【作者】 韩卫东；

【导师】 胡汛；

【作者基本信息】 浙江大学 ， 肿瘤学， 2008， 博士

【摘要】 研究背景肿瘤耐药性是化疗的主要障碍。抗癌药的药效取决于选择性杀死肿瘤细胞同时诱导后者产生耐药性的综合指数。临床化疗药,尽管有不同的靶位和机理,但均引起肿瘤细胞的凋亡。肿瘤细胞通常开始对凋亡诱导较敏感,但最终产生凋亡耐受,表现为抗凋亡蛋白的过表达和凋亡信号传导缺陷。大多数临床化疗药还是药物转运泵的底物,包括P-糖蛋白（P-glycoprotein,P-gp）,多药耐药相关蛋白1（multidrug-resistance associated protein,MRP1）,乳腺癌抗药蛋白（breast cancer resistance protein,BCRP）等。由于药物转运蛋白（如P-gp,MRP1）可以识别多种结构和功能不相关的化疗药,并有效地将后者排出细胞外,使得肿瘤细胞获得广谱的抗药性,称为多药耐药性（MultidrugResistance,MDR）。有些药物转运蛋白如P-gp,除了药泵功能外,还可以抑制caspase依赖的细胞凋亡。目前有几种药理学方法来逆转耐药。从理论上讲,逆转药泵介导的多药耐药是可能的,因为靶点明确且机理清楚。然而,这些特异性的药泵抑制剂在临床试验中药效不佳。克服肿瘤细胞凋亡耐受引起的耐药比逆转药泵要复杂的多。凋亡通路至少由几十个促凋亡和抑制凋亡的成员组成。促凋亡和抑制凋亡的平衡决定细胞生长和死亡的平衡。很多证据说明在肿瘤细胞中往往表现有抑凋亡因子活性升高,促凋亡因子活性下降。如抑制凋亡因子的表达增高（Bcl-2,Bcl-x_L,Mcl-1,c-FLIP,IAPs,heat shock proteins）,促凋亡因子的突变失活（P53,Apaf-1,Bax,FAS,FADD,caspase）,caspases的丢失（caspase 3,caspase 8）。此外,肿瘤细胞中的凋亡通路还被许多癌基因调控。因为存在如此多的潜在靶点,使得治疗凋亡耐受的肿瘤细胞变得非常困难。研究表明,肿瘤细胞内存在多种方式的死亡通路,凋亡不是唯一的程序性死亡方式。肿瘤细胞能通过各种不同的非凋亡性途径死亡,如坏死、衰老、自噬、及丝裂灾变等等。不同的死亡通路在分子机制上各不相同,肿瘤细胞为了躲避凋亡而设置的障碍对另外的死亡途径一般没有影响。因此,不管凋亡途径上有多少问题存在,也不管凋亡耐药是一个多么复杂,动态且难以捉摸的问题,如果一种药物可以引起肿瘤细胞非凋亡的死亡方式,并且这种药物不是P-gp等‘药泵’的诱导剂或底物,就有可能克服由凋亡耐受或P-gp等药泵引起的肿瘤耐药性,从而可以考虑为专门治疗耐药肿瘤的药物。研究目的1.研究紫草素及其衍生物是否具有良好的抗多药耐药肿瘤特性;2.明确紫草素及其衍生物克服肿瘤多药耐药的机理及细节,揭示其可以克服肿瘤多药耐药性的潜在规律;3.提出和证实靶向非凋亡死亡通路避开肿瘤耐药性的理论,并获得一类靶向该死亡通路的新型抗多药耐药肿瘤小分子药物。研究内容1.紫草素克服肿瘤细胞多药耐药性建立各种不同机制的多药耐药肿瘤细胞模型,用不同浓度紫草素处理后,计算其杀伤这些肿瘤细胞的IC₅₀,结果发现紫草素对耐药细胞的IC₅₀值和对亲本敏感细胞的IC₅₀值基本一致。2.紫草素专一性诱导细胞necroptosis克服肿瘤多药耐药性2.1紫草素专一性诱导肿瘤细胞necroptosis紫草素处理MCF—7细胞后,我们用不同实验发现以下结果:PI染色提示MCF-7细胞在死亡早期出现细胞膜破损,但未有细胞核固缩、断裂;透射电镜结果显示细胞内出现大量扩张的线粒体和自噬体;Caspase和凋亡诱导因子（AIF）不参与此死亡过程;JC-1染色后,流式细胞仪检测到细胞内线粒体膜电位迅速下降;小分子抑制剂necrostatin-1可以特异性地抑制细胞膜破损和线粒体膜电位下降;shRNA干扰Atg7基因不能抑制细胞死亡;荧光染料H₂DCFDA显示细胞内ROS水平升高,但活性氧清除剂不能抑制细胞死亡。同时我们发现:紫草素诱导的MCF-7耐药株（高表达P-gp的MCF-7/Adr、高表达BCRP的MCF-7/mx、高表达Bcl-2和Bcl-x_L的MCF-7/Bcl-2及MCF-7/Bcl-x_L）和HEK293及其耐药株细胞（高表达Bcl-2和Bcl-x_L的HEK293/Bcl-2及HEK293/Bcl-x_L）死亡可以被Nec-l所抑制。2.2 Necroptosis体内外克服肿瘤多药耐药性不同浓度紫草素处理MCF-7细胞及其耐药株MCF-7/Adr、MCF-7/mx、MCF-7/Bcl-2、MCF-7/Bcl-x_L细胞后,用PI拒染流式统计细胞死亡率,结果发现耐药株和亲本株的死亡率基本一致。我们进一步用MCF-7和MCF-7/Adr接种荷瘤鼠,2.5 mg/天腹腔连续给药5天,结果显示紫草素可以明显抑制体内肿瘤生长,对MCF-7的瘤重抑制率为43±10%,对MCF-7/Adr的瘤重抑制率为57±14%。透射电镜显示,紫草素引起了体内肿瘤necroptosis样细胞死亡。3.紫草素选择性诱导细胞necroptosis克服肿瘤多药耐药性3.1紫草素选择性诱导细胞凋亡与necroptosis1.25-5μM紫草素处理HL60等细胞后,引起细胞核固缩断裂,细胞内caspase激活,透射电镜显示凋亡样细胞死亡。10—20μM紫草素处理HL60等细胞后,引起的细胞死亡可以被Nec-1抑制,透射电镜示necroptosis样细胞死亡。3.2 Necroptosis避开耐药肿瘤细胞的凋亡耐受性不同浓度紫草素处理HL60及其耐药株（MRP1高表达细胞株HL60/Adr、Bcl-2高表达细胞株HL60/Bcl-2）和K562及其耐药株（P-gp高表达细胞株K562/Adr）,用Hoechst/台盼蓝染色统计细胞凋亡/坏死比率,结果发现:在诱导凋亡的浓度下,耐药细胞凋亡比率明显低于亲本敏感株;在诱导necroptosis的浓度下,耐药细胞死亡率等同于亲本敏感株。3.3紫草素选择性诱导原代白血病细胞necroptosis紫草素处理15例慢性粒细胞性白血病（CML）及急性白血病（AL）病人的原代肿瘤细胞,结果显示紫草素具有良好的抗肿瘤活性,其抗瘤机理为选择性地诱导肿瘤细胞凋亡或者necroptosis。4.Necroptosis分子机理初步探讨4.1 Necrostatin-1可以促进凋亡Nec-1预处理后,不同浓度紫草素处理HL60、HL60/Adr、K562、K562/Adr细胞,用Hoechst/台盼蓝染色统计细胞凋亡/坏死比率,结果发现Nec-1可以使紫草素诱导细胞凋亡的比率增加,坏死比率下降。进一步检测细胞内caspase活性,也显示Nec-1可以增加紫草素处理后细胞内caspase 3,8,9的活性。4.2线粒体在necroptosis过程中扮演的角色不同浓度紫草素处理HL60及HL60/Adr等细胞,JC-1染色后流式细胞仪统计发现高浓度紫草素可以引起线粒体膜电位迅速下降,Calcein-AM荧光淬灭实验和透射电镜结果都提示necroptosis过程中伴有线粒体内膜通透性增大。5.紫草素衍生物诱导肿瘤细胞necroptosis克服肿瘤细胞多药耐药性以紫草素作为先导化合物,设计合成14种衍生物。用该类小分子处理MCF-7细胞,结果发现其诱导的细胞死亡均可以被Nec-1抑制。我们用MTT的方法测定了此类小分子对K562、K562/Adr、MCF-7、MCF-7/Adr、HL60、HL60/Adr细胞的IC₅₀值,结果显示在敏感株和对应耐药株细胞上,其IC₅₀值基本一致。研究结论1.紫草素作为先导化合物可以避开多种机理引起的肿瘤细胞多药耐药。2.紫草素可以专一性地诱导一些肿瘤细胞necroptosis,如MCF-7和HEK293。3.紫草素可以选择性地诱导一些肿瘤细胞凋亡或necroptosis,如HL60和K562,其具体死亡方式选择呈现药物浓度依赖性。4.紫草素诱导的necroptosis可以避开P-gp、MRP1、BCRP等药泵高表达引起的肿瘤多药耐药性。5.紫草素诱导的necroptosis可以避开Bcl-2及Bcl-x_L高表达引起的肿瘤凋亡耐受。6.在紫草素选择性诱导肿瘤细胞necroptosis或凋亡的细胞模型上,Necrostatin-1可以抑制细胞necroptosis,促进细胞凋亡。7.紫草素诱导肿瘤细胞necroptosis过程中伴有线粒体内膜损伤。8.多种紫草素衍生物诱导的necroptosis可以避开肿瘤细胞多药耐药性。9.诱导necroptosis可能是一种克服由凋亡耐受及过表达药泵蛋白导致肿瘤多药耐药性的可选择方法。更多还原

【Abstract】 Background:Cancer drug resistance is a major problem in chemotherapy.The potency of anticancer drugs is largely determined by their efficacies in selectively killing cancer cells and simultaneously inducing drug resistance in cancer cells.Conventional anticancer agents,regardless of their targets and mechanisms,mostly induce apoptosis.Cancer cells are usually sensitive to apoptotic induction initially,but become resistant eventually through dysregulation of apoptotic machinery, manifested by overexpression of anti-apoptotic proteins and defect in apoptotic signaling.Numerous apoptotic inducers are also inducers and substrates of drug transporters,including P-glycoprotein（P-gp）,multidrug-resistance associated protein （MRP1）,and breast cancer resistance protein（BCRP）.Since these drug transporters recognize many structurally and functionally unrelated anticancer agents and efficiently expel intracellular drugs out of cells,the overexpression of these proteins confers cancer cells with a multidrug resistance.Some drug transporters such as P-gp also protect cancer ceils from caspase-dependent cell death.There are several potential pharmacological approaches to overcome cancer drug resistance.To overcome drug transporter（i.g.,P-gp）mediated drug resistance is theoretically achievable,because the targets are few and mechanisms are clear. However,clinically,the efficacies of the specific inhibitors to these drug transporters are yet not conclusive.To overcome the drug resistance relevant to apoptotic defect is much more complicated than to drug transporters.Apoptotic machinery is composed of at least dozens of anti-apoptotic and pro-apoptotic proteins.The balance of anti-and pro-apoptotic proteins contributes to the balance of cell growth and cell death.Many lines of evidence have demonstrated an imbalance with elevated anti-apoptotic and reduced pro-apoptotic activities in cancer cells one way or another,including overexpression of anti-apoptotic proteins（Bcl-2,Bcl-x_L,Mcl-1, c-FLIP,lAPs,heat shock proteins）,mutations of pro-apoptotic proteins（P53,Apaf-1, Bax,FAS,FADD,caspase）,and loss ofcaspases（caspase 3,caspase 8）.In addition, the apoptotic pathways in cancer cells are affected by many oncogenic signals. Therefore,it is highly difficult to treat cancers with apoptotic resistance because of so many potential targets.Many research showed that,besides apoptosis,there are several other programmed cell death pathway distinct form the former.Tumor cells can die by various non-apoptotic programmed cell death pathway,such as necrosis,senescence, autophagy and mitotic catastrophe etc.Since the molecular mechanisms of each death pathway are distinct from each other,no matter how dynamic the resistance occurred along apoptosis,it would be restricted within apoptotic pathway only,and would not affect other death pathways with mechanisms separate from apoptosis. So,it is likely to circumvent apoptotic resistance of cancer cells,if the action of chemotherapeutic agents is to induce non-apoptotic cell death.In addition,this kind of chemotherapeutic agents should not be the substrates of drug transporters（P-gp or MRP1 etc）.To develop such class of agents would be beneficial for treating MDR cancers,considering that there are no clinically available drugs against the latter.Objects:The objects of this study is to investigate（1）if shikonin,a naturally occurring naphthoquinone compound,or its analogues,could circumvent multiple drug resistance;（2）the mechanisms whereby shikonin and its analogues circumvent cancer drug resistance;（3）and the potential of this class of compounds in treating drug resistant cancers.Contents:1.Shikonin Overcomes Cancer Drug ResistanceWe established a variety of drug resistant cancer cell lines with overexpression of P-gp,MRP 1,BCRP,Bcl-2,or Bcl-x_L.After treated with shikonin for 72 hours, we calculated the IC₅0 values of these cancer cells.The results demonstrated that the IC₅0 values of drug resistant cancer cells and their parental sensitive cells were not significantly different from each other,indicating that these drug resistant factors did not hamper the efficacy of shikonin.2.Shikonin Circumvents Cancer Drug Resistance by Exclusive Induction of a Neeroptotic Death2.1 Shikonin Exclusive Induction of NecroptosisAfter treating MCF-7 cells with shikonin（2.5-20μM）,cells permeable to PI increased proportionally to the increment of both concentration and incubation time, indicating the loss of plasma membrane integrity.Hoechst staining showed dying cells did not exhibit apoptotic nuclear fragmentation.Ultrastructure of shikonin-treated cells demonstrated an extensive dilation of mitochondrion and formation of autophagosomes.The death was neither prevented by the pan-caspase inhibitor z-VAD-fmk nor involved a translocation of apoptosis inducing factor（AIF）. Shikonin caused a loss of mitochondrial membrane potential,which was probed by JC-1 and measured by FACScan.When cells were treated with shikonin in the presence of Nec-1,a small molecule specifically inhibiting necroptosis,cells with low mitochondrial membrane potential and positive PI were significantly reduced. The death rate of MCF-7 cells was not significantly inhibited by RNA interference of Atg7--a critical gene in autophagic death.Antioxidants could suppress DCFH oxidation in shikonin-treated cells,but did not prevent cellular PI permeability.Similarly,shikonin could induce a cell death of drug-resistant cells（MCF-7 and HEK293 overexpressing P-gp,BCRP,Bcl-2,or Bcl-X_L）which could be effectively prevented by Nec-1.2.2 Necroptosis Circumvents Cancer Drug Resistance in vitro and in vivoThe potency of shikonin against MCF-7/Adr,MCF-7/mx,MCF-7/Bcl-2, MCF-7/Bcl-x_L was not significant different from MCF-7 in a vitro study.Similar results were obtained with HEK293/Bcl-2 and HEK293/Bcl-X_L.In a pilot animal study,MCF-7 or MCF-7/Adr were injected（s.c.）into one flank of 5-week-old nude female mice.On day 7 after inoculation,mice received vehicle control or shikonin（2.5 mg/kg/d,5 days,i.p.）.The tumor weight inhibition rate of MCF-7 and MCF-7/Adr was 43±10%and 57±14%,respectively,clearly indicating that the potency of shikonin was not significantly affected by P-glycoprotein, consistent with the in vitro data.The dead cancer cells in the xenograft were morphologically similar to necroptosis.3.Shikonin Circumvents Cancer Drug Resistance by Alternative Induction of a Necroptotic Cell Death3.1 Shikonin alternatively induces Apoptosis or NecroptosisWhen treated with 1.25-5μM shikonin,HL60 cells exhibited apoptotic characteristics including nuclear fragmentation,caspase activation,and apoptotic morphology.While treated with 10-20μM shikonin,HL60 cells had necroptotic characteristics such as mitochondrion dilation and formation of autophagosomes,and cell death inhibitable by Nec-1.These results indicated that shikonin induced a dominant apoptotic death at low concentration and induce a dominant necroptotic death at high concentration in certain types of cells.Similar results were obtained with HL60/Adr,HL60/Bcl-2,K562 and K562/Adr.3.2 Necroptosis Circumvents Apoptotic Resistance in Tumor CellsAfter treated with various concentration of shikonin,we used Hoechst/tryphan blue staining method to calculate the apoptotic/necroptotic rates of HL60,HL60/Adr, HL60/Bcl-2,K562 or K562/Adr cells.The results demonstrated that drug resistant tumor cells were more resistant to shikonin-induced apoptosis but had the same susceptibility to shikonin-induced necroptosis versus its parental sensitive cells.3.3 Shikonin Alternatively Induces Necroptosis in Primary Leukemia Cells.Shikonin demonstrated a strong potency against primary leukemia cells which were isolated from bone marrow of 15 CML or AL patients.Like in HL60 or K562, shikonin can induce apoptosis or necroptosis in primary leukemia cells depending on the concentration.4.Preliminary Study on Molecular Mechanism of Necroptosis4.1 Neerostatin-1 Enhances Shikonin-induced ApoptosisAfter treated with shikonin in absence or presence of Nec-1,we calculated the apoptotic/necrotic rates of HL60,HL60/Adr,K562 or K562/Adr cells by Hoechst/tryphan blue stain.The results showed that there was less necroptotic but more apoptotic cell rate in Nec-1 pre-treated cells,which indicated that Nec-1 can convert shikonin induced necroptosis to apoptosis.Furthermore,we found the activity of caspase 3,8,9 in shikonin-treated cells was increased by Nec-1.4.2 Role of Mitochondrion in NecroptosisAfter HL60 or HL60/adr was treated with various concentrations of shikonin, we checked the mitochondria membrane potential（MMP）of cells probed with JC-1 by flow cytometry analyses.The results demonstrated that shikonin-induced neeroptosis was accompanied by a rapid MMP dissipation but not shikonin-induced apoptosis.This MMP dissipation was possibly resulted from inner mitochondrial membrane（IM）permeabilization,as supported by the calcein quenching assay and electron microscope.5.Shikonin Analogues Circumvents Cancer Drug Resistance by Induction of NeeroptosisFourteen shikonin analogues were chemically synthesized on the basis of shikonin as a leader.These chemicals,like shikonin,could overcome cancer drug resistance by induction of neeroptosis.We determined the IC₅₀values of K562, K562/Adr,MCF-7,MCF-7/Adr,HL60 and HL60/Adr against these agents by MTT assay.The results demonstrated that anti-cancer activity of these agents had no significant difference between drug resistant cell lines and parental drug-sensitive lines.Conclusions:1.Shikonin is a leader that has the capacity to circumvents cancer drug resistance.2.Shikonin can exclusively induce neeroptosis in some types of cancer cells,such as MCF-7 and HEK293.3.Shikonin can alternatively induce apoptosis or neeroptosis on cancer cells,such as HL60 and K562.The selection of cell death pathway is dependent on concentration of shikonin.4.Shikonin-induced necroptosis circumvents cancer drug resistance mediated by overexpression of drug transporters,such as P-gp,MRP 1 and BCRP.5.Shikonin-induced necroptosis circumvents cancer drug resistance mediated by apoptotic resistance.6.In the presence of necrostatin-1,a small molecule specifically inhibiting necroptosis,shikonin induced cell death could be converted to apoptosis.7.The mechanism whereby shikonin induces necroptosis is associated with the inner mitochondrial membrane permeabilization.8.Shikonin analogues circumvents cancer drug resistance by induction of necroptosis.9.We propose here necroptosis induction is an alternative choice to circumvents cancer drug resistance mediated by overexpression of drug transporter or apoptotic resistance.更多还原