4-HPR联合应用小白菊内酯对肝癌细胞凋亡的作用及分子机制研究
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摘要
细胞凋亡(apoptosis),又称程序性细胞死亡(programmed cell death,PCD),是由于细胞内外环境变化或死亡信号触发以及在基因调控下所引起的细胞主动死亡的过程。目前已证实多种基因参与了细胞凋亡的调控。研究表明,无论是体外的组织细胞培养还是体内动物实验,细胞凋亡存在于肝细胞癌(hepatocellularcarcinoma,HCC)的发生、发展过程中。现在肝癌的许多非手术性治疗方法,如经导管肝动脉栓塞化疗术(TACE)、经皮穿刺瘤内酒精注射术(PEI)、射频、微波固化(PMCT)、冷冻等都与肝癌细胞凋亡密切相关。因此,通过诱导促凋亡基因表达,可达到治疗目的。
4-羟基维胺酯(N-4-hydroxyphenyl retinoid,4-HPR)是一种类视黄醇的合成化合物,能抑制肿瘤的发展,促进肿瘤细胞凋亡,但其诱导细胞凋亡的机制,尚不清楚。NF-κB是一种重要的核转录激活因子,与肿瘤细胞的耐药性和抗凋亡作用密切相关。芳香植物小白菊(Tanacetumparthenium)以及它的主要生物活性成分一小白菊内酯(Parthenolide),具有抗菌和抗炎作用,并可抑制NF-κB的激活。尽管上述两种药物作用机理方面的研究已涉及到肿瘤细胞分化,信号转导以及逆转耐药等方面,但是4-HPR联合应用小白菊内酯对人肝癌细胞凋亡的作用及其分子机制尚未见报道。
本实验的目的是研究小白菊内酯对4-HPR诱导肿瘤细胞凋亡的影响,检测4-HPR诱导肝癌细胞凋亡中的差异表达基因,筛选小白菊内酯引起的细胞凋亡相关基因候选者并探索其作用途径,为今后肝癌的药物治疗提供依据。
本实验利用TUNEL法及流式细胞仪技术,采用Hep3B和SK-HEP-1两种肝癌细胞株,观察小白菊内酯联合应用4-HPR对肝癌细胞凋亡的作用,并利用凝胶电泳迁移率变化分析(electrophoretic mobility shift assay,EMSA)、抗体胶移位分析(supershift assay)和放射自显影技术检测NF-κB、NF-κB p65、NF-κB p50、c-Rel、p52及RelB结合蛋白的表达;利用DD—PCR技术检测4-HPR处理和未处理的Hep3B差异表达基因,进一步确认和筛选凋亡相关基因候选者,并对筛选的cDNA进行克隆,测定基因序列后利用基因库中的BLAST程序确认;利用半定量反转录酶聚合酶链反应(RT-PCR)和Northern blotting技术,观察4-HPR联合应用小白菊内酯引起人肝癌细胞凋亡过程中差异表达基因,并进行分析。5'-末端快速扩增法(rapid amplification of cDNA ends,RACE)制备上调基因ANKRD1,并克隆到pEGM-T载体,进行鉴定:利用PCR技术扩增ANKRD1基因的正(sense)反(antisense)方向基因,并通过基因重组技术连接到增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因融合表达载体pEGFPC3和pcDNA3.1/HisA载体。再采用脂质体转染法,将重组基因转入Hep3B和SK-HEP-1两种肝癌细胞株,同时在荧光显微镜下观察GFP的表达,对转染后的肝癌细胞用G418(表达载体带有neo基因片断,对G418具有抗药性)筛选阳性克隆并分析克隆集落形成。在荧光显微镜下标记发光良好的单个克隆,分离克隆继续扩增培养建立稳定表达ANKRD1基因的细胞系,同时利用Western blotting技术和免疫细胞化学染色方法检测目的基因蛋白的表达。在激光共聚焦显微镜(laser scanning confocal microscopy,LSCM)下,采用免疫荧光细胞染色法对Hep3B、SK-HEP-1、HepG2和Huh7肝癌细胞进行ANKRD1基因编码蛋白的定位和表达,并观察该基因高表达和低表达时细胞核形态学变化,并利用流式细胞仪检测高表达ANKRD1基因的细胞凋亡率。
本研究的主要实验方法与结果:
1.TUNEL法及流式细胞仪检测凋亡率:Hep3B和SK-HEP-1两种肝癌细胞株,分别给予4-HPR(10uM/ml)和4-HPR(10uM/ml)联合应用小白菊内酯(4uM/ml),72个小时后通过TUNEL染色检测凋亡发生率,可见其凋亡率随着药物的不同升高,4-HPR联合应用小白菊内酯的细胞凋亡率近2倍高于单用4-HPR组,P＜0.01。通过流式细胞仪可观察小白菊内酯增强4-HPR诱导的肿瘤细胞凋亡,M1期细胞凋亡率明显高于对照组和4-HPR组,P＜0.01,但不影响细胞周期阻滞。
2.利用EMSA方法检测药物处理后不同时间NF-κB的表达:4-HPR处理Hep3B和SK-HEP-1细胞0.2,0.5,1,3,6,12及24h后测定NF-κB表达,结果NF-κB随4-HPR作用时间延长增高。12h时NF-κB的活性最强。利用NF-κB家族特定抗体进行Supershift assay结果显示以NF-κB p65在4-HPR处理的Hep 3B细胞中高表达,之后观察4-HPR(10uM/ml)联合应用小白菊内酯(4uM/ml)对不同时间Hep3B肝癌细胞NF-κB的影响,结果小白菊内酯有效地阻断了4-HPR诱导的NF-κB活性,且12h时最明显。
3.利用DD-PCR法检测4-HPR诱导细胞凋亡过程中基因表达:提取4-HPR处理和4-HPR未处理的Hep3B细胞总RNA,利用DD-PCR技术对大约13000个不同cDNA片断进行对照分析,并筛选93个差异表达的cDNA,并克隆到TA载体,测定基因序列后利用基因库中的BLAST程序确认。
4.利用Northern blotting和RT-PCR进一步检测小白菊内酯调控的凋亡相关基因:对DD-PCR检测出的差异表达基因,通过Northern blot和RT-PCR技术进一步检测4-HPR联合应用小白菊内酯时差异表达基因,结果筛选了35个差异表达基因,由小白菊内酯上调18个基因(SNTB1,ALB,PSMD2,RPS23,DNMT,GADD153,SERPIND1,ARPP-19,ND2,ANKRD1和2个未知基因与对照组相比高表达1.5倍)和17个下调基因(Gas5,TF,ND3,CYR61,KTN1和5个未知基因与对照组相比低表达50%),其中GADD153,NACA,TF和CYR61已证明与NF-κB活性有关。
5.克隆基因及确定基因:35个凋亡相关基因,其中一个上调基因HA1A2从肝cDNA中分离克隆测序,结果与Gene Bank中ANKRD1一致,此基因在心肌也称为是CARP基因。
6.构建ANKRD1正(sense,S)反(antisense,AS)方向真核表达载体及在Hep3B和SK-HEP-1细胞内表达:成功克隆了ANKRD1全长基因,并利用PCR技术分别扩增ANKRD1 sense和antisense基因片断,与pEGFPC3和pcDNA3.1/HisA真核表达载体进行重组,成功构建了ANKRD1真核表达载体,且在Hep3B和SK-HEP-1细胞内获得表达,并通过Western blotting和荧光显微镜进行了验证。
7.细胞集落形成:将已构建的真核表达载体转染到Hep3B和SK-HEP-1细胞,用G418筛选阳性克隆时,观察到转染ANKRD1(S)的细胞克隆集落形成的细胞融合灶与pEGFPC3和pcDNA3.1/HisA空载体和ANKRD1(AS)相比减少约50%。
8.ANKRD1蛋白的定位及细胞形态改变:将末端标记有绿色荧光蛋白(GFP)的ANKRD1瞬间转染到Hep3B(S),Hep3B(AS)和SK-HEP-1,HepG2,Huh7细胞并利用鼠的抗ANKRD1的单克隆抗体进行免疫组织化学实验显示,GFP融合ANKRD1蛋白主要在胞浆内表达,在细胞核内很少表达;高表达细胞核膜浓缩、碎片状,引起细胞凋亡;瞬时转染pEGFPC3-ANKRD1基因的Hep3B细胞通过流式细胞仪分析细胞凋亡结果显示,空载体pEGFPC3转染细胞与对照组细胞的凋亡率相比未发生明显改变,但转染pEGFPC3-ANKRD1细胞凋亡率与空载体pEGFPC3转染细胞与对照组细胞相比有明显增高,增高至27.5±2.5%。
以上结果提示:
1.单独应用小白菊内酯不引起肝癌细胞凋亡,但与4-HPR联合应用可增强肝癌细胞凋亡,并且与细胞周期阻滞无关。
2.小白菊内酯增强4-HPR诱导的肝癌细胞凋亡作用与抑制NF-κB活性有关。
3.增加药物引起的细胞凋亡过程中,筛选出肝癌凋亡相关基因35个,其中一个上调基因HA1A2从肝cDNA中分离克隆并证明与ANKRD1一致。
4.成功克隆ANKRD1基因,并构建了ANKRD1真核表达载体及在Hep3B和SK-HEP-1细胞内获得表达,为今后ANKRD1与肿瘤细胞凋亡的进一步研究打下了基础。
5.ANKRD1具有引起肝癌细胞凋亡的潜能,有望成为肝癌治疗的分子作用靶点。
Fenretinide(N-4-hydroxyphenyl retinamide,4HPR),a synthetic anticancer retinoid,is a well-known apoptosis-inducing agent.Recently,we observed that the apoptosis induced by fenretinide could be effectively enhanced in hepatoma cells by concomitant treatment with parthenolide,a known inhibitor of nuclear factor NF-kB Furthermore,treatment with fenretinide triggered the activation of NF-kB.during apoptosis,an effect that could be substantially inhibited by parthenolide,which suggests that NF-κB activation during fenretinide-induced apoptosis might have an anti-apoptotic effect.In this study,we investigated the molecular mechanism of this apoptotic potentiation by NF-κB inhibition.Using the differential display-PCR (DD-PCR) method,and subsequent Northern blot or semiquantitative reverse transcriptase PCR(RT-PCR) analysis we identified genes which are involved in enhanced fenretinide-induced apoptosis by parthenolide.We identified 35 apoptosis-related genes including 12 unknown genes that are up- or down-regulated by parthenolide.Intriguingly,one up-regulated gene(HA1A2) was isolated and cloned from liver cDNA,and turned out to be identical to ANKRD1,which is also referred to as the CARP gene.Ectopic expression of ANKRD1 led to reduced colony formation and to enhanced apoptotic cell death in hepatoma cells,compared with controls treated with an empty vector or with antisense cDNA.These results imply that ANKRD1 and other genes whose expressions were substantially modulated by parthenolide-mediated inhibition of NF-κB activation may play roles in the enhanced drug-induced apoptosis and further,that those identified genes may be useful in anticancer strategies against hepatoma.
The results are as below:
1.Parthenolide enhances fenretinide-induced apoptotic cell death. apoptotic cell death was determined by TUNEL staining in Hep 3B cells and in SK-HEP-1 cells treated for 72 h with 10μM or 15μM fenretinide,respectively,in the presence or absence of 4μM parthenolide(P),or treated with vehicle alone.Vertical bars represent the means±SE of quadruplicate experiments.~(**)P＜0.01,compared with cells treated with fenretinide alone,quantitation of the apoptotic fraction by FCM analysis in Hep3B cells(upper panels) and SK-HEP-1 cells(lower panels) treated with 10μM fenretinide in the presence or absence of 4μM parthenolide(P) for 72 h,or were treated with vehicle alone.The Sub-G_1 fraction was estimates by gating hypodiploid cells in the histogram using the LYSISⅡprogram.DNA contents are plotted on the linear abscissa(M_1,apoptotic fraction).Each value represents the mean±SE of triplicate experiments.~(**) P＜0.01,compared with cells treated with fenretinide alone.
2.Parthenolide effectively inhibits NF-κB activation during fenretinide-induced apoptosis.
Hep 3B cells and SK-HEP cells(1×10~6) were treated with 10μM or 15μM fenretinide,respectively,for the indicated time periods,or were treated with vehicle alone.DNA-binding activity for NF-kB was detected by autoradiography. Experiments were performed at least three times in duplicate,and the result of one representative experiment is shown.
3.Differential display analysis of genes expressed in apoptotic Hep 3B cells. Differential display using three one-base anchored oligo-dT primers(H-T_(11)G,H-T_(11)A and H-T_(11)C) in combination with 80 arbitrary 13-mers.Representative patterns of differentially expressed genes were obtained from DD-PCR analysis using duplicate RIgA samples from control Hep 3B cells(C) and from apoptotic Hep 3B cells(A) induced by treatment with 10μM fenretinide for 72 h using H-T_(11)C in combination with 67-79th-arbitrary 13-mers.The HC70A1,HC71A1,HC75A1 and HC75C2 cDNA fragments are marked by arrows.
4.ANKRD1 overexpression during drug-induced apoptosis
Isolated ankyrin repeat domain 1/cardia ankyrin repeat protein gene(ANKRD1/CARP) cDNA and its up-regulation by the NF-kB inhibitor parthenolide.,Northern blot analysis of ANKRD1 in Hep 3B cells(upper panels) and in SK-HEP-1 cells(middle panels) treated either with fenretinide alone or in combination with parthenolide (4HPR + P) at the indicated time intervals,or treated with vehicle alone.Total RNAs were extracted and fractionated by electrophoresis on 1%agarose gels containing formaldehyde,and were then transferred to membranes.Each blot was stripped and subsequently rehybridized with a probe for 18S cDNA as a loading control. Experiments were performed at least three times,and the result of one representative experiment is shown.Relative densitometric plots on the quantification of ANKRD1 mRNA induction(%of control)(lower panel).
5.Decreased clonogenicity and apoptotic morphology by ectopic ANKRD1 overexpression.
6.Increased drug-susceptibility by ectopic ANKRD1 overexpression. Isolated ankyrin repeat domain 1/cardia ankyrin repeat protein gene(ANKRD1/CARP) cDNA and its up-regulation by the NF-kB inhibitor parthenolide C,immunoblot analysis of ANKRD1 expression in Hep 3B cells(upper panels) and in SK-HEP-1 cells(lower panels) treated either with fenretinide alone or in combination with parthenolide(4HPR + P) at the indicated time intervals,or treated with vehicle alone (C).Thirtyμg of extracted proteins were resolved by 12%SDS-PAGE and were transferred to the membrane.The blots were probed with a polyclonal antibody against ANKRD1 and were then stripped and reprobed with a monoclonal antibody to actin as a loading control.Experiments were performed at least three times,and the result of one representative experiment is shown.
7.We transiently transfected the cells with the ANKRD1 tagged at its amino terminus with the enhanced green fluorescent protein(GFP) and simultaneously performed immunohistochemistry with a mouse monoclonal antibody for ANKRD1. GFP fused to ANKRD1 protein expression is confined to mainly cytoplasm and less nuclei of the Hep 3B,SK-Hep1 and Hep G2,Huh7,which is perfectively merged with immunoreactivity for ANKRD1 expression.
8 The ectopic overexpression of ANKRD1 sensitizes the Hep 3B cells to drug-induced apoptosis.Ectopic over-expression of ANKRD1 sensitizes Hep 3B cells to drug-induced apoptosis.Expression of ANKRD1 protein in stably transfected Hep 3B cells with a sense GFP-ANKRD1(S1 and S6) were compared with antisense GFP-ANKRD1(AS8 and AS13) or with GFP vector control cells(VC2 and VC3) (upper).Ectopic,ectopic expression of ANKRD1 protein.Endogenous,endogenous expression of ANKRD1 protein.The stable transfectants and vector control cells were treated with 10 M fenretinide or 4 M parthenolide(P) for 48 h as indicated. Quantification of apoptotic fractions was performed using a FACScan.The sub-G_1 fraction was estimated by gating hypodiploid cells in the histogram using a C-30 program.DNA contents are plotted on the linear abscissa(M_1,apoptotic fraction). Each value represents the mean±SE of two independent experiments performed in triplicate±SE(~(**) P＜0.01 compared with control).
Our conclusions:
1.Parthenolide enhances fenretinide-induced apoptosis in hepatoma cells,The Sub-G_1 fraction was estimates by gating hypodiploid cells in the histogram using the LYSISⅡprogram.
2.Parthenolide inhibit NF-kB activation induced by fenretinide and enhance fenretinide-induced apoptosis.
3.Identified 35 apoptosis-related genes including 12 tmknown genes that are up- or down-regulated by parthenolide,one up-regulated gene(HA1A2) was isolated and cloned from liver cDNA,and turned out to be identical to ANKRD1,which is also referred to as the CARP gene.
4.Ectopic expression of ANKRD1 led to reduced colony formation and to enhanced apoptotic cell death in hepatoma cells.
5.These results imply that ANKRD1 and other genes whose expressions were substantially modulated by parthenolide-mediated inhibition of NF-kB activation may play roles in the enhanced drug-induced apoptosis and further,that those identified genes may be useful in anticancer strategies against hepatoma.
4-羟基维胺酯(N-4-hydroxyphenyl retinoid,4-HPR)是一种类视黄醇的合成化合物,能抑制肿瘤的发展,促进肿瘤细胞凋亡,但其诱导细胞凋亡的机制,尚不清楚。NF-κB是一种重要的核转录激活因子,与肿瘤细胞的耐药性和抗凋亡作用密切相关。芳香植物小白菊(Tanacetumparthenium)以及它的主要生物活性成分一小白菊内酯(Parthenolide),具有抗菌和抗炎作用,并可抑制NF-κB的激活。尽管上述两种药物作用机理方面的研究已涉及到肿瘤细胞分化,信号转导以及逆转耐药等方面,但是4-HPR联合应用小白菊内酯对人肝癌细胞凋亡的作用及其分子机制尚未见报道。
本实验的目的是研究小白菊内酯对4-HPR诱导肿瘤细胞凋亡的影响,检测4-HPR诱导肝癌细胞凋亡中的差异表达基因,筛选小白菊内酯引起的细胞凋亡相关基因候选者并探索其作用途径,为今后肝癌的药物治疗提供依据。
本实验利用TUNEL法及流式细胞仪技术,采用Hep3B和SK-HEP-1两种肝癌细胞株,观察小白菊内酯联合应用4-HPR对肝癌细胞凋亡的作用,并利用凝胶电泳迁移率变化分析(electrophoretic mobility shift assay,EMSA)、抗体胶移位分析(supershift assay)和放射自显影技术检测NF-κB、NF-κB p65、NF-κB p50、c-Rel、p52及RelB结合蛋白的表达;利用DD—PCR技术检测4-HPR处理和未处理的Hep3B差异表达基因,进一步确认和筛选凋亡相关基因候选者,并对筛选的cDNA进行克隆,测定基因序列后利用基因库中的BLAST程序确认;利用半定量反转录酶聚合酶链反应(RT-PCR)和Northern blotting技术,观察4-HPR联合应用小白菊内酯引起人肝癌细胞凋亡过程中差异表达基因,并进行分析。5'-末端快速扩增法(rapid amplification of cDNA ends,RACE)制备上调基因ANKRD1,并克隆到pEGM-T载体,进行鉴定:利用PCR技术扩增ANKRD1基因的正(sense)反(antisense)方向基因,并通过基因重组技术连接到增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因融合表达载体pEGFPC3和pcDNA3.1/HisA载体。再采用脂质体转染法,将重组基因转入Hep3B和SK-HEP-1两种肝癌细胞株,同时在荧光显微镜下观察GFP的表达,对转染后的肝癌细胞用G418(表达载体带有neo基因片断,对G418具有抗药性)筛选阳性克隆并分析克隆集落形成。在荧光显微镜下标记发光良好的单个克隆,分离克隆继续扩增培养建立稳定表达ANKRD1基因的细胞系,同时利用Western blotting技术和免疫细胞化学染色方法检测目的基因蛋白的表达。在激光共聚焦显微镜(laser scanning confocal microscopy,LSCM)下,采用免疫荧光细胞染色法对Hep3B、SK-HEP-1、HepG2和Huh7肝癌细胞进行ANKRD1基因编码蛋白的定位和表达,并观察该基因高表达和低表达时细胞核形态学变化,并利用流式细胞仪检测高表达ANKRD1基因的细胞凋亡率。
本研究的主要实验方法与结果:
1.TUNEL法及流式细胞仪检测凋亡率:Hep3B和SK-HEP-1两种肝癌细胞株,分别给予4-HPR(10uM/ml)和4-HPR(10uM/ml)联合应用小白菊内酯(4uM/ml),72个小时后通过TUNEL染色检测凋亡发生率,可见其凋亡率随着药物的不同升高,4-HPR联合应用小白菊内酯的细胞凋亡率近2倍高于单用4-HPR组,P＜0.01。通过流式细胞仪可观察小白菊内酯增强4-HPR诱导的肿瘤细胞凋亡,M1期细胞凋亡率明显高于对照组和4-HPR组,P＜0.01,但不影响细胞周期阻滞。
2.利用EMSA方法检测药物处理后不同时间NF-κB的表达:4-HPR处理Hep3B和SK-HEP-1细胞0.2,0.5,1,3,6,12及24h后测定NF-κB表达,结果NF-κB随4-HPR作用时间延长增高。12h时NF-κB的活性最强。利用NF-κB家族特定抗体进行Supershift assay结果显示以NF-κB p65在4-HPR处理的Hep 3B细胞中高表达,之后观察4-HPR(10uM/ml)联合应用小白菊内酯(4uM/ml)对不同时间Hep3B肝癌细胞NF-κB的影响,结果小白菊内酯有效地阻断了4-HPR诱导的NF-κB活性,且12h时最明显。
3.利用DD-PCR法检测4-HPR诱导细胞凋亡过程中基因表达:提取4-HPR处理和4-HPR未处理的Hep3B细胞总RNA,利用DD-PCR技术对大约13000个不同cDNA片断进行对照分析,并筛选93个差异表达的cDNA,并克隆到TA载体,测定基因序列后利用基因库中的BLAST程序确认。
4.利用Northern blotting和RT-PCR进一步检测小白菊内酯调控的凋亡相关基因:对DD-PCR检测出的差异表达基因,通过Northern blot和RT-PCR技术进一步检测4-HPR联合应用小白菊内酯时差异表达基因,结果筛选了35个差异表达基因,由小白菊内酯上调18个基因(SNTB1,ALB,PSMD2,RPS23,DNMT,GADD153,SERPIND1,ARPP-19,ND2,ANKRD1和2个未知基因与对照组相比高表达1.5倍)和17个下调基因(Gas5,TF,ND3,CYR61,KTN1和5个未知基因与对照组相比低表达50%),其中GADD153,NACA,TF和CYR61已证明与NF-κB活性有关。
5.克隆基因及确定基因:35个凋亡相关基因,其中一个上调基因HA1A2从肝cDNA中分离克隆测序,结果与Gene Bank中ANKRD1一致,此基因在心肌也称为是CARP基因。
6.构建ANKRD1正(sense,S)反(antisense,AS)方向真核表达载体及在Hep3B和SK-HEP-1细胞内表达:成功克隆了ANKRD1全长基因,并利用PCR技术分别扩增ANKRD1 sense和antisense基因片断,与pEGFPC3和pcDNA3.1/HisA真核表达载体进行重组,成功构建了ANKRD1真核表达载体,且在Hep3B和SK-HEP-1细胞内获得表达,并通过Western blotting和荧光显微镜进行了验证。
7.细胞集落形成:将已构建的真核表达载体转染到Hep3B和SK-HEP-1细胞,用G418筛选阳性克隆时,观察到转染ANKRD1(S)的细胞克隆集落形成的细胞融合灶与pEGFPC3和pcDNA3.1/HisA空载体和ANKRD1(AS)相比减少约50%。
8.ANKRD1蛋白的定位及细胞形态改变:将末端标记有绿色荧光蛋白(GFP)的ANKRD1瞬间转染到Hep3B(S),Hep3B(AS)和SK-HEP-1,HepG2,Huh7细胞并利用鼠的抗ANKRD1的单克隆抗体进行免疫组织化学实验显示,GFP融合ANKRD1蛋白主要在胞浆内表达,在细胞核内很少表达;高表达细胞核膜浓缩、碎片状,引起细胞凋亡;瞬时转染pEGFPC3-ANKRD1基因的Hep3B细胞通过流式细胞仪分析细胞凋亡结果显示,空载体pEGFPC3转染细胞与对照组细胞的凋亡率相比未发生明显改变,但转染pEGFPC3-ANKRD1细胞凋亡率与空载体pEGFPC3转染细胞与对照组细胞相比有明显增高,增高至27.5±2.5%。
以上结果提示:
1.单独应用小白菊内酯不引起肝癌细胞凋亡,但与4-HPR联合应用可增强肝癌细胞凋亡,并且与细胞周期阻滞无关。
2.小白菊内酯增强4-HPR诱导的肝癌细胞凋亡作用与抑制NF-κB活性有关。
3.增加药物引起的细胞凋亡过程中,筛选出肝癌凋亡相关基因35个,其中一个上调基因HA1A2从肝cDNA中分离克隆并证明与ANKRD1一致。
4.成功克隆ANKRD1基因,并构建了ANKRD1真核表达载体及在Hep3B和SK-HEP-1细胞内获得表达,为今后ANKRD1与肿瘤细胞凋亡的进一步研究打下了基础。
5.ANKRD1具有引起肝癌细胞凋亡的潜能,有望成为肝癌治疗的分子作用靶点。
Fenretinide(N-4-hydroxyphenyl retinamide,4HPR),a synthetic anticancer retinoid,is a well-known apoptosis-inducing agent.Recently,we observed that the apoptosis induced by fenretinide could be effectively enhanced in hepatoma cells by concomitant treatment with parthenolide,a known inhibitor of nuclear factor NF-kB Furthermore,treatment with fenretinide triggered the activation of NF-kB.during apoptosis,an effect that could be substantially inhibited by parthenolide,which suggests that NF-κB activation during fenretinide-induced apoptosis might have an anti-apoptotic effect.In this study,we investigated the molecular mechanism of this apoptotic potentiation by NF-κB inhibition.Using the differential display-PCR (DD-PCR) method,and subsequent Northern blot or semiquantitative reverse transcriptase PCR(RT-PCR) analysis we identified genes which are involved in enhanced fenretinide-induced apoptosis by parthenolide.We identified 35 apoptosis-related genes including 12 unknown genes that are up- or down-regulated by parthenolide.Intriguingly,one up-regulated gene(HA1A2) was isolated and cloned from liver cDNA,and turned out to be identical to ANKRD1,which is also referred to as the CARP gene.Ectopic expression of ANKRD1 led to reduced colony formation and to enhanced apoptotic cell death in hepatoma cells,compared with controls treated with an empty vector or with antisense cDNA.These results imply that ANKRD1 and other genes whose expressions were substantially modulated by parthenolide-mediated inhibition of NF-κB activation may play roles in the enhanced drug-induced apoptosis and further,that those identified genes may be useful in anticancer strategies against hepatoma.
The results are as below:
1.Parthenolide enhances fenretinide-induced apoptotic cell death. apoptotic cell death was determined by TUNEL staining in Hep 3B cells and in SK-HEP-1 cells treated for 72 h with 10μM or 15μM fenretinide,respectively,in the presence or absence of 4μM parthenolide(P),or treated with vehicle alone.Vertical bars represent the means±SE of quadruplicate experiments.~(**)P＜0.01,compared with cells treated with fenretinide alone,quantitation of the apoptotic fraction by FCM analysis in Hep3B cells(upper panels) and SK-HEP-1 cells(lower panels) treated with 10μM fenretinide in the presence or absence of 4μM parthenolide(P) for 72 h,or were treated with vehicle alone.The Sub-G_1 fraction was estimates by gating hypodiploid cells in the histogram using the LYSISⅡprogram.DNA contents are plotted on the linear abscissa(M_1,apoptotic fraction).Each value represents the mean±SE of triplicate experiments.~(**) P＜0.01,compared with cells treated with fenretinide alone.
2.Parthenolide effectively inhibits NF-κB activation during fenretinide-induced apoptosis.
Hep 3B cells and SK-HEP cells(1×10~6) were treated with 10μM or 15μM fenretinide,respectively,for the indicated time periods,or were treated with vehicle alone.DNA-binding activity for NF-kB was detected by autoradiography. Experiments were performed at least three times in duplicate,and the result of one representative experiment is shown.
3.Differential display analysis of genes expressed in apoptotic Hep 3B cells. Differential display using three one-base anchored oligo-dT primers(H-T_(11)G,H-T_(11)A and H-T_(11)C) in combination with 80 arbitrary 13-mers.Representative patterns of differentially expressed genes were obtained from DD-PCR analysis using duplicate RIgA samples from control Hep 3B cells(C) and from apoptotic Hep 3B cells(A) induced by treatment with 10μM fenretinide for 72 h using H-T_(11)C in combination with 67-79th-arbitrary 13-mers.The HC70A1,HC71A1,HC75A1 and HC75C2 cDNA fragments are marked by arrows.
4.ANKRD1 overexpression during drug-induced apoptosis
Isolated ankyrin repeat domain 1/cardia ankyrin repeat protein gene(ANKRD1/CARP) cDNA and its up-regulation by the NF-kB inhibitor parthenolide.,Northern blot analysis of ANKRD1 in Hep 3B cells(upper panels) and in SK-HEP-1 cells(middle panels) treated either with fenretinide alone or in combination with parthenolide (4HPR + P) at the indicated time intervals,or treated with vehicle alone.Total RNAs were extracted and fractionated by electrophoresis on 1%agarose gels containing formaldehyde,and were then transferred to membranes.Each blot was stripped and subsequently rehybridized with a probe for 18S cDNA as a loading control. Experiments were performed at least three times,and the result of one representative experiment is shown.Relative densitometric plots on the quantification of ANKRD1 mRNA induction(%of control)(lower panel).
5.Decreased clonogenicity and apoptotic morphology by ectopic ANKRD1 overexpression.
6.Increased drug-susceptibility by ectopic ANKRD1 overexpression. Isolated ankyrin repeat domain 1/cardia ankyrin repeat protein gene(ANKRD1/CARP) cDNA and its up-regulation by the NF-kB inhibitor parthenolide C,immunoblot analysis of ANKRD1 expression in Hep 3B cells(upper panels) and in SK-HEP-1 cells(lower panels) treated either with fenretinide alone or in combination with parthenolide(4HPR + P) at the indicated time intervals,or treated with vehicle alone (C).Thirtyμg of extracted proteins were resolved by 12%SDS-PAGE and were transferred to the membrane.The blots were probed with a polyclonal antibody against ANKRD1 and were then stripped and reprobed with a monoclonal antibody to actin as a loading control.Experiments were performed at least three times,and the result of one representative experiment is shown.
7.We transiently transfected the cells with the ANKRD1 tagged at its amino terminus with the enhanced green fluorescent protein(GFP) and simultaneously performed immunohistochemistry with a mouse monoclonal antibody for ANKRD1. GFP fused to ANKRD1 protein expression is confined to mainly cytoplasm and less nuclei of the Hep 3B,SK-Hep1 and Hep G2,Huh7,which is perfectively merged with immunoreactivity for ANKRD1 expression.
8 The ectopic overexpression of ANKRD1 sensitizes the Hep 3B cells to drug-induced apoptosis.Ectopic over-expression of ANKRD1 sensitizes Hep 3B cells to drug-induced apoptosis.Expression of ANKRD1 protein in stably transfected Hep 3B cells with a sense GFP-ANKRD1(S1 and S6) were compared with antisense GFP-ANKRD1(AS8 and AS13) or with GFP vector control cells(VC2 and VC3) (upper).Ectopic,ectopic expression of ANKRD1 protein.Endogenous,endogenous expression of ANKRD1 protein.The stable transfectants and vector control cells were treated with 10 M fenretinide or 4 M parthenolide(P) for 48 h as indicated. Quantification of apoptotic fractions was performed using a FACScan.The sub-G_1 fraction was estimated by gating hypodiploid cells in the histogram using a C-30 program.DNA contents are plotted on the linear abscissa(M_1,apoptotic fraction). Each value represents the mean±SE of two independent experiments performed in triplicate±SE(~(**) P＜0.01 compared with control).
Our conclusions:
1.Parthenolide enhances fenretinide-induced apoptosis in hepatoma cells,The Sub-G_1 fraction was estimates by gating hypodiploid cells in the histogram using the LYSISⅡprogram.
2.Parthenolide inhibit NF-kB activation induced by fenretinide and enhance fenretinide-induced apoptosis.
3.Identified 35 apoptosis-related genes including 12 tmknown genes that are up- or down-regulated by parthenolide,one up-regulated gene(HA1A2) was isolated and cloned from liver cDNA,and turned out to be identical to ANKRD1,which is also referred to as the CARP gene.
4.Ectopic expression of ANKRD1 led to reduced colony formation and to enhanced apoptotic cell death in hepatoma cells.
5.These results imply that ANKRD1 and other genes whose expressions were substantially modulated by parthenolide-mediated inhibition of NF-kB activation may play roles in the enhanced drug-induced apoptosis and further,that those identified genes may be useful in anticancer strategies against hepatoma.
引文
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