Notch1在人舌鳞癌中的作用及其与表皮生长因子受体信号的交互作用
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摘要
Notch信号途径在哺乳动物细胞增殖、分化和凋亡等命运决定中起关键作用。其异常调控可引起组织发育异常并导致肿瘤等多种疾病的发生。研究表明,Notch信号途径的受体之一Notch1在许多肿瘤中存在异常表达,根据肿瘤的组织来源和细胞类型,Notch1可起促癌或抑癌作用。目前,Notch1已成为潜在的肿瘤治疗靶点。舌鳞癌是最常见的口腔癌,Notch1在舌鳞癌中的表达和作用至今仍未明确。
近年来的研究表明,Notch1与表皮生长因子受体(epidermal growth factorreceptor,EGFR)信号途径在许多肿瘤的发生、发展中存在交互作用。根据肿瘤的组织来源和细胞类型,两者可相互协同或相互拮抗。EGFR在舌鳞癌等多种肿瘤中过表达,导致相关信号途径异常活化,促进肿瘤生长、抑制细胞凋亡、导致放化疗敏感性下降、引起复发及预后不良。抑制EGFR表达和(或)阻断其相关信号途径可抑制舌鳞癌等肿瘤的生长。目前,EGFR已成为极具发展潜力的肿瘤治疗靶点,通过单克隆抗体、小分子酪氨酸激酶抑制剂等药物靶向治疗头颈部鳞癌等肿瘤的研究已取得较大进展,但仍存在着药物敏感性不高、肿瘤细胞耐药以及药物不良反应等问题。研究EGFR与其他信号途径的交互作用,可能为肿瘤的多靶点分子靶向治疗开辟新的途径。Notch1与EGFR信号途径在舌鳞癌中的交互作用至今仍未明确。
本研究探讨了Notch1在人舌鳞癌中的表达和作用及其与EGFR信号途径的交互作用,为揭示口腔癌的分子病理机制及口腔癌的多靶点分子靶向治疗研究提供实验基础。
第一部分Notch1和EGFR在人正常舌黏膜、舌黏膜癌前病变和舌鳞癌中的表达
目的研究Notch1和EGFR在人正常舌黏膜、舌黏膜癌前病变和舌鳞癌中的表达。
方法免疫组织化学方法检测15例人正常舌黏膜、59例舌黏膜白斑和65例舌鳞癌标本中Notch1和EGFR的表达。
结果(1)在正常舌黏膜和舌黏膜白斑标本中,Notch1的阳性表达主要位于角化层,部分颗粒层和棘层细胞也有表达,基底层细胞无表达;随上皮异常增生程度的加重,Notch1的阳性细胞率和表达水平逐渐降低(P<0.05)。EGFR的阳性表达主要位于基底层,少见于棘层,颗粒层及角化层无表达;随上皮异常增生程度的加重,EGFR的阳性细胞率和表达水平逐渐增高(P<0.05)。(2)在舌鳞癌中,Notch1的阳性表达仅见于高分化和中分化标本癌巢中鳞状化生的角化细胞及类棘层细胞,癌巢周边细胞无表达;低分化标本中无Notch1表达;分化程度越高,Notch1的阳性细胞率和表达水平越高(P<0.05);TNM分期越高,Notch1的阳性细胞率和表达水平越低(P<0.05)。EGFR的阳性表达主要位于癌巢周边细胞,处于鳞状化生的角化细胞及类棘层细胞则无表达;分化程度越高,EGFR的阳性细胞率和表达水平越低(P<0.05);TNM分期越高,EGFR的阳性细胞率和表达水平越高(P<0.05)。
结论
(1)在舌黏膜上皮中Notch1的表达诱导细胞分化。随上皮异常增生程度的加重Notch1的表达逐渐下调。
(2)在舌鳞癌的发生、发展中Notch1表达下调,可能起抑癌作用;而鳞状化生部位Notch1的表达可能与诱导癌细胞的分化有关。
(3)根据Notch1和EGFR的表达特点,提示两者在舌鳞癌的发生、发展中可能存在交互作用。
第二部分Notch1组成性活化对人舌鳞癌细胞体外生长及EGFR信号的影响
目的研究Notch1组成性活化对人舌鳞癌细胞体外生长及EGFR信号的影响。
方法用脂质体Lipofectamine~(TM) 2000分别将编码Notch1胞内域的真核表达质粒pRAMIC-IRES2-EGFP和对照质粒pIRES2-EGFP体外瞬时转染人舌鳞癌细胞系Tca8113细胞,用RT-PCR检测Notch1和EGFR的mRNA表达变化,用Western Blot检测Notch1及其效应子Hes1、EGFR及其下游信号分子p-ERK、以及p53蛋白的表达变化,用MTT法和流式细胞术分别检测细胞增殖活性和凋亡情况,用免疫细胞化学检测Tca8113细胞Notch1和EGFR蛋白的表达变化。
结果转染48h后,目的质粒转染组与未转染组相比,Notch1的mRNA和蛋白分别上调3.2倍和6.1倍,其效应子Hes1蛋白上调6.9倍,EGFR的mRNA和蛋白则分别下调12.7倍和9.3倍,其下游信号分子p-ERK蛋白下调16.6倍,而p53蛋白则上调9.7倍(均以β-actin作为内参)。目的质粒转染组与对照质粒转染组和未转染组相比,Notch1的mRNA和蛋白及Hes1蛋白显著上调(P<0.05),而EGFR的mRNA和蛋白及p-ERK蛋白显著下调(P<0.05),p53蛋白则显著上调(P<0.05)。目的质粒瞬时转染后,MTT法检测显示Tca8113细胞的增殖受到明显抑制(P<0.05)。转染48h后,流式细胞术检测显示细胞早期凋亡率显著增高(P<0.05),免疫细胞化学检测显示Tca8113细胞Notch1蛋白的表达上调而EGFR蛋白的表达下调。
结论
(1) Notch1组成性活化抑制人舌鳞癌细胞体外增殖、诱导细胞凋亡。结合第一部分实验,提示Notch1在舌鳞癌中起抑癌作用,可能成为潜在的治疗靶点。
(2) Notch1组成性活化在上调人舌鳞癌细胞Notch1及其效应子Hes1的同时,下调EGFR及其下游信号分子p-ERK蛋白,并上调p53蛋白。结合第一部分实验,提示人舌鳞癌中存在Notchl对EGFR信号的负向调控。
第三部分EGFR基因沉默及阻断EGFR信号途径对人舌鳞癌细胞体外生长及Notch1信号的影响
目的研究EGFR基因沉默及阻断EGFR信号途径对人舌鳞癌细胞体外生长及Notch1信号的影响。
方法(1)实验一:构建靶向人EGFR基因的短发卡RNA(short hairpin RNA,shRNA)真核表达质粒shEGFR,用脂质体Lipofectamine~(TM) 2000分别将shEGFR和作为对照的非相关序列shRNA表达质粒shNC体外瞬时转染人舌鳞癌细胞系Tca8113细胞;(2)实验二:用5μM和10μM浓度的受体酪氨酸激酶抑制剂AG1478分别阻断Tca8113细胞的EGFR信号途径;(3)对实验一、二,用RT-PCR检测EGFR和Notch1 mRNA的表达变化,用Western Blot检测EGFR及其下游信号分子p-ERK、Notch1及其效应子Hes1、以及p53蛋白的表达变化,用免疫细胞化学检测Tca8113细胞Notch1和EGFR蛋白的表达变化;(4)对实验一,用MTT法和流式细胞术分别检测细胞增殖活性和凋亡情况,
结果(1)转染48h后,shEGFR转染组与未转染组相比,EGFR的mRNA和蛋白分别下调13.4倍和10.0倍,其下游信号分子p-ERK蛋白下调10.6倍,而Notch1的mRNA和蛋白则分别上调2.2倍和7.0倍,其效应子Hes1蛋白上调13.9倍,p53蛋白上调12.5倍。shEGFR组与shNC组和未转染组相比,EGFR的mRNA和蛋白及p-ERK蛋白显著下调(P<0.05),而Notch1的mRNA和蛋白及Hes1蛋白显著上调(P<0.05),p53蛋白显著上调(P<0.05)。瞬时转染shEGFR后,MTT法检测显示Tca8113细胞的增殖受到明显抑制(P<0.05);转染48h后,流式细胞术检测显示细胞早期凋亡率显著增高(P<0.05),细胞免疫化学显示Tca8113细胞的EGFR蛋白表达下调而Notch1蛋白表达上调。(2)在5μM和10μM的AG1478处理后,与未处理组相比,Tca8113细胞的EGFR mRNA和蛋白的表达均无明显改变(P>0.05),p-ERK蛋白分别下调2.0倍和3.5倍(P<0.05),Notch1 mRNA则分别上调1.8倍和2.7倍(P<0.05),Notch1蛋白分别上调1.3倍和2.9倍(P<0.05),Hes1蛋白分别上调4.4倍和14.8倍(P<0.05),p53蛋白分别上调1.8倍和13.2倍(P<0.05)(均以β-actin作为内参),免疫细胞化学检测显示Tca8113细胞的EGFR表达无明显变化而Notch1蛋白表达上调。
结论
(1)短发卡RNA介导的EGFR基因沉默下调人舌鳞癌细胞EGFR及其下游信号分子p-ERK的表达,上调p53蛋白的表达,抑制细胞体外增殖,诱导细胞凋亡。
(2)受体酪氨酸激酶抑制剂阻断人舌鳞癌细胞EGFR信号途径,对EGFR的表达无明显影响,但下调其下游信号分子p-ERK蛋白的表达,而上调p53蛋白的表达。
(3) EGFR基因沉默及阻断EGFR信号途径,均上调Notch1及其效应子Hes1蛋白的表达,提示人舌鳞癌中存在EGFR对Notch1信号的负向调控。
(4)结合第一、第二部分实验,提示人舌鳞癌中可能存在Notch1与EGFR信号之间双向调控、相互拮抗的交互作用。
Notch signalling plays a decisive role in mammalian cell fate determination including cell proliferation,differentiation and apoptosis.Perturbation of Notch signalling can manifest as tissue abnormalities ultimately leading to disease states such as cancer. Accumulating evidence demonstrated that Notch1,the main form of Notch receptors,is deregulated in various malignancies.It can either promote or suppress tumors depending on type and context of cells.It might be a potential therapeutic target in oncological applications.Up to now,there is no report on the tissue-specific expression of Notchl in tongue squamous cell carcinoma(TSCC),the most common oral cancer, and the potential role of Notchl in TSCC remains to be elucidated.
Recently,increasing evidence indicated that the interplay between Notchl and epidermal growth factor receptor(EGFR) was involved in carcinogenesis of many cancers where the two pathways can be either antagonistic or synergistic,depending on type and context of cells.EGFR,as a member of erbB family of receptor tyrosine kinases(RTKs) has been extensively investigated.It is overexpressed in a large number of tumors including TSCC.Aberrant activation of EGFR signalling is frequently associated with transformation of primary cells,tumor progression,poor prognosis and decreased survival.EGFR signalling is considered a prime target for development of novel antineoplastic agents.Small molecule inhibitors of RTKs and monoclonal antibodies targeting EGFR signalling have been developed and clinically investigated for treatment of various cancers including head and neck cancers.However,the exact molecular mechanisms underlying sensitivity and resistance of tumor cells to EGFR inhibition remain to be elucidated.Hence,the identification of the potential interplay between EGFR and other signalling in cancers is of considerable significance.To the best of our knowledge,there is no report on the correlation between Notch1 and EGFR signalling in TSCC.
This study was to investigate the tissue-specific expression and the role of Notch1 in TSCC,and to explore the potential cross-talk between Notch1 and EGFR signalling in TSCC.The study might provide significant clues to developing new and promising strategies of multitargeted molecular therapy for the treatment of oral cancers.
PartⅠ
Expression of Notch1 and EGFR in human normal tongue mucosa, precancerous lesion and tongue squamous cell carcinoma
Objective To investigate the expression of Notch1 and EGFR in human normal tongue mucosa,precancerous lesion and tongue squamous cell carcinoma(TSCC).
Methods The expression of Notch1 and EGFR was detected in human normal tongue mucosa(n=15),tongue leukoplakia(LP)(n=59) and TSCC(n=65) by immunohistochemistry.
Results(1) In normal tongue mucosa and LP,the positive staining of Notchl was mainly distributed in stratum corneum,partially in stratum granulosum and stratum spinosum,but not in stratum basale.With increase of the degree of epithelial dysplasia, the proportion of positive cells and expression levels of Notch1 gradually decreased(P < 0.05 ).The positive staining of EGFR was mainly distributed in stratum basale,rarely in stratum spinosum,but not in stratum granulosum and stratum corneum.With increase of the degree of epithelial dysplasia,the proportion of positive cells and expression levels of EGFR gradually increased(P < 0.05 ).(2) In TSCC,Notch1 expression was abrogated in peripheral cells of carcinomas except locations of squamous metaplasia of well-differentiated and moderately differentiated specimens where Notchl expression was elevated in stratifying cells.In poorly differentiated specimens,Notchl expression is negative in carcinomas.The higher the degree of differentiation of TSCC was,the higher the proportion of positive cells and expression levels of Notchl was(P<0.05). The higher the stage of TNM of TSCC was,the lower the proportion of positive cells and expression levels of Notchl was(P<0.05).EGFR expression was detected mainly in peripheral cells of carcinomas,but not in locations of squamous metaplasia.The higher the degree of differentiation of TSCC was,the lower the proportion of positive cells and expression levels of EGFR was(P<0.05).The higher the stage of TNM of TSCC was,the higher the proportion of positive cells and expression levels of EGFR was(P<0.05).
Conclusions(1) The distribution of positive cells of Notchl was obviously different from that of EGFR in human normal tongue mucosa,precancerous lesion and tongue squamous cell carcinoma.(2) Notchl was down-regulated in the process of development and progression of tongue squamous cell carcinoma,while EGFR was up-regulated.
PartⅡ
Effects of constitutive activation of Notch1 on cell growth and EGFR signalling in human tongue squamous cell carcinoma cells in vitro
Objective To investigate effects of constitutive activation of Notchl in human tongue squamous cell carcinoma(TSCC) cells on cell growth and EGFR signalling in vitro.
Methods Human TSCC cell line Tca8113 cells were transiently transfected with the eukaryotic expression plasmid pRAMIC-IRES2-EGFP encoding exogenous intracellular fragment of Notchl and control plasmid pIRES2-EGFP by Lipofectamine~(TM)2000,respectively.Untransfected parental Tca8113 cells served as control.The mRNA of Notchl and EGFR in Tca8113 cells was detected by reverse transcriptase polymerase chain reaction(RT-PCR).The protein levels of Notchl and its effector Hes1,EGFR and its downstream signaling molecule p-ERK,and p53 in Tca8113 cells were detected by Western Blot.The cell proliferation was evaluated by methyl thiazolyl tetrazolium(MTT) assay.The apoptosis was assessed by flow cytometry.The expression of Notch1 and EGFR protein in Tca8113 cells was detected by immunocytochemistry.
Results After transfected with pRAMIC-IRES2-EGFP for 48h,when compared with untransfected parental Tca8113 cells,Notchl expression in Tca8113 cells significantly increased at mRNA(3.2-fold)(P<0.05) and protein level(6.1-fold)(P<0.05),and its effector Hes1 expression significantly increased at protein level(6.9-fold)(P<0.05), while EGFR expression significantly decreased at mRNA(12.7-fold)(P<0.05) and protein level(9.3-fold)(P<0.05),and its downstream signaling molecule p-ERK significantly decreased at protein level(16.6-fold)(P<0.05),and p53 significantly increased at protein level(9.7-fold)(P<0.05).The control plasmid pIRES2-EGFP had no effect.β-actin was used as an internal control.MTT assay showed that the cell proliferation of Tca8113 cells transfected with pRAMIC-IRES2-EGFP was significantly inhibited as compared with controls(P<0.05).After transfected with pRAMIC-IRES2-EGFP for 48h,the rate of early apoptosis(%) of Tca8113 cells was significantly higher than that of Tca8113 cells transfected with pIRES2-EGFP and untransfected Tca8113 cells(P<0.05),and immunocytochemistry showed that the expression of Notch1 significantly up-regulated in Tca8113 cells transfected with pRAMIC-IRES2-EGFP,while EGFR expression significantly down-regulated,as compared with controls.
Conclusions(1) Constitutive activation of Notch1 up-regulated Notch1 signaling molecules and down-regulated EGFR signaling molecules in TSCC cells in vitro.(2) Constitutive activation of Notch1 inhibited cell proliferation,induced apoptosis and up-regulated p53 protein in TSCC cells in vitro.
PartⅢ
Effects of EGFR gene silencing and blocking EGFR signalling on cell growth and Notchl signalling in human tongue squamous cell carcinoma cells in vitro
Objective To investigate effects of EGFR gene silencing and blocking EGFR signalling on cell growth and Notchl signalling in human tongue squamous cell carcinoma(TSCC) cells in vitro.
Methods(1) Experiment 1:Human TSCC cell lines Tca8113 cells were transiently transfected with the eukaryotic expression plasmid shEGFR encoding the specific short hairpin RNA(shRNA) targeting EGFR and the control plasmid shNC encoding shRNA targeting unrelated sequence by Lipofectamine~(TM)2000,respectively.Untransfected parental Tca8113 cells served as control.(2) Experiment 2:Tca8113 cells were treated with AG1478,an inhibitor of receptor tyrosine kinases,at a total concentration of 5 or 10μM,respectively.(3) In Experiment 1 and 2,the mRNA of EGFR and Notchl in Tca8113 cells was detected by reverse transcriptase polymerase chain reaction (RT-PCR).The protein levels of EGFR and its downstream signaling molecule p-ERK, Notch1 and its effector Hes1,and p53 in Tca8113 cells were detected by Western Blot. The expression of Notch1 and EGFR protein in Tca8113 cells was detected by immunocytochemistry.(4) In Experiment 1,the cell proliferation was evaluated by methyl thiazolyl tetrazolium(MTT) assay,and the apoptosis was assessed by flow cytometry,and
Results(1) After transfection with shEGFR for 48h,when compared with untransfected parental Tca8113 cells,EGFR expression in Tca8113 cells significantly decreased at mRNA level(13.4-fold)(P<0.05) and protein level(10.0-fold)(P<0.05), and its downstream signaling molecule p-ERK significantly decreased at protein level (10.6-fold)(P<0.05),while Notchl expression in Tca8113 cells significantly increased at mRNA(2.2-fold)(P<0.05) and protein level(7.0-fold)(P<0.05),and its effector Hesl expression significantly increased at protein level(13.9-fold)(P<0.05),and p53 significantly increased at protein level(12.5-fold)(P<0.05).The control vector shNC had no effect.β-actin was used as an internal control.MTT assay showed that the cell proliferation of Tca8113 cells transfected with shEGFR was significantly inhibited as compared with controls(P<0.05).After transfected with shEGFR for 48h,the rate of early apoptosis(%) of Tca8113 cells was significantly higher than that of Tca8113 cells transfected with shNC and untransfected Tca8113 cells(P<0.05),and immunocytochemistry showed that the expression of EGFR protein significantly down-regulated,while Notchl expression significantly up-regulated in Tca8113 cells transfected with shEGFR,as compared with controls.(2) After treatment with AG1478 at a total concentration of 5 or 10μM in Tca8113 cells for 4h,when compared with untreated cells,the expression of mRNA and protein of EGFR had no significant difference,but its downstream signaling molecule p-ERK significantly decreased at protein level(2.0 or 3.5-fold)(P<0.05),while Notchl expression significantly increased at mRNA level(1.8 or 2.7-fold)(P<0.05) and protein level(1.3 or 2.9-fold)(P<0.01), and its effector Hesl expression significantly increased at protein level(4.4 or 14.8-fold) (P<0.05),and p53 significantly increased at protein level(1.8 or 13.2-fold)(P<0.05).β-actin was used as an internal control.Immunocytochemistry showed that the expression of EGFR protein had no obvious difference,while Notchl expression obviously up-regulated in Tca8113 cells treated with AG1478 at a total concentration of 5 or 10μM,as compared with controls.
Conclusion(1) EGFR gene silencing mediated by shRNA down-regulated EGFR signaling molecules,up-regulated Notchl signaling molecules,inhibited cell proliferation,induced apoptosis and up-regulated p53 protein in TSCC cells in vitro.(2) Blocking EGFR signalling had no effect on the level of EGFR expression,but down-regulated its downstream signaling molecule p-ERK and up-regulated Notchl signaling molecules and p53 protein in TSCC cells in vitro.
近年来的研究表明,Notch1与表皮生长因子受体(epidermal growth factorreceptor,EGFR)信号途径在许多肿瘤的发生、发展中存在交互作用。根据肿瘤的组织来源和细胞类型,两者可相互协同或相互拮抗。EGFR在舌鳞癌等多种肿瘤中过表达,导致相关信号途径异常活化,促进肿瘤生长、抑制细胞凋亡、导致放化疗敏感性下降、引起复发及预后不良。抑制EGFR表达和(或)阻断其相关信号途径可抑制舌鳞癌等肿瘤的生长。目前,EGFR已成为极具发展潜力的肿瘤治疗靶点,通过单克隆抗体、小分子酪氨酸激酶抑制剂等药物靶向治疗头颈部鳞癌等肿瘤的研究已取得较大进展,但仍存在着药物敏感性不高、肿瘤细胞耐药以及药物不良反应等问题。研究EGFR与其他信号途径的交互作用,可能为肿瘤的多靶点分子靶向治疗开辟新的途径。Notch1与EGFR信号途径在舌鳞癌中的交互作用至今仍未明确。
本研究探讨了Notch1在人舌鳞癌中的表达和作用及其与EGFR信号途径的交互作用,为揭示口腔癌的分子病理机制及口腔癌的多靶点分子靶向治疗研究提供实验基础。
第一部分Notch1和EGFR在人正常舌黏膜、舌黏膜癌前病变和舌鳞癌中的表达
目的研究Notch1和EGFR在人正常舌黏膜、舌黏膜癌前病变和舌鳞癌中的表达。
方法免疫组织化学方法检测15例人正常舌黏膜、59例舌黏膜白斑和65例舌鳞癌标本中Notch1和EGFR的表达。
结果(1)在正常舌黏膜和舌黏膜白斑标本中,Notch1的阳性表达主要位于角化层,部分颗粒层和棘层细胞也有表达,基底层细胞无表达;随上皮异常增生程度的加重,Notch1的阳性细胞率和表达水平逐渐降低(P<0.05)。EGFR的阳性表达主要位于基底层,少见于棘层,颗粒层及角化层无表达;随上皮异常增生程度的加重,EGFR的阳性细胞率和表达水平逐渐增高(P<0.05)。(2)在舌鳞癌中,Notch1的阳性表达仅见于高分化和中分化标本癌巢中鳞状化生的角化细胞及类棘层细胞,癌巢周边细胞无表达;低分化标本中无Notch1表达;分化程度越高,Notch1的阳性细胞率和表达水平越高(P<0.05);TNM分期越高,Notch1的阳性细胞率和表达水平越低(P<0.05)。EGFR的阳性表达主要位于癌巢周边细胞,处于鳞状化生的角化细胞及类棘层细胞则无表达;分化程度越高,EGFR的阳性细胞率和表达水平越低(P<0.05);TNM分期越高,EGFR的阳性细胞率和表达水平越高(P<0.05)。
结论
(1)在舌黏膜上皮中Notch1的表达诱导细胞分化。随上皮异常增生程度的加重Notch1的表达逐渐下调。
(2)在舌鳞癌的发生、发展中Notch1表达下调,可能起抑癌作用;而鳞状化生部位Notch1的表达可能与诱导癌细胞的分化有关。
(3)根据Notch1和EGFR的表达特点,提示两者在舌鳞癌的发生、发展中可能存在交互作用。
第二部分Notch1组成性活化对人舌鳞癌细胞体外生长及EGFR信号的影响
目的研究Notch1组成性活化对人舌鳞癌细胞体外生长及EGFR信号的影响。
方法用脂质体Lipofectamine~(TM) 2000分别将编码Notch1胞内域的真核表达质粒pRAMIC-IRES2-EGFP和对照质粒pIRES2-EGFP体外瞬时转染人舌鳞癌细胞系Tca8113细胞,用RT-PCR检测Notch1和EGFR的mRNA表达变化,用Western Blot检测Notch1及其效应子Hes1、EGFR及其下游信号分子p-ERK、以及p53蛋白的表达变化,用MTT法和流式细胞术分别检测细胞增殖活性和凋亡情况,用免疫细胞化学检测Tca8113细胞Notch1和EGFR蛋白的表达变化。
结果转染48h后,目的质粒转染组与未转染组相比,Notch1的mRNA和蛋白分别上调3.2倍和6.1倍,其效应子Hes1蛋白上调6.9倍,EGFR的mRNA和蛋白则分别下调12.7倍和9.3倍,其下游信号分子p-ERK蛋白下调16.6倍,而p53蛋白则上调9.7倍(均以β-actin作为内参)。目的质粒转染组与对照质粒转染组和未转染组相比,Notch1的mRNA和蛋白及Hes1蛋白显著上调(P<0.05),而EGFR的mRNA和蛋白及p-ERK蛋白显著下调(P<0.05),p53蛋白则显著上调(P<0.05)。目的质粒瞬时转染后,MTT法检测显示Tca8113细胞的增殖受到明显抑制(P<0.05)。转染48h后,流式细胞术检测显示细胞早期凋亡率显著增高(P<0.05),免疫细胞化学检测显示Tca8113细胞Notch1蛋白的表达上调而EGFR蛋白的表达下调。
结论
(1) Notch1组成性活化抑制人舌鳞癌细胞体外增殖、诱导细胞凋亡。结合第一部分实验,提示Notch1在舌鳞癌中起抑癌作用,可能成为潜在的治疗靶点。
(2) Notch1组成性活化在上调人舌鳞癌细胞Notch1及其效应子Hes1的同时,下调EGFR及其下游信号分子p-ERK蛋白,并上调p53蛋白。结合第一部分实验,提示人舌鳞癌中存在Notchl对EGFR信号的负向调控。
第三部分EGFR基因沉默及阻断EGFR信号途径对人舌鳞癌细胞体外生长及Notch1信号的影响
目的研究EGFR基因沉默及阻断EGFR信号途径对人舌鳞癌细胞体外生长及Notch1信号的影响。
方法(1)实验一:构建靶向人EGFR基因的短发卡RNA(short hairpin RNA,shRNA)真核表达质粒shEGFR,用脂质体Lipofectamine~(TM) 2000分别将shEGFR和作为对照的非相关序列shRNA表达质粒shNC体外瞬时转染人舌鳞癌细胞系Tca8113细胞;(2)实验二:用5μM和10μM浓度的受体酪氨酸激酶抑制剂AG1478分别阻断Tca8113细胞的EGFR信号途径;(3)对实验一、二,用RT-PCR检测EGFR和Notch1 mRNA的表达变化,用Western Blot检测EGFR及其下游信号分子p-ERK、Notch1及其效应子Hes1、以及p53蛋白的表达变化,用免疫细胞化学检测Tca8113细胞Notch1和EGFR蛋白的表达变化;(4)对实验一,用MTT法和流式细胞术分别检测细胞增殖活性和凋亡情况,
结果(1)转染48h后,shEGFR转染组与未转染组相比,EGFR的mRNA和蛋白分别下调13.4倍和10.0倍,其下游信号分子p-ERK蛋白下调10.6倍,而Notch1的mRNA和蛋白则分别上调2.2倍和7.0倍,其效应子Hes1蛋白上调13.9倍,p53蛋白上调12.5倍。shEGFR组与shNC组和未转染组相比,EGFR的mRNA和蛋白及p-ERK蛋白显著下调(P<0.05),而Notch1的mRNA和蛋白及Hes1蛋白显著上调(P<0.05),p53蛋白显著上调(P<0.05)。瞬时转染shEGFR后,MTT法检测显示Tca8113细胞的增殖受到明显抑制(P<0.05);转染48h后,流式细胞术检测显示细胞早期凋亡率显著增高(P<0.05),细胞免疫化学显示Tca8113细胞的EGFR蛋白表达下调而Notch1蛋白表达上调。(2)在5μM和10μM的AG1478处理后,与未处理组相比,Tca8113细胞的EGFR mRNA和蛋白的表达均无明显改变(P>0.05),p-ERK蛋白分别下调2.0倍和3.5倍(P<0.05),Notch1 mRNA则分别上调1.8倍和2.7倍(P<0.05),Notch1蛋白分别上调1.3倍和2.9倍(P<0.05),Hes1蛋白分别上调4.4倍和14.8倍(P<0.05),p53蛋白分别上调1.8倍和13.2倍(P<0.05)(均以β-actin作为内参),免疫细胞化学检测显示Tca8113细胞的EGFR表达无明显变化而Notch1蛋白表达上调。
结论
(1)短发卡RNA介导的EGFR基因沉默下调人舌鳞癌细胞EGFR及其下游信号分子p-ERK的表达,上调p53蛋白的表达,抑制细胞体外增殖,诱导细胞凋亡。
(2)受体酪氨酸激酶抑制剂阻断人舌鳞癌细胞EGFR信号途径,对EGFR的表达无明显影响,但下调其下游信号分子p-ERK蛋白的表达,而上调p53蛋白的表达。
(3) EGFR基因沉默及阻断EGFR信号途径,均上调Notch1及其效应子Hes1蛋白的表达,提示人舌鳞癌中存在EGFR对Notch1信号的负向调控。
(4)结合第一、第二部分实验,提示人舌鳞癌中可能存在Notch1与EGFR信号之间双向调控、相互拮抗的交互作用。
Notch signalling plays a decisive role in mammalian cell fate determination including cell proliferation,differentiation and apoptosis.Perturbation of Notch signalling can manifest as tissue abnormalities ultimately leading to disease states such as cancer. Accumulating evidence demonstrated that Notch1,the main form of Notch receptors,is deregulated in various malignancies.It can either promote or suppress tumors depending on type and context of cells.It might be a potential therapeutic target in oncological applications.Up to now,there is no report on the tissue-specific expression of Notchl in tongue squamous cell carcinoma(TSCC),the most common oral cancer, and the potential role of Notchl in TSCC remains to be elucidated.
Recently,increasing evidence indicated that the interplay between Notchl and epidermal growth factor receptor(EGFR) was involved in carcinogenesis of many cancers where the two pathways can be either antagonistic or synergistic,depending on type and context of cells.EGFR,as a member of erbB family of receptor tyrosine kinases(RTKs) has been extensively investigated.It is overexpressed in a large number of tumors including TSCC.Aberrant activation of EGFR signalling is frequently associated with transformation of primary cells,tumor progression,poor prognosis and decreased survival.EGFR signalling is considered a prime target for development of novel antineoplastic agents.Small molecule inhibitors of RTKs and monoclonal antibodies targeting EGFR signalling have been developed and clinically investigated for treatment of various cancers including head and neck cancers.However,the exact molecular mechanisms underlying sensitivity and resistance of tumor cells to EGFR inhibition remain to be elucidated.Hence,the identification of the potential interplay between EGFR and other signalling in cancers is of considerable significance.To the best of our knowledge,there is no report on the correlation between Notch1 and EGFR signalling in TSCC.
This study was to investigate the tissue-specific expression and the role of Notch1 in TSCC,and to explore the potential cross-talk between Notch1 and EGFR signalling in TSCC.The study might provide significant clues to developing new and promising strategies of multitargeted molecular therapy for the treatment of oral cancers.
PartⅠ
Expression of Notch1 and EGFR in human normal tongue mucosa, precancerous lesion and tongue squamous cell carcinoma
Objective To investigate the expression of Notch1 and EGFR in human normal tongue mucosa,precancerous lesion and tongue squamous cell carcinoma(TSCC).
Methods The expression of Notch1 and EGFR was detected in human normal tongue mucosa(n=15),tongue leukoplakia(LP)(n=59) and TSCC(n=65) by immunohistochemistry.
Results(1) In normal tongue mucosa and LP,the positive staining of Notchl was mainly distributed in stratum corneum,partially in stratum granulosum and stratum spinosum,but not in stratum basale.With increase of the degree of epithelial dysplasia, the proportion of positive cells and expression levels of Notch1 gradually decreased(P < 0.05 ).The positive staining of EGFR was mainly distributed in stratum basale,rarely in stratum spinosum,but not in stratum granulosum and stratum corneum.With increase of the degree of epithelial dysplasia,the proportion of positive cells and expression levels of EGFR gradually increased(P < 0.05 ).(2) In TSCC,Notch1 expression was abrogated in peripheral cells of carcinomas except locations of squamous metaplasia of well-differentiated and moderately differentiated specimens where Notchl expression was elevated in stratifying cells.In poorly differentiated specimens,Notchl expression is negative in carcinomas.The higher the degree of differentiation of TSCC was,the higher the proportion of positive cells and expression levels of Notchl was(P<0.05). The higher the stage of TNM of TSCC was,the lower the proportion of positive cells and expression levels of Notchl was(P<0.05).EGFR expression was detected mainly in peripheral cells of carcinomas,but not in locations of squamous metaplasia.The higher the degree of differentiation of TSCC was,the lower the proportion of positive cells and expression levels of EGFR was(P<0.05).The higher the stage of TNM of TSCC was,the higher the proportion of positive cells and expression levels of EGFR was(P<0.05).
Conclusions(1) The distribution of positive cells of Notchl was obviously different from that of EGFR in human normal tongue mucosa,precancerous lesion and tongue squamous cell carcinoma.(2) Notchl was down-regulated in the process of development and progression of tongue squamous cell carcinoma,while EGFR was up-regulated.
PartⅡ
Effects of constitutive activation of Notch1 on cell growth and EGFR signalling in human tongue squamous cell carcinoma cells in vitro
Objective To investigate effects of constitutive activation of Notchl in human tongue squamous cell carcinoma(TSCC) cells on cell growth and EGFR signalling in vitro.
Methods Human TSCC cell line Tca8113 cells were transiently transfected with the eukaryotic expression plasmid pRAMIC-IRES2-EGFP encoding exogenous intracellular fragment of Notchl and control plasmid pIRES2-EGFP by Lipofectamine~(TM)2000,respectively.Untransfected parental Tca8113 cells served as control.The mRNA of Notchl and EGFR in Tca8113 cells was detected by reverse transcriptase polymerase chain reaction(RT-PCR).The protein levels of Notchl and its effector Hes1,EGFR and its downstream signaling molecule p-ERK,and p53 in Tca8113 cells were detected by Western Blot.The cell proliferation was evaluated by methyl thiazolyl tetrazolium(MTT) assay.The apoptosis was assessed by flow cytometry.The expression of Notch1 and EGFR protein in Tca8113 cells was detected by immunocytochemistry.
Results After transfected with pRAMIC-IRES2-EGFP for 48h,when compared with untransfected parental Tca8113 cells,Notchl expression in Tca8113 cells significantly increased at mRNA(3.2-fold)(P<0.05) and protein level(6.1-fold)(P<0.05),and its effector Hes1 expression significantly increased at protein level(6.9-fold)(P<0.05), while EGFR expression significantly decreased at mRNA(12.7-fold)(P<0.05) and protein level(9.3-fold)(P<0.05),and its downstream signaling molecule p-ERK significantly decreased at protein level(16.6-fold)(P<0.05),and p53 significantly increased at protein level(9.7-fold)(P<0.05).The control plasmid pIRES2-EGFP had no effect.β-actin was used as an internal control.MTT assay showed that the cell proliferation of Tca8113 cells transfected with pRAMIC-IRES2-EGFP was significantly inhibited as compared with controls(P<0.05).After transfected with pRAMIC-IRES2-EGFP for 48h,the rate of early apoptosis(%) of Tca8113 cells was significantly higher than that of Tca8113 cells transfected with pIRES2-EGFP and untransfected Tca8113 cells(P<0.05),and immunocytochemistry showed that the expression of Notch1 significantly up-regulated in Tca8113 cells transfected with pRAMIC-IRES2-EGFP,while EGFR expression significantly down-regulated,as compared with controls.
Conclusions(1) Constitutive activation of Notch1 up-regulated Notch1 signaling molecules and down-regulated EGFR signaling molecules in TSCC cells in vitro.(2) Constitutive activation of Notch1 inhibited cell proliferation,induced apoptosis and up-regulated p53 protein in TSCC cells in vitro.
PartⅢ
Effects of EGFR gene silencing and blocking EGFR signalling on cell growth and Notchl signalling in human tongue squamous cell carcinoma cells in vitro
Objective To investigate effects of EGFR gene silencing and blocking EGFR signalling on cell growth and Notchl signalling in human tongue squamous cell carcinoma(TSCC) cells in vitro.
Methods(1) Experiment 1:Human TSCC cell lines Tca8113 cells were transiently transfected with the eukaryotic expression plasmid shEGFR encoding the specific short hairpin RNA(shRNA) targeting EGFR and the control plasmid shNC encoding shRNA targeting unrelated sequence by Lipofectamine~(TM)2000,respectively.Untransfected parental Tca8113 cells served as control.(2) Experiment 2:Tca8113 cells were treated with AG1478,an inhibitor of receptor tyrosine kinases,at a total concentration of 5 or 10μM,respectively.(3) In Experiment 1 and 2,the mRNA of EGFR and Notchl in Tca8113 cells was detected by reverse transcriptase polymerase chain reaction (RT-PCR).The protein levels of EGFR and its downstream signaling molecule p-ERK, Notch1 and its effector Hes1,and p53 in Tca8113 cells were detected by Western Blot. The expression of Notch1 and EGFR protein in Tca8113 cells was detected by immunocytochemistry.(4) In Experiment 1,the cell proliferation was evaluated by methyl thiazolyl tetrazolium(MTT) assay,and the apoptosis was assessed by flow cytometry,and
Results(1) After transfection with shEGFR for 48h,when compared with untransfected parental Tca8113 cells,EGFR expression in Tca8113 cells significantly decreased at mRNA level(13.4-fold)(P<0.05) and protein level(10.0-fold)(P<0.05), and its downstream signaling molecule p-ERK significantly decreased at protein level (10.6-fold)(P<0.05),while Notchl expression in Tca8113 cells significantly increased at mRNA(2.2-fold)(P<0.05) and protein level(7.0-fold)(P<0.05),and its effector Hesl expression significantly increased at protein level(13.9-fold)(P<0.05),and p53 significantly increased at protein level(12.5-fold)(P<0.05).The control vector shNC had no effect.β-actin was used as an internal control.MTT assay showed that the cell proliferation of Tca8113 cells transfected with shEGFR was significantly inhibited as compared with controls(P<0.05).After transfected with shEGFR for 48h,the rate of early apoptosis(%) of Tca8113 cells was significantly higher than that of Tca8113 cells transfected with shNC and untransfected Tca8113 cells(P<0.05),and immunocytochemistry showed that the expression of EGFR protein significantly down-regulated,while Notchl expression significantly up-regulated in Tca8113 cells transfected with shEGFR,as compared with controls.(2) After treatment with AG1478 at a total concentration of 5 or 10μM in Tca8113 cells for 4h,when compared with untreated cells,the expression of mRNA and protein of EGFR had no significant difference,but its downstream signaling molecule p-ERK significantly decreased at protein level(2.0 or 3.5-fold)(P<0.05),while Notchl expression significantly increased at mRNA level(1.8 or 2.7-fold)(P<0.05) and protein level(1.3 or 2.9-fold)(P<0.01), and its effector Hesl expression significantly increased at protein level(4.4 or 14.8-fold) (P<0.05),and p53 significantly increased at protein level(1.8 or 13.2-fold)(P<0.05).β-actin was used as an internal control.Immunocytochemistry showed that the expression of EGFR protein had no obvious difference,while Notchl expression obviously up-regulated in Tca8113 cells treated with AG1478 at a total concentration of 5 or 10μM,as compared with controls.
Conclusion(1) EGFR gene silencing mediated by shRNA down-regulated EGFR signaling molecules,up-regulated Notchl signaling molecules,inhibited cell proliferation,induced apoptosis and up-regulated p53 protein in TSCC cells in vitro.(2) Blocking EGFR signalling had no effect on the level of EGFR expression,but down-regulated its downstream signaling molecule p-ERK and up-regulated Notchl signaling molecules and p53 protein in TSCC cells in vitro.
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[1]Weng AP,Aster JC.Multiple niches for Notch in cancer:context is everything.Curr Opin Genet Dev,2004,14(1):48-54
[2]Miele L.Notch signaling.Clin Cancer Res,2006,12(4):1074-1079.
[3]Radtke F.,Raj K.The role of Notch in tumorigenesis:oncogene or tumor suppressor?Nat Rev Cancer,2003,3(10):756-767.
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[5]Dotto GP.Notch tumor suppressor function.Oncogene,2008,27(38):5115-5123.
[6]Leong KG,Karsan A.Recent insights into the role of Notch signaling in tumorigenesis.Blood,2006,107(6):2223-2233.
[7]Hiraishi Y,Wada T,Nakatani K,et al.Immunohistochemical expression of EGFR and p-EGFR in oral squamous cell carcinomas.Pathol Oncol Res,2006,12(2):87-91.
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[12]Meera V.Sundaram.The love-hate relationship between Ras and Notch.Genes Dev,2005,19(16):1825-1839.
[13]Lai EC.Notch signaling:control of cell communication and cell fate.Development,2004,131(5):965-973.
[14]Bianchi S,Dotti MT,Federico A.Physiology and pathology of notch signalling system.J Cell Physiol,2006,207(2):300-308.
[15]Lefort K,Dotto GP.Notch signaling in the integrated control of keratinocyte growth/differentiation and tumor suppression.Semin Cancer Biol,2004,14(5):374-386.
[16]Lowell S,Jones P,Le Roux I,et al.Stimulation of human epidermal differentiation by delta-notch signaling at the boundaries of stem-cell clusters.Curr Biol,2000,10(9):491-500.
[17]Baldi A,De Falco M,De Luca L,Cottone G,Paggi MG,Nickoloff BJ,et al.Characterization of tissue specific expression of Notch-1 in human tissues.Biol Cell,2004,96(4):303-311.
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[21]Parr C,Watkins G,Jiang WG.The possible correlation of Notch-1 and Notch-2 with clinical outcome and tumour clinicopathological parameters in human breast cancer.Int J Mol Med,2004,14(5):779-786.
[22]Talora C,Sgroi DC,Crum CP,et al.Specific down-modulation of Notch 1 signaling in cervical cancer cells is required for sustained HPV-E6/E7 expression and late steps of malignant transformation.Genes Dev,2002,16(17):2252-2263.
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[25]Masuda S,Kumano K,Shimizu K,et al.Notchl oncoprotein antagonizes TGF-beta/Smad-mediated cell growth suppression via sequestration of coactivator p300.Cancer Sci,2005,96(5):274-282.
[26]Zavadil J,Cermak L,Soto-Nieves N,et al.Integration of TGF-beta/Smad and Jagged 1/Notch signalling in epithelial-to-mesenchymal transition.EMBO J,2004,23(5):1155-1165.
[27]Doroquez DB,Rebay I.Signal integration during development:mechanisms of EGFR and Notch pathway function and cross-talk.Crit Rev Biochem Mol Biol,2006,41(6):339-385.
[28]Kolev V,Mandinova A,Guinea-Viniegra J,et al.EGFR signalling as a negative regulator of Notch 1 gene transcription and function in proliferating keratinocytes and cancer.Nat Cell Biol,2008,10(8):902-911.
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[1]Lefort K,Dotto GP.Notch signaling in the integrated control of keratinocyte growth/differentiation and tumor suppression.Semin Cancer Biol,2004,14(5):374-386.
[2]Sundaram MV.The love-hate relationship between Ras and Notch.Genes Dev,2005,19(6):1825-1839.
[3]Doroquez DB,Rebay I.Signal integration during development:mechanisms of EGFR and Notch pathway function and cross-talk.Crit Rev Biochem Mol Biol,2006,41(6):339-385.
[4]Kolev V,Mandinova A,Guinea-Viniegra J,et al.EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.Nat Cell Biol,2008,10(8):902-911.
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[7]Miele L.Notch signaling.Clin Cancer Res,2006,12(4):1074-1079.
[8]Bianchi S,Dotti MT,Federico A.Physiology and pathology of notch signalling system.J Cell Physiol,2006,207(2):300-308.
[9]Wu L,Griffin JD.Modulation of Notch signaling by mastermind-like (MAML) transcriptional co-activators and their involvement in tumorigenesis.Semin Cancer Biol,2004,14(5):348-356.
[10]Anne Wilsona,Freddy Radtke.Multiple functions of Notch signaling in self-renewing organs and cancer.FEBS Letters,2006,580(12):2860-2868.
[11]Jonas Sjolund,Christina Manetopoulos,Marie-Therese Stockhausen,et al.The Notch pathway in cancer:Differentiation gone awry.European Journal of Cancer,2005,41(17):2620-2629.
[12]Claudio Taloraa,Samantha Cialfia,Oreste Segattob,et al.Constitutively active Notch 1 induces growth arrest of HPV-positive cervical cancer cells via separate signaling pathways.Experimental Cell Research,2005,305(2):343-354.
[13]Radtke R,Raj K.The role of Notch in tumorigenesis:oncogene or tumor suppressor? Nat Rev Cancer,2003,3(10):756-767.
[14]Weng AP,Aster JC.Multiple niches for Notch in cancer:context is everything.Curr Opin Genet Dev,2004,14(1):48-54.
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[17]Leong KG,Karsan A.Recent insights into the role of Notch signaling in tumorigenesis.Blood,2006,107(6):2223-2233.
[18]Nickoloff BJ,Osborne BA,Miele L.Notch signaling as a therapeutic target in cancer:a new approach to the development of cell fate modifying agents. Oncogene,2003,22(42):6598-6608.
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[20]Sriuranpong V,Borges MW,Ravi RK,et al.Notch signaling induces cell cycle arrest in small cell lung cancer cells.Cancer Res,2001,61(7):3200-3205.
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[23]Yao J,Duan L,Fan M,Wu X.Gamma-secretase inhibitors exerts antitumor activity via down-regulation of Notch and Nuclear factor kappa B in human tongue carcinoma cells.Oral Dis,2007,13(6):555-563.
[24]Purow BW,Sundaresan TK,Burdick MJ,et al.Notch-1 regulates transcription of the epidermal growth factor receptor through p53.Carcinogenesis,2008,29(5):918-925.
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[6]Claudio Taloraa,Samantha Cialfia,Oreste Segattob,et al.Constitutively active Notch1 induces growth arrest of HPV-positive cervical cancer cells via separate signaling pathways.Experimental Cell Research,2005,305(2):343-354.
[7]Wu L,Griffin JD.Modulation of Notch signaling by mastermind-like(MAML)transcriptional co-activators and their involvement in tumorigenesis.Semin Cancer Biol,2004,14(5):348-356.
[8]Anne Wilsona,Freddy Radtke.Multiple functions of Notch signaling in self-renewing organs and cancer.FEBS Letters,2006,580(12):2860-2868.
[9]Jonas Sj(o|¨)lund,Christina Manetopoulos,Marie-Thérése Stockhausen,et al.The Notch pathway in cancer:Differentiation gone awry.European Journal of Cancer,2005,41(17):2620-2629.
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[20]Talora C,Sgroi DC,Crum CP,et al.Specific down-modulation of Notch 1 signaling in cervical cancer cells is required for sustained HPV-E6/E7 expression and late steps of malignant transformation.Genes Dev,2002,16(17):2252-2263.
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[22]Sriuranpong V,Borges MW,Ravi RK,et al.Notch signaling induces cell cycle arrest in small cell lung cancer cells.Cancer Res,2001,61(7):3200-3205.
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[1]Weng AP,Aster JC.Multiple niches for Notch in cancer:context is everything.Curr Opin Genet Dev,2004,14(1):48-54
[2]Miele L.Notch signaling.Clin Cancer Res,2006,12(4):1074-1079.
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