突变p53及Pgp介导肝癌砷剂耐受的新推断与Nutlin靶向逆转耐药的实验研究

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.

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。

三氧化二砷是我国传统中药砒霜的主要成分，上世纪70年代初，哈尔滨医科大学张亭栋教授等人创造性地将其应用于急性早幼粒细胞白血病的治疗，取得显著疗效。与此同时，三氧化二砷对实体肿瘤的抑制作用吸引了国内外的广泛关注。然而，近期台湾学者发表的一项II期临床研究指出，单纯经静脉输注三氧化二砷对进展期肝癌患者的5年生存率没有影响，并得出单用三氧化二砷对此类肝癌患者无效的结论。如此尴尬的结论在使众多对三氧化二砷治疗肝癌报以希望的肿瘤学者们失望的同时，也使人们开始关注实体瘤对三氧化二砷耐药问题。针对三氧化二砷临床治疗肝癌的瓶颈局面及肝癌三氧化二砷耐药研究尚且缺乏的现状，本课题组在国内较早的把重点转移到各因素对三氧化二砷肝癌化疗敏感性及耐药机制研究上。本项目研究成果表明：①p53的获得性突变可能是导致肝癌三氧化二砷耐药主要原因，进一步机制可能是对Pgp的激活以及对p73的抑制，并筛选出了相应的功能性突变靶点；②发现低浓度三氧化二砷长期暴露可诱导肝癌细胞p53突变导致耐药，高浓度的三氧化二砷却可以降解突变p53，而Nutlin-3可以通过竞争性抑制Pgp对三氧化二砷的转运，升高细胞内三氧化二砷的浓度促进其对突变p53蛋白的降解，进而激活p73逆转肝癌对三氧化二砷的耐受。本项目通过对肝癌耐受三氧化二砷的研究探索肝癌的耐药特点，为寻找三氧化二砷肝癌化疗的方向提供线索，为传统中药对肝脏肿瘤的治疗寻求有效途径。本项目的主体研究成果现已发表于国际知名肿瘤学杂志Molecular Cancer, 影响因子5.3 (Mol Cancer. 2014 May 31;13:133.)。在该项目基金的支持下，本人以第一、并列第一及通讯作者完成的论文相继发表于Hepatology (Hepatology. 2014;59(3):935-46.)、Oncotarget (Oncotarget. 2015;6(19):17206-20.)、BMC Cancer (BMC Cancer. 2014;14:783.)、Oncotarget (Oncotarget. 2014;5(18):8478-91.)、Molecular Cancer (Mol Cancer. 2013;12(1):114. )、Scientific Reports(Sci Rep. 2015;5:17567.)等杂志。