Notch信号通路对乳腺癌细胞增殖的影响及其机制研究
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摘要
第一部分乳腺癌标本及乳腺细胞系中Notch信号通路的表达、活化及γ分泌酶抑制剂对乳腺癌细胞增殖的影响
研究背景:
Notch信号传导通路是在进化过程中高度保守的信号传导系统,由Notch受体和配体组成。Notch基因编码4种跨膜蛋白分子,分别为Notch1,Notch2,Notch3,Notch 4,作为受体与相应配体(Delta-like 1,Delta-like 3,Delta-like 4,Jagged 1和Jagged 2)结合。位于细胞膜表面的Notch受体与相应配体结合后,引发分别由TACE(TNF-α-converting enzyme)和γ-secretase/presenilin complex介导的水解过程,释放其活性片段N-IC.N-IC转至细胞核内,与DNA结合蛋白CBF1/Su(H)/Lag1(CSF)结合后,激活其下游靶基因Hes、Hey等的转录。Notch信号通路主要介导细胞的分化抑制信号,在胚胎发育、T细胞发育、肿瘤形成等生理病理过程中发挥重要作用。近年来研究发现,Notch信号的异常表达与乳腺癌的发病相关。在对MMTV(mouse mammary tumor virus)导致的CzechⅡ小鼠乳腺肿瘤模型的研究中发现,Notch4及Notch1基因存在MMTV的插入位点。病毒的插入导致Notch蛋白跨膜区及胞内区的异常表达,提示Notch通路参与乳腺癌的发生。进一步研究证实,外源组成性活化表达的Notch1或Notch4可以使正常鼠和人乳腺上皮细胞发生恶性转化。在对人类乳腺癌标本的研究中发现,50%的乳腺癌标本存在Numb蛋白(Notch通路的负性调节因子)的缺失,在过表达H-ras的乳腺癌标本中发现Notch1蛋白的高表达,这种高表达与病人不良预后相关。
研究目的:
研究在人类乳腺癌标本中Notch通路表达与活化情况,用γ-分泌酶抑制剂阻断Notch的活化,观测其对乳腺癌增殖的影响。
研究方法:
1.收集乳腺癌患者肿瘤组织标本,以癌旁正常乳腺组织作为对照。Trizol方法提取组织总RNA,在一定的反应体系中逆转录成cDNA,PCR方法检测Notch1,Notch3,Notch4,Jagged1和Delta-like 4在mRNA水平的表达情况
2.体外培养乳腺癌细胞系MDA-MB-231,MDA-MB-435。Trizol方法提取组织总RNA,在一定的反应体系中逆转录成cDNA,PCR方法检测Notch1,Notch3,Notch4,Jagged1和Delta-like 4在mRNA水平的表达情况,
3.免疫化学染色:收集10例乳腺癌组织和4例癌旁正常乳腺组织石蜡切片,脱腊,抗原修复后用抗N1-IC抗体对标本进行免疫组化染色;或体外培养乳腺癌细胞系MDA-MB-231和MDA-MB-435,细胞爬片,固定后用N1-IC抗体对标本进行免疫染色,以检测Notch1在乳腺癌组织和细胞中的活化情况。
4.γ分泌酶抑制剂对乳腺癌细胞增殖的影响:取指数生长期细胞消化计数后按4×10~3/孔接种于6孔板中,次日(待细胞70%-80%融合)加入3μmol/L的γ分泌酶抑制剂,阻断Notch的活化,以相同体积的DMSO做对照。作用24小时后MTT方法检测细胞增殖情况。
5.流式细胞仪检测γ分泌酶抑制剂对乳腺癌细胞周期的影响:取指数生长期细胞消化计数后按2×10~5/孔接种于6孔板中,次日(待细胞70%-80%融合)加入3μmol/L的γ分泌酶抑制剂,阻断Notch的活化,以相同体积的DMSO做对照。作用24小时后收集细胞,PI染色后流式细胞仪检测细胞周期变化。
6.流式细胞仪检测γ分泌酶抑制剂对细胞凋亡率的影响:取指数生长期细胞消化计数后按2×10~5/孔接种于6孔板中,次日(待细胞70%-80%融合)加入3μmol/L的γ分泌酶抑制剂,阻断Notch的活化,以相同体积的DMSO做对照。作用24小时后收集细胞,AnnexinV/PI染色后流式细胞仪检测细胞凋亡率变化
研究结果:
1.Notch受体及其配体在乳腺癌组织和细胞中呈高表达:共检测62例乳腺癌标本及其癌旁正常乳腺组织。Notch1,3,4,Jagged1和Delta-like 4在乳腺癌标本中的阳性表达率分别是98%,35%,8%,15%,81%,而在癌旁正常组织中的阳性表达率为73%,0%,0%,0%,0%。所检测Notch受体及其配体在乳腺癌中的mRNA表达阳性率显著高于癌旁组织;
在乳腺癌细胞系MDA-MB-231、MDA-MB-435中Notch-1,3和Jagged1均呈阳性表达。
2.乳腺癌组织及细胞中存在Notch1通路的活化:利用特异性识别Notch1活化形式N-IC的抗体,通过免疫组化和免疫细胞染色方法,检测Notch1通路的活化情况。共检测10例乳腺癌标本和4例正常乳腺组织。在9/10例乳腺癌组织的细胞核中发现N-IC的聚集,提示Notch1的活化,而在正常乳腺组织均呈阴性染色;
在乳腺癌细胞系MDA-MB-231、MDA-MB-435均发现Notch1的活化。
3.γ分泌酶抑制剂能够抑制乳腺癌细胞的增殖:γ分泌酶抑制剂作用于乳腺癌细胞MDA-MB-231、MDA-MB-435 24小时后显著抑制其增殖,并且呈剂量依赖性。
4.γ分泌酶抑制剂能够使乳腺癌细胞周期阻滞于G2/M期。γ分泌酶抑制剂作用于乳腺癌细胞24小时后,与对照组比较,其G2/M期细胞分别为29.9%vs15.99%(MDA-MB-231).and 45.5%vs 16.63%(MDA-MB-435),有显著性差异。
5.γ分泌酶抑制剂能够增加乳腺癌细胞凋亡率:γ分泌酶抑制剂作用于乳腺癌细胞24小时后,与对照组比较,其凋亡率分别是3.03%vs 0.94%(MDA-MB-231).和4.33%vs 0.23%(MDA-MB-435)。提示γ分泌酶抑制剂能够诱导乳腺癌细胞的凋亡。
结论:
1.Notch通路分子在人类乳腺癌及乳腺癌细胞系中广泛表达和活化
2.γ分泌酶抑制剂阻断其活化后可以抑制乳腺癌细胞的增殖,导致乳腺癌细胞周期阻滞和诱导其凋亡。
因此,Notch信号通路可以作为抗乳腺癌治疗的潜在靶点。
第二部分siRNA介导的Notch1下调对乳腺细胞增殖及药物敏感性的影响
研究背景:
RNA干扰(RHA interference)是一种由双链RNA诱发的基因沉默。在此过程中,与双链RNA有同源序列的信使RNA(mRNA)被降解,从而抑制了该基因的表达。长于30个碱基对的双链RNA常常会激活蛋白激酶而诱发对蛋白质合成的非特异抑制,而小干扰性RNA(small interfering RNA,siRNA)是长度为21-23bp左右的RNA片段,一般不会在哺乳类细胞中诱发这种非特异性抑制。用人工合成的siRNA可特异性地抑制哺乳类细胞中外源性或内源性基因的表达。siRNA诱发的基因抑制具有高度的序列特异性。在siRNA上一个错配的碱基对即可使其失去原有的抑制基因表达的活性。换言之,只有与siRNA高度同源的mRNA才会被降解。有研究证实,siRNA可以阻断90%以上的目的基因的表达。siRNA的高度特异性和高效性在将RNA干扰技术用于治疗人类疾病时极为重要,为减少或避免抑制不相关基因奠定了基础。
我们在前面的研究中发现,Notch1在乳腺癌组织和乳腺癌细胞系中广泛表达和活化,用γ分泌酶抑制剂阻断其活化后可以抑制乳腺癌细胞的增殖,导致其周期阻滞和凋亡。但γ分泌酶抑制剂是一种具有多种活性的化学合成药物,对γ分泌酶抑制剂的一期临床实验显示,患者可能对之产生不可耐受的胃肠道反应。除此之外,γ分泌酶抑制剂还可能具有脱靶效应。为了进一步阐明Notch信号通路对乳腺癌细胞增殖的影响,我们探讨了其他阻断Notch信号通路的方法——即siRNA。我们用针对Notch 1的siRNA来阻断Notch1的表达,观测其对乳腺癌细胞增殖及对化疗药物敏感性的影响。并且探讨了Notch信号通路与另一影响细胞增殖与分化的重要信号系统-NF-kappaB的关系。
研究目的:
用siRNA阻断Notch1的表达后检测Notch信号对乳腺癌细胞增殖的影响,并阐明其分子机制,为乳腺癌的分子靶向治疗提供新的作用位点。
研究方法:
1.siRNA片段的转染:取指数生长期细胞消化计数后按1.2×10~5个/孔接种于6孔细胞培养板中,24小时后应用Lipofectamine2000将针对Notch1的siRNA片段转染乳腺癌细胞MDA-MB-231和MCF7。72小时后real-time PCR方法检测siRNA片段对Notch1 mRNA表达的阻断效率。
2.MTT方法检测siRNA片段对乳腺癌细胞增殖的影响:取指数生长期细胞消化计数后按4×10~3个/孔接种于96孔细胞培养板中,24小时后转染siRNA片段。转染后72小时MTT方法检测小干扰片段对细胞的增殖的影响。
3.流式细胞仪检测siRNA片段对细胞周期的影响:取指数生长期细胞消化计数后按1.2×10~5个/孔接种于6孔细胞培养板中,24小时后转染siRNA片段,转染后72小时收集细胞,PI染色后利用流式细胞仪检测细胞周期的变化。
4.流式细胞仪检测siRNA片段对细胞凋亡率的影响:取指数生长期细胞消化计数后按1.2×10~5个/孔接种于6孔细胞培养板中,24小时后转染siRNA片段,转染后72小时收集细胞,AnnexinV/PI双染后利用流式细胞仪检测细胞凋亡率的变化。
5.MTT方法检测siRNA片段对乳腺癌细胞化疗药物敏感性的影响:取指数生长期细胞消化计数后按4×10~3个/孔接种于96孔细胞培养板中,24小时后转染siRNA片段。转染后24小时加入1.5nM docetaxel或75μg/ml doxorubicin抚育48小时,MTT检测细胞增殖情况。
6.Western-blot检测通路相关蛋白表达情况:取指数生长期细胞消化计数后以2×10~5接种于10cm培养皿,24小时后转染siRNA片段,转染后72小时收集细胞,提取细胞总蛋白,Western-blot检测通路相关蛋白的表达情况。
7.EMSA检测NF-KappaB活性变化:取指数生长期细胞消化计数后按1.2×10~5个/孔接种于6孔细胞培养板中,24小时后转染siRNA片段,转染后72小时收集细胞,提取细胞核蛋白,EMSA检测NF-KappaB活性变化
研究结果:
1.针对Notch1的siRNA片段可以有效下调Notch1的表达:siRNA转染后72小时,干扰组Notch1和其下游基因Hes1 mRNA的表达较对照组下降90%,Notch1和Hes1蛋白表达同样显著下调。
2.下调Notch1表达能够抑制乳腺癌细胞的增殖:MDA-MB-231转染siRNA后72小时,MTT检测其吸光度值,干扰组为0.332±0.0563,对照组为0.559±0.044,P值为0.0053,有显著统计学意义;MCF7在转染后72小时MTT检测其吸光度值,干扰组为0.3060±0.073 1,对照组为0.6077±0.0049,P值为0.0020,有显著统计学意义。siRNA介导的Notch 1表达下调能够显著抑制乳腺癌细胞的增殖。
3.下调Notch 1的表达能够引起乳腺癌细胞的周期阻滞:MCF7转染siRNA后72小时,流式细胞仪检测细胞周期分布,干扰组为:G1:78.5%,G2:5.34%,S:19.54%:而对照组细胞的周期分布为:G1:54.29%,G2:8%,S:38.7%,与对照组细胞比较,下调Notch 1的表达能够导致MCF7的细胞周期阻滞于S期,而对MDA-MB-231细胞的周期无明显作用。
4.下调Notch 1的表达能够增加乳腺癌细胞的凋亡率:MDA-MB-231细胞转染siRNA后72小时,AnnexinV/PI双染后流式细胞仪检测细胞凋亡率,干扰组为5.64%,对照组凋亡率为1.25%:MCF7转染siRNA后72小时,干扰组凋亡率为2.88%,对照组为0.94%。下调Notch 1的表达能够显著增加乳腺癌细胞的凋亡率。
5.下调Notch 1的表达能够增加乳腺癌细胞对docetaxel或doxorubicin的药物敏感性:MDA-MB-231和MCF7细胞转染后24小时,加入1.5nM docetaxel或75ng/ml doxorubicin作用48小时。结果显示docetaxel或doxorubicin对干扰组MDA-MB-231和MCF7细胞的增殖抑制效率较对照组分别增加50%或70%。对其机制的研究显示,Notch 1下调后可以增强docetaxel或doxorubicin对MDA-MB-231和MCF7细胞的凋亡诱导效率。
6.下调Notch 1的表达能够降低NF-KappaB活性:MDA-MB-231和MCF7细胞转染后72小时,EMSA检测显示干扰组NF-KappaB活性较对照组明显降低。
结论:
1.我们所用siRNA可以有效阻断Notch1的表达
2.siRNA介导的Notch1表达下调能够引起细胞周期阻滞和细胞凋亡率增加,从而抑制乳腺癌细胞的增殖,
3.siRNA介导的Notch1表达下调能够增加乳腺癌细胞对docetaxel或doxorubicin的药物敏感性。
4.siRNA介导的Notch1表达下调可以抑制NK-kappaB的DNA结合活性。
因此,Notch1是治疗乳腺癌的有效分子靶点,并且可以和传统的化疗药物联合应用,以降低化疗药物毒副作用,增强其抗肿瘤治疗效果。
SectionⅠ: The expression and activation of Notch signaling in human breast cancer and the effect ofγ-secretase inhibitor on the proliferation of breast cancer
Background:
Notch signaling is a pathway highly conserved through evolution which regulates various physiological processes,including stem cell maintenance, differentiation,proliferation and apoptosis.In mammals,key components of the Notch pathway include four transmembrane receptors(Notch 1-4)and five ligands(Delta-like 1,Delta-like 3,Delta-like 4,Jagged 1,Jagged 2).Direct binding of a ligand from a signaling cell to a Notch receptor on the membrane of the receiving cell initiates two successive proteolytic cleavages by TACE(TNF-α-converting enzyme)and theγ-secretase/presenilin complex,which ultimately results in the release of the intracellular domain(N-IC).N-IC then translocates into the nucleus and directly interacts with the DNA binding protein CBF 1/Su(H)/Lag 1(CSF)that activates the transcription of target genes including the Hes(hairy/enhancer-of-split)and hey.It was found recently that Notch signaling is related with the etiology of breast cancer. The first indication that Notch signaling might play a role in neoplastic development of the mammary gland came from the characterization of a common insertion site for the mouse mammary tumor virus in CzechⅡmice.In 20%of these tumors,the mouse mammary tumor virus was inserted within the Notch4/Notch1 locus.At both loci,insertion of the provirus leads to expression of Notch protein that consists of the transmembrane and intracellular domains only,suggesting that deregulated Notch signaling related to tumor.Several studies show that over expression of consititutively activated notch1 or notch4 in normal human breast epithelial cells can induce transformation in vitro.Notch1 protein was highly expressed in four breast tumors that over expressed H-ras,and the expression was associated with adverse prognosis.
Aim:
To detect systematically the expression of Notch signaling moleculars and the aberrant activation of Notch signaling in human breast cancer.Explore the role ofγ -secretase inhibitor on the proliferation of breast cancer cells.
Meterial and Methods:
1.Collect breast cancer specimens and normal breast tissues from the margin of tumor sections as control.Total RNA was extracted by TRIZOL according to the manufacturers instructions.Synthesis of first-strand cDNA was carried out with Revert Aid~(TM)First Strand cDNA Synthesis Kit.Notch1,Notch3,Notch4 and Jagged1,Delta-like4 were amplified.
Immunostaining was used to detect the activated form of Notch1(N-IC)in formalin-fixed,paraffin-embedded tissue sections
2.Cell culture.Human breast cancer cell lines MDA-MB-231,MDA-MB-435 were cultured in RPMI medium 1640.RT-PCR was used to detect the expression of. Notch 1,Notch3,Notch4 and Jagged 1,Delta-like4.Immunostaining was used to detect the activated form of Notch1(N-IC)in these cell lines.
3.Immunostaining.10 of breast cancer tissues and 4 of normal breast tissues at the margin of breast cancer were collected.Formalin-fixed,paraffin-embedded tissue sections(5μm thick)were dewaxed,rehydrated and optimized antigen retrieval.
Alternatively,cultured cells grown on cover slides were fixed in 95%ethanol. Sections were then used to detect Notch1-IC using Histostain-plus kit according to the manufacturer's instructions.
4.Inhibition assay byγ-secretase inhibitor.4×10~4 of MDA-MB-231 cells and MDA-MB-435 cells were plated in a 96-well plate and allowed to proliferate overnight.The cells were then treated with increasing concentration ofγ-secretase inhibitor.24 hours after treatment,the proliferation of the cells were measured by MTT method.
5.Analysis of Cell cycle.Briefly,1×10~6 cells were plated in 100 ml culture flasks and allowed to proliferate till 70%-80%confluent.Then,the cells were treated withγ-secretase inhibitor(3μmol/L)or DMSO(15μl)as a control,and after 24h, cells were harvested and stained with PI.The DNA content was analyzed by FACS Calibur
6.Detection of apoptosis.Cells were treated withγ-secretase inhibitor(3μmol/L)or DMSO(15μl)as described above.24 hours after treatment,cells were collected and labeled with 1:500 Annexin V-biotin conjugated with fluorescent isothiocyanate(FITC)followed by 1:1000 PI.Annexin V-PI were measured by FACS Calibur
Results:
1.Notch receptors and ligands are over expressed in human breast cancer.The expression rates of Notch1,3,4,Jagged1 and Delta-like4 gene mRNA in cancer specimen and normal breast tissue at the margin of tumor sections are 98%,35%, 8%,15%,81%and 73%,0%,0%,0%,0%,respectively.The expression rate of Notch signaling moleculars mRNA in the cancer specimens is significantly higher than that in the specimens at the margin of tumor sections.Notch1,3 and Jagged1 were all expressed in human breast cancer cell lines MDA-MB-231 and MDA-MB-435.
2.Notch1 is activated in human breast cancer.We used the antibody which recognizes only the activated form of Notch1(N1-ICD,the cleaved intracellular form)to detect its activation,we saw accumulation of N1-ICD in most(9 in 10) tumor samples analyzed,only 1 sample showed negative staining.Whereas,no N1-ICD staining was detected in all the normal breast tissues.In all the cell lines, we observed a clear accumulation of N1-ICD in both the cytoplasm and nuclear, with the later finding being indicative of Notch activation.
3.γ-secretase inhibitor can inhibit the proliferation of breast cancer cells.γ-secretase inhibitor,a pharmacologic agent known to block effectively Notch activation,was used to evaluate the effect of Notch inhibition on breast cancer cells. We observed the proliferation of both cell lines decreased with the increasing dosage ofγ-secretase inhibitor I(1-5μmol/L).
4.γ-secretase inhibitor can cause cell cycle arrest in breast cancer cell lines.γ-secretase inhibitor I-treated cells of MDA-MB-231 and MDA-MB-435 showed a higher proportion of cells in G2/M phase compared with control(29.9%vs 15.99%, and 45.5%vs 16.63%in MDA-MB-231 and MDA-MB-435 respectively).
5.γ-secretase inhibitor can cause apoptosis in breast cancer cell lines.The fraction of apoptotic cells inγ-secretase inhibitor -treated populations was significantly higher than that observed in controls(0.94%vs 3.03%,0.23%vs 4.33%in MDA-MB-231 and MDA-MB-435 respectively,p＜0.05,data are mean percentage of apoptotic cells from three independent experiments,demonstrating that inhibition of Notch signaling could also induce apoptosis in breast cancer cells.
Conclusion:
Notch signaling is over expressed and highly activated in human breast cancer. Blockage of Notch signaling byγ-secretaseinhibitor can inhibit the growth of breast cancer cells,causing cell cycle arrest and apoptosis.Thus,Notch signaling can be a therapeutic target for human breast cancer.
SectionⅡThe effect of down-regualtion of Notch1 mediated by siRNA on the proliferation of breast cancer cells
Background:
RNA interference(RNAi)is a process of gene silencing induced by double strand RNA(dsRNA).Basically,the process of RNAi is triggered by dsRNA precursors. These dsRNA precursors are processed into small interfering RNAs(siRNAs)that vary in length from 21-23 nucleotides(nt).The siRNAs are subsequently incorporated into a multiprotein complex.This complex is known as the RNA-induced silencing complex(RISC).siRNAs in the RISC complex guide degradation that is highly sequence-specific,of the complementary mRNAs.The gene silencing effect is highly efficient,as reported as high as 90%expression can be blocked.The high specificity and efficiency are very useful for human disease treatment as it can specifically knock down its target gene without affecting other gene's expression.
Previously we have found that Notch signaling is over expressed and highly activated in human breast cancer,andγ-secretase inhibitor can inhibit the proliferation by causing cell cycle arrest and apoptosis.Butγ-secretase inhibitor is a compound that has many activities including block the activation of Notch signaling.Phase I clinical study showed that it also has severe gastrointestinal side effect.So we further explored more specific method to knock down the expression of Notch1.We use siRNA that can specific binds to Notch1 to down regulate its expression and examine the effect on proliferation of breast cancer cells.Also we detected whether Notch signaling is related to chemosensitivity and the relation between Notch and NK-kappaB.
Meterial and Methods:
1.SiRNA Transfection.1.2×10~5 of MDA-MB-231 and MCF-7 Cells were seeded in 6-well plates(or 4000 cells/well in 96-well plates).After overnight incubation,cells (30-50%confluence)were treated with 40nM siRNA/control siRNA.Seventy-two hours after siRNA transfection,cells were used for MTT,real-time PCR,western blot,and other experiments.
2.Cell growth inhibition by MTT assay.MDA-MB-231 and MCF-7 Cells were incubated overnight at a density of 4,000 cells/well in 96-well plates,and subsequently transfected with Notch- 1 siRNA or control siRNA.72 hours after tansfection,20μl of MTT was added each well.48 additional hours later,color development was measured on a microplate reader at 570 nm.
3.Flow Cytometry and Cell Cycle Analysis.Notchl siRNA,or control siRNA transfected MDA-MB-231 and MCF7 cells were collected and stained with PI. DNA content was detected on a FACS Calibur.
4.Apoptosis Assay.72 hours after transfection,Notch1 siRNA/Control siRNA transfected MDA-MB-231 and MCF-7 cells were collected and were labeled with annexin V-biotin followed by PI.Annexin V-PI were measured by FACS Calibur and analyzed with the Modfit Software.
For the chemotherapeutic drug assays,24 hours after transfection,cells were exposed to 1.5 nM docetaxel or 75ug/ml doxorubicin respectively for 48 hours, then cells were collected and analyzed as above.
5.Western Blot Analysis.Total protein was extrated from transfected/control cells. Total proteins were fractionated using SDS-PAGE and transferred onto nitrocellulose membrane.After incubation with blocking buffer,the membrane was incubated with primary and secondary antibodies,the protein bands were detected using the enhanced chemiluminesence detection system.
6.Electrophoresis Mobility Shift Assay for Measuring NF-KappaB Activity.Nuclear extracts from MDA-MB-231 and MCF-7 cells were prepared.For EMSA,32 fmol of labeled probes were incubated for 20 min at 25℃with 2μg of nuclear extracts. Competition experiments were performed using EMSA conditions similar to those described above,except that the protein extracts were incubated with the probe in the presence of 250-fold molar excess of unlabeled double-stranded oligonucleotides as competitors.Subsequently,the DNA-protein complexes were separated from the free probes by electrophoresis through a 5%non-denaturing polyacrylamide gel,the gel was transferred to a PVDF membrane and chemiluminescent detection was performed.
Results:
1.Notch 1 siRNA effectively down-regulated the expression level of Notch 1 in human breast cancer cells.We observed that Notch1 and Hes1 mRNA decreased by about 90%in both MDA-MB-231 and MCF7 cells.Protein levels were also greatly reduced in Notch1 siRNA transfected cells compared with control siRNA transfected cells
2.Down-regulation of Notch1 expression by siRNA inhibited the growth of human breast cancer cells.72 hours after transfection with Notch1 siRNA or control siRNA,cell viability was determined by the MTT assay.We found that the down-regulation of Notch1 expression caused approximately a 40%reduction of cell growth in both breast cancer cell lines.
3.Down-regulation of Notch1 expression by siRNA induced S phase cell cycle arrest in MCF7 Cells.The Notch1 siRNA transfected MCF7 cells demonstrated an S phase arrest pattern(38.7%vs 19.54%)at 72 hours after transfection as compared with control cells.No alteration in cell cycle distribution was observed in MDA-MB-231 cells.
4.Down-regulation of Notch-1 expression by siRNA led to apoptosis in human breast cancer.72 hours after transfection,cells were stained with Annexin V/PI,and analyzed by flow cytometry.The apoptosis rates were 5.64%and 2.88%in Notch1 siRNA transfected MDA-MB-231 and MCF7 respectively,compared with 1.25% and 0.94%in control siRNA transfected cells.These data suggested that the growth inhibition induced by Notch1 siRNA was partially due to an increase in cell apoptosis.
5.Down-regulation of Notch-1 expression by siRNA increased chemosensitivity.We found that down-regulation of Notch-1 expression in combination with docetaxel or doxorubicin treatment led to a 50%or 70%enhancement in growth inhibition respectively as compared to single chemotherapeutic treatment in MDA-MB-231 cells.Similar results were found in MCF7 cells.In addition,the Notch-1 transfected MDA-MB-231 and MCF-7 cells were significantly more sensitive to docetaxel and doxorubicin-induced apoptosis,this may explain the enhanced growth inhibition.
6.Down-regulation of Notch-1 expression by siRNA reduces NF-κB DNA-binding activity.Down-regulation of Notch-1 significantly inhibited NF-κB DNA-binding activity compared with control cells in both MDA-MB-231 and MCF7 cells as detected by EMSA.These results provide evidence that cross-talk exists between Notch1 and NF-κB in breast cancer and that Notch signaling is the upstream regulator.
Conclusion: Down-regualtion of Notch-1 mediated by siRNA can inhibit the growth of breast cancer cells and increased chemosensitivity to doxorubicin and docetaxel.This effect may through the inactivation of NF-kappaB.Our results suggested that Notch signaling may be a promising target for breast cancer treatment.
研究背景:
Notch信号传导通路是在进化过程中高度保守的信号传导系统,由Notch受体和配体组成。Notch基因编码4种跨膜蛋白分子,分别为Notch1,Notch2,Notch3,Notch 4,作为受体与相应配体(Delta-like 1,Delta-like 3,Delta-like 4,Jagged 1和Jagged 2)结合。位于细胞膜表面的Notch受体与相应配体结合后,引发分别由TACE(TNF-α-converting enzyme)和γ-secretase/presenilin complex介导的水解过程,释放其活性片段N-IC.N-IC转至细胞核内,与DNA结合蛋白CBF1/Su(H)/Lag1(CSF)结合后,激活其下游靶基因Hes、Hey等的转录。Notch信号通路主要介导细胞的分化抑制信号,在胚胎发育、T细胞发育、肿瘤形成等生理病理过程中发挥重要作用。近年来研究发现,Notch信号的异常表达与乳腺癌的发病相关。在对MMTV(mouse mammary tumor virus)导致的CzechⅡ小鼠乳腺肿瘤模型的研究中发现,Notch4及Notch1基因存在MMTV的插入位点。病毒的插入导致Notch蛋白跨膜区及胞内区的异常表达,提示Notch通路参与乳腺癌的发生。进一步研究证实,外源组成性活化表达的Notch1或Notch4可以使正常鼠和人乳腺上皮细胞发生恶性转化。在对人类乳腺癌标本的研究中发现,50%的乳腺癌标本存在Numb蛋白(Notch通路的负性调节因子)的缺失,在过表达H-ras的乳腺癌标本中发现Notch1蛋白的高表达,这种高表达与病人不良预后相关。
研究目的:
研究在人类乳腺癌标本中Notch通路表达与活化情况,用γ-分泌酶抑制剂阻断Notch的活化,观测其对乳腺癌增殖的影响。
研究方法:
1.收集乳腺癌患者肿瘤组织标本,以癌旁正常乳腺组织作为对照。Trizol方法提取组织总RNA,在一定的反应体系中逆转录成cDNA,PCR方法检测Notch1,Notch3,Notch4,Jagged1和Delta-like 4在mRNA水平的表达情况
2.体外培养乳腺癌细胞系MDA-MB-231,MDA-MB-435。Trizol方法提取组织总RNA,在一定的反应体系中逆转录成cDNA,PCR方法检测Notch1,Notch3,Notch4,Jagged1和Delta-like 4在mRNA水平的表达情况,
3.免疫化学染色:收集10例乳腺癌组织和4例癌旁正常乳腺组织石蜡切片,脱腊,抗原修复后用抗N1-IC抗体对标本进行免疫组化染色;或体外培养乳腺癌细胞系MDA-MB-231和MDA-MB-435,细胞爬片,固定后用N1-IC抗体对标本进行免疫染色,以检测Notch1在乳腺癌组织和细胞中的活化情况。
4.γ分泌酶抑制剂对乳腺癌细胞增殖的影响:取指数生长期细胞消化计数后按4×10~3/孔接种于6孔板中,次日(待细胞70%-80%融合)加入3μmol/L的γ分泌酶抑制剂,阻断Notch的活化,以相同体积的DMSO做对照。作用24小时后MTT方法检测细胞增殖情况。
5.流式细胞仪检测γ分泌酶抑制剂对乳腺癌细胞周期的影响:取指数生长期细胞消化计数后按2×10~5/孔接种于6孔板中,次日(待细胞70%-80%融合)加入3μmol/L的γ分泌酶抑制剂,阻断Notch的活化,以相同体积的DMSO做对照。作用24小时后收集细胞,PI染色后流式细胞仪检测细胞周期变化。
6.流式细胞仪检测γ分泌酶抑制剂对细胞凋亡率的影响:取指数生长期细胞消化计数后按2×10~5/孔接种于6孔板中,次日(待细胞70%-80%融合)加入3μmol/L的γ分泌酶抑制剂,阻断Notch的活化,以相同体积的DMSO做对照。作用24小时后收集细胞,AnnexinV/PI染色后流式细胞仪检测细胞凋亡率变化
研究结果:
1.Notch受体及其配体在乳腺癌组织和细胞中呈高表达:共检测62例乳腺癌标本及其癌旁正常乳腺组织。Notch1,3,4,Jagged1和Delta-like 4在乳腺癌标本中的阳性表达率分别是98%,35%,8%,15%,81%,而在癌旁正常组织中的阳性表达率为73%,0%,0%,0%,0%。所检测Notch受体及其配体在乳腺癌中的mRNA表达阳性率显著高于癌旁组织;
在乳腺癌细胞系MDA-MB-231、MDA-MB-435中Notch-1,3和Jagged1均呈阳性表达。
2.乳腺癌组织及细胞中存在Notch1通路的活化:利用特异性识别Notch1活化形式N-IC的抗体,通过免疫组化和免疫细胞染色方法,检测Notch1通路的活化情况。共检测10例乳腺癌标本和4例正常乳腺组织。在9/10例乳腺癌组织的细胞核中发现N-IC的聚集,提示Notch1的活化,而在正常乳腺组织均呈阴性染色;
在乳腺癌细胞系MDA-MB-231、MDA-MB-435均发现Notch1的活化。
3.γ分泌酶抑制剂能够抑制乳腺癌细胞的增殖:γ分泌酶抑制剂作用于乳腺癌细胞MDA-MB-231、MDA-MB-435 24小时后显著抑制其增殖,并且呈剂量依赖性。
4.γ分泌酶抑制剂能够使乳腺癌细胞周期阻滞于G2/M期。γ分泌酶抑制剂作用于乳腺癌细胞24小时后,与对照组比较,其G2/M期细胞分别为29.9%vs15.99%(MDA-MB-231).and 45.5%vs 16.63%(MDA-MB-435),有显著性差异。
5.γ分泌酶抑制剂能够增加乳腺癌细胞凋亡率:γ分泌酶抑制剂作用于乳腺癌细胞24小时后,与对照组比较,其凋亡率分别是3.03%vs 0.94%(MDA-MB-231).和4.33%vs 0.23%(MDA-MB-435)。提示γ分泌酶抑制剂能够诱导乳腺癌细胞的凋亡。
结论:
1.Notch通路分子在人类乳腺癌及乳腺癌细胞系中广泛表达和活化
2.γ分泌酶抑制剂阻断其活化后可以抑制乳腺癌细胞的增殖,导致乳腺癌细胞周期阻滞和诱导其凋亡。
因此,Notch信号通路可以作为抗乳腺癌治疗的潜在靶点。
第二部分siRNA介导的Notch1下调对乳腺细胞增殖及药物敏感性的影响
研究背景:
RNA干扰(RHA interference)是一种由双链RNA诱发的基因沉默。在此过程中,与双链RNA有同源序列的信使RNA(mRNA)被降解,从而抑制了该基因的表达。长于30个碱基对的双链RNA常常会激活蛋白激酶而诱发对蛋白质合成的非特异抑制,而小干扰性RNA(small interfering RNA,siRNA)是长度为21-23bp左右的RNA片段,一般不会在哺乳类细胞中诱发这种非特异性抑制。用人工合成的siRNA可特异性地抑制哺乳类细胞中外源性或内源性基因的表达。siRNA诱发的基因抑制具有高度的序列特异性。在siRNA上一个错配的碱基对即可使其失去原有的抑制基因表达的活性。换言之,只有与siRNA高度同源的mRNA才会被降解。有研究证实,siRNA可以阻断90%以上的目的基因的表达。siRNA的高度特异性和高效性在将RNA干扰技术用于治疗人类疾病时极为重要,为减少或避免抑制不相关基因奠定了基础。
我们在前面的研究中发现,Notch1在乳腺癌组织和乳腺癌细胞系中广泛表达和活化,用γ分泌酶抑制剂阻断其活化后可以抑制乳腺癌细胞的增殖,导致其周期阻滞和凋亡。但γ分泌酶抑制剂是一种具有多种活性的化学合成药物,对γ分泌酶抑制剂的一期临床实验显示,患者可能对之产生不可耐受的胃肠道反应。除此之外,γ分泌酶抑制剂还可能具有脱靶效应。为了进一步阐明Notch信号通路对乳腺癌细胞增殖的影响,我们探讨了其他阻断Notch信号通路的方法——即siRNA。我们用针对Notch 1的siRNA来阻断Notch1的表达,观测其对乳腺癌细胞增殖及对化疗药物敏感性的影响。并且探讨了Notch信号通路与另一影响细胞增殖与分化的重要信号系统-NF-kappaB的关系。
研究目的:
用siRNA阻断Notch1的表达后检测Notch信号对乳腺癌细胞增殖的影响,并阐明其分子机制,为乳腺癌的分子靶向治疗提供新的作用位点。
研究方法:
1.siRNA片段的转染:取指数生长期细胞消化计数后按1.2×10~5个/孔接种于6孔细胞培养板中,24小时后应用Lipofectamine2000将针对Notch1的siRNA片段转染乳腺癌细胞MDA-MB-231和MCF7。72小时后real-time PCR方法检测siRNA片段对Notch1 mRNA表达的阻断效率。
2.MTT方法检测siRNA片段对乳腺癌细胞增殖的影响:取指数生长期细胞消化计数后按4×10~3个/孔接种于96孔细胞培养板中,24小时后转染siRNA片段。转染后72小时MTT方法检测小干扰片段对细胞的增殖的影响。
3.流式细胞仪检测siRNA片段对细胞周期的影响:取指数生长期细胞消化计数后按1.2×10~5个/孔接种于6孔细胞培养板中,24小时后转染siRNA片段,转染后72小时收集细胞,PI染色后利用流式细胞仪检测细胞周期的变化。
4.流式细胞仪检测siRNA片段对细胞凋亡率的影响:取指数生长期细胞消化计数后按1.2×10~5个/孔接种于6孔细胞培养板中,24小时后转染siRNA片段,转染后72小时收集细胞,AnnexinV/PI双染后利用流式细胞仪检测细胞凋亡率的变化。
5.MTT方法检测siRNA片段对乳腺癌细胞化疗药物敏感性的影响:取指数生长期细胞消化计数后按4×10~3个/孔接种于96孔细胞培养板中,24小时后转染siRNA片段。转染后24小时加入1.5nM docetaxel或75μg/ml doxorubicin抚育48小时,MTT检测细胞增殖情况。
6.Western-blot检测通路相关蛋白表达情况:取指数生长期细胞消化计数后以2×10~5接种于10cm培养皿,24小时后转染siRNA片段,转染后72小时收集细胞,提取细胞总蛋白,Western-blot检测通路相关蛋白的表达情况。
7.EMSA检测NF-KappaB活性变化:取指数生长期细胞消化计数后按1.2×10~5个/孔接种于6孔细胞培养板中,24小时后转染siRNA片段,转染后72小时收集细胞,提取细胞核蛋白,EMSA检测NF-KappaB活性变化
研究结果:
1.针对Notch1的siRNA片段可以有效下调Notch1的表达:siRNA转染后72小时,干扰组Notch1和其下游基因Hes1 mRNA的表达较对照组下降90%,Notch1和Hes1蛋白表达同样显著下调。
2.下调Notch1表达能够抑制乳腺癌细胞的增殖:MDA-MB-231转染siRNA后72小时,MTT检测其吸光度值,干扰组为0.332±0.0563,对照组为0.559±0.044,P值为0.0053,有显著统计学意义;MCF7在转染后72小时MTT检测其吸光度值,干扰组为0.3060±0.073 1,对照组为0.6077±0.0049,P值为0.0020,有显著统计学意义。siRNA介导的Notch 1表达下调能够显著抑制乳腺癌细胞的增殖。
3.下调Notch 1的表达能够引起乳腺癌细胞的周期阻滞:MCF7转染siRNA后72小时,流式细胞仪检测细胞周期分布,干扰组为:G1:78.5%,G2:5.34%,S:19.54%:而对照组细胞的周期分布为:G1:54.29%,G2:8%,S:38.7%,与对照组细胞比较,下调Notch 1的表达能够导致MCF7的细胞周期阻滞于S期,而对MDA-MB-231细胞的周期无明显作用。
4.下调Notch 1的表达能够增加乳腺癌细胞的凋亡率:MDA-MB-231细胞转染siRNA后72小时,AnnexinV/PI双染后流式细胞仪检测细胞凋亡率,干扰组为5.64%,对照组凋亡率为1.25%:MCF7转染siRNA后72小时,干扰组凋亡率为2.88%,对照组为0.94%。下调Notch 1的表达能够显著增加乳腺癌细胞的凋亡率。
5.下调Notch 1的表达能够增加乳腺癌细胞对docetaxel或doxorubicin的药物敏感性:MDA-MB-231和MCF7细胞转染后24小时,加入1.5nM docetaxel或75ng/ml doxorubicin作用48小时。结果显示docetaxel或doxorubicin对干扰组MDA-MB-231和MCF7细胞的增殖抑制效率较对照组分别增加50%或70%。对其机制的研究显示,Notch 1下调后可以增强docetaxel或doxorubicin对MDA-MB-231和MCF7细胞的凋亡诱导效率。
6.下调Notch 1的表达能够降低NF-KappaB活性:MDA-MB-231和MCF7细胞转染后72小时,EMSA检测显示干扰组NF-KappaB活性较对照组明显降低。
结论:
1.我们所用siRNA可以有效阻断Notch1的表达
2.siRNA介导的Notch1表达下调能够引起细胞周期阻滞和细胞凋亡率增加,从而抑制乳腺癌细胞的增殖,
3.siRNA介导的Notch1表达下调能够增加乳腺癌细胞对docetaxel或doxorubicin的药物敏感性。
4.siRNA介导的Notch1表达下调可以抑制NK-kappaB的DNA结合活性。
因此,Notch1是治疗乳腺癌的有效分子靶点,并且可以和传统的化疗药物联合应用,以降低化疗药物毒副作用,增强其抗肿瘤治疗效果。
SectionⅠ: The expression and activation of Notch signaling in human breast cancer and the effect ofγ-secretase inhibitor on the proliferation of breast cancer
Background:
Notch signaling is a pathway highly conserved through evolution which regulates various physiological processes,including stem cell maintenance, differentiation,proliferation and apoptosis.In mammals,key components of the Notch pathway include four transmembrane receptors(Notch 1-4)and five ligands(Delta-like 1,Delta-like 3,Delta-like 4,Jagged 1,Jagged 2).Direct binding of a ligand from a signaling cell to a Notch receptor on the membrane of the receiving cell initiates two successive proteolytic cleavages by TACE(TNF-α-converting enzyme)and theγ-secretase/presenilin complex,which ultimately results in the release of the intracellular domain(N-IC).N-IC then translocates into the nucleus and directly interacts with the DNA binding protein CBF 1/Su(H)/Lag 1(CSF)that activates the transcription of target genes including the Hes(hairy/enhancer-of-split)and hey.It was found recently that Notch signaling is related with the etiology of breast cancer. The first indication that Notch signaling might play a role in neoplastic development of the mammary gland came from the characterization of a common insertion site for the mouse mammary tumor virus in CzechⅡmice.In 20%of these tumors,the mouse mammary tumor virus was inserted within the Notch4/Notch1 locus.At both loci,insertion of the provirus leads to expression of Notch protein that consists of the transmembrane and intracellular domains only,suggesting that deregulated Notch signaling related to tumor.Several studies show that over expression of consititutively activated notch1 or notch4 in normal human breast epithelial cells can induce transformation in vitro.Notch1 protein was highly expressed in four breast tumors that over expressed H-ras,and the expression was associated with adverse prognosis.
Aim:
To detect systematically the expression of Notch signaling moleculars and the aberrant activation of Notch signaling in human breast cancer.Explore the role ofγ -secretase inhibitor on the proliferation of breast cancer cells.
Meterial and Methods:
1.Collect breast cancer specimens and normal breast tissues from the margin of tumor sections as control.Total RNA was extracted by TRIZOL according to the manufacturers instructions.Synthesis of first-strand cDNA was carried out with Revert Aid~(TM)First Strand cDNA Synthesis Kit.Notch1,Notch3,Notch4 and Jagged1,Delta-like4 were amplified.
Immunostaining was used to detect the activated form of Notch1(N-IC)in formalin-fixed,paraffin-embedded tissue sections
2.Cell culture.Human breast cancer cell lines MDA-MB-231,MDA-MB-435 were cultured in RPMI medium 1640.RT-PCR was used to detect the expression of. Notch 1,Notch3,Notch4 and Jagged 1,Delta-like4.Immunostaining was used to detect the activated form of Notch1(N-IC)in these cell lines.
3.Immunostaining.10 of breast cancer tissues and 4 of normal breast tissues at the margin of breast cancer were collected.Formalin-fixed,paraffin-embedded tissue sections(5μm thick)were dewaxed,rehydrated and optimized antigen retrieval.
Alternatively,cultured cells grown on cover slides were fixed in 95%ethanol. Sections were then used to detect Notch1-IC using Histostain-plus kit according to the manufacturer's instructions.
4.Inhibition assay byγ-secretase inhibitor.4×10~4 of MDA-MB-231 cells and MDA-MB-435 cells were plated in a 96-well plate and allowed to proliferate overnight.The cells were then treated with increasing concentration ofγ-secretase inhibitor.24 hours after treatment,the proliferation of the cells were measured by MTT method.
5.Analysis of Cell cycle.Briefly,1×10~6 cells were plated in 100 ml culture flasks and allowed to proliferate till 70%-80%confluent.Then,the cells were treated withγ-secretase inhibitor(3μmol/L)or DMSO(15μl)as a control,and after 24h, cells were harvested and stained with PI.The DNA content was analyzed by FACS Calibur
6.Detection of apoptosis.Cells were treated withγ-secretase inhibitor(3μmol/L)or DMSO(15μl)as described above.24 hours after treatment,cells were collected and labeled with 1:500 Annexin V-biotin conjugated with fluorescent isothiocyanate(FITC)followed by 1:1000 PI.Annexin V-PI were measured by FACS Calibur
Results:
1.Notch receptors and ligands are over expressed in human breast cancer.The expression rates of Notch1,3,4,Jagged1 and Delta-like4 gene mRNA in cancer specimen and normal breast tissue at the margin of tumor sections are 98%,35%, 8%,15%,81%and 73%,0%,0%,0%,0%,respectively.The expression rate of Notch signaling moleculars mRNA in the cancer specimens is significantly higher than that in the specimens at the margin of tumor sections.Notch1,3 and Jagged1 were all expressed in human breast cancer cell lines MDA-MB-231 and MDA-MB-435.
2.Notch1 is activated in human breast cancer.We used the antibody which recognizes only the activated form of Notch1(N1-ICD,the cleaved intracellular form)to detect its activation,we saw accumulation of N1-ICD in most(9 in 10) tumor samples analyzed,only 1 sample showed negative staining.Whereas,no N1-ICD staining was detected in all the normal breast tissues.In all the cell lines, we observed a clear accumulation of N1-ICD in both the cytoplasm and nuclear, with the later finding being indicative of Notch activation.
3.γ-secretase inhibitor can inhibit the proliferation of breast cancer cells.γ-secretase inhibitor,a pharmacologic agent known to block effectively Notch activation,was used to evaluate the effect of Notch inhibition on breast cancer cells. We observed the proliferation of both cell lines decreased with the increasing dosage ofγ-secretase inhibitor I(1-5μmol/L).
4.γ-secretase inhibitor can cause cell cycle arrest in breast cancer cell lines.γ-secretase inhibitor I-treated cells of MDA-MB-231 and MDA-MB-435 showed a higher proportion of cells in G2/M phase compared with control(29.9%vs 15.99%, and 45.5%vs 16.63%in MDA-MB-231 and MDA-MB-435 respectively).
5.γ-secretase inhibitor can cause apoptosis in breast cancer cell lines.The fraction of apoptotic cells inγ-secretase inhibitor -treated populations was significantly higher than that observed in controls(0.94%vs 3.03%,0.23%vs 4.33%in MDA-MB-231 and MDA-MB-435 respectively,p＜0.05,data are mean percentage of apoptotic cells from three independent experiments,demonstrating that inhibition of Notch signaling could also induce apoptosis in breast cancer cells.
Conclusion:
Notch signaling is over expressed and highly activated in human breast cancer. Blockage of Notch signaling byγ-secretaseinhibitor can inhibit the growth of breast cancer cells,causing cell cycle arrest and apoptosis.Thus,Notch signaling can be a therapeutic target for human breast cancer.
SectionⅡThe effect of down-regualtion of Notch1 mediated by siRNA on the proliferation of breast cancer cells
Background:
RNA interference(RNAi)is a process of gene silencing induced by double strand RNA(dsRNA).Basically,the process of RNAi is triggered by dsRNA precursors. These dsRNA precursors are processed into small interfering RNAs(siRNAs)that vary in length from 21-23 nucleotides(nt).The siRNAs are subsequently incorporated into a multiprotein complex.This complex is known as the RNA-induced silencing complex(RISC).siRNAs in the RISC complex guide degradation that is highly sequence-specific,of the complementary mRNAs.The gene silencing effect is highly efficient,as reported as high as 90%expression can be blocked.The high specificity and efficiency are very useful for human disease treatment as it can specifically knock down its target gene without affecting other gene's expression.
Previously we have found that Notch signaling is over expressed and highly activated in human breast cancer,andγ-secretase inhibitor can inhibit the proliferation by causing cell cycle arrest and apoptosis.Butγ-secretase inhibitor is a compound that has many activities including block the activation of Notch signaling.Phase I clinical study showed that it also has severe gastrointestinal side effect.So we further explored more specific method to knock down the expression of Notch1.We use siRNA that can specific binds to Notch1 to down regulate its expression and examine the effect on proliferation of breast cancer cells.Also we detected whether Notch signaling is related to chemosensitivity and the relation between Notch and NK-kappaB.
Meterial and Methods:
1.SiRNA Transfection.1.2×10~5 of MDA-MB-231 and MCF-7 Cells were seeded in 6-well plates(or 4000 cells/well in 96-well plates).After overnight incubation,cells (30-50%confluence)were treated with 40nM siRNA/control siRNA.Seventy-two hours after siRNA transfection,cells were used for MTT,real-time PCR,western blot,and other experiments.
2.Cell growth inhibition by MTT assay.MDA-MB-231 and MCF-7 Cells were incubated overnight at a density of 4,000 cells/well in 96-well plates,and subsequently transfected with Notch- 1 siRNA or control siRNA.72 hours after tansfection,20μl of MTT was added each well.48 additional hours later,color development was measured on a microplate reader at 570 nm.
3.Flow Cytometry and Cell Cycle Analysis.Notchl siRNA,or control siRNA transfected MDA-MB-231 and MCF7 cells were collected and stained with PI. DNA content was detected on a FACS Calibur.
4.Apoptosis Assay.72 hours after transfection,Notch1 siRNA/Control siRNA transfected MDA-MB-231 and MCF-7 cells were collected and were labeled with annexin V-biotin followed by PI.Annexin V-PI were measured by FACS Calibur and analyzed with the Modfit Software.
For the chemotherapeutic drug assays,24 hours after transfection,cells were exposed to 1.5 nM docetaxel or 75ug/ml doxorubicin respectively for 48 hours, then cells were collected and analyzed as above.
5.Western Blot Analysis.Total protein was extrated from transfected/control cells. Total proteins were fractionated using SDS-PAGE and transferred onto nitrocellulose membrane.After incubation with blocking buffer,the membrane was incubated with primary and secondary antibodies,the protein bands were detected using the enhanced chemiluminesence detection system.
6.Electrophoresis Mobility Shift Assay for Measuring NF-KappaB Activity.Nuclear extracts from MDA-MB-231 and MCF-7 cells were prepared.For EMSA,32 fmol of labeled probes were incubated for 20 min at 25℃with 2μg of nuclear extracts. Competition experiments were performed using EMSA conditions similar to those described above,except that the protein extracts were incubated with the probe in the presence of 250-fold molar excess of unlabeled double-stranded oligonucleotides as competitors.Subsequently,the DNA-protein complexes were separated from the free probes by electrophoresis through a 5%non-denaturing polyacrylamide gel,the gel was transferred to a PVDF membrane and chemiluminescent detection was performed.
Results:
1.Notch 1 siRNA effectively down-regulated the expression level of Notch 1 in human breast cancer cells.We observed that Notch1 and Hes1 mRNA decreased by about 90%in both MDA-MB-231 and MCF7 cells.Protein levels were also greatly reduced in Notch1 siRNA transfected cells compared with control siRNA transfected cells
2.Down-regulation of Notch1 expression by siRNA inhibited the growth of human breast cancer cells.72 hours after transfection with Notch1 siRNA or control siRNA,cell viability was determined by the MTT assay.We found that the down-regulation of Notch1 expression caused approximately a 40%reduction of cell growth in both breast cancer cell lines.
3.Down-regulation of Notch1 expression by siRNA induced S phase cell cycle arrest in MCF7 Cells.The Notch1 siRNA transfected MCF7 cells demonstrated an S phase arrest pattern(38.7%vs 19.54%)at 72 hours after transfection as compared with control cells.No alteration in cell cycle distribution was observed in MDA-MB-231 cells.
4.Down-regulation of Notch-1 expression by siRNA led to apoptosis in human breast cancer.72 hours after transfection,cells were stained with Annexin V/PI,and analyzed by flow cytometry.The apoptosis rates were 5.64%and 2.88%in Notch1 siRNA transfected MDA-MB-231 and MCF7 respectively,compared with 1.25% and 0.94%in control siRNA transfected cells.These data suggested that the growth inhibition induced by Notch1 siRNA was partially due to an increase in cell apoptosis.
5.Down-regulation of Notch-1 expression by siRNA increased chemosensitivity.We found that down-regulation of Notch-1 expression in combination with docetaxel or doxorubicin treatment led to a 50%or 70%enhancement in growth inhibition respectively as compared to single chemotherapeutic treatment in MDA-MB-231 cells.Similar results were found in MCF7 cells.In addition,the Notch-1 transfected MDA-MB-231 and MCF-7 cells were significantly more sensitive to docetaxel and doxorubicin-induced apoptosis,this may explain the enhanced growth inhibition.
6.Down-regulation of Notch-1 expression by siRNA reduces NF-κB DNA-binding activity.Down-regulation of Notch-1 significantly inhibited NF-κB DNA-binding activity compared with control cells in both MDA-MB-231 and MCF7 cells as detected by EMSA.These results provide evidence that cross-talk exists between Notch1 and NF-κB in breast cancer and that Notch signaling is the upstream regulator.
Conclusion: Down-regualtion of Notch-1 mediated by siRNA can inhibit the growth of breast cancer cells and increased chemosensitivity to doxorubicin and docetaxel.This effect may through the inactivation of NF-kappaB.Our results suggested that Notch signaling may be a promising target for breast cancer treatment.
引文
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2. Dear R, Wilcken N, Shannon J, et, al. Beyond chemotherapy-demystifying the new 'targeted' cancer treatments. Aust Fam Physician. 2004 37(1-2): 45-9
3. D'Addario G, Rauch D, Stupp R, et, al. Multicenter phase II trial of gefitinib first-line therapy followed by chemotherapy in advanced non-small-cell lung cancer (NSCLC): SAKK protocol 19/03. Ann Oncol. 2008 19(4):739-45.
4. Gajraj E, Chung H, Longson C, et, al. Rituximab for follicular non-Hodgkin lymphoma. Lancet Oncol. 2008 9(4):320-l.
5. Gidzinska B Notchl receptor: structure and role during T-cell development Postepy Hig Med Dosw. 2003;57(4):369-76.
6. Xia W, Wolfe MS Intramembrane proteolysis by presenilin and presenilin-like proteases..J Cell Sci. 2003 116:2839-44.
7. Black RA, White JM. ADAMs: focus on the protease domain. Curr Opin Cell Biol. 1998 10(5):654-9.
8. kogeat F, BessiaC, Bron C, et al. The Notchl receptoris cleaved constitutively by a furin-like convertase. Proc Natl AcadSci USA. 1998 95(14): 8108-8112.
9. Miller LA. Bigas A. Notch as a mediator of cell fate determination in hematopoiesis: evidence and speculation. Blood. 1999 93(8): 2431-2448.
10. Mumm JS, Schroeter EH, Saxena MT, et, al. A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notchl. Mol Cell. 2000 5(2): 197-206.
11 Gray GE, Mann R.S, et al. Human ligands of the Notch receptor. Am.J.Pathol. 154(1999): 785-794
12. Artavanis Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science. 1999 284(5415): 770-776.
13. Ordentlich P, Lin A, Shen CP, et al. Notch inhibition of FAT supports the existence of a novel signaling pathway. Mol Cel Biol. 1998 18(4): 2230- 2339.
14. Katsube K, Sakamoto KInt J Notch in vertebrates-molecular aspects of the signal. Dev Biol. 2005 49(2-3):369-74.
15. Laky K, Fleischacker C, Fowlkes B. TCR and Notch signaling in CD4 and CD8 T-cell development. J Immunol Rev. 2006 209:274-83.
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