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。
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.
引文
[1]Jemal A,Siegel R,Ward E,et al.Cancer statistics,2006[J].CA Cancer J Clin,2006;56(2):106-130.
[2]El-Serag H B and Rudolph K L.Hepatocellular carcinoma:epidemiology and molecular carcinogenesis[J].Gastroenterology,2007;132(7):2557-2576.
[3]Hussain S A,Ferry D R,El-Gazzaz G,et al.Hepatocellular carcinoma[J].Ann Oncol,2001;12(2):161-172.
[4]Llovet J M and Bruix J.Molecular targeted therapies in hepatocellular carcinoma[J].Hepatology,2008;48(4):1312-1327.
[5]Llovet J M,Di Bisceglie A M,Bruix J,et al.Design and endpoints of clinical trials in hepatocellular carcinoma[J].J Natl Cancer Inst,2008;100(10):698-711.
[6]Zhu A X.Systemic therapy of advanced hepatocellular carcinoma:how hopeful should we be?[J].Oncologist,2006;11(7):790-800.
[7]Dean M,Fojo T,and Bates S.Tumour stem cells and drug resistance[J].NatRev Cancer,2005;5(4):275-284.
[8]Llovet J M,Ricci S,Mazzaferro V,et al.Sorafenib in advanced hepatocellular carcinoma[J].N Engl J Med,2008;359(4):378-390.
[9]Wilhelm S,Carter C,Lynch M,et al.Discovery and development of sorafenib:a multikinase inhibitor for treating cancer[J].Nat Rev Drug Discov,2006;5(10):835-844.
[10]Hayes J D and Wolf C R.Molecular mechanisms of drug resistance[J].Biochem J,1990;272(2):281-295.
[11]Borst P and Elferink R O.Mammalian ABC transporters in health and disease[J].Annu Rev Biochem,2002;71:537-592.
[12]Leslie E M,Deeley R G,and Cole S P.Multidrug resistance proteins:role of P-glycoprotein,MRP1,MRP2,and BCRP (ABCG2) in tissue defense[J].Toxicol Appl Pharmacol,2005;204(3):216-237.
[13]Gillet J P,Efferth T,and Remacle J.Chemotherapy-induced resistance by ATP-binding cassette transporter genes[J].Biochim Biophys Acta,2007; 1775(2):237-262.
[14]Chandra P and Brouwer K L.The complexities of hepatic drug transport:current knowledge and emerging concepts[J].Pharm Res,2004;21(5):719-735.
[15]Hyde S C,Emsley P,Hartshorn M J,et al.Structural model of ATP-binding proteins associated with cystic fibrosis,multidrug resistance and bacterial transport[J].Nature,1990;346(6282):362-365.
[16]Konig J,Rost D,Cui Y,et al.Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane[J].Hepatology,1999;29(4):1156-1163.
[17]Grude P,Conti F,Mennecier D,et al.MDR1 gene expression in hepatocellular carcinoma and the peritumoral liver of patients with and without cirrhosis[J].Cancer Lett,2002;186(1):107-113.
[18]Silverman J A and Schrenk D.Hepatic canalicular membrane 4:expression of the multidrug resistance genes in the liver[J].FASEB J,1997;11(5):308-313.
[19]Doyle L A,Yang W,Abruzzo L V,et al.A multidrug resistance transporter from human MCF-7 breast cancer cells[J].Proc Natl Acad Sci U S A,1998;95(26):15665-15670.
[20]Sarkadi B,Ozvegy-Laczka C,Nemet K,et al.ABCG2--a transporter for all seasons[J].FEBS Lett,2004;567(1):116-120.
[21]Robey R W,To K K,Polgar O,et al.ABCG2:a perspective[J].Adv Drug Deliv Rev,2009;61(1):3-13.
[22]Reya T,Morrison S J,Clarke M F,et al.Stem cells,cancer,and cancer stem cells[J].Nature,2001;414(6859):105-111.
[23]Wicha M S,Liu S,and Dontu G.Cancer stem cells:an old idea--a paradigm shift[J].Cancer Res,2006;66(4):1883-1890;discussion 1895-1886.
[24]Hirschmann-Jax C,Foster A E,Wulf G G,et al.A distinct“side population”of cells with high drug efflux capacity in human tumor cells[J].Proc Natl Acad Sci U S A,2004;101(39):14228-14233.
[25]Goodell M A,Brose K,Paradis G,et al.Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo[J].J Exp Med, 1996;183(4):1797-1806.
[26]Challen G A and Little M H.A side order of stem cells:the SP phenotype[J].Stem Cells,2006;24(1):3-12.
[27]Kondo T,Setoguchi T,and Taga T.Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line[J].Proc Natl Acad Sci U S A,2004;101(3):781-786.
[28]Chiba T,Kita K,Zheng Y W,et al.Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties[J].Hepatology,2006;44(1):240-251.
[29]Allikmets R,Schriml L M,Hutchinson A,et al.A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug resistance[J].Cancer Res,1998;58(23):5337-5339.
[30]Rabindran S K,He H,Singh M,et al.Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C[J].Cancer Res,1998;58(24):5850-5858.
[31]Bailey-Dell K J,Hassel B,Doyle L A,et al.Promoter characterization and genomic organization of the human breast cancer resistance protein (ATP-binding cassette transporter G2) gene[J].Biochim Biophys Acta,2001;1520(3):234-241.
[32]Kage K,Tsukahara S,Sugiyama T,et al.Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization[J].Int J Cancer,2002;97(5):626-630.
[33]Xu J,Peng H,Chen Q,et al.Oligomerization domain of the multidrug resistance-associated transporter ABCG2 and its dominant inhibitory activity[J].Cancer Res,2007;67(9):4373-4381.
[34]Robey R W,Polgar O,Deeken J,et al.ABCG2:determining its relevance in clinical drug resistance[J].Cancer Metastasis Rev,2007;26(1):39-57.
[35]Mao Q and Unadkat J D.Role of the breast cancer resistance protein (ABCG2) in drug transport[J].AAPS J,2005;7(1):E118-133.
[36]Scharenberg C W,Harkey M A,and Torok-Storb B.The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors[J].Blood,2002;99(2):507-512.
[37]Zhou S,Schuetz J D,Bunting K D,et al.The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype[J].Nat Med,2001;7(9):1028-1034.
[38]Ho M M,Ng A V,Lam S,et al.Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells[J].Cancer Res,2007;67(10):4827-4833.
[39]Patrawala L,Calhoun T,Schneider-Broussard R,et al.Side population is enriched in tumorigenic,stem-like cancer cells,whereas ABCG2+ and ABCG2-cancer cells are similarly tumorigenic[J].Cancer Res,2005;65(14):6207-6219.
[40]Szotek P P,Pieretti-Vanmarcke R,Masiakos P T,et al.Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness[J].Proc Natl Acad Sci U S A,2006;103(30):11154-11159.
[41]Wang J,Guo L P,Chen L Z,et al.Identification of cancer stem cell-like side population cells in human nasopharyngeal carcinoma cell line[J].Cancer Res,2007;67(8):3716-3724.
[42]Zhang L,Hu J,Hong T P,et al.Monoclonal side population progenitors isolated from human fetal pancreas[J].Biochem Biophys Res Commun,2005;333(2):603-608.
[43]Wulf G G,Wang R Y,Kuehnle I,et al.A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia[J].Blood,2001;98(4):1166-1173.
[44]Shimano K,Satake M,Okaya A,et al.Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1[J].Am J Pathol,2003;163(1):3-9.
[45]Ros J E,Roskams T A,Geuken M,et al.ATP binding cassette transporter gene expression in rat liver progenitor cells[J].Gut,2003;52(7):1060-1067.
[46]Vander Borght S,Libbrecht L,Katoonizadeh A,et al.Breast cancer resistance protein (BCRP/ABCG2) is expressed by progenitor cells/reactive ductules and hepatocytes and its expression pattern is influenced by disease etiology and species type:possible functional consequences[J].J Histochem Cytochem,2006;54(9):1051-1059.
[47]Zen Y,Fujii T,Yoshikawa S,et al.Histological and culture studies with respect to ABCG2 expression support the existence of a cancer cell hierarchy in human hepatocellular carcinoma[J].Am J Pathol,2007;170(5):1750-1762.
[48]Yin S,Li J,Hu C,et al.CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity[J].Int J Cancer,2007;120(7):1444-1450.
[49]Krishan A,Sauerteig A,Gordon K,et al.Flow cytometric monitoring of cellular anthracycline accumulation in murine leukemic cells[J].Cancer Res,1986;46(4 Pt 1):1768-1773.
[50]Minderman H,Suvannasankha A,O'Loughlin K L,et al.Flow cytometric analysis of breast cancer resistance protein expression and function[J].Cytometry,2002;48(2):59-65.
[51]Zhou J,Wulfkuhle J,Zhang H,et al.Activation of the PTEN/mTOR/STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance[J].Proc Natl Acad Sci U S A,2007;104(41):16158-16163.
[52]Lee G Y,Kenny P A,Lee E H,et al.Three-dimensional culture models of normal and malignant breast epithelial cells[J].Nat Methods,2007;4(4):359-365.
[53]Haraguchi N,Utsunomiya T,Inoue H,et al.Characterization of a side population of cancer cells from human gastrointestinal system[J].Stem Cells,2006;24(3):506-513.
[54]Fojo A T,Shen D W,Mickley L A,et al.Intrinsic drug resistance in human kidney cancer is associated with expression of a human multidrug-resistance gene[J].J Clin Oncol,1987;5(12):1922-1927.
[55]Park J G,Kramer B S,Lai S L,et al.Chemosensitivity patterns and expression of human multidrug resistance-associated MDR1 gene by human gastric and colorectal carcinoma cell lines[J].J Natl Cancer Inst,1990;82(3):193-198.
[56]To K K,Zhan Z,and Bates S E.Aberrant promoter methylation of the ABCG2 gene in renal carcinoma[J].Mol Cell Biol,2006;26(22):8572-8585.
[57]Platet N,Mayol J F,Berger F,et al.Fluctuation of the SP/non-SP phenotype in the C6 glioma cell line[J].FEBS Lett,2007;581(7):1435-1440.
[58]Bonnet D.Normal and leukemic CD34-negative human hematopoietic stem cells[J].Rev Clin Exp Hematol,2001;5(1):42-61.
[59]Zhong Y,Zhou C,Ma W,et al.Most MCF7 and SK-OV3 cells were deprived of their stem nature by Hoechst 33342[J].Biochem Biophys Res Commun,2007;364(2):338-343.
[60]Simpson C,Pearce D J,Bonnet D,et al.Out of the blue:a comparison of Hoechst side population(SP) analysis of murine bone marrow using 325,363and 407 nm excitation sources[J].J Immunol Methods,2006;310(1-2):171-181.
[61]Montanaro F,Liadaki K,Schienda J,et al.Demystifying SP cell purification:viability,yield,and phenotype are defined by isolation parameters[J].Exp Cell Res,2004;298(1):144-154.
[62]Jordan C T.Cancer stem cells:controversial or just misunderstood?[J].Cell Stem Cell,2009;4(3):203-205.
[63]Mogi M,Yang J,Lambert J F,et al.Akt signaling regulates side population cell phenotype via Bcrp1 translocation[J].J Biol Chem,2003;278(40):39068-39075.
[64]Vivanco I and Sawyers C L.The phosphatidylinositol 3-Kinase AKT pathway in human cancer[J].Nat Rev Cancer,2002;2(7):489-501.
[65]Kim S K and Novak R F.The role of intracellular signaling in insulin-mediated regulation of drug metabolizing enzyme gene and protein expression[J].Pharmacol Ther,2007;113(1):88-120.
[66]Vlahos C J,Matter W F,Hui K Y,et al.A specific inhibitor of phosphatidylinositol 3-kinase,2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one(LY294002)[J].J Biol Chem,1994;269(7):5241-5248.
[67]Petroulakis E,Mamane Y,Le Bacquer O,et al.mTOR signaling:implications for cancer and anticancer therapy[J].Br J Cancer,2006;94(2):195-199.
[68]Hay N and Sonenberg N.Upstream and downstream of mTOR[J].Genes Dev,2004;18(16):1926-1945.
[69]Jiang Z Y,Zhou Q L,Coleman K A,et al.Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing[J].Proc Natl Acad Sci U S A,2003;100(13):7569-7574.
[70]Alessi D R,Andjelkovic M,Caudwell B,et al.Mechanism of activation of protein kinase B by insulin and IGF-1[J].EMBO J,1996;15(23):6541-6551.
[71]Song G,Ouyang G,and Bao S.The activation of Akt/PKB signaling pathway and cell survival[J].J Cell Mol Med,2005;9(1):59-71.
[72]Boyault S,Rickman D S,de Reynies A,et al.Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[J].Hepatology,2007;45(1):42-52.
[73]Bleau A M,Hambardzumyan D,Ozawa T,et al.PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells[J].Cell Stem Cell,2009;4(3):226-235.
[74]Foster L J,Li D,Randhawa V K,et al.Insulin accelerates inter-endosomal GLUT4 traffic via phosphatidylinositol 3-kinase and protein kinase B[J].J Biol Chem,2001;276(47):44212-44221.
[75]Kipp H and Arias I M.Newly synthesized canalicular ABC transporters are directly targeted from the Golgi to the hepatocyte apical domain in rat liver[J].J Biol Chem,2000;275(21):15917-15925.
[76]Gatmaitan Z C,Nies A T,and Arias I M.Regulation and translocation of ATP-dependent apical membrane proteins in rat liver[J].Am J Physiol,1997;272(5 Pt 1):G1041-1049.
[77]Misra S,Ujhazy P,Gatmaitan Z,et al.The role of phosphoinositide 3-kinase in taurocholate-induced trafficking of ATP-dependent canalicular transporters in rat liver[J].J Biol Chem,1998;273(41):26638-26644.
[78]Kipp H and Arias I M.Trafficking of canalicular ABC transporters in hepatocytes[J].Annu Rev Physiol,2002;64:595-608.
[79]Nakanishi T,Shiozawa K,Hassel B A,et al.Complex interaction of BCRP/ABCG2 and imatinib in BCR-ABL-expressing cells:BCRP-mediated resistance to imatinib is attenuated by imatinib-induced reduction of BCRP expression[J].Blood,2006;108(2):678-684.
[80]Semenza G L.Targeting HIF-1 for cancer therapy[J].Nat Rev Cancer,2003;3(10):721-732.
[81]Martin C M,Ferdous A,Gallardo T,et al.Hypoxia-inducible factor-2alpha transactivates Abcg2 and promotes cytoprotection in cardiac side population cells[J].Circ Res,2008;102(9):1075-1081.
[82]Krishnamurthy P,Ross D D,Nakanishi T,et al.The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with heme[J].J Biol Chem,2004;279(23):24218-24225.
[83]Reya T,Duncan A W,Ailles L,et al.A role for Wnt signalling in self-renewal of haematopoietic stem cells[J].Nature,2003;423(6938):409-414.
[84]Niwa H,Burdon T,Chambers I,et al.Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3[J].Genes Dev,1998;12(13):2048-2060.
[85]Kern S E and Shibata D.The fuzzy math of solid tumor stem cells:a perspective[J].Cancer Res,2007;67(19):8985-8988.
[86]Tang C,Ang B T,and Pervaiz S.Cancer stem cell:target for anti-cancer therapy [J].Faseb J,2007;21(14):3777-3785.
[87]Li L and Neaves W B.Normal stem cells and cancer stem cells:the niche matters[J].Cancer Res,2006;66(9):4553-4557.
[88]Pang R W and Poon R T.From molecular biology to targeted therapies for hepatocellular carcinoma:the future is now[J].Oncology,2007;72 Suppl 1:30-44.
[89]Mamane Y,Petroulakis E,LeBacquer O,et al.mTOR,translation initiation and cancer[J].Oncogene,2006;25(48):6416-6422.
[90]Kneteman N M,Oberholzer J,Al Saghier M,et al.Sirolimus-based immunosuppression for liver transplantation in the presence of extended criteria for hepatocellular carcinoma[J].Liver Transpl,2004;10(10):1301-1311.
[91]Treiber G.mTOR inhibitors for hepatocellular cancer:a forward-moving target [J].Expert Rev Anticancer Ther,2009;9(2):247-261.
[92]Piguet A C,Semela D,Keogh A,et al.Inhibition of mTOR in combination with doxorubicin in an experimental model of hepatocellular carcinoma[J].J Hepatol,2008;49(1):78-87.
[93]Ozben T.Mechanisms and strategies to overcome multiple drug resistance in cancer[J].FEBS Lett,2006;580(12):2903-2909.
[94]Juliano R L and Ling V.A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants[J].Biochim Biophys Acta,1976;455(1):152-162.
[95]Gros P,Croop J,and Housman D.Mammalian multidrug resistance gene:complete cDNA sequence indicates strong homology to bacterial transport proteins[J].Cell,1986;47(3):371-380.
[96]Gerlach J H,Endicott J A,Juranka P F,et al.Homology between P-glycoprotein and a bacterial haemolysin transport protein suggests a model for multidrug resistance[J].Nature,1986;324(6096):485-489.
[97]Chaudhary P M and Roninson I B.Expression and activity of P-glycoprotein,a multidrug efflux pump,in human hematopoietic stem cells[J].Cell,1991;66(1):85-94.
[98]Sato T,Laver J H,and Ogawa M.Reversible expression of CD34 by murine hematopoietic stem cells[J].Blood,1999;94(8):2548-2554.
[99]Beachy P A,Karhadkar S S,and Berman D M.Tissue repair and stem cell renewal in carcinogenesis[J].Nature,2004;432(7015):324-331.
[100]Lapidot T,Sirard C,Vormoor J,et al.A cell initiating human acute myeloid leukaemia after transplantation into SCID mice[J].Nature,1994;367(6464):645-648.
[101]Bonnet D and Dick J E.Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell[J].Nat Med,1997;3(7):730-737.
[102]Singh S K,Clarke I D,Terasaki M,et al.Identification of a cancer stem cell in human brain tumors[J].Cancer Res,2003;63(18):5821-5828.
[103]Al-Hajj M,Wicha M S,Benito-Hernandez A,et al.Prospective identification of tumorigenic breast cancer cells[J].Proc Natl Acad Sci U S A,2003;100(7):3983-3988.
[104]Dean M.ABC transporters,drug resistance,and cancer stem cells[J].J Mammary Gland Biol Neoplasia,2009;14(1):3-9.
[105]Modok S,Mellor H R,and Callaghan R.Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer[J].Curr Opin Pharmacol,2006;6(4):350-354.
[106]Roche-Lestienne C,Soenen-Cornu V,Grardel-Duflos N,et al.Several types of mutations of the Ab1 gene can be found in chronic myeloid leukemia patients resistant to STI571,and they can pre-exist to the onset of treatment[J].Blood,2002;100(3):1014-1018.
[107]Sell S.The hepatocyte:heterogeneity and plasticity of liver cells[J].Int J Biochem Cell Biol,2003;35(3):267-271.
[108]Sell S.Cellular origin of hepatocellular carcinomas[J].Semin Cell Dev Biol,2002;13(6):419-424.
[109]Brill S,Zvibel I,and Reid L M.Maturation-dependent changes in the regulation of liver-specific gene expression in embryonal versus adult primary liver cultures[J].Differentiation,1995;59(2):95-102.
[110]Shafritz D A and Dabeva M D.Liver stem cells and model systems for liver repopulation[J].J Hepatol,2002;36(4):552-564.
[111]Dabeva M D and Shafritz D A.Hepatic stem cells and liver repopulation[J].Semin Liver Dis,2003;23(4):349-362.
[112]Overturf K,al-Dhalimy M,Ou C N,et al.Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes[J].Am J Pathol,1997;151(5):1273-1280.
[113]Evarts R P,Nagy P,Nakatsukasa H,et al.In vivo differentiation of rat liver oval cells into hepatocytes[J].Cancer Res,1989;49(6):1541-1547.
[114]Theise N D,Saxena R,Portmann B C,et al.The canals of Hering and hepatic stem cells in humans[J].Hepatology,1999;30(6):1425-1433.
[115]Roskams T.Liver stem cells and their implication in hepatocellular and cholangiocarcinoma[J].Oncogene,2006;25(27):3818-3822.
[116]Clarke M F,Dick J E,Dirks P B,et al.Cancer stem cells--perspectives on current status and future directions:AACR Workshop on cancer stem cells[J].Cancer Res,2006;66(19):9339-9344.
[117]Ma S,Chan K W,Hu L,et al.Identification and characterization of tumorigenic liver cancer stem/progenitor cells[J].Gastroenterology,2007;132(7):2542-2556.
[118]Shmelkov S V,St Clair R,Lyden D,et al.AC133/CD133/Prominin-l[J].Int J Biochem Cell Biol,2005;37(4):715-719.
[119]Yin A H,Miraglia S,Zanjani E D,et al.AC133,a novel marker for human hematopoietic stem and progenitor cells[J].Blood,1997;90(12):5002-5012.
[120]O'Brien C A,Pollett A,Gallinger S,et al.A human colon cancer cell capable of initiating tumour growth in immunodeficient mice[J].Nature,2007;445(7123):106-110.
[121]Collins A T,Berry P A,Hyde C,et al.Prospective identification of tumorigenic prostate cancer stem cells[J].Cancer Res,2005;65(23):10946-10951.
[122]Craig C E,Quaglia A,Selden C,et al.The histopathology of regeneration in massive hepatic necrosis[J].Semin Liver Dis,2004;24(1):49-64.
[123]Yang Z F,Ho D W,Ng M N,et al.Significance of CD90+ cancer stem cells in human liver cancer[J].Cancer Cell,2008;13(2):153-166.
[124]Ma S,Lee T K,Zheng B J,et al.CD133(+) HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway[J].Oncogene,2007.
[125]Hill R P.Identifying cancer stem cells in solid tumors:case not proven[J].Cancer Res,2006;66(4):1891-1895;discussion 1890.
[126]Zhu L,Hu C,Li J,et al.Real-time imaging nuclear translocation of Akt1 in HCC cells[J].Biochem Biophys Res Commun,2007;356(4):1038-1043.