title: The role of cytochrome c on apoptosis induced by Anagrapha falcifera multiple nuclear polyhedrosis virus in insect Spodoptera litura cells.
abstract: There are conflicting reports on the role of cytochrome c during insect apoptosis. Our previous studies have showed that cytochrome c released from the mitochondria was an early event by western blot analysis and caspase-3 activation was closely related to cytochrome c release during apoptosis induced by baculovirus in Spodoptera litura cells (Sl-1 cell line). In the present study, alteration in mitochondrial morphology was observed by transmission electron microscopy, and cytochrome c release from mitochondria in apoptotic Sl-1 cells induced with Anagrapha falcifera multiple nuclear polyhedrosis virus (AfMNPV) has further been confirmed by immunofluoresence staining protocol, suggesting that structural disruption of mitochondria and the release of cytochrome c are important events during Lepidoptera insect cell apoptosis. We also used Sl-1 cell-free extract system and the technique of RNA interference to further investigate the role of cytochrome c in apoptotic Sl-1 cells induced by AfMNPV. Caspase-3 activity in cell-free extracts supplemented with exogenous cytochrome c was determined and showed an increase with the extension of incubation time. DsRNA-mediated silencing of cytochrome c resulted in the inhibition of apoptosis and protected the cells from AfMNPV-induced cell death. Silencing of expression of cytochrome c had a remarkable effect on pro-caspase-3 and pro-caspase-9 activation and resulted in the reduction of caspase-3 and caspase-9 activity in Sl-1 cells undergoing apoptosis. Caspase-9 inhibitor could inhibit activation of pro-caspase-3, and the inhibition of the function of Apaf-1 with FSBA blocked apoptosis, hinting that Apaf-1 could be involved in Sl-1 cell apoptosis induced by AfMNPV. Taken together, these results strongly demonstrate that cytochrome c plays an important role in apoptotic signaling pathways in Lepidopteran insect cells.
authors.affiliation: Institute of Entomology, Central China Normal University, Wuhan, People's Republic of China.
authors.forename: Kaiyu
authors.initials: K
authors.lastname: Liu
authors.forename: Duanyang
authors.initials: D
authors.lastname: Shu
authors.forename: Na
authors.initials: N
authors.lastname: Song
authors.forename: Zhongchao
authors.initials: Z
authors.lastname: Gai
authors.forename: Yuan
authors.initials: Y
authors.lastname: Yuan
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Shuying
authors.initials: S
authors.lastname: Guo
authors.forename: Jianxin
authors.initials: J
authors.lastname: Peng
authors.forename: Huazhu
authors.initials: H
authors.lastname: Hong
mesh_terms: D000818:Animals; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1 / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002474:Cell-Free System; D045304:Cytochromes c / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D020868:Gene Silencing; D007313:Insecta; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000378:metabolism*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism
chemical_list: D053477:Apoptotic Protease-Activating Factor 1; D012330:RNA, Double-Stranded; D003609:Dactinomycin; D045304:Cytochromes c; D053148:Caspase 3; D053453:Caspase 9