Anticancer Effect And Cellular Mechanisms Of Novel Oxadiazolo[3,4-d] Pyrimidine Nucleoside Derivatives

A kind of novel oxdiazolo[3,4-d]pyrimidine nucleoside derivatives(1-15) were synthesized to investigate their anti-tumor effect and possible mechanisms.Fifteen pyrimidine nucleoside derivatives exposed to four human cancer cell lines including Hela,ECA109,HepG2 and A459 cells. Cancer cell proliferation were detected by MTT assay and the 50%inhibitory concentration(IC₅₀) were calculated;NO content was detected by nitrate reductase assay;HepG2 cells were staining with hoechest 33342 and nucleus morphological changes were observed.Annexin-V/PI staining were detected; accumulation of LC3 protein was examined by western blot analysis;the formation of autophagic vacuoles was assessed by staining cells with MDC; formation of ROS were detected by DCFH-DA staining;mitochondrial membrane potential was observed by JC-1 staining;ATP content was detected by luciferase assay;intracellular free Ca²⁺ content were detected by Fluo-3 staining;cell cycle were analyzed by PI staining.The results showed that 4-(2,3,5-Tri-O-benzoyl-1-β-D-ribofuranosyl)-[1,2,5]-oxadiazolo[3,4-d]pyrimidine-5(4H),7(6H)-dione1-oxide,named compound 7 and 6-Methyl-4-(2-fluoro-3,5-di-O-benzoyl-1-β-D-ribofuranosyl) -[1,2,5]oxadiazolo[3,4-d]pyrimidine-5(4H),7(6H)-dione1-oxide, named compound 10 exhibited significant inhibition on cancer cell proliferation.The IC₅₀ are around 30-70μmol/L.They exhibited dose- and time-dependent effect on HepG2 proliferation.Both compounds could release nitric oxide(NO),as a NO donor.Treated with compound 7,the results showed that nucleus morphology were not change and the apoptotic cells were not significantly increased.But the LC3 protein expression was up-regulated and punctuate staining structures appeared in compound 7-treated HepG2 cells.The results indicated that compound 7 induced HepG2 cells autophagy.Moreover,compound 7 elevated ROS,intracellular Ca²⁺ overloaded,induced mitochondrial membrane potential and ATP level decrease.Compound 7 induced cancer cells G2/M phase arrest could be reversed by PTIO.Treated with compound 10,the results showed that apoptotic rate increased in a concentration- and time-dependent manner and compound 10 induced DNA fragmentation of HepG2 cells.In addition,compound 10 caused a loss of mitochondrial membrane potential in a time and dose-dependent manner,elevated ROS, intracellular Ca²⁺ overloading.These data suggest that compound 7 inhibited cancer cell proliferation and induced HepG2 cell autophagy.The mechanism was related to NO-induced mitochondrial dysfunction and G2/M phase arrest.Anticancer effects of compound 10 result from apoptosis induction,which may be related with ROS increasing,NO releasing,mitochondrial membrane potential dissipation.