Study On Schisandrin B Enhance The Chemosensitivity Of U251 Cells Through DNA Impair/Repair Pathway

ObjectiveGlioblastoma as a high maligant and lethal tumor in central neverous system,radiotherapy combines chemotherapy is the most common treatment by now,but resistance impact the effect.We are looking for a effective and low side-effect compound to assist the treatment,and we found a lignans compound-Schisandrin B.Study reported that Schisandrin B could against various cancer cells but no evidence in indirect reaction.Our study focus on the mechanism of enhance the sensitivity of Temozolomide via Schisandrin B.We aim at FOXM1,a kind of transcription factor,regulates DNA impair/repair balance and how the drugs cause apoptosis.Methods1. The effect of Temozolomid/Schisandrin B on viability of U251The human glioblastoma U251 cell line were treated with different concentration of Temozolomid and Schisandrin B to verify the terminal treatment time and concentration by CCK-8 assay,and the statistic were treat with regression analysis and IC₅₀ were calculated.2. The effect of Temozolomid/Schisandrin B on apoptosis of U251We detected the morphology and rates of U251 apoptosis cells by Hoechst33342/PI staining and flow cytometric assay;We measured the Caspase3/8/9 protein activity and apoptosis protein Bcl-2 and Bax by ultraviolet spectrophotometry and western blotting.3.The effect of Temozolomid/Schisandrin B on DNA impair/repair protein expression of U251We detected the 53BP1,γH2AX and Rad51 protein expression and rates of U251apoptosis cells by immunofluorescent and western blotting.4.The effect of Temozolomid/Schisandrin B on FOXM1 gene and protein expression of U251We detected the FOXM1 gene and protein expression by q PCR and western blotting.Results1.The effect of Temozolomid/Schisandrin B on viability of U251The results from CCK-8 assay showed that the IC50 of Temozolomide for 48h and 72h on U251 cells were 793μM and 550μM respectively.According to these results,the concentration of Temozolomide were identified as 550μM and 72h.The results showed that the IC50 of Schisandrin B for 72h on U251 cells were 68μM.But Schisandrin B considered as sensitizing agent,we chose the concentration of 10μM and 20μM for 72h.The results of combination of Temozolomide and Schisandrin B showed that cell viability of TMZ group was 44.6%and the combination groups were 33.8%and 30.9%seperately after 5 days,indicated the proliferation of cells were inhibited.2.The effect of Temozolomid/Schisandrin B on apoptosis of U251The chromatin of U251 apoptosis cells by Hoechst33342/PI staining were cracked into blue pieces and highlighted shrinkage.Flow cytometric assay showed that the apoptosis rate of combination group were 19.0%and 28.2%respectively,the apoptosis rate of TMZ group was 16.5%,nevertheless the control group were 9.1%;The results showed in combination group that Caspase3 and 9 protein activity were increased;The western blotting showed that anti-apoptosis protein Bcl-2 were decreased and promoting apoptosis Bax were increased.These results indicated cells were performed as mitochondrion-mediated apoptosis.3. The effect of Temozolomid/Schisandrin B on DNA impair/repair protein expression of U251Immunofluorescent assay showed that 53BP1 protein expression were increased.Western Blottting showed thatγH2AX protein expression were increased and Rad51 protein expression were decreased in combination group.4. The effect of Temozolomid/Schisandrin B on FOXM1 gene and protein expression of U251The qPCR and western blotting experiment showed that gene and protein expression of FOXM1 were decreased.ConclusionIn our study,Temozolomide combined with Schisandrin B could enhance the effect of agents.The enhancement mainly influenced the FOXM1 gene,therefore inhibited the FOXM1 protein expression.The inhibition of FOXM1 probably increased the impair protein and decreased the repair protein.After the balance of DNA impair/repair damaged,the mitochondrion-mediated apoptosis started and caused the inhibition of cell proliferation.