title: Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1-6 cells.
abstract: Staphylococcal enterotoxin C2 (SEC2), a member of bacterial superantigen, is one of the most potent known activators of T lymphocytes. With this property, SEC2 has already been used in clinic as a tumor immunotherapy agent in China. To increase the antitumor activity, a SEC2 mutant named ST-4 (GKVTG102-106WWH) with amino acid substitutions in T cell receptor (TCR)-binding domain was generated by site-directed mutagenesis, and the molecular mechanism of the enhanced antitumor activity was investigated. Results showed that ST-4 could activate much more Vβ 8.2 and 8.3 T cells and NK cells compared with SEC2, and exhibited significantly enhanced immunocyte stimulation and antitumor activity in vitro. The synthetic peptide sequencing the residues of mutant TCR-binding domain could competitively inhibit the immunocyte stimulation activity of ST-4. Most importantly, ST-4 up-regulated granzyme B and perforin at both mRNA and protein levels. We also found that expression of proapoptotic proteins cytochrome c, BAX and activation of caspase-3, 9 was up-regulated, and antiapoptotic protein Bcl-xL was down-regulated in the treatment with either ST-4 or SEC2. When granzyme B inhibitor or perforin inhibitor is presented, tumor cell viability was significantly rescued. Taken together, we demonstrate that increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. These activated cells released up-regulated granzyme B and perforin, which induced the enhanced tumor cells apoptosis by mitochondrial apoptotic pathway, and ultimately led to enhanced tumor cell growth inhibition. ST-4 may be a promising candidate for antitumor clinic usage in future.
authors.affiliation: Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning, China; University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Guojun
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning, China. Electronic address: mkxu@iae.ac.cn.
authors.forename: Mingkai
authors.initials: M
authors.lastname: Xu
authors.affiliation: Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning, China.
authors.forename: Huiwen
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning, China; University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Yubo
authors.initials: Y
authors.lastname: Song
authors.affiliation: Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.affiliation: Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning, China.
authors.forename: Chenggang
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000235:genetics / Q000633:toxicity*; D005260:Female; D053804:Granzymes / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D054353:Perforin / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D004768:Enterotoxins; D054353:Perforin; C034959:enterotoxin C, staphylococcal; D053804:Granzymes
keywords: Apoptosis; Cytotoxicity; Granzyme B; Perforin; Staphylococcal enterotoxins