The Mechanism Of Cycle Arrest And Apoptosis In Human Glioblastoma U251 Cells Induced By Diallyl Disulfide

Objective: To investigate molecular mechanism of the inhibiting proliferation, cycle arrest and apoptosis induced by diallyl disulfide (DADS) in human glioblastoma U251 Cells.Methods: Morphological analysis, MTT assay, flow cytometry, Real Time PCR, western blot and immunocytochemical technique were used to observe the effects of growth inhibition, cell cycle, apoptosis and its expression of related genes and proteins in human glioblastoma U251 Cells induced by DADS.Results: MTT assay showed that 15, 30, 45, 60mg L-1 DADS significantly inhibited proliferation of U251 cells at 48 hours, its inhibition ratio were 22.01%, 38.82%, 49.23% and 55.27%, respectively, in dose-dependent manner (P<0.05). Flow cytometry analysis revealed that U251 cells treated with 15, 30, 45, 60mg L-1 DADS for 48 hours significantly accumulated cells in the G2/M phase, which were 14.1±1.2, 25.0±0.6, 41.2±1.4, 53.5±3.3, respectively, and more than 9.6±0.9 of untreated cells in a dose-dependent model (P<0.05). Immuocytochemistry detect revealed that expression down-regulation of cyclin B1 and c-myc proteins were 0.52±0.09 and 0.56±0.08 than obviously decrease with 0.23±0.02 and 0.39±0.04 of untreated cells in average optical value (P<0.05). qRT-PCR showed that expression of Cyclin B1 and c-myc mRNA decreased 0.42 and 0.63 times (P<0.05). Western blot showed that expression downregulation of cyclin B1 and c-myc proteins were 1.1±0.14 and 1.2±0.15 that lower than 1.7±0.15 and 2.2±0.19 of untreated cells in gray scale value (P<0.05).After exposure to DADS, partial U251 cells presented characteristic morphological changes of apoptosis under the light microscopy, including cell rounding, kytoplasm strong acidophily, karyopyknosis and anachromasis, and nuclear chromatin to accumulate in the caryotheca. Flow cytometry analysis showed that U251 cells treated with 15, 30 and 45mgL-1 DADS for 48 hours significantly increased the percentage of apoptosis cells, its apoptosis rate were 5.36±0.87%, 28.36±3.15% and 44.58±3.95%, respectively, higher than 2.14±0.45% of untreated cells (P<0.05). Immuocytochemistry detect revealed that expression of Bcl-2 decreased from 0.75±0.06 to 0.34±0.03, and Bax and Caspase 3 expression increased from 0.26±0.03 and 0.19±0.02 to 0.63±0.04 and 0.41±0.05, respectively, in average optical value (P<0.01). Western blot showed that downregulation of Bcl-2 expression was 0.87±0.09 and upregulation of Bax and Caspase 3 was 0.87±0.09 and 0.73±0.06, higher than 0.31±0.04, while lower 0.31±0.04 and 0.41±0.05 of untreated cells, respectively, in gray scale value (P<0.05).Conclusion:3,DADS could inhibit the proliferation and induce cycle arrest in G2/M in dose-dependent manner in U251 cells, and its molecular mechanism concerned with expression downregulation of cyclin B1 and c-Myc.4,DADS could induce apoptosis of U251 cells related to expression of Bcl-2 downregulate and upregulation of Bax and Caspase 3.