The Cytotoxic Effect Of Bortezomib On Endothelial Cells And Its Effect On Cell Migration

PARTⅠ:The Cytotoxic Effect of Bortezomib on endothelial cell line HMEC-1 and its effect on cell migrationObjective To investigate the cytotoxic effect of 0,2.5,5.0,10 nmol / L bortezomib on endothelial cell line HMEC-1 and its effect on cell migration. Methods Cell count kit CCK-8 was used to determine the relative proliferation activity of endothelial cells treated by bortezomib in different concentration for 12h and 24h, respectivly; The cell apoptosis rate was detected by AnnexinV / PI double staining; Transwell model was uesd to detect the migration rate of cells. Results After being treated by bortezomib in 2.5,5.0nmol/L for 12h, the proliferation activity of HMEC-1 cells was not significantly suppressed, which, on the contrary, was significantly suppressed with bortezomib in 10nmol/L (0.874±0.062) (P=0.024). Bortezomib had higher suppressive effect on proliferation activity of HMEC-1 cells when treated for 24h,48h, and the proliferation activity of HMEC-1 cells was 0.635±0.030, 0.164±0.009(P<0.0001), respectively. Compared with the control group (3.5%), apoptosis rate of cells treated by bortezomib in 2.5,5.0,10nmol/L for 12h was 2.0%,1.6%,2.7%, respectively, which is not higher. While apoptosis rate of cells treated by bortezomib in 10nmol/L for 24h was 18.1%. However, the migration rate of such cells, treated by bortezomib in 2.5, 5.0, 10nmol/L for 12h, were significantly inhibited (P<0.05). conclusions Bortezomib had some extent cytotoxic effect on endothelial cell line HMEC-1 in higher concentration for a longer time ;Meanwhile, Bortezomib could inhibit migration of endothelial cell line HMEC-1,and compared with the cytotoxic effect, the effect of migration inhibitory appeared earlier. PARTⅡ: Possible Mechanisms of the inhibiting effect of Bortezomib on migration of endothelial cell line HMEC-1Objective To investigate the expression of angiogenesis-related molecules VEGF and Annexin A2 of endothelial cell line HMEC-1 treated by bortezomib in 2.5,5.0,10nmol/L for 12h, in order to discuss the possible mechanisms of the inhibiting effect of Bortezomib on migration of endothelial cell line HMEC-1; And to determine the changes of the transcriptional regulation activity of hypoxia-inducible factor 1α(HIF-1α), in order to explore the reasons for the expression changes of VEGF. Methods Expression levels of VEGF and Annexin A2 genes were determined by real-time quantitative PCR, and Annexin A2 protein was validated by western blotting;Reverse transcription-PCR (RT-PCR) was employed to exame the expression level of CAIX gene, a symbol of HIF-1αtranscriptional regulation activity. Results The expression of VEGF and Annexin A2 gene of endothelial cell line HMEC-1, treated by bortezomib in 2.5, 5.0, 10nmol/L for 12h, were both significantly inhibited (P<0.05), and conspicuous downregulation of Annexin A2 protein was also confirmed by Western Blotting; RT-PCR showed that bortezomib also significantly inhibited the expression intensity of CAIX gene. Conclusions Bortezomib could inhibits migration of endothelial cell line HMEC-1 through downregulating the expression of VEGF and Annexin A2; while downregulation of VEGF gene may be related to inhibition of the transcriptional regulation activity of HIF-1α.