title: Apoptosis of NG108-15 cells induced by buprenorphine hydrochloride occurs via the caspase-3 pathway.
abstract: Apoptosis of NG108-15 neuroblastoma x glioma hybrid cells (NG108-15 cells) is induced by a morphine alkaloid derivative, buprenorphine hydrochloride (Bph). In a previous report, we used various apoptosis inhibitors to identify the "death pathway," and found that caspase inhibitors Ac-YVAD-CHO (Ac-Tyr-Val-Ala-Asp-CHO) and Ac-DEVD-CHO (Ac-Asp-Glu-Val-Asp-CHO) did not inhibit this particular apoptosis. Here, we tested Z-VAD-FMK (Z-Val-Ala-Asp[OMe]-CH2F) and Z-DEVD-FMK (Z-Asp[OMe]-Glu-[OMe]Val-Asp[OMe]-CH2F) for their ability to inhibit Bph-induced NG108-15 apoptosis. These tri- or tetra-peptide caspase inhibitors have a fluoromethyl ketone in their C-terminus instead of an aldehyde, and thus are more permeable than Ac-YVAD-CHO and AC-DEVD-CHO. Our observations of DNA ladder formation, cell morphology changes, and caspase-3 activities all indicated that these cell membrane-permeable caspase inhibitors completely inhibited the apoptosis, providing strong evidence that this apoptosis occurs through the caspase cascade "death pathway." Our previous report also showed that pretreatment of NG108-15 cells with TPCK (N-tosyl-L-phenylalanyl chloromethyl ketone) prevented DNA fragmentation and decreased the cell viability in Bph-induced apoptosis. The comparison of caspase-3 activities in Bph-induced samples with or without TPCK pretreatment revealed that caspase-3 was activated in both samples. Taken together, these results indicate that the Bph-induced apoptosis of NG108-15 cells occurs via the conventional caspase-dependent death pathway and that TPCK pretreatment results in a DNA ladder-deficient apoptosis.
authors.affiliation: Department of Biological Pharmaceutical Sciences, College of Pharmacy, Nihon University, Funabashi, Chiba, Japan. jfkugawa@pha.nihon-u.ac.jp
authors.forename: F
authors.initials: F
authors.lastname: Kugawa
authors.forename: A
authors.initials: A
authors.lastname: Ueno
authors.forename: M
authors.initials: M
authors.lastname: Aoki
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002047:Buprenorphine / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D006801:Humans; D006822:Hybrid Cells; D011494:Protein Kinases / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D002047:Buprenorphine; D011494:Protein Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases