DAPT对骨肉瘤U2OS细胞增殖和凋亡影响的实验研究

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Abstract Objective To observe the effect of gamma secretase inhibitor DAPT on proliferation and apoptosis of human osteosarcoma cell line U2OS, and to explore the possible mechanism of DAPT inhibiting osteosarcoma proliferation and inducing apoptosis. Methods Osteosarcoma cell line U2OS was cultured in vitro. Cell proliferation was tested by cell counting kit-8 (CCK-8) assay. Double stained by Annexin V-FITC and Propidium Iodide (PI), the cells were detected by flow cytometry (FCM) for apoptosis. Hoechst 33258 was used to detect the morphological change of typical apoptotic cells. The apoptosis related mRNA and protein levels in U2OS were detected by RT-qPCR and Western blot. The activities of Caspase-3, Caspase-8 and Caspase-9 were measured with Caspase activity assay kit. Results CCK-8 assay showed that 5-50 μmol/L DAPT inhibited the proliferation of human osteosarcoma U2OS cells and in a time- and dose-dependent manners. Flow cytometry showed that 5, 10 and 20 μmol/L DAPT could significantly induce apoptosis of osteosarcoma U2OS cells, compared to the control group, the differences were statistically significant (P < 0.05). Apoptotic body could be observed by Hoechst 33258 staining after treatment with 5, 10 and 20 μmol/L DAPT. RT-PCR and Western blot analysis showed that after treatment with DAPT (5, 10, 30 μmol/L), the expression of Caspase-3, 8 ,9 mRNA were higher than those of control group (P < 0.05), the pro-apoptotic protein Bax increased (P < 0.05), and anti-apoptosis protein Bcl-2 protein decreased (P < 0.05). The activities of Caspase-3, Caspase-8 and Caspase-9 promoted obviously after treatment with DAPT (5, 10, 20, 30 μmol/L) when compared with the control group (P < 0.05). Conclusion DAPT can significantly inhibit the proliferation and induce apoptosis of human osteosarcoma cell line U2OS. Its mechanism of action may be related to activation of Caspase dependent apoptosis pathway.

Key words： γ-secretase inhibitor DAPT Osteosarcoma Proliferation Apoptosis

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	HU Guohai DAI Guo LIU Gaiwei SONG Qi YU Ling YANG Jian GUO Weichun

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HU Guohai DAI Guo LIU Gaiwei SONG Qi YU Ling YANG Jian GUO Weichun. The effects of γ-secretase inhibitor DAPT on proliferation and apoptosis of U2OS[J]. 中国医药导报, 2017, 14(28): 4-8,12.

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http://www.yiyaodaobao.com.cn/EN/ OR http://www.yiyaodaobao.com.cn/EN/Y2017/V14/I28/4

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