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目的研究组蛋白去乙酰化酶抑制剂PCI-24781诱导耐甲氨蝶呤骨肉瘤细胞凋亡的作用及机制。方法四甲基偶氮唑蓝(MTT)法及平板克隆形成实验检测PCI-24781对骨肉瘤细胞U-20S/MTX300增殖的影响,hochest33258染色检测PCI-24781处理后细胞核形态改变,流式细胞仪检测凋亡,Western blot法检测cleaved-PARP、P53表达量及组蛋白H3、H4乙酰化水平变化。结果PCI-24781对耐甲氨蝶呤U2-OS/MT 300细胞呈剂量依赖性增殖抑制,其48 h IC50值为(0.55±0.03)μmol.L-1,该值与甲氨蝶呤敏感U2-OS细胞IC50值之间无统计学差异(P>0.05)。0.5μmol.L-1PCI-24781可显著抑制细胞克隆形成,其抑制率达(61±7)%。PCI-24781处理48 h后细胞内出现典型凋亡小体,流式细胞仪检测凋亡率达(29±4)%。Western blot检测可见随PCI-24781浓度升高cleaved-PARP,组蛋白H3、H4乙酰化水平及P53显著升高。结论组蛋白去乙酰化酶抑制剂PCI-24781可显著抑制耐甲氨蝶呤骨肉瘤细胞增殖并诱导细胞凋亡,其作用机制与上调组蛋白乙酰化水平,促进抑癌基因P53表达有关。
Objective To investigate the effect and mechanism of histone deacetylase inhibitor PCI-24781 on apoptosis of methotrexate-resistant osteosarcoma cells. Methods Methyl thiazolyl tetrazolium (MTT) assay and plate clone formation assay were used to detect the effect of PCI-24781 on the proliferation of U-20S / MTX300 osteosarcoma cells. Histochitosan 33258 staining was used to detect the morphological changes of the nuclei after PCI-24781 treatment. Flow cytometry Apoptosis was detected. The expression of cleaved-PARP, P53 and histone H3, H4 acetylation levels were detected by Western blot. Results PCI-24781 inhibited the proliferation of methotrexate-resistant U2-OS / MT 300 cells in a dose-dependent manner. The IC50 value at 48 h was (0.55 ± 0.03) μmol.L-1, which was significantly higher than that of methotrexate-sensitive U2 There was no significant difference (P> 0.05) between the IC50 values of -OS cells. 0.5μmol.L-1PCI-24781 could significantly inhibit cell clone formation with the inhibition rate of (61 ± 7)%. Typical apoptotic bodies appeared within 48 h after PCI-24781 treatment, and apoptosis rate was (29 ± 4)% by flow cytometry. Western blot showed cleaved-PARP increased with the concentration of PCI-24781, histone H3, H4 acetylation levels and P53 was significantly increased. Conclusion The histone deacetylase inhibitor PCI-24781 can significantly inhibit the proliferation and induce the apoptosis of methotrexate-induced osteosarcoma cells. Its mechanism may be related to the upregulation of histone acetylation and the expression of the suppressor gene P53.