ABSTRACT
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 VSVΔM51 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, VSVΔM51 group, rapamycin +VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+VSVΔM51 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 VSVΔM51 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 VSVΔM51 group, rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+ VSVΔM51 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 VSVΔM51 group, BID-1870+VSVΔM51 group and BI2536+VSVΔM51 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 VSVΔM51; 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 VSVΔM51 group, the rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+VSVΔM51 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 VSVΔM51 group, but obvious increased amount of apoptotic corpuscles in the rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+ VSVΔM51 group; Western blotting indicated that GL261 and CT2A cells from the control group, rapamycin group, BI-D1870 group, BI-2536 group, and VSVΔM51 group had lower cleaved PARP expression level than those from the rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+VSVΔM51 group. The results of Annexin V-FITC/propidium iodide double staining were consistent with those of cleaved Caspase-3 staining. (2) As compared with VSVΔM51 group and rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group and BI2536+VSVΔM51 group had significantly increased GFP expression and statistically higher intensity of virus luciferase (P<0.05). (3) CT2A cells in the VSVΔM51 group, BID-1870+VSVΔM51 group and BI2536+VSVΔM51 group had increased intensity of virus luciferase successively, with significant differences (P<0.05); GL261 cells in the VSVΔM51 group, BID-1870+VSVΔM51 group and BI2536+VSVΔM51 group had increased virus titers successively, with significant differences (P<0.05). Conclusion Both small molecule inhibitors promote the replication of VSVΔM51 virus and enhance the killing effect on glioma cells, and its synergistic effect is obviously better than rapamycin.
ABSTRACT
Objective To investigate the impact of AG490 on the blood-brain barrier (BBB ) permeability and the expression of interleukin-6 (IL-6 )and tumor necrosis factor-α(TNF-α)after traumatic brain injury (TBI)in rats. Methods A total of 144 healthy male SD rats were randomly divided into a control group,a trauma group,and an AG490 intervention group (n=48 in each group). The rats in each group were redivided into four subgroups (4 h,1 d,3 d,and 7 d subgroups)according to the time points after cerebral injury (n=12 in each subgroup). A brain trauma models were induced by hydraulic shock method. Evans blue was used to determine the changes of the BBB permeability after cerebral injury in each group. Real-time fluorescence quantitative PCR was to detect the expression levels of TNF-αand IL-6 mRNA in rat brain tissue. Immunohistochemistry was used to detect the expression of human phospho tyrosine kinase (P-JAK2). Results (1)The permeability of BBB:The permeability of BBB increased at 4 h,1 d,3 d and 7 d after brain injury in the trauma group (Evans blue permeation:10. 4 ± 1. 2,16. 0 ± 1. 4,22. 3 ± 2. 0,and 8. 4 ± 0. 9μg/g,respectively). Compared with the control group, there were significant differences (all P<0. 01). The Evans blue permeation of the AG490 intervention group were 9. 1 ± 1. 0,12. 8 ± 1. 1,17. 5 ± 1. 4 and 7. 1 ± 0. 8μg/g,respectively at each time point,and they were all significantly lower than those of the trauma group (all P<0. 01). (2)The expression of IL-6 and TNF-α mRNA:The expression levels of IL-6 mRNA and TNF-α mRNA at 4 h,1 d,3 d and 7 d after traumatic brain injury in the trauma group were 2. 31 ± 0. 35,2. 73 ± 0. 35,3. 32 ± 0. 29,2. 14 ± 0. 24 and 7. 46 ± 1. 18,9. 42 ± 1. 54,13. 76 ± 1. 89,and 6. 28 ± 1. 00,respectively,they were all significantly higher than those of the control group (all P<0. 01). The expression levels of IL-6 mRNA and TNF-α mRNA of the AG490 intervention group were 1. 14 ± 0. 22,1. 54 ± 0. 23,1. 94 ± 0. 32,1. 26 ± 0. 21 and 5. 57 ± 0. 88, 7. 78 ± 1. 02,11. 51 ± 1. 29,and 5. 05 ± 0. 97,respectively,they were all lower than those of the trauma group,but they still higher than the control group. There were significant differences (all P<0. 01). (3 )The expression of P-JAK2:The expression levels of P-JAK2-positive cells at each time point after traumatic brain injury in the trauma group were significantly higher than the control group (all P<0. 01),they were 17. 4 ± 2. 7,56. 2 ± 6. 7,26. 1 ± 5. 4,and 15. 3 ± 2. 5,respectively;those of the AG490 intervention group were 12. 2 ± 1. 4,41. 5 ± 4. 6,19. 4 ± 4. 1,and 9. 6 ± 2. 0,respectively,they were all lower than those of the trauma group,but still higher than the control group. There were significant differences (all P<0. 01). Conclusion During the acute phase after TBI,AG490 may activate the factor signaling pathways by inhibiting the non-receptor tyrosine kinase/signal transduction and transcription,significantly inhibit the expression of brain tissue inflammatory cytokines IL-6 IL-6 and TNF-α,reduce the BBB damage,and help to reduce secondary brain injury.