Enhanced cystatin C and lysosomal protease expression following 6-hydroxydopamine exposure.
6-Hydroxydopamine (6-OHDA) is a selective neurotoxin used to induce apoptosis in catecholamine-containing neurons. Although biochemical products and reactive oxygen species (ROS) of 6-OHDA have been well documented, the activation of cellular pathways following exposure are not well understood. Apoptosis in PC12 (Pheochromocytoma) cells was induced by 6-OHDA in a dose (10-150 microM) and time-dependent (24-72 h) manner compared to experimental controls (no treatment). PC 12 cells exposed to 50 microM 6-OHDA demonstrated the involvement of caspase 3 and lysosomal protease alterations. Following 6-OHDA exposure, the caspase 3-like inhibitor Ac-DEVD-CHO significantly decreased 6-OHDA induced cell death. In addition, alterations in expression of the lysosomal cysteine and aspartic proteases, cathepsin B (CB) and cathepsin D (CD) and the endogenous cysteine protease inhibitor cystatin C were observed utilizing immunocytochemical analysis at 24, 48, and 72 h following 6-OHDA exposure. Furthermore, CB and CD and cystatin C immuno-like reactivity was more pronounced in TUNEL positive cells. Moreover, Western blot analysis confirmed a significant increase in protein expression for CB and CD at 72 h and a temporal and concentration dependent increase in cystatin C in response to 6-OHDA. Cells treated with pepstatin A, an inhibitor for CD, showed a significant decrease in cell death, however, CA-074ME, a specific inhibitor for CB, failed to protect cells from 6-OHDA induced cell death. Thus, these results suggest that apoptosis induced by 6-OHDA exposure is mediated in part through caspase 3 activation and lysosomal protease CD.
Lee
Daniel C
DC
College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA.
Close
Fran T
FT
Goodman
Carl B
CB
Jackson
Inneke M
IM
Wight-Mason
Ceceile
C
Wells
Lateesha M
LM
Womble
Tracy A
TA
Palm
Donald E
DE
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002401:Cathepsin B / Q000096:biosynthesis; D002402:Cathepsin D / Q000096:biosynthesis; D016923:Cell Death / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D055316:Cystatin C; D015891:Cystatins / Q000096:biosynthesis*; D045304:Cytochromes c / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology*; D018384:Oxidative Stress / Q000187:drug effects; D016627:Oxidopamine / Q000633:toxicity*; D016716:PC12 Cells; D010447:Peptide Hydrolases / Q000096:biosynthesis*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051381:Rats; D013268:Stimulation, Chemical; D013565:Sympatholytics / Q000633:toxicity*
C525030:CST3 protein, human; C525032:Cst3 protein, rat; D055316:Cystatin C; D015891:Cystatins; D013565:Sympatholytics; D016627:Oxidopamine; D045304:Cytochromes c; D004247:DNA; D007770:L-Lactate Dehydrogenase; D011065:Poly(ADP-ribose) Polymerases; D010447:Peptide Hydrolases; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D002401:Cathepsin B; D002402:Cathepsin D