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 de-tected 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 differentiate 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 upregu-lated, 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 collectively showed that 100 nmol/L wortmanin can be used to study the role of PI3k pathway on the preadipocytes differention without affecting the cell proliferation and apoptosis.
GUO Yun-Xue, MO De-Lin, CHEN Yao-Sheng, ZHANG Yue, ZHANG Yun, XIAO Shu-Qi, LIU Xiao-Hong
. Effects of PI3K/AKT inhibitor wortmannin on proliferation and apoptosis of primary porcine preadipocytes[J]. Hereditas(Beijing), 2012
, 34(10)
: 1282
-1290
.
DOI: 10.3724/SP.J.1005.2012.01282
[1] Wolin KY, Carson K, Colditz GA. Obesity and cancer. Oncologist, 2010, 15(6): 556- 565.
[2] Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism. Nature, 2001, 414(6865): 799-806.
[3] Sakaue H, Ogawa W, Matsumoto M, Kuroda S, Takata M, Sugimoto T, Spiegelman BM, Kasuga M. Posttranscriptional control of adipocyte differentiation through activation of phosphoinositide 3 -kinase. J Biol Chem, 1998, 273 (44): 28945-28952.
[4] Cross MJ, Stewart A, Hodgkin MN, Kerr DJ, Wakelam MJO. Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. J Biol Chem, 1995, 270(43): 25352- 25355.
[5] Tomiyama K, Nakata H, Sasa H, Arimura S, Nishio E, Watanabe Y. Wortmannin, a specific phosphatidylinositol 3-kinase inhibitor, inhibits adipocytic differentiation of 3T3-L1 cells. Biochem Biophys Res Commun, 1995, 212(1): 263-269.
[6] Xia X, Serrero G. Inhibition of adipose differentiation by phosphatidylinositol 3-kinase inhibitors. J Cell Physiol, 1999, 178(1): 9-16.
[7] Aubin D, Gagnon A, Sorisky A. Phosphoinositide 3-kinase is required for human adipocyte differentiation in culture. Int J Obesity, 2005, 29(8): 1006-1009.
[8] Cantley LC. The phosphoinositide 3-kinase pathway. Science, 2002, 296(5573): 1655-1657.
[9] 王晓南, 吴青, 张连生, 吴一品, 舒砚文. PI3K/Akt抑制剂渥曼青霉素对白血病细胞增殖和凋亡的影响. 中国肿瘤生物治疗杂志, 2010, 17(1): 82-87.
[10] 石丹, 范德生, 甄蕾, 张红. PI3K/Akt信号通路抑制剂在舌鳞癌细胞系增殖和凋亡中的作用. 口腔颌面 外科杂志, 2010, 20(5): 324-326.
[11] Ihle NT, Lemos R Jr, Wipf P, Yacoub A, Mitchell C, Siwak D, Mills GB, Dent P, Kirkpatrick DL, Powis G. Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor PX-866 whereas oncogenic ras is a dominant predictor for resistance. Cancer Res, 2009, 69(1): 143-150.
[12] Varticovski L, Lu ZR, Mitchell K, de Aos I, Kopecek J. Water-soluble HPMA copolymer- wortmannin conjugate retains phosphoinositide 3-kinase inhibitory activity in vitro and in vivo. J Controlled Rel, 2001, 74(1-3): 275-281.
[13] Nobusue H, Kano K. Establishment and characteristics of porcine preadipocyte cell lines derived from mature adipocytes. J Cell Biochem, 2010, 109(3): 542-552.
[14] 王丽, 那威, 王宇祥, 王彦博, 王宁, 王启贵, 李玉茂, 李辉. 鸡PPARγ基因的表达特性及其对脂肪细 胞增殖分化的影响. 遗传, 2012, 34(5): 454-464.
[15] 谢霞, 高青, 王艳丽, 田翀. 抑制PI3K/PKB信号通路提高胃癌细胞化疗敏感性的研究. 中国药理学通报 , 2008, 24(12): 1666-1670.
[16] Arcaro A, Wymann MP. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochemical J, 1993, 296(Pt2): 297-301.
[17] Yano H, Nakanishi S, Kimura K, Hanai N, Saitoh Y, Fukui Y, Nonomura Y, Matsuda Y. Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3- kinase in RBL-2H3 cells. J Biol Chem, 1993, 268(34): 25846-25856.
[18] Prevo R, Deutsch E, Sampson O, Diplexcito J, Cengel K, Harper J, O'Neill P, McKenna WG, Patel S, Bernhard EJ. Class I PI3 kinase inhibition by the pyridinylfuranopyrimidine inhibitor PI -103 enhances tumor radiosensitivity. Cancer Res, 2008, 68(14): 5915-5923.
