贝利司他抑制骨肉瘤细胞活力的分子机制

Molecular mechanisms of Belinostat inhibiting viability of osteosarcoma cells

目的:观察组蛋白脱乙酰酶抑制剂贝利司他对骨肉瘤细胞活力的影响,并初步研究其作用机制。方法:用不同浓度的贝利司他处理体外培养的骨肉瘤细胞系SAOS-2和U2OS后,采用MTT法检测细胞的活力,荧光探针和ELISA分别检测caspase-3/-7的酶活性以及DNA片段化以观察贝利司他对骨肉瘤细胞凋亡的影响,并采用Western blot检测组蛋白乙酰化水平、caspase-3、Bcl-xL和PTEN的表达,以及Akt和糖原合成酶激酶3β(GSK-3β)的磷酸化水平。用不同浓度的贝利司他和多柔比星(又称阿霉素)共同孵育U2OS和SAOS-2细胞,MTT观察其对细胞活力的影响,并计算其联合指数(CI)。结果:0.5、1、2.5和5μmol/L贝利司他处理U2OS和SAOS-2细胞48 h后,均可呈剂量依赖性方式抑制其活力,诱导DNA片段化,增强caspase-3/-7的活性,上调cleaved caspase-3水平,减少Bcl-xL的表达,促进细胞内组蛋白H3和H4乙酰化。Western blot结果显示,贝利司他处理后,U2OS和SAOS-2细胞中磷酸化Akt和GSK-3β水平显著降低(P<0.01)。MTT结果显示,贝利司他和阿霉素联合作用后,可进一步降低细胞的活力,两者之间具有协同作用(CI<1)。结论:贝利司他能协同阿霉素抑制骨肉瘤细胞活力,其机制可能与抑制Akt信号通路有关。

AIM: To observe the effect of histone deacetylase inhibitor(HDACi) Belinostat on the viability of osteosarcoma cells and to study the underlying mechanism. METHODS: Osteosarcoma cell lines SAOS-2 and U2 OS were incubated with Belinostat at different concentrations in vitro. The viability of the cells was measured by MTT assay. The activity of caspase-3/-7 and the DNA fragmentation were detected by fluorescence probe and ELISA, respectively. Western blot was used to detect the levels of histone acetylation, expression of PTEN, caspase-3, Bcl-xL and Akt, and phosphorylation of glycogen synthetase kinase 3β(GSK-3β) and Akt. Finally, the cells were incubated with Belinostat and doxorubicin at different concentrations, and then the combination index(CI) was calculated by MTT. RESULTS: Belinostat at 0.5, 1, 2.5 and 5 μmol/L inhibited the viability of U2 OS cells and SAOS-2 cells in a dose-dependent manner, induced DNA fragmentation, enhanced caspase-3/-7 activity, and promoted the activation of caspase-3. At the same time, in the SAOS-2 cells, the expression of Bcl-xL was reduced, and the acetylation of histones H3 and H4 was increased. The results of Western blot showed that phosphorylation levels of Akt and GSK-3β in U2 OS cells and SAOS-2 cells were decreased significantly after treatment with Belinostat(P<0.05). MTT results showed that combination of Belinostat and doxorubicin further reduced the viability of U2 OS and SAOS-2 cells(CI<1). CONCLUSION: Belinostat inhibits the viability of osteosarcoma cells treated with doxorubicin, and the mechanism may be related to the inhibition of Akt signaling pathway.