尿多酸肽抑制多发性骨髓瘤细胞株RPIMI 8226增殖及其机制研究

Uroacitides(CDA-2) Inhibited the Proliferation of Multiple Myeloma Cell Line RPMI 8226

关键词:  尿多酸肽  RPMI8226  增殖抑制

Key words:  uroacitides(CDA-2)  RPMI8226  proliferative inhibition

摘要:

目的 探讨尿多酸肽(CDA-2)抑制多发性骨髓瘤细胞株RPMI8226增殖作用及其机制。方法 采用四甲基偶氮唑(MTT)比色法检测CDA-2对RPMI8226抑制作用并筛选研究浓度；通过Hoechst33258、Annexin-V/PI、细胞周期分析、DNA琼脂糖凝胶电泳等方法检测CDA-2诱导RPMI8226凋亡作用；使用Western Blot法检测caspase-8、caspase-3及其激活物的表达改变；半定量RT-PCR法检测TNF、FADD、TRAF3 mRNA表达。结果 CDA-2能够抑制RPMI8226细胞株增殖，并呈浓度依赖性，IC50为1.64 mg·mL^-1；经CDA-2作用后，Hoechst33258荧光染色提示细胞核浓集及出现凋亡小体，Annexin-V/PI分析示早期凋亡细胞比例呈时间依赖增加，细胞周期分析提示呈浓度依赖上调凋亡峰及下调G1期比例，DNA凝胶电泳可见断裂成180~200 bp及其倍数的片断梯形条带，故CDA-2可诱导RPMI8226细胞株凋亡；Western Blot检测发现随药物作用时间延长caspase-8、caspase-3表达明显下降，而active-caspase8、active-caspase3表达上升；半定量RT-PCR证实凋亡相关基因TNF、FADD、TRAF3 mRNA表达上调。结论 CDA-2可抑制RPMI8226增殖，且可通过死亡受体途径诱导细胞凋亡。

Abstract:

OBJECTIVE To explore the effects of uroacitides(CDA-2) on the proliferation of RPMI 8226 cells and its mechanisms. METHODS The proliferative inhibition of CDA-2 on RPMI 8226 cells was detected by MTT and the drug concentrations for further researches were screened out. The apoptosis of RPMI8226 cells after treating with CDA-2 was analyzed by Hoechst33258 staining, Annexin-V/PI staining, PI staining, and DNA gel electrophoresis. Changes in the expression of caspase-8, caspase-3 and their active forms were tested by Western Blot. The expression of TNF, ADD and TRAF3 mRNA were detected by semi-quantitative RT-PCR. RESULTS CDA-2 inhibited the proliferation of RPMI8226 cells in a dose-dependent manner with the IC50 1.64 mg·mL^-1. Condensed nuclei and apoptotic body were found via Hoechst33258 fluorescence staining when cells were treated with CDA-2. Annexin-V/PI analysis showed that the proportion of early apoptotic cells raised in a time-dependent manner. Cell cycle analysis showed that the apoptotic peak was up-regulated and G1 phase was decreased in a dose-dependent manner. DNA gel electrophoresis revealed integer multiples of 180~200 bp “ladder” bands. Western blot revealed that the expression of caspase-8, caspase-3 was down-regulated, while the expression of active caspase-8, activecaspase-3 was increased as the exposed time extended. The semi-quantitative RT-PCR showed up-regulation of TNF, FADD, TRAF3 mRNA, which were associated with apoptosis. CONCLUSION CDA-2 inhibited the proliferation of RPMI8226 cells and induced apoptosis via death receptor pathway.