【作者】 胡晨；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 外科学， 2009， 博士

【摘要】 肝细胞肝癌是世界上最为高发的恶性肿瘤之一,而且有超过半数患者在诊断时已经处于疾病晚期而失去手术治愈的机会。目前晚期患者治疗方案的选择相当有限,针对这些患者的药物治疗方案正在不断地研究和试验之中。肝癌细胞对化疗药物的耐受性常常导致常规化疗方案的失败,而肝癌细胞高表达ATP-bindingcassette(ABC)转运蛋白被认为是肿瘤耐药的主要原因之一。ABC转运蛋白造成耐药的主要机制是这些膜蛋白能够借助ATP水解的能量,将细胞内的多种药物分子主动地“泵”到细胞外,使肿瘤细胞内毒性药物的浓度降低而逃避多种药物的杀伤作用。ABCG2(或BRCP,乳腺癌耐药蛋白)是在阿霉素耐药的MCF-7乳腺癌细胞系中克隆得到的多药耐药基因,也是ABC转运蛋白家族的重要成员。既往研究已经证实ABCG2参与多种肿瘤多药耐药的形成,而且在肝癌组织中亦能检测到ABCG2的表达,但对ABCG2在肝癌耐药中的确切作用和机制还没有完全了解。近年来ABCG2蛋白在肿瘤中的作用随着侧群(Side population,SP)细胞和肿瘤干细胞研究的进展而愈加受到研究者重视。SP细胞的显著特点是能够外排Hoechst 33342染料而显示为低荧光染色,这一细胞亚群最早在骨髓造血细胞Hoechst染色的流式细胞分析中被发现和定义。随后研究发现,SP细胞不仅在正常组织中存在,而且在多种肿瘤中存在并具有肿瘤干细胞的特性。肿瘤干细胞通常在肿瘤中比例极小,却具有类似干细胞特性,能够保持自我更新,是肿瘤发生和复发的根源。ABCG2转运蛋白是Hoechst 33342染料的外排泵,该蛋白的高度表达是SP细胞外排能力的基础,同时也赋予SP细胞多药耐药的能力。实验研究已经证实SP细胞亚群存在于肝癌中,并提示这一细胞亚群可能是肝癌细胞致瘤能力和耐药特性的根源。然而ABCG2表达和SP细胞亚群在肝癌多药耐药中的确切影响和功能调控,目前的了解还不够充分,因此我们着力于深入地研究这些问题。在本研究中,我们采用较大样本量的肝癌组织微阵列来检测ABCG2在肝癌中的表达水平,发现ABCG2在肝癌组织中的表达水平呈现零散型集中表达和弥漫型大量表达等不同的亚型,而且临床病理指标分析提示弥漫型高表达ABCG2可能与肝硬化的基础疾病密切相关。我们的结果提示ABCG2的表达并不一定局限于少量的“干细胞”亚群,而可能存在过度的弥漫型表达,而这一类型恰与肝癌组织的内在性耐药特性十分符合。我们进一步在肝癌细胞系中研究ABCG2表达和SP细胞亚群的情况,发现肝癌细胞系中也同样存在不同类型的ABCG2表达,而ABCG2的表达水平与各细胞系中SP细胞亚群的比例及阿霉素的外排能力呈正相关。在ABCG2高表达的MHCC-97L肝癌细胞系中,我们发现SP细胞亚群的比例代表了细胞总体中具有外排活性的细胞数量,也能够反映了ABCG2阳性细胞转运活力的变化。采用SP检测和流式细胞分析术,我们观察到血清刺激能够增加MHCC-97L肝癌细胞系中SP细胞的比例,而且SP细胞亚群具有更高的PI3K/Akt通路活性。进一步实验则发现采用药物抑制或SiRNA干扰PI3K/Akt通路的活性能够减少SP细胞的比例。对MHCC-97L细胞SP及non-SP亚群进行PI3K/Akt通路活性和ABCG2表达的研究之后,我们发现PI3K/Akt通路活性与SP细胞中ABCG2的细胞内定位相关,即PI3K/Akt通路可能通过改变ABCG2的亚细胞定位来调控SP细胞的外排能力。我们的发现提示抑制或干扰PI3K/Akt通路的活化,可以减弱ABCG2阳性细胞的药物外排能力。我们在体外实验中也证实LY294002和Rapamycin等PI3K/Akt/mTOR通路抑制剂可以增强阿霉素对MHCC-97L肝癌细胞的杀伤作用,为Rapamycin等分子靶向药物与传统化疗药物联合应用于肝癌治疗提供了实验依据。本研究更全面地提供了ABCG2在肝癌组织中的表达情况,强调了ABC转运蛋白的表达水平对肝癌内在耐药特性的影响;而在MHCC-97L肝癌细胞系中的实验揭示了SP细胞ABCG2功能活性的变化以及PI3K/Akt通路对其调控的可能机制,为针对ABC转运蛋白外排活性的药物治疗策略提供了新的思路和初步的实验依据。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of most common cancers worldwide, and a large proportion of HCC patients present with late stage cancer at diagnosis. Currently, the treatment is limited for patients with advanced HCC, and the drug resistance to conventional chemotherapy is a major impediment to successful treatment. In HCC, one of leading causes for chemotherapeutic failure has been attributed to the fact that a large proportion of cancer cells express relatively high levels of several ATP-binding cassette (ABC) transporters, which actively "pump" out a broad spectrum of clinical relevant compounds and decrease the intracellular drug accumulation. The ABCG2 transporter (ATP-binding cassette subfamily G, member 2), also known as breast cancer resistance protein (BCRP), was first identified in a doxorubicin selected drug-resistant MCF7 cancer cell line and have been shown to mediate multiple drug resistance (MDR) in various cancers. Previous studies found the ABCG2 expression was upregulated HCCs, suggesting the role of ABCG2 in protecting HCC cells against chemotherapy.Recently, the ABCG2 transporter and its function in Hoechst dye efflux were identified in the "side population" cells in many cancers. The side population, a rare subset of cells distinguished by their low Hoechst dye staining in flow cytometry, was first described by Goodell et al in identifying hematopoietic stem cells in bone marrow. Unlike the common methods by recognizing cell surface markers, side population is a functional phenotype that defined by the cells’ performance in "pumping" out DNA binding dye Hoechst 33342. Later studies in leukemia and some solid tumors found this population could be a source of cancer stem cells, though it appeared heterogeneous in terms of cell surface marker profile. Current evidences support ABCG2 is the primary transporter for the Hoechst dye efflux, and the elevated ABCG2 expression on side population was proved to confer intrinsic resistance to chemotherapeutic agents.In HCC cell lines, previous studies had reported the existence of a distinct side population displaying extreme tumorigenicity. However, ABCG2 expression, side population and their relevance to chemotherapy resistance in HCC still need further investigation. In this study, we examined the ABCG2 expression patterns in HCC tissues, and found the diffused ABCG2 expression pattern suggesting the existence of intrinsic drug resistance in HCC. We demonstrated that the ABCG2 expression patterns had great impact on side population fraction and doxorubicin efflux transport in HCC cell lines, especially in the MHCC-97L cell line with intrinsic ABCG2 expression. Further, we found the serum stimulation could elevated the SP proportion in MHCC-97L cells, and the isolated MHCC-97L SP cells exhibited relatively higher Akt signaling activities than the non-SP cells, which prompted us to study the contribution of Akt signaling to the SP fraction. The results showed the Akt signaling blockade by inhibitiors or by siRNA approach could significantly modulate the SP proportion in the MHCC-97L cell line. Further investigation revealed that the Akt signaling inhibition could regulate ABCG2 function by promoting its subcellular redistribution. Moreover, we found inhibiting Akt signaling in MHCC-97L cells could block the doxorubicin efflux and potentiate the drug efficacy on cancer cells. Our findings would improve the existing understanding for ABCG2 and its functional modulation in HCC cells, and provide some clues for clinical regimens targeting HCC cells with inherent drug resistance.更多还原