卡介苗和猪苓多糖对膀胱癌细胞TLR2/4-NF-κB信号通路的影响
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摘要
卡介苗(Bacillus Calmette-Guerin,BCG)膀胱内灌注是目前治疗和预防浅表性膀胱癌较成功的免疫治疗,其作用认为与BCG诱导并增强了免疫反应尤其是与局部的免疫细胞有关,但这种机理尚不能完整阐释临床实践中BCG很少用于其它肿瘤治疗,用于其它肿瘤治疗疗效也不如用于治疗浅表性膀胱癌,这表明BCG治疗膀胱癌不只是激活癌组织局部天然免疫细胞,可能还存在其它途径和机制发挥抗肿瘤效应。近年文献报道尿路上皮细胞有TLR2和TLR4分布,可被BCG诱导表达,有研究报道膀胱癌细胞也表达TLRs,因此我们推测卡介苗也可能直接刺激膀胱移行上皮细胞TLRs,通过TLR-NF-κB信号通路产生免疫活化因子,提高肿瘤免疫原性,增强BCG抗肿瘤免疫反应。所以本课题拟探讨BCG对膀胱癌细胞(T739)的TLR2/4—NF-κB信号通路相关基因的影响。
首先应用实时定量PCR技术(qRT-PCR)探讨卡介苗对膀胱癌细胞T739 NF-κB信号通路相关基因mRNA表达影响,发现BCG对NF-κB信号通路相关基因TLR2、TLR4、CD14,MD-2、CHUK、MAP3K1、IKBKB、NFKB1(NF-κB p50)、Rel A(NF-κBp65)、CCL2和ICAM1等基因mRNA水平有显著上调。未检测出该通路抑制基因Nfkbia和Nfkbib的表达,结合后续实验NF-κB p65的DNA结合活性的实验结果,表明T739细胞NF-κB p65是持续活化的,符合多数哺乳动物肿瘤细胞NF-κB分子持续性活化的现象;BCG对NFKB1、Rel A表达有影响,对NFKB2表达无明显影响,推测NF-κB分子组成形式为Rel A/NFKB1。用TLR2和TLR4抗体阻断的方法发现MAP3K1、IKBKB、Rel A(NF-κB p65)、CCL2和ICAM1 mRNA表达都与TLR2或TLR4有关,即阻断TLR2或TLR4可以抑制BCG对上述基因的上调作用。
为证明TLR2和TLR4基因表达在介导BCG治疗膀胱癌方面的作用,我们选用基于载体表达的RNAi技术,针对小鼠TLR2基因编码区(ORF)不同位置的靶序列,设计3对miRNA前体序列,成功构建3个miRNA表达载体,并筛选出瞬时干扰效果最佳的载体pcDNA~(TM)6.2-GW/EmGFPmiR-TLR2.949。转染该载体,应用Blasticidin抗性筛选出TLR2沉默的稳转细胞株T739-TLR2A,连续传代1个月,T739-TLR2A细胞TLR2表达维持在稳定下调的水平,从而为将来实验提供了基础。针对小鼠TLR4基因,应用相同的方法,筛选并建立稳定细胞株T739-TLR4A。
用TLR2或TLR4基因稳定沉默细胞株研究发现BCG对MAP3K1、IKBKB mRNA上调主要经TLR2介导;对Rel A、CCL2 mRNA上调与TLR2或TLR4均相关;ICAM1mRNA的上调主要是经TLR4介导。在蛋白质水平,应用免疫印迹(Western blotting)和流式细胞仪(FCM)技术探讨卡介苗对IKKβ、CCL2和ICAM1表达与TLR2或TLR4关系,结果显示CCL2表达上调与TLR2或TLR4均相关,与CCL2 mRNA表达结果一致。IKKβ表达上调主要经TLR2介导,此与IKKβmRNA表达的实验结果吻合。ICAM1表达上调主要与TLR2相关,此与BCG对ICAM1 mRNA表达趋势不完全吻合。BCG对T739细胞NF-κB p65的活性或核移位表达的增强效应主要经TLR2介导,与TLR4也有一定程度相关。
猪苓多糖(polyporud polysaccharide,PPS)为葡萄糖与乳糖多聚体,是中药猪苓的主要有效成分。临床用于治疗乙型肝炎,改善机体的免疫功能,也具有抑制肿瘤生长和调节荷瘤动物及肿瘤病人免疫功能的作用,可减轻化疗药物所至的毒副反应,但PPS抗肿瘤效应并不十分清楚。NF-κB信号通路参与了病毒复制的调控、自身免疫性疾病、炎症反应、肿瘤发生和凋亡等,与肿瘤的发生发展、转移等密切相关,多数肿瘤细胞NF-κB通路是组成性或持续性活化,NF-κB抑制剂可用于肿瘤的辅助治疗。天然免疫系统TOLL样受体(TLRs)的发现为我们认识多糖作用机理研究提供新的思路,目前有文献表明植物多糖也是TLRs的配体之一。有研究报道膀胱上皮细胞也表达TLR2、TLR4,并且能对相应配体产生效应。我们以往的研究发现PPS可显著增加体外培养膀胱癌细胞p53蛋白表达,有抑制膀胱癌细胞增殖作用且能阻滞细胞由S期进入G2期。另有研究表明猪苓可使亚硝胺(BBN)诱发的膀胱癌的发生率显著降低,临床观察也显示猪苓可使膀胱癌复发率由对照组的65.1%降至33.3%。因此,根据我们以往关于猪苓多糖对膀胱癌研究的初步结果,推测PPS可能可以直接通过TLRs抑制膀胱癌细胞NFκB信号通路过度活化或某一(些)激酶或信号转导分子,调控细胞增殖与分化、细胞周期和凋亡相关基因表达,而发挥抗肿瘤作用。基于此,本课题设计应用RNAi技术沉默TLR2或TLR4,探讨猪苓多糖是否经TLR2/4介导对膀胱癌细胞NF-κB通路的调节作用。
应用qRT-PCR技术探讨PPS对膀胱癌细胞T739 NF-κB信号通路相关基因mRNA表达影响,发现部分膜表面分子TLR2、TLR4、CD14和MD-2等表达均增加,推测PPS可能主要通过TLRs向胞内转导信号。TRAF6、TIRAP、IRAK1为Toll信号通路中通用的接头分子,表达均有不同程度变化。IKBKB(IKKβ)mRNA表达显著减少,随着时间延长下降更为明显,Ratio值由0.32降至0.07。目前有研究显示,IKKs异常表达在肿瘤的发生发展过程中起着重要的作用,IKKs高表达和高活性赋予肿瘤细胞恶性生长特性和抗凋亡能力。PPS引起IKKβ表达显著下降,可能与我们以往研究PPS有抑制膀胱癌细胞增殖作用且能阻滞细胞由S期进入G2期这一结果有关。
T739细胞在PPS作用后,NF-κB p65mRNA表达下降。ICAM1和CCL2为信号通路下游的应答基因,CCL2为分泌性蛋白,具有单核细胞趋化作用,ICAM1为细胞间黏附分子,具有促使炎症细胞与靶细胞黏附。T739细胞在PPS作用后ICAM1和CCL2 mRNA表达均有不同程度下降,推测PPS具有抑制T739细胞NF-κB通路的部分基因表达水平和NF-κB活性;但PPS抑制T739细胞NF-κB通路的细胞生物学功能的改变尚未探讨。
进一步探讨了PPS与TLR2和TLR4的关系,发现TLR4可介导PPS下调T739细胞IKBKB mRNA和蛋白水平,而TLR2介导IKKβ蛋白下调。TLR4介导CCL2 mRNA下调,而TLR2介导CCL2蛋白下调。TLR2主要介导ICAM1mRNA表达下调,但PPS对ICAM1蛋白表达无明显影响,因此不能判断介导ICAM1蛋白表达的受体。PPS可下调Rel AmRNA表达,沉默TLR2或TLR4均可以部分抑制Rel A mRNA表达,表明TLR2和TLR4均介导Rel A mRNA表达下调。以上结果显示上述基因的表达水平下调主要由TLR4介导,TLR2也参与该效应。
PPS对胞核NF-κB p65的DNA结合活性与胞核表达的影响,发现PPS可使T739细胞NF-κB p65的DNA结合活性减弱,NF-κB p65胞核表达下调。在与TLR2/4关系探讨方面,PPS可显著降低T739-TLR2A细胞NF-κB p65的DNA结合活性,对T739-TLR4A细胞NF-κB p65的DNA结合活性无显著变化;在NF-κB p65胞核表达实验也反应出这一趋势,即PPS可显著降低T739-TLR2Δ细胞NF-κB p65胞核表达,对T739-TLR4Δ细胞NF-κB p65胞核表达无显著影响。上述实验结果均表明PPS主要经TLR4介导T739细胞NF-κB p65的DNA结合活性降低与胞核表达。
由于卡介苗和猪苓多糖均显示有抗膀胱癌的作用,且两者对NF-κB信号通路不同的调节趋势,因此拟观察两者同时作用膀胱癌细胞后该通路主要相关基因的效应。结果显示PPS可拮抗BCG上调T739细胞IKBKB、ICAM1、CCL2、NF-κB p65表达水平和NF-κB p65的DNA结合活性;其中PPS对NF-κB p65的DNA结合活性的拮抗作用尤为显著。
卡介苗抗膀胱癌效果很好,但副作用多,如膀胱炎等,这些症状与过度免疫有关,加入PPS后可调节上述免疫反应,减少毒性反应,为今后临床二者的联合用药减毒增效提供有重要价值的实验和理论依据。
Intravesical instillation of Bacillus Calmette-Guerin(BCG) is currently a comparatively effective immunotherapy for treating and preventing superficial bladder cancer and it is considered that the mechanism of the effect lies in the immune response induced and enhanced by BCG and especially its action on local immune cells.Whereas, the above mechanism is unable to completely explain the fact that the BCG is rarely applied and has much less effect on other types of cancer than on superficial bladder cancer.Thus it is highly probable that apart from activation of local natural immune cells by BCG in cancer tissue,there are some other underlying pathways and mechanisms which are still uncovered involved in its antitumor activity.In the recent years,it is reported that TLR2 and TLR4 were found in epithelial cells in urine and the expression can be induced by BCG. Considering bladder cancer cells also express TLRs,possibly,it is BCG that directly stimulates TLRs levels in the bladder transitional epithelial cells and subsequently causes the release immune activating factors via TLR-NF-κB signaling pathway,thereby strengthens the immunogenicity of tumor and promotes the antitumor immune reaction. The current study was designed to determine the role of BCG in regulating the genes expression via TLR2/4—NF-κB signaling pathway in mouse bladder cancer cell line T739.
First,the changes of mRNA expressionin of NF-κB signaling pathway associated-genes in BCG-treated bladder cancer cells T739 were analyzed by real-time PCR and some of the genes such as TLR2,TLR4,CD14,MD-2,CHUK,MAP3K1, LKBKB,NFKB1(NF-κB p50),Rel A(NF-κB p65),CCL2 and ICAM1 significantly increased after BCG treatment.The expression of the suppressor genes in NF-κB pathway Nfkbia and Nfkbibof could not be detected.Through the NF-κB p65 DNA-binding activity assay in the following experiment,it was revealed the fact that NF-κB p65 in T739 cells was constitutively activated,which corresponded to the common feature of NF-κB molecules in major mammalian tumor cells.BCG treatment had an effect on the expression of NFKB1 and RelA but not on the expression of NFKB2,thus,it seems reasonable to conjecture that the molecular form of NF-κB is Rel A/NFKB1.Using the antibody blocking assay,we discovered that the levels of MAP3K1,IKBKB,Rel A(NF-κB p65), CCL2 and ICAM1 mRNA rose after TLR2 and TLR4 were blocked.
Secondly,we applied vector based RNAi technology for investigating the effect of the expression of TLR2 and TLR4 on the BCG-mediated treatment for bladder cancer.Three pairs of miRNA precursor sequences were designed based on the different target sequences in mice coding region(ORF) and we constructed three miRNA expression vectors rom which the vector pcDNA~(TM)6.2-GW/EmGFPmiR- TLR2.949 were selected for its best transient interference effect.The vector was transfected into the cells and the TLR2-silencing stable cell line T739-TLR2Δwere selected by Blasticidin-resistant selection and passaged continually for a month.The TLRs expression maintained at a low level in T739-TLR2Δcells for the future experiments.Another stable cell line T739-TLR4Δ, silenced mouse gene TLR4,was established in the same way.
By using the two stale cell lines mentioned above,it was shown that BCG increased MAP3K1,IKBKB mRNAs via TLR2,up-modulated ICAM1 mRNAs via TLR4, up-regulated mRNA levels of Rel A and CCL2 via either TLR2 or TLR4.In a further study,the protein expression of IKKβ,CCL2 and ICAM1 were measured by western blot and flow cytometry to determine their relation with TLR2 and TLR4 following the BCG treatment.The results indicated that,in consistent with its mRNA expression,the increase in CCL2 protein levels associated both with TLR2 or TLR4.Up-regulation of IKKβprotein level mediated by TLR2 also matched up with the result of its mRNA.While inconsistent with its regulation of mRNA expression,ICAM1 protein level up-modulation mainly related with TLR2.The enhancement effect of BCG on NF-κB p65 activity and nuclear translocation mostly mediated by TLR2 and correlated with TLR4 to a certain extent.
Polyporus polysaccharide(PPS),as a main active component of polyporus,is a polymer of glucose and lactose.It has been applied clinically on the treatment of type B hepatitis and immunity enhancement.It can also inhibit the growth of carcinoma cells, modulate the immune function of tumor animals and patients,and reduce toxicity as well as side effect of chemotherapeutics.However,the mechanism of its anti-tumor activity is still unclear.NF-κB signaling pathway plays important roles in regulation of virus replication, autoimmune diseases,inflammatory response,tumorigenesis and apoptosis etc,and closely relates to the tumor development and metastasis.NF-κB signaling pathway in most tumor cells is activated constitutively or continuously.The inhibitor of NF-κB has been used as an adjuvant therapy for tumor.The finding of Toll-like receptor(TLRs) in innate immune system turns out to be a new way to study the mechanism of anti-tumor effect of polysaccharide.Recently,it has been reported that the plant polysaccharide is one of the ligands of TLRs.Bladder epithelial cells expressed TLR2 and TLR4 that can respond to corresponding ligands.Our previous research indicates that PPS can significantly increase p53 expression in the bladder cancer cells which culture in vitro and inhibit their proliferation by blocking the S-G2 phase transition in cell cycle.Other researcher reported that Polyporus could reduce the incidence of BBN-induced bladder cancer and the recurrence rate of bladder cancer can drop from 65.1%to 33.3%in contrast to the control group after administration of Polyporus.Therefore,according to the previous study about the role of Polyporus in the treatment of bladder cancer,we suppose that PPS may regulate some genes expression in proliferation,differentiation,cell cycle and apoptosis by means of direct inhibitation of the NF-κB signaling pathway in bladder cancer cells and excessive activation of some kinases or signal transduction molecules.Based on the assumption above,we utilized the RNAi technology to silence the genes of TLR2 and TLR4 so that whether the regulation of the PPS to NF-κB pathway in bladder cancer cells was mediated by TLR2 of TLR4 could be tested.
The increased expression of some membrane molecules such as TLR2,TLR.4,CD14 and MD-2 was detected by using qRT-PCR to study the effect of the PPS on the NF-κB signaling pathway-associated genes in bladder cancer cell T739,thus we think that PPS might transfer signals into cells through TLRs.As the adaptor molecules in Toll-like signaling pathway,TRAF6,TIRAP and IRAK1 had varying changes in their gene expression.The mRNA of IKBKB(IKKβ) decreased greatly and progressively over time and the ration declined from 0.32 to 0.07.Abnormal expression of IKKs is reported to have an important role in the tumorigenesis and tumor progression,its overexpression and overactivity gives a rise to malignant growth characteristic and anti-apoptosis capability of tumor.The decreased expression of IKKβdue to PPS could be explained by the our previous finding that PPS had an inhibitory effect on the proliferation of bladder cancer cells and S-G2 phase transition in cell cycle.
NF-κB p65 mRNA in T739 cells decrease after PPS treatment.ICAM1 and CCL2 act as downstream genes of the NF-κB pathway.CCL2 is a secretory protein involving in monocyto chemotaxis.ICAM1 is an intercellular adhesion molecule that can promote adhesion between inflammatory cells and target cells.The decreased expression of ICAM1 and CCL2 mRNA suggests that PPS has the ability to down-regulate some of the gene expression in NF-κB pathway and inhibit the activity of NF-κB,however,the inhibitory effect PPS on NF-κB pathway in T739 cells have not been studied yet at biological functional level.
Our further experiment demonstrated that the IKBKB mRNA and protein could be down-regulated by PPS via TLR4,but TLR2 only mediated the down-regulation of IKKβprotein.CCL2 mRNA and protein are down-modulated respectively via TLR4 and TLR2.PPS decreased the ICAM1 mRNA expression via TLR2 while had no significant effect on ICAM1 at protein level.The silencing of TLR2 or TLR4 gene can reduce the mRNA expression of Rel A,it shows that TLR2 and TLR4 are involved in the procedure of PPS down-regulating Rel A mRNA.The evidence above indicates that the genes we mentioned here are mainly mediated by TLR4.TLR2 also works on it in some way.
The DNA-binding activity of NF-κB p65 and its nuclear expression in T739 cells can be reduced by PPS.The DNA-binding activity of NF-κB p65 in T739-TLR2Δcells could be brought down by PPS,but the counterpart in T739-TLR4Δhad no significant response to this.Moreover,through nuclear expression assay,the effect of PPS on reducing NF-κB p65 nuclear expression in T739-TLR2Δcells but not in T739-TLR4Δwas exhibited.The results above show that the PPS reduce both DNA-binding activity and nuclear expression of NF-κB p65 in T739 cells mainly via TLR4.
Both BCG and PPS have the effect against bladder cancer and they regulate NF-κB pathway in different ways,so we focus on determining the effect of the both chemicals on the expression of main NF-κB pathway-related genes in bladder cancer cells.Our data show that PPS can antagonize the action of BCG on increasing the levels of IKBKB,ICAM1,CCL2 and NF-κB p65 and the DNA-binding activity of NF-κB p65,the antagonism is extremely significant to the latter one,DNA-bind activity of NF-κB p65.
BCG works well on against bladder cancer but it has some side effects,such as haematuria and pollakisuria,which are correlated with hyperimmunization.PPS can attenuate the above immune reaction and toxicity reaction when it is added to the treatment. Considering the combined medication with PPS can reduce the toxicity and increase the efficacy of BCG,this research will provide a crucial and helpful experimental and theoretical basis for the future clinical practice.
首先应用实时定量PCR技术(qRT-PCR)探讨卡介苗对膀胱癌细胞T739 NF-κB信号通路相关基因mRNA表达影响,发现BCG对NF-κB信号通路相关基因TLR2、TLR4、CD14,MD-2、CHUK、MAP3K1、IKBKB、NFKB1(NF-κB p50)、Rel A(NF-κBp65)、CCL2和ICAM1等基因mRNA水平有显著上调。未检测出该通路抑制基因Nfkbia和Nfkbib的表达,结合后续实验NF-κB p65的DNA结合活性的实验结果,表明T739细胞NF-κB p65是持续活化的,符合多数哺乳动物肿瘤细胞NF-κB分子持续性活化的现象;BCG对NFKB1、Rel A表达有影响,对NFKB2表达无明显影响,推测NF-κB分子组成形式为Rel A/NFKB1。用TLR2和TLR4抗体阻断的方法发现MAP3K1、IKBKB、Rel A(NF-κB p65)、CCL2和ICAM1 mRNA表达都与TLR2或TLR4有关,即阻断TLR2或TLR4可以抑制BCG对上述基因的上调作用。
为证明TLR2和TLR4基因表达在介导BCG治疗膀胱癌方面的作用,我们选用基于载体表达的RNAi技术,针对小鼠TLR2基因编码区(ORF)不同位置的靶序列,设计3对miRNA前体序列,成功构建3个miRNA表达载体,并筛选出瞬时干扰效果最佳的载体pcDNA~(TM)6.2-GW/EmGFPmiR-TLR2.949。转染该载体,应用Blasticidin抗性筛选出TLR2沉默的稳转细胞株T739-TLR2A,连续传代1个月,T739-TLR2A细胞TLR2表达维持在稳定下调的水平,从而为将来实验提供了基础。针对小鼠TLR4基因,应用相同的方法,筛选并建立稳定细胞株T739-TLR4A。
用TLR2或TLR4基因稳定沉默细胞株研究发现BCG对MAP3K1、IKBKB mRNA上调主要经TLR2介导;对Rel A、CCL2 mRNA上调与TLR2或TLR4均相关;ICAM1mRNA的上调主要是经TLR4介导。在蛋白质水平,应用免疫印迹(Western blotting)和流式细胞仪(FCM)技术探讨卡介苗对IKKβ、CCL2和ICAM1表达与TLR2或TLR4关系,结果显示CCL2表达上调与TLR2或TLR4均相关,与CCL2 mRNA表达结果一致。IKKβ表达上调主要经TLR2介导,此与IKKβmRNA表达的实验结果吻合。ICAM1表达上调主要与TLR2相关,此与BCG对ICAM1 mRNA表达趋势不完全吻合。BCG对T739细胞NF-κB p65的活性或核移位表达的增强效应主要经TLR2介导,与TLR4也有一定程度相关。
猪苓多糖(polyporud polysaccharide,PPS)为葡萄糖与乳糖多聚体,是中药猪苓的主要有效成分。临床用于治疗乙型肝炎,改善机体的免疫功能,也具有抑制肿瘤生长和调节荷瘤动物及肿瘤病人免疫功能的作用,可减轻化疗药物所至的毒副反应,但PPS抗肿瘤效应并不十分清楚。NF-κB信号通路参与了病毒复制的调控、自身免疫性疾病、炎症反应、肿瘤发生和凋亡等,与肿瘤的发生发展、转移等密切相关,多数肿瘤细胞NF-κB通路是组成性或持续性活化,NF-κB抑制剂可用于肿瘤的辅助治疗。天然免疫系统TOLL样受体(TLRs)的发现为我们认识多糖作用机理研究提供新的思路,目前有文献表明植物多糖也是TLRs的配体之一。有研究报道膀胱上皮细胞也表达TLR2、TLR4,并且能对相应配体产生效应。我们以往的研究发现PPS可显著增加体外培养膀胱癌细胞p53蛋白表达,有抑制膀胱癌细胞增殖作用且能阻滞细胞由S期进入G2期。另有研究表明猪苓可使亚硝胺(BBN)诱发的膀胱癌的发生率显著降低,临床观察也显示猪苓可使膀胱癌复发率由对照组的65.1%降至33.3%。因此,根据我们以往关于猪苓多糖对膀胱癌研究的初步结果,推测PPS可能可以直接通过TLRs抑制膀胱癌细胞NFκB信号通路过度活化或某一(些)激酶或信号转导分子,调控细胞增殖与分化、细胞周期和凋亡相关基因表达,而发挥抗肿瘤作用。基于此,本课题设计应用RNAi技术沉默TLR2或TLR4,探讨猪苓多糖是否经TLR2/4介导对膀胱癌细胞NF-κB通路的调节作用。
应用qRT-PCR技术探讨PPS对膀胱癌细胞T739 NF-κB信号通路相关基因mRNA表达影响,发现部分膜表面分子TLR2、TLR4、CD14和MD-2等表达均增加,推测PPS可能主要通过TLRs向胞内转导信号。TRAF6、TIRAP、IRAK1为Toll信号通路中通用的接头分子,表达均有不同程度变化。IKBKB(IKKβ)mRNA表达显著减少,随着时间延长下降更为明显,Ratio值由0.32降至0.07。目前有研究显示,IKKs异常表达在肿瘤的发生发展过程中起着重要的作用,IKKs高表达和高活性赋予肿瘤细胞恶性生长特性和抗凋亡能力。PPS引起IKKβ表达显著下降,可能与我们以往研究PPS有抑制膀胱癌细胞增殖作用且能阻滞细胞由S期进入G2期这一结果有关。
T739细胞在PPS作用后,NF-κB p65mRNA表达下降。ICAM1和CCL2为信号通路下游的应答基因,CCL2为分泌性蛋白,具有单核细胞趋化作用,ICAM1为细胞间黏附分子,具有促使炎症细胞与靶细胞黏附。T739细胞在PPS作用后ICAM1和CCL2 mRNA表达均有不同程度下降,推测PPS具有抑制T739细胞NF-κB通路的部分基因表达水平和NF-κB活性;但PPS抑制T739细胞NF-κB通路的细胞生物学功能的改变尚未探讨。
进一步探讨了PPS与TLR2和TLR4的关系,发现TLR4可介导PPS下调T739细胞IKBKB mRNA和蛋白水平,而TLR2介导IKKβ蛋白下调。TLR4介导CCL2 mRNA下调,而TLR2介导CCL2蛋白下调。TLR2主要介导ICAM1mRNA表达下调,但PPS对ICAM1蛋白表达无明显影响,因此不能判断介导ICAM1蛋白表达的受体。PPS可下调Rel AmRNA表达,沉默TLR2或TLR4均可以部分抑制Rel A mRNA表达,表明TLR2和TLR4均介导Rel A mRNA表达下调。以上结果显示上述基因的表达水平下调主要由TLR4介导,TLR2也参与该效应。
PPS对胞核NF-κB p65的DNA结合活性与胞核表达的影响,发现PPS可使T739细胞NF-κB p65的DNA结合活性减弱,NF-κB p65胞核表达下调。在与TLR2/4关系探讨方面,PPS可显著降低T739-TLR2A细胞NF-κB p65的DNA结合活性,对T739-TLR4A细胞NF-κB p65的DNA结合活性无显著变化;在NF-κB p65胞核表达实验也反应出这一趋势,即PPS可显著降低T739-TLR2Δ细胞NF-κB p65胞核表达,对T739-TLR4Δ细胞NF-κB p65胞核表达无显著影响。上述实验结果均表明PPS主要经TLR4介导T739细胞NF-κB p65的DNA结合活性降低与胞核表达。
由于卡介苗和猪苓多糖均显示有抗膀胱癌的作用,且两者对NF-κB信号通路不同的调节趋势,因此拟观察两者同时作用膀胱癌细胞后该通路主要相关基因的效应。结果显示PPS可拮抗BCG上调T739细胞IKBKB、ICAM1、CCL2、NF-κB p65表达水平和NF-κB p65的DNA结合活性;其中PPS对NF-κB p65的DNA结合活性的拮抗作用尤为显著。
卡介苗抗膀胱癌效果很好,但副作用多,如膀胱炎等,这些症状与过度免疫有关,加入PPS后可调节上述免疫反应,减少毒性反应,为今后临床二者的联合用药减毒增效提供有重要价值的实验和理论依据。
Intravesical instillation of Bacillus Calmette-Guerin(BCG) is currently a comparatively effective immunotherapy for treating and preventing superficial bladder cancer and it is considered that the mechanism of the effect lies in the immune response induced and enhanced by BCG and especially its action on local immune cells.Whereas, the above mechanism is unable to completely explain the fact that the BCG is rarely applied and has much less effect on other types of cancer than on superficial bladder cancer.Thus it is highly probable that apart from activation of local natural immune cells by BCG in cancer tissue,there are some other underlying pathways and mechanisms which are still uncovered involved in its antitumor activity.In the recent years,it is reported that TLR2 and TLR4 were found in epithelial cells in urine and the expression can be induced by BCG. Considering bladder cancer cells also express TLRs,possibly,it is BCG that directly stimulates TLRs levels in the bladder transitional epithelial cells and subsequently causes the release immune activating factors via TLR-NF-κB signaling pathway,thereby strengthens the immunogenicity of tumor and promotes the antitumor immune reaction. The current study was designed to determine the role of BCG in regulating the genes expression via TLR2/4—NF-κB signaling pathway in mouse bladder cancer cell line T739.
First,the changes of mRNA expressionin of NF-κB signaling pathway associated-genes in BCG-treated bladder cancer cells T739 were analyzed by real-time PCR and some of the genes such as TLR2,TLR4,CD14,MD-2,CHUK,MAP3K1, LKBKB,NFKB1(NF-κB p50),Rel A(NF-κB p65),CCL2 and ICAM1 significantly increased after BCG treatment.The expression of the suppressor genes in NF-κB pathway Nfkbia and Nfkbibof could not be detected.Through the NF-κB p65 DNA-binding activity assay in the following experiment,it was revealed the fact that NF-κB p65 in T739 cells was constitutively activated,which corresponded to the common feature of NF-κB molecules in major mammalian tumor cells.BCG treatment had an effect on the expression of NFKB1 and RelA but not on the expression of NFKB2,thus,it seems reasonable to conjecture that the molecular form of NF-κB is Rel A/NFKB1.Using the antibody blocking assay,we discovered that the levels of MAP3K1,IKBKB,Rel A(NF-κB p65), CCL2 and ICAM1 mRNA rose after TLR2 and TLR4 were blocked.
Secondly,we applied vector based RNAi technology for investigating the effect of the expression of TLR2 and TLR4 on the BCG-mediated treatment for bladder cancer.Three pairs of miRNA precursor sequences were designed based on the different target sequences in mice coding region(ORF) and we constructed three miRNA expression vectors rom which the vector pcDNA~(TM)6.2-GW/EmGFPmiR- TLR2.949 were selected for its best transient interference effect.The vector was transfected into the cells and the TLR2-silencing stable cell line T739-TLR2Δwere selected by Blasticidin-resistant selection and passaged continually for a month.The TLRs expression maintained at a low level in T739-TLR2Δcells for the future experiments.Another stable cell line T739-TLR4Δ, silenced mouse gene TLR4,was established in the same way.
By using the two stale cell lines mentioned above,it was shown that BCG increased MAP3K1,IKBKB mRNAs via TLR2,up-modulated ICAM1 mRNAs via TLR4, up-regulated mRNA levels of Rel A and CCL2 via either TLR2 or TLR4.In a further study,the protein expression of IKKβ,CCL2 and ICAM1 were measured by western blot and flow cytometry to determine their relation with TLR2 and TLR4 following the BCG treatment.The results indicated that,in consistent with its mRNA expression,the increase in CCL2 protein levels associated both with TLR2 or TLR4.Up-regulation of IKKβprotein level mediated by TLR2 also matched up with the result of its mRNA.While inconsistent with its regulation of mRNA expression,ICAM1 protein level up-modulation mainly related with TLR2.The enhancement effect of BCG on NF-κB p65 activity and nuclear translocation mostly mediated by TLR2 and correlated with TLR4 to a certain extent.
Polyporus polysaccharide(PPS),as a main active component of polyporus,is a polymer of glucose and lactose.It has been applied clinically on the treatment of type B hepatitis and immunity enhancement.It can also inhibit the growth of carcinoma cells, modulate the immune function of tumor animals and patients,and reduce toxicity as well as side effect of chemotherapeutics.However,the mechanism of its anti-tumor activity is still unclear.NF-κB signaling pathway plays important roles in regulation of virus replication, autoimmune diseases,inflammatory response,tumorigenesis and apoptosis etc,and closely relates to the tumor development and metastasis.NF-κB signaling pathway in most tumor cells is activated constitutively or continuously.The inhibitor of NF-κB has been used as an adjuvant therapy for tumor.The finding of Toll-like receptor(TLRs) in innate immune system turns out to be a new way to study the mechanism of anti-tumor effect of polysaccharide.Recently,it has been reported that the plant polysaccharide is one of the ligands of TLRs.Bladder epithelial cells expressed TLR2 and TLR4 that can respond to corresponding ligands.Our previous research indicates that PPS can significantly increase p53 expression in the bladder cancer cells which culture in vitro and inhibit their proliferation by blocking the S-G2 phase transition in cell cycle.Other researcher reported that Polyporus could reduce the incidence of BBN-induced bladder cancer and the recurrence rate of bladder cancer can drop from 65.1%to 33.3%in contrast to the control group after administration of Polyporus.Therefore,according to the previous study about the role of Polyporus in the treatment of bladder cancer,we suppose that PPS may regulate some genes expression in proliferation,differentiation,cell cycle and apoptosis by means of direct inhibitation of the NF-κB signaling pathway in bladder cancer cells and excessive activation of some kinases or signal transduction molecules.Based on the assumption above,we utilized the RNAi technology to silence the genes of TLR2 and TLR4 so that whether the regulation of the PPS to NF-κB pathway in bladder cancer cells was mediated by TLR2 of TLR4 could be tested.
The increased expression of some membrane molecules such as TLR2,TLR.4,CD14 and MD-2 was detected by using qRT-PCR to study the effect of the PPS on the NF-κB signaling pathway-associated genes in bladder cancer cell T739,thus we think that PPS might transfer signals into cells through TLRs.As the adaptor molecules in Toll-like signaling pathway,TRAF6,TIRAP and IRAK1 had varying changes in their gene expression.The mRNA of IKBKB(IKKβ) decreased greatly and progressively over time and the ration declined from 0.32 to 0.07.Abnormal expression of IKKs is reported to have an important role in the tumorigenesis and tumor progression,its overexpression and overactivity gives a rise to malignant growth characteristic and anti-apoptosis capability of tumor.The decreased expression of IKKβdue to PPS could be explained by the our previous finding that PPS had an inhibitory effect on the proliferation of bladder cancer cells and S-G2 phase transition in cell cycle.
NF-κB p65 mRNA in T739 cells decrease after PPS treatment.ICAM1 and CCL2 act as downstream genes of the NF-κB pathway.CCL2 is a secretory protein involving in monocyto chemotaxis.ICAM1 is an intercellular adhesion molecule that can promote adhesion between inflammatory cells and target cells.The decreased expression of ICAM1 and CCL2 mRNA suggests that PPS has the ability to down-regulate some of the gene expression in NF-κB pathway and inhibit the activity of NF-κB,however,the inhibitory effect PPS on NF-κB pathway in T739 cells have not been studied yet at biological functional level.
Our further experiment demonstrated that the IKBKB mRNA and protein could be down-regulated by PPS via TLR4,but TLR2 only mediated the down-regulation of IKKβprotein.CCL2 mRNA and protein are down-modulated respectively via TLR4 and TLR2.PPS decreased the ICAM1 mRNA expression via TLR2 while had no significant effect on ICAM1 at protein level.The silencing of TLR2 or TLR4 gene can reduce the mRNA expression of Rel A,it shows that TLR2 and TLR4 are involved in the procedure of PPS down-regulating Rel A mRNA.The evidence above indicates that the genes we mentioned here are mainly mediated by TLR4.TLR2 also works on it in some way.
The DNA-binding activity of NF-κB p65 and its nuclear expression in T739 cells can be reduced by PPS.The DNA-binding activity of NF-κB p65 in T739-TLR2Δcells could be brought down by PPS,but the counterpart in T739-TLR4Δhad no significant response to this.Moreover,through nuclear expression assay,the effect of PPS on reducing NF-κB p65 nuclear expression in T739-TLR2Δcells but not in T739-TLR4Δwas exhibited.The results above show that the PPS reduce both DNA-binding activity and nuclear expression of NF-κB p65 in T739 cells mainly via TLR4.
Both BCG and PPS have the effect against bladder cancer and they regulate NF-κB pathway in different ways,so we focus on determining the effect of the both chemicals on the expression of main NF-κB pathway-related genes in bladder cancer cells.Our data show that PPS can antagonize the action of BCG on increasing the levels of IKBKB,ICAM1,CCL2 and NF-κB p65 and the DNA-binding activity of NF-κB p65,the antagonism is extremely significant to the latter one,DNA-bind activity of NF-κB p65.
BCG works well on against bladder cancer but it has some side effects,such as haematuria and pollakisuria,which are correlated with hyperimmunization.PPS can attenuate the above immune reaction and toxicity reaction when it is added to the treatment. Considering the combined medication with PPS can reduce the toxicity and increase the efficacy of BCG,this research will provide a crucial and helpful experimental and theoretical basis for the future clinical practice.
引文
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42)Balachandran P,Pugh ND,Ma G,etc.Toll-like receptor 2-dependent activation of monocytes by Spirulina polysaccharide and its immune enhancing action in mice.Int Immunopharmacol,2006;6(12):1808-14.
43)Yoon YD,Han SB,Kang JS,etc.Toll-like receptor 4-dependent activation of macrophages by polysaccharide isolated from the radix of Platycodon grandiflorum.Int Immunopharmacol,2003;3(13-14):1873-82.
44)Han SB,Yoon YD,Ahn HJ,etc.Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide isolated from cell culture of Acanthopanax senticosus.Int Immunopharmacol,2003;3(9):1301-12.
45)Ahn JY,Choi IS,Shim JY,etc.The immunomodulator ginsan induces resistance to xperimental sepsis by inhibiting Toll-like receptor-mediated inflammatory signals.Eur J Immunol,2006;36(1):37-45.
46)Ahn JY,Song JY,Yun YS,etc.Protection of Staphylococcus aureus-infected septic mice by suppression of early acute inflammation and enhanced antimicrobial activity by ginsan.FEMS Immunol Med Microbiol,2006;46(2):187-197.
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50)Mayo MW,Wang CY,Cogswell PC,etc.Requirement of NF-kappaB activation to suppress p53-independent apoptosis induced by oncogenic Ras.Science,1997;278(5344):1812-5.
51)Wang CY,Mayo MW,Baldwin AS Jr.TNF-and cancer therapy-induced apoptosis:potentiation by inhibition of NF-kappaB.Science,1996;274(5288):784-7.
52)Fracchiolla NS,Lombardi L,Salina M,etc.Structural alterations of the NF-kappa B transcription factor lyt-10 in lymphoid malignancies.Oncogene,1993;8(10):2839-2845.
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55)Tai DI,Tsai SL,Chen YM,etc.Activation of nuclear factor kappaB in hepatitis C virus infection:implications for pathogenesis and hepatocarcinogenesis.Hepatology,2000;31(3):656-64.
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73)梅玉屏,曾量.重组卡介苗及猪苓多糖对荷瘤小鼠巨噬细胞NO释放量的影响.细胞与分子免疫学杂志,2002;18(3):292-295
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