腺花素对多发性骨髓瘤细胞的生物学效应及其机制研究

Biological effects and mechanism of adenanthin on multiple myeloma cells

目的·探讨腺花素(Aden)对多发性骨髓瘤(MM)细胞的生物学效应与作用机制。方法·不同浓度Aden处理MM细胞H929、U266不同时间后,采用锥虫蓝排斥测试法检测细胞密度与活率;不同浓度Aden处理H929、U266细胞24 h后,采用CCK8检测细胞增殖变化,Annexin V-APC/PI双染后流式细胞术检测细胞凋亡率,Western blotting检测凋亡相关蛋白的表达、NF-κB信号通路相关蛋白以及NF-κB调控的下游蛋白变化;采用CETSA方法检测Aden对IKKβ蛋白热力学稳定性的影响。结果·锥虫蓝排斥测试法结果显示:Aden呈时间和剂量依赖性降低H929、U266细胞的活率及抑制细胞的增殖;在同样浓度的Aden处理下,U266较H929细胞更敏感。CCK8结果显示Aden呈剂量依赖性抑制H929、U266细胞的增殖。流式细胞术表明Aden可引起MM细胞轻微凋亡。Western blotting结果显示:H929细胞经Aden处理后未检测到PARP、caspase3的剪切;U266细胞经Aden处理后总蛋白PARP和caspase3下调,同时也可检测到PARP、caspase3的剪切;2株MM细胞经Aden处理后NF-κB信号通路蛋白p-p65、p-IκBα的表达均减少。CETSA结果显示Aden能使IKKβ蛋白热力学稳定性下降。结论·Aden能明显抑制MM细胞H929、U266增殖,但不能显著诱导其凋亡;Aden对MM细胞增殖抑制效应可能与其结合IKKβ进而抑制NF-κB信号通路的活化有关。

Objective- To explore the biologic effect and mechanism of adenanthin （Aden） on multiple myeloma （MM） cells. Methods· MM cells, H929 and U266 were treated with various dose of Aden for different time, and the density and viability of MM cells were detected by trypan blue exclusion assay. After H929 and U266 cells were treated with various dose of Aden for 24 hours, cell growth inhibition was examined by CCK8 assay, and cell apoptosis was examined by AnnexinV-APC/PI staining assay. Apoptosis related proteins, NF-κB signaling pathway associated proteins and the NF- κB regulated proteins were detected by Western blotting. The effect of Aden on the thermal stability of IKK[3 protein was determined by CETSA assay. Results · Trypan blue exclusion results showed that Aden inhibited cell growth and reduced cell viability in concentration and time dependent manners. U266 was more sensitive than H929 when exposed to the same concentration of Aden. The CCK8 results showed that Aden inhibited the growth of H929 and U266 cells in a concentration dependent manner. Flow cytometry results suggested that Aden induced a low apoptosis rate of MM cells. Moreover, cleavage of caspase3 and PARP were detected in U266 cells but not in H929 cells. CETSA assay indicated that Aden decreased the thermal stability of IKKβ. Expression of p-p65 and p-IκBα proteins decreased in MM cells treated with Aden. Conclusion · Aden significantly inhibits MM cell proliferation by inhibiting NF-κB activation through interacting with IKKβ. Aden has little effect on apoptosis of MM cells.