Abstract 3546: DNA-damaging reagent -0404 effectively inhibits hepatocellular carcinoma through p53-dependent autophagy

Background On a worldwide basis, the prevalence of hepatocellular carcinoma (HCC) is twice as much in the past two decades, which is the third most common cause in cancer deaths. Therefore there is still an urgent need for new drugs with greater validity against HCC. Autophagy, which is essential in the intracellular clearance of many proteins and organelles, has been closely related to cancer generation and development. Generally, p53 is recognized as a pro-autophagic factor in a transcription-dependent pattern and also an important factor in the progress of HCC. As a selective substrate for autophagy, p62 is a key factor in tumorigenesis on account of its crucial roles in the control of cellular events. Now that small molecule compound-0404 was defined as a DNA-damaging reagent by the p53 pathway, our work aims to examine whether 0404 can affect p53-dependent autophagy and its positive effect in the treatment of HCC. Methods The anti-cancer activity of 0404 was examined in vitro. In the HepG2 cells and Huh7 cells, the apoptosis was detected by Flow Cytometry, while cell viability was measured by the CCK8 Assay. The Hela eGFP-LC3 cells, which express fusion protein combined light chain 3 (LC3) with enhanced Green Fluorescent Protein (eGFP), were treated with 0404 at various concentrations for various time points. HepG2 cells were treated with 0404 at various concentrations (5.0 nM, 20 nM, and 50 nM) for 24h. Autophagy was identified by eGFP-labeled LC3. And the expression levels of autophagy-associated proteins, such as mammalian target of rapamycin (mTOR), phospho-mTOR (p-mTOR), p62 and LC3B, were tested by Western Blotting. Results We confirmed that 0404 could inhibit the proliferation of HepG2 and Huh7 in a dose-dependent manner, and compared to Huh7 cells with mutant p53, HepG2 cells with wild-type p53 were more sensitive to 0404 treatment for apoptosis. We also found that 0404 promoted the development of autophagy in a dose- and time-dependent manner. Compared with untreated group, LC3 in 0404-treated groups resided primarily in the cytoplasm including cytoplasmic puncta indicative of autophagosomes. Simultaneously, the expression levels of mTOR and p62 were down-regulation, while the LC3B expression level was up-regulation by 0404-treatment. And the expression level of p-mTOR was down-regulation in a dose-dependent in HepG2 cells. Conclusion These results show clearly that DNA-damaging reagent-0404 can effectively inhibit HCC through p53-dependent autophagy. Acknowledgment This work was supported by the National Natural Science Foundation of China (81272679) and the Major national ST 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3546.