib-induced autophagic cell death occurred via formation of reactive oxygen species in RA-FLS. Taken together, this study demonstrates that both apoptosis and autophagy contribute to celecoxib-induced cell death in RA-FLS.

류마티스관절염은 활막의 비정상적 증식이 특징이다. 최근, 류마티스관절염 활막세포에서 자가포식은 세포의 증식과 사멸의 저항성을 조절한다고 알려진 바 있다. 본 연구에서는 류마티스관절염 활막세포에서 셀레콕십이 세포사을 유도함에 있어 어떠한 영향을 미치는지에 대하여 알아보고자 하였다. 세포사는 MTS assay와 세포수를 측정하였다. 자가포식 현상의 발현은 western blot으로, 세포사멸 활성은 caspase 활성 assay를 이용하여 측정하였다. 류마티스관절염 활막세포에서 셀레콕십은 농도 의존적으로 세포생존을 감소시켰으며, IC50값은 120 μM로 확인하였다. Caspase 억제제는 셀레콕십으로 유도된 세포 사멸을 억제시켰다. 셀레콕십은 류마티스관절염 활막세포에서 LC3, Atg5, Beclin1의 발현을 증가시켰다. 자가포식억제제인 3-methyladenine 처리와 Atg5와 Beclin1 유전자의 siRNA처리에 의한 자가포식의 억제는 셀레콕십으로 감소된 세포생존을 증가시켰다. 또한 셀레콕십은 활성산소을 생성하여 자가포식을 통한 세포사를 유도하였다. 결론적으로, 류마티스관절염 활막세포에서 셀레콕십이 유도하는 세포사는 세포사멸과 자가포식이 모두 관여하는 것으로 유추된다.