title: [Effects of PI3K/AKT inhibitor wortmannin on proliferation and apoptosis of primary porcine preadipocytes].
abstract: The purpose of this study was to determine the proper concentration of wortmannin that effectively inhibits PI3K/AKT but does not affect the proliferation and apoptosis of primary porcine preadipocytes. Firstly, primary porcine preadipocytes were isolated and their abilities to be induced to differentiation into mature adipocytes were evaluated. The preadipocytes were then treated with different concentrations of wortmannin, and the proliferation of the cells was detected with methanethiosulfonate (MTS). Annexin V- FITC/PI double-staining was used to detect the level of cell apoptosis. The apoptosis-related gene expressions were also quantified by qRT-PCR. At the same time, single cell electrophoresis was used to examine the extent of cellular DNA damage. Our data demonstrated that the primary porcine preadipocytes could differ-entiate into mature adipocytes. Up to 200 nmol/L of wortmannin had no effect on the proliferation ability of primary porcine preadipocytes (P>0.05). Results from the flow cytometry Annexin V- FITC/PI double-staining showed that 200 nmol/L wortmannin significantly induced apoptosis of the primary porcine preadipocytes (P<0.05). QRT-PCR results also showed that the expressions of caspase8, TNFR1, GZMB, and Bcl-x1 were significantly upregulated, while the expression of GZMA and cFLIP were not significantly affected when treated with 200 nmol/L wortmannin. In addition, results from the single cell gel electrophoresis indicated that 100 nmol/L wortmannin did not induce DNA damage. In conclusion, our results col-lectively showed that 100 nmol/L wortmanin can be used to study the role of PI3k pathway on the preadipocytes differen-tion without affecting the cell proliferation and apoptosis.
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510006 China. yunxueguo@gmail.com
authors.forename: Yun-Xue
authors.initials: YX
authors.lastname: Guo
authors.forename: De-Lin
authors.initials: DL
authors.lastname: Mo
authors.forename: Yao-Sheng
authors.initials: YS
authors.lastname: Chen
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Shu-Qi
authors.initials: SQ
authors.lastname: Xiao
authors.forename: Xiao-Hong
authors.initials: XH
authors.lastname: Liu
mesh_terms: D017667:Adipocytes / Q000187:drug effects* / Q000502:physiology; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors*; D013234:Stem Cells / Q000187:drug effects* / Q000502:physiology; D013552:Swine; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin