Mechanism Of TMZ Analogs On DNA Damage And Apoptosis

Temozolomide is the current clinical drug of glioma.In the process of using TMZ came the problem of drug resistance,which is related to O6-methylguanine methyl transferase and mismatch repair.lt also limits the clinical application of TMZ.So the development and research temozolomide analogues which independent on MGMT and MMR is of great significance,new temozolomide analogues has been successfully synthesized,which is proved independent on MGMT and MMR by cell activity experiment,but its mechanism is unclear.In order to study the effect of 377 and 465 to the MGMT high expression cell in proliferation for a long time,we did a clone forming experiments.The result indicate that IC50 of TMZ to SNB19V and SNB19M is 32.2?M and 854.65?M,IC50 of 377 to SNB19V and SNB19M is 17.86±7.3?M and>100?M,the IC50 of465 to SNB19V and SNB19M is 14.34±2.92?M and 31.83 ±8.66?M,which indicate that MGMT can repair the damage produced by TMZ and 377,and the effect of 465 is independent of MGMT.In order to study the effect of 377 and 465 on the cell proliferation of endogenous high expression of MGMT,MTT assay was performed.The result indicate that IC50 of 377 and 465 to T98G is 62.5±6.93?M and 33.09±3.42?M.which indicate that 377 and 465 compound can inhibit T98G cell proliferation.In order to explore other DNA repair pathways influence on TMZ analogues,We use the combination 465 and PARP inhibitors NU1025 to observation base excision repair(BER)pathway on 465 activity,MTT data show that before and after the combination of NU1025,the IC50 of 465 to SNB19V,SNB19M and HCT116 is 7.16±2.19 ? M and 5.49±1.57 ? M,8.39±1.43 ? M and 6.03±1.99 ? M,29.99±0.21 ? M and 23.50±2.28? M.Next,DNA damage experiments proved that the 377 and 465 compounds can make TMZ resistant HCT116 base mismatch repair defects in colon cancer cells and high expression of MGMT T98G glioma cells to produce double-stranded DNA break,And this kind of double-stranded DNA break is not caused by DNA cross-linking.Then,in order to study the homologous recombination repair(HR)way impact on the activity of 377 and 465 compounds,We through the method of RNA interference on the key protein Rad51 HR way,MTT data indicate that before and after the Rad51 interference,the IC50 of 377 to HCT116 is 28.68±2?M and 27.74± 1.51 ?M,the IC50 of 465 to HCT116 is 3.6±0.92 ? M and 3.08±0.13 ? M,which show that HR has no effect on antitumor activity of 377 and 465 compounds,Western Blot Results showed that 465 compounds in HCT116 cells produced by double-stranded DNA break eventually caused the cell apoptosis,led to the decrease of the mitochondrial membrane potential,mitochondria may be related to the intrinsic apoptotic pathways,at the same time,we found that TMZ analogues of 465 can downregulate MGMT and Rad51 expression of HCT116 cells.Above all,TMZ analogues antitumor activity of 377 and 465 compounds is independent on the MGMT and MMR,which can inhibit tumor cell proliferation,generate DNA damage and lead to cell apoptosis,and that 465 compounds can downregulate MGMT and Rad51 expression.