溶瘤病毒联合新型小分子抑制剂治疗胶质瘤的实验研究

Treatment of gliomas with combination of viral hemolytic virus and novel small molecule inhibitor

目的研究新型水疱口炎病毒VSV^ΔM51联合新型小分子抑制剂—核糖体S6激酶1(RSK1)抑制剂BI-D1870和Polo样激酶1(PLK1)抑制剂BI2536对胶质瘤细胞的体外杀伤效果。方法(1)将体外培养的GL261、CT2A、HS68细胞分为对照组、雷帕霉素组、BI-D1870组、BI-2536组、VSV^ΔM51组、雷帕霉素+VSV^ΔM51组、BI-D1870+VSV^ΔM51组、BI2536+VSV^ΔM51组,分别用100 nmol/L雷帕霉素,10 μmol/L BI-D1870,100 nmol/L BI-2536预处理2 h后感染0.1感染复数(MOI) VSV^△M51病毒,72 h后采用Alarma Blue法测定细胞的存活率;分别用上述药物预处理1 h后感染10 MOI VSV^△M51病毒,24 h后活化Caspase-3染色检测GL261细胞的凋亡;Western blotting检测细胞凋亡蛋白聚ADP-核糖聚合酶(PARP)的表达;Annexin V-FITC/碘化丙啶(PI)双染法检测细胞凋亡。(2)将GL261、CT2A细胞细胞分为VSV^ΔM51组、雷帕霉素+VSV^ΔM51组、BI-D1870+VSV^ΔM51组、BI2536+VSV^ΔM51组,分别用上述药物预处理1 h后感染0.1 MOI VSV^△M51病毒,48 h后荧光显微镜下观察细胞中绿色荧光蛋白(GFP)的表达;IVIS200活体成像系统检测4组细胞病毒荧光素酶的变化;(3)15只CT2A细胞颅内种植胶质瘤模型小鼠,按随机数字表法分为VSV^△M51组、BID-1870+VSV^△M51组和BI2536+VSV^ΔM51组,每组5只。后2组小鼠分别腹腔注射BI-1870(100 mg/kg)、静脉注射BI-2536(20 mg/kg),24 h后3组小鼠均静脉注射病毒VSV^△M51,24 h、72 h后IVIS200活体成像系统检测病毒荧光素酶;15只GL261细胞颅内种植胶质瘤模型小鼠的分组和处理同上,48 h后荧光显微镜下观察病毒GFP的表达;病毒噬斑法检测小鼠的病毒滴度。结果(1)与对照组、雷帕霉素组、VSV^ΔM51组、BI-D1870组、BI-2536组比较,雷帕霉素+VSV^ΔM51组、BI-D1870+VSV^ΔM51组、BI2536+VSV^ΔM51组细胞存活率显著降低,差异有统计学意义(P<0.05)。活化Caspase-3染色检测显示对照组无凋亡,雷帕霉素组、BI-D1870组、BI-2536组、VSV^ΔM51组可见少量凋亡小体,但雷帕霉素+VSV^ΔM51组、BI-D1870+VSV^ΔM51组、BI2536+VSV^ΔM51组凋亡小体明显增多。Western blotting检测显示对照组、雷帕霉素组、BI-D1870组、BI-2536组、VSV^ΔM51组GL261、CT2A细胞活化PARP蛋白的表达少于雷帕霉素+VSV^ΔM51组、BI-D1870+VSV^ΔM51组、BI2536+VSV^ΔM51组。Annexin V-FITC/PI染色结果与活化Caspase-3染色结果一致。(2)与VSV^ΔM51组、雷帕霉素+VSV^ΔM51组比较,BI-D1870+VSV^ΔM51组、BI2536+VSV^ΔM51组细胞GFP的表达增强、病毒荧光素酶更亮,差异均有统计学意义(P<0.05)。(3)72 h后VSV^△M51组、BID-1870+VSV^△M51组、BI2536+VSV^△M51组CT2A细胞颅内种植胶质瘤模型小鼠荧光素酶亮度依次增加,差异有统计学意义(P<0.05)。48 h后VSV^△M51组、BID-1870+VSV^ΔM51组、BI2536+VSV^△M51组GL261细胞颅内种植胶质瘤模型小鼠的病毒滴度依次增加,差异均有统计学意义(P<0.05)。结论2种小分子抑制剂都在一定程度上促进了VSV^△M51病毒的复制,进而增强了对胶质瘤细胞的杀伤作用,并且其增效效果明显优于雷帕霉素。

Objective To study the in vitro killing effect of novel small molecule inhibitors, ribosomal S6 kinase1 (RSK1) inhibitor (BI-D1870) and polo-like kinase 1 (PLK1) inhibitor (BI2536), combined with recombinant attenuated vesicular stomatitis virus VSV^ΔM51 on various glioma cells. Methods (1) In vitro cultured GL261, CT2A and HS68 cells were divided into control group, rapamycin group, BI-D1870 group, BI-2536 group, VSV^ΔM51 group, rapamycin+VSV^ΔM51 group, BI-D1870+VSV^ΔM51 group, and BI2536+VSV^ΔM51 group;pretreatments with 100 nmol/L rapamycin, 10 μmol/L BI-D1870, and 100 nmol/L BI-2536 for 2 h were given to the cells from the above groups, respectively, and then, they were infected with VSV^ΔM51 virus at 0.1 mutiplicity of infection (MOI);at 72 h after treatments, the cell survival rate was determined by Alarma Blue method;VSV^△M51 virus was infected at 10 MOI one h after pretreatment with the above drugs, apoptosis of GL261 cells was detected by cleaved caspase-3 staining 24 h after that;the expression of apoptotic protein polyadp-ribosomal polymerase (PARP) was detected by Western blotting;Annexin V-FITC/propidium iodide double staining was used to detect the cell apoptosis.(2) GL261 and CT2A cells were divided into VSV^ΔM51 group, rapamycin+VSV^ΔM51 group, BI-D1870+VSV^ΔM51 group, and BI2536+ VSV^ΔM51 group;VSV^△M51 virus was infected at 0.1 MOI one h after pretreatment with the above drugs,;48 h after treatments, fluorescence microscope was used to detect the expression of green fluorescent protein (GFP);IVIS200 in vivo imaging system was used to detect the changes of cell virus luciferase in the 4 groups.(3) Fifteen CT2A intracranial implanted glioma model mice were divided into VSV^ΔM51 group, BID-1870+VSV^ΔM51 group and BI2536+VSV^ΔM51 group according to random number table method (n=5);mice in the latter two groups were intraperitoneally injected with BI-1870 (100 mg/kg) or intravenously injected with BI-2536 (20 mg/kg);24 h after that, mice in the three groups were intravenously injected with virus VSV^ΔM51;virus luciferase was detected by IVIS200 in vivo imaging system 24 and 72 h after treatments;the grouping and treatments of GL261 intracranial glioma model mice were the same as above, the expression of virus GFP was observed under fluorescence microscope 48 h after treatments, and virus titers of these mice were detected by virus plaque assay. Results (1) As compared with the control group, rapamycin group, BI-D1870 group, BI-2536 group, and VSV^ΔM51 group, the rapamycin+VSV^ΔM51 group, BI-D1870+VSV^ΔM51 group, and BI2536+VSV^ΔM51 group had significantly lower cell survival rate (P<0.05);cleaved Caspase-3 staining showed no cell apoptosis in the control group, a small amount of apoptotic corpuscles in the rapamycin group, BI-D1870 group, BI-2536 group, and VSV^ΔM51 group, but obvious increased amount of apoptotic corpuscles in the rapamycin+VSV^ΔM51 group, BI-D1870+VSV^ΔM51 group, and BI2536+ VSV^ΔM51 group;Western blotting indicated that GL261 and CT2A cells from the control group, rapamycin group, BI-D1870 group, BI-2536 group, and VSV^ΔM51 group had lower cleaved PARP expression level than those from the rapamycin+VSV^ΔM51 group, BI-D1870+VSV^ΔM51 group, and BI2536+VSV^ΔM51 group. The results of Annexin V-FITC/propidium iodide double staining were consistent with those of cleaved Caspase-3 staining.(2) As compared with VSV^ΔM51 group and rapamycin+VSV^ΔM51 group, BI-D1870+VSV^ΔM51 group and BI2536+VSV^ΔM51 group had significantly increased GFP expression and statistically higher intensity of virus luciferase (P<0.05).(3) CT2A cells in the VSV^ΔM51 group, BID-1870+VSV^ΔM51 group and BI2536+VSV^ΔM51 group had increased intensity of virus luciferase successively, with significant differences (P<0.05);GL261 cells in the VSV^ΔM51 group, BID-1870+VSV^ΔM51 group and BI2536+VSV^ΔM51 group had increased virus titers successively, with significant differences (P<0.05). Conclusion Both small molecule inhibitors promote the replication of VSV^ΔM51 virus and enhance the killing effect on glioma cells, and its synergistic effect is obviously better than rapamycin.