力达霉素联合TRAIL对非小细胞肺癌的协同作用及其机制

Synergistic effect and its possible mechanisms of lidamycin in combination with TRAIL in NSCLC

研究力达霉素(LDM)与肿瘤坏死因子相关的凋亡诱导配体(TRAIL)联合作用对非小细胞肺癌细胞株H460细胞的增效作用及其作用机制。MTT法观察两药联合对H460细胞增殖的抑制作用。AnnexinV-FITC/PI双染、流式细胞术和Hoechst33342荧光染色检测两药联合对细胞凋亡的影响。Westernblotting检测凋亡通路中PARP、Caspase-3和Caspase-8分子的变化以及LDM对TRAIL受体DR4、DR5表达的影响。MTT结果显示,LDM与TRAIL的IC50值分别为4.603×10-10mol·L-1和915.3ng·mL-1,在TRAIL(50及100ng·mL-1)作用下LDM的IC50值分别为3.064×10-11和1.611×10-11mol·L-1。两药相互作用指数CDI<1。荧光显微镜下观察及流式细胞术检测两药联合细胞凋亡作用增强。联合用药组中Caspase-3和Caspase-8的激活作用明显增强。LDM能够增强TRAIL受体DR5的表达,不仅具有剂量依赖性,且作用时间越长表达量越高。研究结果提示,LDM对人非小细胞肺癌细胞株H460细胞具有生长抑制作用,可能通过诱导DR5表达上调而促进TRAIL诱导的肿瘤细胞凋亡,抑制细胞增殖,从而增加细胞对TRAIL的敏感性。

This study is to investigate the effect and its possible mechanisms of lidamycin （LDM） combined with tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） in human non-small cell lung cancer （NSCLC） cells. MTT assay was used to determine the growth inhibition of the two ingredients on H460 cells. Apoptosis was examined by Annexin V-FITC/PI staining, flow cytometry assay and DNA-specific dye Hoechst 33342 staining. The level of TRAIL receptor and apoptosis-associated protein expression was detected by Western blotting analysis. The results showed that the IC50 value of LDM and TRAIL for H460 cells was 4.603×10^-10 mol·L^-1 and 915.3 ng·mL^-1 respectively, but the IC50 value of LDM was 3.064×10^-11 mol·L^-1 and 1.611×10^-10 mol·L^-1 when different concentrations of LDM was combined with 50 and 100 ng·mL^-1 TRAIL respectively. And the CDI value was less than 1. The apoptosis ratios also increased in the combination group relative to the single-agent treatment and the untreated control. Furthermore, the induction of the cleavage of PARP and the activation of Caspase-3 and Caspase-8 by the combination were more effective than LDM or TRAIL alone. At last, the level of death receptor 5 （DR5） expressions increased in a dose-dependent manner and time-related pattern. The data indicate that LDM inhibits the growth of H460 cells in vitro. DR5 induction contributes to enhancement of TRAIL-induced apoptosis by LDM in human non-smaU lung cancer cells.