Background Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that leads to cellular proliferation in cancer cells by stabilizing c-Myc protein. The effect of CIP2A in stabilizing c-Myc by inhibition of protein phosphatase 2A activity is a prerequisite step in tumor cell growth and in vivo tumor formation. We have previously shown that CIP2A is expressed in RA FLS and its expression is strongly associated with histopathological synovitis score and invasive function of RA FLS. However, the effect of CIP2A in association with c-Myc on the apoptosis of RA FLS is unknown.
Objectives This study was undertaken to investigate the effect of CIP2A and c-myc on the apoptosis of RA FLS and to determine the signaling pathway through which dysfunctional apoptosis is facilitated.
Methods Proliferation and apoptotic activity of RA FLS following treatment with CIP2A siRNA or control siRNA were analyzed using MTT assays and Cell Death Detection ELISA kit. c-Myc expression from RA FLS after treatment with CIP2A siRNA or control at 3, 6, 9 days interval was also determined using Western blot analysis. To evaluate which signal transduction pathways were engaged in apoptosis, caspase-3 activity and phosphorylation of the Akt kinase were also analyzed by Western blot.
Results Survival of RA FLS as measured by MTT assay was significantly lower in CIP2A siRNA -treated group compared to control after 7 days (n=5, each, p=0.0221). Apoptosis of RA FLS as measured by DNA fragmentation was significantly higher in CIP2A siRNA treated group compared to control when incubated for 3, 6 and 9 days (p=0.029, p=0.021, p=0.043, respectively). C-Myc expression measured by Western blot did not change during the incubation period. Apoptosis of CIP2A siRNA-treated RA FLS was induced by activation of caspase-3 and dephosphorylation of Akt kinase, but these findings were not observed in the control siRNA-treated RA FLS.
Conclusions Our data show that CIP2A expression in RA FLS is an important mediator of dysfunctional apoptosis that is independent of c-Myc stabilization. CIP2A may induce dysfunctional apoptosis of RA FLS by activation of Akt and deactivation of caspase-3. These results warrant further investigation of CIP2A as a potential pathogenetic factor and therapeutic target in RA.
Disclosure of Interest None Declared
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