三氧化二砷是我国在白血病治疗领域里的伟大发现，但由于其毒副作用大及先天或获得性耐药等原因，将其应用于临床肝癌治疗的研究一直停滞不前。小分子抑制剂Nutlin因可以激活野生型p53、p73及抑制P糖蛋白（Pgp）对药物转运而备受关注。课题组预实验中发现Pgp及MDM2高表达与p53突变可能导致肝癌砷剂耐受。针对砷剂治疗临床肝癌患者效果较差及国际上对突变p53功能研究已日趋成为热点的现状，本项目将以自主建立的肝癌砷剂耐药细胞系为平台，开展以下研究：应用基因测序法分析耐药细胞中p53突变情况；通过慢病毒干扰突变p53研究其在耐药中的作用；研究p73在砷剂诱导耐药细胞凋亡中的作用；砷剂联和Nutlin降解突变p53，抑制Pgp，激活p73进而逆转肝癌砷剂耐受的细胞及原位肝癌实验研究。本项目将首次从p53突变，p73受抑及Pgp上调的角度阐明肝癌砷剂耐受的机制，为实现砷剂对肝癌的成功治疗寻找新思路。

Arsenic trioxide (ATO) has been demonstrated as an effective anticancer drug against acute promyelocytic leukemia and other tumors in vitro and in vivo. As we know, most of the exogenous materials are metabolized in liver for the purpose of detoxication, and most inorganic arsenicals are also biotransferred in liver. These give us a sense that ATO would kill liver cancer cells as a result of aggregation of intracellular arsenicals. However, a phase II trial showed that single agent ATO is poorly active against advanced liver cancer, which might be the reason of primary or acquired drug resistance due to the overexpression of Pgp, MDM2 and mutation of p53 from our previous results. Unlike p53, another member of the p53 family-p73 is rarely mutated in liver cancer and few stimuli have been identified that induce p73 and apoptosis, including ATO. We hypothesize that the induction of p73 might be the mechanisms of the apoptosis mediated by ATO in p53 mutant liver cancer cells. However, mutant p53 and MDM2 could suppress the apoptotic function of p73, which might contribute to the ATO resistance. Importantly, another important function of mutant p53 is to upregulate the expression of Pgp. Based upon these evidence and our previous findings, we hypothesize that the problems of p53 mutation and drug efflux could be ideal targets to restore the sensitivity of liver cancer resistant cells to ATO. In the present study, using the ATO resistant cell lines we established, we will perform the following studies: 1) confirm the effects of p53 mutation on the sensitivity of liver cancer cells to ATO 2) investigate whether stepwise selections with increasing concentrations of ATO could induce the mutation and inactivation of p53 3) use Lenti-virus mediated specific shRNA targeting p53 to investigate the exact role of mutant p53 in ATO resistance 4) investigate the mechanisms of p73 in ATO mediated apoptosis in resistant cells 5) investigate whether Nutlin-3 could overcome the ATO resistance through the inhibition of mutant p53, Pgp and activation of p73 in vitro and in vivo (iorthotopic hepatic tumor in nude mice). We anticipate that Nutlin-3 could increase intracellular arsenicals through the inhibition of drug efflux of Pgp and induce mutant p53 degradation when combined with ATO. And the Nutlin-3/ATO combination might show the most effective anticancer effect in the liver cancer cells and could be a more potential way in reversal of liver cancer drug resistance.