Ecstasy‐induced Apoptosis and Lysosomal Protease Alterations in Differentiated PC12 Cells

Abstract

Ecstasy (MDMA), has been shown to cause oxidative stress-induced apoptosis. MDMA induces apoptosis via the caspase. However, the role of cystatin C (CC), cathepsin B (CB) and cathepsin D (CD) has not been investigated. The objective of this study is to characterize alterations in CB, CD and CC following MDMA exposure. Cell viability with differentiated PC12 cells exposed to MDMA. Immunocytochemistry, western blotting analysis and TUNEL staining were performed to confirm apoptosis and the presence of CC, CB and CD. MDMA reduced cell viability in a dose and time dependent manner. Positive TUNEL staining confirmed apoptosis and immunocytochemical analysis demonstrated the presence of CC-immuno-like reactivity (IR), CB-IR, and CD-IR in PC12 cells treated with MDMA. Additionally, enzyme analysis indicated alterations in CC, CB and CD activity and expression, respectively, verses the untreated control. Following 3.5mM MDMA exposure at 24 and 48 h, CB activity decreased significantly (64.79%, p<0.001) and (53.13% p<0.001)respectively compared to untreated control. Papain was used to access CC activity. Papain activity decreased significantly at 24 and 48 h (18.33%, p<0.001) and 34.35%, p<0.001) respectively with 3.5mM MDMA, compared to untreated control. Alterations in these proteases may suggest involvement of CC, CB and CD in an MDMA-induced apoptotic pathway. Research supported by NIH/NIGMS/MBRS S06GM08111 and RCMI/NIH RR03020