title: ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication.
abstract: ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy.
authors.affiliation: Department of Surgery, Shinshu University School of Medicine, Matsumoto, Japan.|Department of Molecular Oncology, Shinshu University Graduate School of Medicine, Matsumoto, Japan.
authors.forename: Masato
authors.identifier: http://orcid.org/0000-0001-7737-7592
authors.initials: M
authors.lastname: Kitazawa
authors.affiliation: Department of Molecular and Cellular Health Science, Nagoya City University Graduate School of Pharmaceutical Sciences, Mizuho-ku, Nagoya, Japan.
authors.forename: Shigeaki
authors.initials: S
authors.lastname: Hida
authors.affiliation: Department of Molecular Oncology, Shinshu University Graduate School of Medicine, Matsumoto, Japan.
authors.forename: Chifumi
authors.initials: C
authors.lastname: Fujii
authors.affiliation: Department of Molecular Oncology, Shinshu University Graduate School of Medicine, Matsumoto, Japan.
authors.forename: Shun'ichiro
authors.initials: S
authors.lastname: Taniguchi
authors.affiliation: Department of Molecular Oncology, Shinshu University Graduate School of Medicine, Matsumoto, Japan.
authors.forename: Kensuke
authors.initials: K
authors.lastname: Ito
authors.affiliation: Department of Molecular Oncology, Shinshu University Graduate School of Medicine, Matsumoto, Japan.
authors.forename: Tomio
authors.initials: T
authors.lastname: Matsumura
authors.affiliation: Department of Molecular Oncology, Shinshu University Graduate School of Medicine, Matsumoto, Japan.
authors.forename: Nagisa
authors.initials: N
authors.lastname: Okada
authors.affiliation: Department of Surgery, Shinshu University School of Medicine, Matsumoto, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sakaizawa
authors.affiliation: Department of Surgery, Shinshu University School of Medicine, Matsumoto, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kobayashi
authors.affiliation: Department of Surgery, Shinshu University School of Medicine, Matsumoto, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Takeoka
authors.affiliation: Department of Surgery, Shinshu University School of Medicine, Matsumoto, Japan.
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Miyagawa
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D053468:CARD Signaling Adaptor Proteins; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism*; D002450:Cell Communication / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival; D018031:Connexin 43 / Q000235:genetics; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D020741:rho GTP-Binding Proteins / Q000235:genetics
chemical_list: D053468:CARD Signaling Adaptor Proteins; D018031:Connexin 43; D003598:Cytoskeletal Proteins; C402202:PYCARD protein, human; D034741:RNA, Small Interfering; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D053453:Caspase 9; D020741:rho GTP-Binding Proteins