title: Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells.
abstract: Hyperglycemia, which occurs under the diabetic condition, is widely recognized as the causal link between diabetes and its serious complications. Diabetic neuropathies, which are among the most frequent complications of diabetes, affect sensory, motor, and autonomic nerves. The exact molecular mechanisms of high glucose-induced toxicity on neuronal cells, is still unclear. We previously reported that high glucose can induce apoptosis in PC12 cells, as evidenced by DNA fragmentation and high Bax/Bcl-2 ratio. The present study examined the involvement of caspase-3, the executioner, and two initiators of apoptosis, caspase-8 and caspase-9, during high glucose-induced apoptosis in PC12 cells, a neuronal cell line. Cells were exposed to high glucose with or without z-VAD-fmk, a pan-caspase inhibitor. Cell viability was measured by MTT assay. Caspase activity was determined spectrophotometrically using enzyme specific substrates. To correlate and confirm the caspase activity with changes in protein expression, procaspase-8, -9, and -3 were evaluated by Western blot analysis. The DNA-fragmentation was determined by DNA ladder using gel electrophoresis. The PC12 cell viability on high glucose exposure was decreased compared to controls, which was reversed by z-VAD-fmk. The activities of caspase-8, -9, and -3 were significantly increased in treated cells compared to controls. Moreover, high glucose exposure induced a significant decrease in protein levels of procaspases, indicating conversion of pro-form into the mature caspases. Finally, DNA fragmentation (Ladder) was shown in treated cells by high glucose. Based on the current data, it could be concluded that high glucose-induced apoptosis in PC12 cells is mediated, in part, by activation of caspase-8, -9, and -3 dependent pathways.
authors.affiliation: Department of Pharmacology and Cellular and Molecular Research Center, School of Medicine, Iran University of Medical Sciences, P.O. Box 14155-6183, Tehran, Iran. sharifal@yahoo.com
authors.forename: Ali M
authors.initials: AM
authors.lastname: Sharifi
authors.forename: Habib
authors.initials: H
authors.lastname: Eslami
authors.forename: Bagher
authors.initials: B
authors.lastname: Larijani
authors.forename: Jamshid
authors.initials: J
authors.lastname: Davoodi
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000008:administration & dosage; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism*; D061945:Caspase Inhibitors; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D053938:DNA Fragmentation; D004587:Electrophoresis, Agar Gel; D005947:Glucose / Q000633:toxicity*; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D018696:Neuroprotective Agents / Q000008:administration & dosage; D016716:PC12 Cells; D051381:Rats
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D018696:Neuroprotective Agents; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D005947:Glucose