Research On Anti-Tumor Effect And Molecular Mechanisms Of A Novel Naphthalimide Derivative B1

Amonafide, a naphthalimide derivative, although selected for exploratory clinical trials for its potent anticancer activity, has long been challenged by its unpredictable side effects. In the course of screening a series of amonafide analogues, B1 (N-(2-(Dimethylamino) ethyl)-2-aminothiazonaphthalimide) in this series that significantly caused damage on DNA and cancer cell growth was identified.Research and development of novel naphthalimide derivatives are of great significance for the development of new anticancer drugs and treatment methods. Our research was designed to elucidate the anti-tumor effect and mechanisms of a novel amonafide analogue B1. We assessed the anti-tumor activity of B1 on a panel of established cancer cell lines by MTT assay and colony forming assay, among which Hela human cervix carcinoma cells were the most striking. For the first time, our investigation demonstrates that B1 inhibited the growth of HeLa cells by the induction of cell cycle arrest and apoptosis. Analysis of flow cytometry and western blots of HeLa cells treated with B1 revealed an appreciable cell cycle arrest and apoptotic induction in dose and time-dependent manner via the p53-dependent pathway. Furthermore, the release of cytochrome c from mitochondria was detected using confocal microscopy in HeLa cells treated with B1. Accordingly, these data demonstrate that the anticancer activity of B1 is associated with the activation of p53 and the release of cytochrome c, which suggest that B1 might have therapeutic potential against cervix carcinoma as an effective lead compound.The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report here that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found significant increase of p53 binding to P2 promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2. Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment.In some preclinical systems, Bcl-2 overexpression has been shown to induce survival, or to restore the clonogenic potential of malignant progenitor cells. On the other hand, anti-apoptotic factors, including Bcl-2, impair the ability to achieve remission and cure with chemotherapy, protecting the tumor cells from the apoptotic effects of various anti-neoplastic agents. Therefore, overcoming the resistance conferred by anti-apoptotic factors such as Bcl-2 represents an attractive therapeutic strategy against leukemia cells. We examined whether B1 overcomes the resistance conferred by Bcl-2, since overcoming the resistance conferred by Bcl-2 represents an attractive therapeutic strategy against cancer. Our viability assay showed that B1 overcomes the resistance conferred by Bcl-2 in human promyelocytic leukemia HL60 cells. Various apoptosis assessment assays demonstrated that B1 overcomes the resistance conferred by Bcl-2 in HL60 cells by inducing apoptosis. Noticeably, we elucidated the marked downregulation of 14-3-3a protein by B1, indicating that B1 overcomes the resistance conferred by Bcl-2 in HL60 cells via 14-3-3σ. Analysis of ChIP assay indicated that MBD2 was associated with the methylated 14-3-3σpromoter-associated CpG Island, and thus, interfered with transcriptional activity of the methylated promoter. Furthermore, knock-down of MBD2 using siRNA transfection inhibited B1-induced apoptosis and overcome the resistance conferred by Bcl-2. Accordingly, these data demonstrated the involvement of MBD2 in B1-induced apoptosis and overcome the resistance conferred by Bcl-2. which suggested that MBD2 might guide the development of future anticancer agents.In conclusion, our study provides further insight into the molecular mechanism of a novel amonafide analogue B1. The results show that B1 induces HeLa and MCF-7 cells apoptosis via p53 activation and Bcl-2 reduction, and overcomes the resistance conferred by Bcl-2 in human promyelocytic leukemia HL60 cells by MBD2 and 14-3-3σ/Bad signal pathways. These results provide an experimental basis for the clinical studies of Bland further research of anti-tumor drugs.