Research On Anti-tumor Effect And Molecular Mechanisms Of A Novel Naphthalimide Derivative7d In P53-deficient K562Cells

P53is distinguished as the most frequently mutated gene in human cancer. Almost50%tumors have being p53mutated or lost. P53alterations have also been held responsible for the failure of most cancers to respond to chemotherapy. Naphthalimide anlogs have been considered as a promising group of anticancer agents by intercalating deoxyribonucleic acid. Though, earlier researches just focused on its anticancer effects in p53-wild cancer cells, little attention has been down on the underlying mechanism of anticancer effects of naphthalimide analogs in p53-deficient cells. Based on MTT and clone formation assay, a amonafide analogue,7d (2-(3-(2-(Dimethylamino)ethylamino)propyl)-6-(dodecylamino)-1H-benzo[de]is oquinoline-1,3(2H)-dione) with a high level of security exhibited high antitumor activity against p53-deficient human Chronic Myelogenous Leukemia K562cells.PI staining, Hoechst staining, DNA ladder and Annexin-V/PI staining assay showed that7d could cause an appreciable arrest in the G2/M phase and apoptosis in a dose-and time-dependent manner. Western blotting assay showed that p73/p21/cyclinBl pathway mediated G2/M arrest, and the apoptotic effect of7d should owing to mitochondria related proteins such as bax/bcl-2, cyt c, caspase-3increase and NF-κB nuclear translocation inhibition. E2F1could substitued p53to arrest cell cycle and induce apoptosis in p53deficient cells. To further investegated the E2F1roles in7d induced anticancer effects in p53-deficient K562cells. Western and real-time PCR assays were used to detect the E2F1protein and mRNA change. In our current study, both E2F1and E2F1-dependent down-stream proteins levels and mRNA levels were increased in K562cells. Moreover, the depletion of E2F1decreased p73, Apaf-1and p21Cip1/WAF1expression, reactivated cell cycle progression and inhibited7d-induced apoptosis in K562cells. The above results revealed E2F1/p73/p21and E2F1p53-independent apoptosis pathway are involved in G2/M arrest and apoptosis by7d. ATM/ATR singnaling pathway regulates E2F1stability and moreover connected E2F1induction with apoptosis in response to DNA damage. In our recent research,7d treated K562cells showed significant’comet’ migration pattern characteristic and an increase of y-H2AX. Pretreatment of caffeine, the ATM/ATR inhibitor antagonized7d induced G2-M arrest and apoptosis, abated H2AX-ser139phosphorylation, E2F1-ser364phosphorylation and E2F1accumulation.Moreover, E2F1-ser364phosphorylation disrupted endogenous E2F1/HDM2 binding. The results indicated that ATM/ATR pathway should be responsible for E2F1signaling activation.In a word, E2F1apoptotic signaling pathway mediated7d induced G2/M arrest and apoptosis, then ATM/ATR signaling act upstream signal in activating E2F1.