Zhou Xiu-Jie, Zhou Yu-Hong, Chen Xiao-Hui, Qian Wen-Bin
Department of Hematology, First Affiliated Hospital, Zhejiang University of Chinese Medicine, Hangzhou 310053, China.
Zhongguo Zhong Xi Yi Jie He Za Zhi. 2013 Jun;33(6):834-9.
To clarify the effects and mechanisms of homoharringtonine (HHT) monomer therapy or combination therapy with arsenic trioxide (ATO) on human multiple myeloma (MM) cell line RPMI 8226 in in vitro researches.
Effects of HHT, ATO, and HHT combined ATO on the growth of MM cell line RPMI 8226 were detected using MTT assay. The morphological changes of cell apoptosis were detected by Hoechst staining. The early apoptosis rate was detected using flow cytometry. Expressions of Caspase-3, Caspase-9, poly-ADP-ribose polymerase (PARP), Bcl-2, Mcl-1, Bcl-xl, and AKT protein were detected by Western blot.
HHT and ATO inhibited the proliferation of RPM1 8226 cell line in a time- and dose-dependent manner (P < 0.05). Synergistic effects was shown in the combination group (Cl < 1). HHT and ATO induced the apoptosis of RPMI 8226 in a dose-dependent manner with typical morphological changes of apoptosis and higher early stage apoptosis rate. The enhancement in apoptotic induction was seen when two agents were combined. HHT activated expressions of Caspase-3 and PARP in a dose dependent manner at 24 h. HHT at 40 ng/mL and ATO at 8.5 micromol/L could significantly activate expressions of Caspase-3 and Caspase-9, and down-regulate expressions of anti-apoptotic proteins Bcl-xl and Mcl-1. In addition, the combination therapy of HHT at 40 ng/mL and ATO at 8.5 micromol/L inhibited phosphorylation of AKT in a time-dependent manner.
HTT, ATO, and combination therapy of HHT and ATO induced the apoptosis of RPMI 8226 cell line possibly through activating Caspase pathways, regulating expressions of Bcl-2 families, and inhibiting phosphorylation of AKT.
在体外研究中阐明高三尖杉酯碱（HHT）单体疗法或与三氧化二砷（ATO）联合疗法对人多发性骨髓瘤（MM）细胞系RPMI 8226的作用及机制。
采用MTT法检测HHT、ATO及HHT联合ATO对MM细胞系RPMI 8226生长的影响。通过Hoechst染色检测细胞凋亡的形态学变化。采用流式细胞术检测早期凋亡率。通过蛋白质免疫印迹法检测半胱天冬酶-3（Caspase-3）、半胱天冬酶-9（Caspase-9）、聚ADP核糖聚合酶（PARP）、Bcl-2、髓细胞白血病-1（Mcl-1）、Bcl-xl及AKT蛋白的表达。
HHT和ATO以时间和剂量依赖性方式抑制RPMI 8226细胞系的增殖（P<0.05）。联合组显示出协同作用（协同系数<1）。HHT和ATO以剂量依赖性方式诱导RPMI 8226细胞凋亡，伴有典型的凋亡形态学变化及较高的早期凋亡率。两种药物联合使用时，凋亡诱导作用增强。HHT在24小时时以剂量依赖性方式激活Caspase-3和PARP的表达。40 ng/mL的HHT和8.5 μmol/L的ATO可显著激活Caspase-3和Caspase-9的表达，并下调抗凋亡蛋白Bcl-xl和Mcl-1的表达。此外，40 ng/mL的HHT和8.5 μmol/L的ATO联合疗法以时间依赖性方式抑制AKT的磷酸化。
HHT、ATO以及HHT与ATO联合疗法可能通过激活Caspase途径、调节Bcl-2家族的表达以及抑制AKT的磷酸化来诱导RPMI 8226细胞系凋亡。
