【作者】 张英；

【导师】 朴炳奎； 林洪生；

【作者基本信息】 中国中医科学院 ， 中西医结合肿瘤， 2009， 博士

【摘要】 最新研究发现,肿瘤干细胞的存在是肿瘤复发和转移的主要原因,它们在常规治疗后残存下来成为肿瘤复发和转移的种子细胞。怎样靶向调控肿瘤干细胞从而提高恶性肿瘤的治愈率是当前亟待研究的课题。长期的临床实践证明,中医中药在预防肿瘤复发专一方面有着独特的优势和特色。但是中医药预防肿瘤复发转移的具体机制至今尚不明了。所以本研究首次将肿瘤干细胞研究与中医药防治肿瘤研究结合在一起,旨在探索中医药是否能以肿瘤干细胞为靶点,通过控制肿瘤干细胞的生物学行为发挥其预防肿瘤复发转移的生物学效应。本研究根据肿瘤干细胞高表达ABCG2受体从而能外排核染料Hochest33342的特性来进行人乳腺癌细胞MCF-7细胞系肿瘤干细胞的分离纯化,以研究该细胞系肿瘤干细胞的体外增殖能力、再分化能力及与干细胞自我更新密切相关的Wnt信号转导通路中的信号物质的表达等相关生物学行为。由于中药苦参的有效成分氧化苦参碱(OM,OM)有抑制肿瘤的增殖和转移,诱导肿瘤细胞分化、促进肿瘤细胞凋亡等作用。所以在对肿瘤干细胞的生物学行为进行研究的基础上,本研究又进行了中药单体氧化苦参碱对MCF-7肿瘤干细胞生物学行为的干预作用的研究,以揭示氧化苦参碱对于实体肿瘤干细胞是否存在调控作用,并进一步揭示其干预调控作用是否与Wnt通路的活性有关,并进一步就其干预Wnt信号转导通路的分子水平作用机制进行研究,为该药特异性靶向调控肿瘤干细胞提供实验依据。1 MCF-7肿瘤干细胞的分离与生物学行为研究1.1方法取对数生长期MCF-7细胞消化为细胞悬液,分为两组,一组加入Hoechst33342至终浓度为5μg/ml,另一组同时加入维拉帕米至终浓度100uM,37℃水浴90min,加入PI至终浓度1μg/ml,流式细胞仪分析SP(side population,侧群细胞,肿瘤干细胞样细胞)亚群细胞含量,并据此进行SP亚群的分选培养,以研究SP亚群细胞的增殖能力,集落形成能力,再分化能力等细胞水平的生物学特点。并用Western-Blot,免疫荧光法检测细胞内总β-catenin的表达,流式细胞仪检测磷酸化β-catenin的表达水平,荧光定量PCR检测β-catenin、c-myc、cyclin D1等Wnt通路有关信号物质的mRNA水平等等。以明确肿瘤干细胞的Wnt信号转导通路的特征性表现。1.2结果MCF-7细胞系中SP细胞所占比例为4.02±0.18%;SP、non-SP(non-sidepopulation,非侧群细胞,非肿瘤干细胞样细胞)细胞的再培养提示在开始的两天二者增殖速度未见明显差异,而第3、4天SP细胞的增殖速度明显快于non-SP细胞,二者相比有显著性差异;集落形成能力提示,SP细胞的集落形成为30.67±2.57%,而non-SP细胞的集落形成能力13.33±1.76%,二者相比有显著性差异;在Wnt通路关键蛋白质β-catenin的表达方面,SP细胞中总β-catenin的表达显著高于non-SP细胞,而磷酸化β-catenin表达下调;与non-SP细胞相比,该通路的下游信号物质c-myc、cyclin D1的表达在SP细胞中亦呈上调。差异均有统计学意义。1.3结论MCF-7细胞系中包含SP亚群;SP细胞经培养后可继续分化成SP和non-SP细胞,说明其具有多向分化潜能;与non-SP细胞相比,具有更高的增殖能力,更强的自我更新能力;说明该细胞系的SP亚群可以代表该细胞系的肿瘤干细胞。而且Wnt通路在该细胞SP亚群细胞中呈活化状态,从另一个侧面说明该通路在肿瘤干细胞的自我更新等其它生物学行为的调控中起重要作用。2氧化苦参碱对MCF-7细胞生物学行为的调控作用2.1方法MTT法检测氧化苦参碱对MCF-7细胞系增殖能力的体外干预作用;AV-PI法进行氧化苦参碱诱导MCF-7细胞凋亡作用的研究;同时以在乳腺癌治疗中常用的DDP为对照,研究了氧化苦参碱对Wnt通路中的信号物质β-catenin,c-myc及cyclinD1的表达的干预作用。2.2结果氧化苦参碱在50ug/ml以上对MCF-7细胞的增殖有抑制作用,呈剂量及时间依赖性;该药能诱导MCF-7细胞的凋亡,表现为核染色质浓缩,核固缩及凋亡小体的生成等等;但这些作用均较DDP弱,然而与DDP相比,氧化苦参碱能明显下调Wnt通路的活性,如减少细胞内总β-catenin的表达,增加磷酸化β-catenin的表达,下调c-myc及cyclinD1的表达等等。2.3结论氧化苦参碱可以在体外抑制MCF-7细胞的增殖,诱导其凋亡,并能抑制Wnt通路的活性,这有可能是氧化苦参碱抑制MCF-7细胞增殖的原因。3氧化苦参碱对MCF-7细胞系肿瘤干细胞生物学行为的调控作用3.1方法分选得到SP细胞和non-SP细胞进行再培养,分别用MTT法研究氧化苦参碱对这两种不同细胞亚群增殖能力的体外干预作用;流式细胞法检测该药对MCF-7细胞系中SP细胞(干细胞池)数量的干预作用;软琼脂集落形成试验研究氧化苦参碱对不同细胞亚群的自我更新的干预作用;同时以DDP为对照,研究了氧化苦参碱对不同细胞亚群中Wnt通路中的信号物质β-catenin,c-myc及cyclinD1的表达的干预作用。3.2结果氧化苦参碱,DDP对不同的细胞亚群的生长均有抑制作用,然而与DDP比较,氧化苦参碱对SP细胞的抑制作用更强(P<0.01),对non-SP的抑制作用弱于DDP;在对SP细胞百分比(干细胞池)的影响方面,氧化苦参碱能明显减少MCF-7细胞系中SP细胞的含量,抑制率达到90%;与DDP相比,氧化苦参碱能明显干预SP细胞的克隆形成,减少细胞内总β-catenin的表达,增加磷酸化β-catenin的表达,下调c-myc及cyclinD1的表达等。3.3结论氧化苦参碱可以特异性地、剂量依赖地在体外抑制MCF-7细胞系中肿瘤干细胞样细胞(SP细胞)的增殖,减少SP细胞百分比,降低其自我更新能力,并能下调与肿瘤干细胞生物学行为密切相关的Wnt通路的活性,这提示氧化苦参碱有可能成为清除肿瘤干细胞的靶向药物。更多还原

【Abstract】 The cancer stem ceils(CSCs) play a critical role in both cancer initiation and relapse as they are resistant to most of cytotoxic agents and able to proliferate indefinitely.The plant alkaloid oxymatrine(OM) has many biological activities including the ability to induce cell cycle arrest and apoptosis,which makes it a potentially useful agent for targeting cancer cells.So in order to determine whether it has beneficial pharmacological properties to eradicate CSCs,we have analyzed the effects of OM on MCF-7 breast cancer cells.Cytotoxicity and cancer stem-like cells (side population,SP) identification and sorting were performed.Inhibitory effect was evaluated on the sorted SP and non-SP cells.SP cells were identified with percentage (4.02±0.18%) in MCF-7 cells.OM caused a dose-dependent reduction in the proliferation of MCF-7 cells and a decrease in SP cells relative to untreated controls. As conon-SPared to non-SP cells,inhibitory effect in the SP cells was higher.Wnt/β-catenin signaling pathway was also examined by analyzing the expression of total P-catenin and phosphorylated p-catenin in cytoplasm,OM treatment was associated with an inhibition of Wnt/p-catenin signaling pathway relative to untreated controls.These results indicate that the growth inhibitory effects of OM treatment on MCF-7 cells may be partly via effects on SP and Wnt/p-catenin signaling pathway. Further work is warranted to explore whether OM may be a useful novel therapeutic drug for targeting breast CSCs.1.Isolating and characterizing SP cells from MCF-7 cells1.1 METHODS:Cells were trpsinized and re-suspended at 10~6cells/ml in conon-SPlete RPMI1640.Hoechst33342 was added to a final concentration of 5μg/ml with or without 100uM verapamil hydrochloride and cells were incubated for 90 minutes in a 37℃water bath.The MCF-7 and the SP/non-SP cells from MCF-7 cultured 7 days were analyzed and sorted by FACS.β-catenin,phosphorylatedp-catenin,c-myc and cyclinDl were also detected by fluorescence,FACS,Western blot and anon-SPlisensor quantitative PCR among SP and non-SP,MCF-7 cells.1.2 RESULTS:SP is contained in MCF-7 for about 4.02±0.18%.The SP cells perform a higher speed proliferation rate and higher clone formation rate than non-SP cells,and SP cells can be separated into SP and non-SP cells after being cultured 7 days.There is no difference between the percentages of SP in the parental cells and the descendant cells.As we speculated,the expressions of p-catenin,c-myc and cyclinD1 were higher,and phosphorylated p-catenin was lower,as compared with non-SP cells.1.3 CONCLUSION:The percentage of SP in MCF-7 cells is 4.01±0.18%.The ability of SP cells developing into SP and non-SP means that SP is in the higher hierarchy.And the activity of Wnt signaling pathway is over-regulated in SP cells.2.The effects of OM on MCF-7 cells2.1 METHODS:MTT experiments were performed to determine whether OM can inhibit the viability of MCF-7 cells.In order to determine whether OM can induce the apoptosis of MCF-7 cells,the cells treated with different concentrations OM for 48h were stained with AV-PI,and analyzed by FACS,at the same time,the fluorescence microscope detection were performed,β-catenin,phosphorylated p-catenin,c-myc and cyclinD1 were also detected by fluorescence microscope,FACS,Western blot and anon-SPlisensor quantitative PCR among OM treated MCF-7 and the control one. Cisplatin was the positive control drug of these experiments.2.2 RESULTS:There was a dose and time depended anti-proliferative activity of OM ranging from 62.5ug/ml to 4mg /ml(Fig.4A) and 0.5mg/ml from 6 to 48h.The percentages of Phosphorylatedβ-catenin positive cells by FACS.The percentages were 26.26%,41.50%,and 62.77%in 0.25mg/ml,0.5 mg/ml and 1mg/ml OM treated cells and 18.52%of untreated cells,respectively.The expressions ofβ-catenin,c-myc and cyclinD1 were all decreased in the OM treated cells,as compared with untreated cells.2.3 CONCLUSION:OM could decrease MCF-7 cell viability,induce MCF-7 cells apoptosis,decrease the expression of totalβ-catenin and its nuclear location,decrease the mRNA of P-catenin,c-myc and cyclinD1 and increase the phosphorylatedβ-catenin in cytoplasm.Most of these effects were mediated in a dose-dependent manner.3 The effects of OM on cancer stem like cells(SP cells) from MCF-7 cells3.1 METHODS:MTT experiments were performed to determine whether OM can inhibit the viability of SP cells from MCF-7cells.In order to determine whether OM can decrease the percentage of SP cells in the total of MCF-7 cells,the cells treated with different concentrations OM for 48h were restained with Hochest33342 and analysis by FACS.At the same time,β-catenin,phosphorylatedβ-catenin,c-myc and cyclinD1 were also detected by fluorescence microscope,FACS,Western blot and anon-SPlisensor quantitative PCR among OM treated MCF-7 and the control one. Cisplatin was the positive control drug of these experiments.3.2 RESULTS:The inhibitory effects of OM on SP cells from MCF-7 cells were significant difference among different concentrations of OM.Conon-SPared with cisplatin,OM showed a higher inhibitory effect in breast CSCs(SP),and a lower inhibitory effect in breast cancer cells(non-SP).In OM-treated cells,the SP population was 3.1%,1.7%,and 0.2%,at 0.25mg/ ml,0.5mg/ml,1mg/ml OM, respectively.The staining intensity of cytoplasm and nuclearβ-catenin of the re-cultured SP cells treated with OM was significiantly decreased,andβ-catenin was typically found at the cell membrane only conon-SPared with controls.The expression of phosphorylatedβ-catenin of re-cultured SP cell treated with OM positive cells were increased and the expression of c-myc and cyclinD1 were all decreased,as compared with untreated cells,but this effect was not observed in cisplatin treated group. 3.3 CONCLUSION:This result showed that the breast cancer stem-like cells(SP) were more sensitive to OM than cisplatin,it leads to a dramatic decrease in the SP population and down regulating the activity of Wnt/β-catenin signaling pathway更多还原