We found that NSC176327, but not ellipticine, induced p73 protein expression in p53-mutant and -null cells and knockdown of TAp73 reduced the cytotoxicity of NSC176327 in p53-null cells (Fig

We found that NSC176327, but not ellipticine, induced p73 protein expression in p53-mutant and -null cells and knockdown of TAp73 reduced the cytotoxicity of NSC176327 in p53-null cells (Fig.6). treatment caused a significant increase in p53-activated reporter signal in HCT116, SW620 and HCT116 p53/cells and upregulated DR5 and p21 Mupirocin protein expression. NSC176327 treatment also resulted in increased p73 protein expression and knockdown of transactivating isoforms of p73 in HCT116 p53/cells showed significant resistance to drug treatment. These results demonstrate an important role of p73 in the anti-tumor effects of NSC176327, and suggest that a close analog of ellipticine may act by a non-genotoxic mechanism targeting the p53/p73 pathway as compared with the original parent compound that targets the same pathway. Keywords:NSC176327, ellipticine, p53, p73, colon cancer, HCT116, SW620 == Introduction == p53 has been the subject of intense study over the past 30 y because of the findings that more KITH_HHV1 antibody than half of all human tumors have Mupirocin lost normal p53 gene function.1The tumor suppressor role of p53 is mainly mediated by its response to stress signals and functions as a transcription factor to induce target genes.2p53 can be activated by DNA damage, hypoxia or oncogenes3and orchestrates a number of cellular effects by transactivating genes such asp21which is involved in cell cycle arrest;4p53R2andp48which are involved in DNA repair5and apoptotic genes likeBax,Death Receptor 5 (DR5),PumaandNoxa.6-8Mutant p53 or defective p53-depedent pathways are associated with tumor development and resistance to conventional radiation and chemotherapy.9Therefore p53 and p53-dependent pathways are attractive therapeutic targets for cancer therapy. In recent years several strategies have been developed to target p53 including: (1) delivery of wild-type p53 to cancer cells by gene therapy;10(2) elimination of mutant p53 with adenovirus such as ONYX-015, which is undergoing clinical trials;11(3) restoration of wild-type function of mutant p53 which led to discovery of small-molecule drugs including CP-31398,12and PRIMA-1,13and (4) inhibition of p53 degradation by small-molecule drugs such as nutlins which inhibit p53 binding to E3 ligase mouse double minute 2 (MDM2).14Of note, p73, a p53 family protein that shares significant sequence homology with p53 Mupirocin and transactivates p53 target genes, has recently become an interesting therapeutic target.15p73 has been shown to Mupirocin substitute for p53 function in p53 mutant cells and peptides and small molecules that activate p73 were reported to suppress mutant p53-bearing tumor cells in vitro and in mouse xenografts.16,17 The discovery of novel therapeutic agents will rely on not only further understanding of p53-dependent pathways but also technology advancement. For example, we previously developed a real-time noninvasive bioluminescence imaging strategy of p53 transcriptional activity in vitro and in vivo.18Utilizing this high through-put imaging system, we performed a screen for small molecules that trigger p53-like transcriptional responses in p53-deficient tumor cells with National Cancer Institute (NCI) diversity set compounds and identified a group of small molecules that activate p53-responsive transcriptional activity in p53-deficient SW480 colon cancer cells.19However, the anti-tumor effects and mechanisms of these compounds remain to be fully examined. Therefore in this study we evaluated the anti-tumor potential of selected compounds by assessing viability and apoptosis in tumor cell lines after compound treatment. We also determined whether DNA damage signaling was involved by monitoring protein expression of H2AX after compound treatment. We found that NSC176327, a derivative of ellipticine, exhibited potent anti-neoplastic effects in vitro. Ellipticine, 5,11-dimethyl-6H-pyrido[4,3-b] carbazole, was originally isolated from the leaves of the evergreen treeochrosia ellipticaLabill. Ellipticine was found to possess great anti-cancer activity in a variety of tumors.20In addition, several derivatives of ellipticine have been identified or synthesized and demonstrated to be active against brain tumors.21Although the exact mechanism of action remains unclear, ellipticine was shown to intercalate with DNA and inhibit topoisomerase II activity.22However, recently a number of studies suggested that alternative mechanisms also play an important role in ellipticines anti-neoplastic effects and that derivatives of ellipticine do not necessarily act by the same mechanism as the.