跨膜型TNF-α反向信号通路及其保护肿瘤抵抗凋亡的分子机制
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摘要
跨膜型TNF-α(transmembrane TNF-α, tmTNF-α)不但可作为配体与靶细胞TNFR结合,向靶细胞传递正向信号,对靶细胞发挥效应；也可作为受体与可溶性或膜TNFR结合,向效应细胞本身传递反向信号,介导效应细胞的生物学效应。tmTNF-α反向信号可上调活化T细胞分泌IFN-γ,下调IL-4的产生；可抑制LPS诱导的单核细胞分泌致凋亡因子和促炎细胞因子TNF-α、IL-1、IL-6等,但却上调IL-12的产生。此外,tmTNF-α反向信号还可诱导HTLV感染的T细胞系或PHA活化的CD4+T细胞表达E-selectin。本室前期工作证实肿瘤细胞高表达tmTNF-α可通过其反向信号保护肿瘤细胞抵抗分泌性TNF-α(secretory TNF-α, sTNF-α)诱导的凋亡。但是对其反向信号通路却知之甚少。
本实验室前期用自己制备的特异性针对tmTNF-α抗体进行免疫共沉淀,沉淀到与tmTNF-α胞内段发生作用的IKKa、TRAF1和NF-κBp52。本研究主要探讨tmTNF-α反向信号复合物成员之间的相互作用,该复合物与线粒体之间的联系,以及tmTNF-α与肿瘤耐药的关系并深入探索其分子机制,为临床干预肿瘤耐药提供新的分子靶点和理论依据。
主要实验结果如下：
一、TNF-LS、IKKα和TRAF1原核表达、真核表达和荧光融合表达载体的构建和鉴定
1. TNF-LS、IKKα和TRAF1原核表达、真核表达和荧光融合表达载体的构建和鉴定：用PCR和分子克隆技术成功构建pTriEx-4C-TNF-LS、pTriEx-4C-TRAF1、pTrieX-4C-IKKα、pET28a-his-TRAF1和pET28α-his-IKKα原核表达重组体；pEGFPN1-TNF-LS、pDsRedN1-TNF-LS、pCFPN1-TNF-LS和pEGFPC1-IKKα等荧光融合表达载体；pTriEx-4C-TNF-LS、pTriEx-4C-TRAF1、pTriEx-4C-IKKα、pcDNA3.1-IKKα等真核表达载体。
2. TNF-LS缺失突变体的构建和鉴定：用重组PCR定位突变技术成功构建了△-75～-69-TNFLS和△-68～-62-TNFLS突变体,并分别连接至pcDNA3.1和pEGFPN1载体上。
二、tmTNF-α反向信号复合物主要成员TNF-LS、TRAF1与IKKα的细胞亚定位和相互关系的确定
1. tmTNF-α与IKK-α, TRAF-1和NF-κBp52发生共沉淀：采用仅识别tmTNF-α的单克隆抗体进行IP-Wetern,结果可共沉淀TRAF1、IKKα和NF-κBp52,但不能与TNFR1/2、TRAF2/3、NIK、SODD、FADD和NF-κBp65等分子共沉淀。反之,分别用TRAF1、IKKα和NF-κBp52抗体可与tmTNF-α共沉淀。提示TRAF-1、IKKα和NF-κBp52可能与tmTNF-α胞浆段形成信号复合物参与NF-κB的活化。
2. TNF-LS与TRAF1直接相互作用：用体外Pull down证实了TRAF1和TNF-LS可直接相互作用；共转染TRAF1和tmTNF-α或TNF-LS可共沉淀到大量TRAF1,当TRAF1与缺失胞浆段的tmTNF-α共转,则不能募集TRAF1;此外,共转TRAF1和TNF-LS可使在原本胞膜周围弥散性分布的TNF-LS改变为与TRAF1共定位,且呈局限性聚集。
3. TNF-LS N端的-75～-69位氨基酸决定了TRAF1和TNF-LS的相互作用：共转△-75～-69-TNF-LS和TRAF1,并不能导致共沉淀的TRAF1增加,提示该位点可能为募集TRAF1所必需。
4. TRAF1与IKKα直接相互作用：用体外Pull down证实TRAF1可与IKKα直接相互作用。共转TRAF1和IKKα使共沉淀的IKKα量明显高于单转TRAF1或者IKKα,并使胞浆内弥散性分布的IKKα向TRAF1分布的地方聚集。
5. TNF-LS不能与IKKα直接相互作用：用体外Pull down实验证实TNF-LS和IKKα之间无直接相互作用。
6.抑制CKI对tmTNF-α胞内段磷酸化导致反向信号复合物募集,NF-κB活化,并减低对ADM和sTNF-α杀伤的敏感性：用CKI抑制剂D4476抑制CKI对tmTNF-α-75～-69位点的磷酸化,促进tmTNF-α与TRAF1、IKKα和NF-κBp52形成复合物增多,NF-κB进一步活化,并保护肿瘤细胞抵抗sTNF-α和阿霉素诱导的凋亡。
三、tmTNF-α反向信号复合物与线粒体的关系
1. TRAF1诱导线粒体聚集,并和线粒体共定位：293T细胞线粒体呈点网状分布,转染TRAF1后,线粒体呈局限性点状分布,且和TRAF1共定位。
2. tmTNF-α反向信号复合物存在于线粒体：提取线粒体蛋白,用Western blot证实在线粒体中可检测到tmTNF-α及其反向信号复合物成员TRAF1、IKKα和NF-κBp52；共转293T细胞TNF-LS和TRAF1可导致线粒体聚集,且聚集的线粒体与TNF-LS和TRAF1发生共定位；用IP证实tmTNF-α和NF-KBp52可在线粒体中发生免疫共沉淀,提示tmTNF-α反向信号复合物可能存在于线粒体中。
3. tmTNF-α及其反向信号复合物成员可在胞核中检出：提取核蛋白,用Western blot在核蛋白中检出完整的tmTNF-α分子及其反向信号复合物成员TRAF1、IKKα和NF-κBp52,但是,它们与tmTNF-α之间的关系尚待研究。
四、高表达TNF-LS诱导MCF-7细胞抵抗sTNF-α的胞毒作用
1.高表达tmTNF-α保护乳腺癌细胞抵抗sTNF-α的胞毒效应：高表达tmTNF-α的MDA-MB-231乳腺癌细胞NF-κB持续性活化,对sTNF-α导致的胞毒效应发生抵抗；而低表达tmTNF-α的MCF-7乳腺癌细胞NF-κB无激活,且对sTNF-α胞毒效应敏感。
2.下调tmTNF-α可逆转MDA-MB-231细胞对sTNF-α的敏感性：用siRNA下调MDA-MB-231细胞tmTNF-α的基因表达,可抑制NF-κB活化,逆转其对sTNF-α杀伤的敏感性。
3.稳转TNF-LS使MCF-7细胞由sTNF-α敏感株变为耐受株：稳转TNF-LS的MCF-7细胞高表达TNF-LS,且TNF-LS主要分布在胞膜上；稳转TNF-LS使MCF-7细胞NF-κB持续性活化,并由sTNF-α敏感细胞株转变为耐受株。用PDTC抑制NF-κB活化,可抑制抗凋亡分子cIAP1的基因表达,并逆转稳转TNF-LS的MCF-7细胞对sTNF-α的敏感性。
4. TNF-LS介导tmTNF-α的反向信号：瞬转TNF-LS和wt-tmTNF-α均可导致NF-κB活化,对sTNF-α胞毒效应耐受；而瞬转缺失胞浆段的△cs-tmTNF-α则几乎不引起NF-κB活化,并保留亲本细胞对sTNF-α的敏感性。提示TNF-LS介导tmTNF-α的反向信号。
5.抑制TNF-LS内吞促进MCF-7细胞对sTNF-α的耐受：用MDC抑制内吞可增加细胞表面TNF-LS的表达,导致NF-κB活性进一步增加,使MCF-7细胞对sTNF-α的耐受增强。提示tmTNF-α的反向信号不需要该分子内化。
6.抑制TNF-LS胞浆段剪切促进MCF-7细胞对sTNF-α的耐受：用ZLL抑制信号肽肽酶对TNF-LS胞浆段的剪切,也可进一步增加TNF-LS在细胞表面的表达,进而促进NF-κB活化,增强MCF-7细胞对sTNF-α的耐受。提示tmTNF-α的反向信号依赖其跨膜性。
五、tmTNF-α反向信号诱导乳腺癌细胞抵抗化疗及其分子机制
1.高表达tmTNF-α导致乳腺癌细胞卡铂耐药：用胞毒实验证实高表达tmTNF-α乳腺癌细胞系MDA-MB-435抵抗卡铂杀伤效应,其NF-κB组成性活化；而低表达tmTNF-α乳腺癌细胞系T47D的NF-κB无活化,对卡铂杀伤则敏感。瞬转tmTNF-α可明显抑制T47D细胞对卡铂的敏感性,用PDTC抑制NF-κB活性则能在一定程度上逆转该转染细胞对卡铂的敏感性。
2. tmTNF-α反向信号与乳腺癌细胞耐药相关：比较3株稳转缺失胞外段的TNF-LS的MCF-7细胞,因TNF-LS表达量不同而对卡铂的敏感性不同,即TNF-LS表达量越高的MCF-7细胞对卡铂的敏感性越差(IC50>400μg/ml),而亲本MCF-7细胞的IC50则为231μg/ml。用PDTC特异性抑制NF-κB的活性,可以在一定程度上逆转高表达TNF-LS的MCF-7细胞对卡铂的敏感性。提示tmTNF-α反向信号与乳腺癌细胞耐药相关。
3. tmTNF-α反向信号导致ERK组成性磷酸化：转染tmTNF-α可导致T47D细胞ERK组成性磷酸化增加,卡铂对其无明显影响；但是卡铂可明显诱导未转染T47D细胞ERK的磷酸化。
4. tmTNF-α反向信号抑制卡铂诱导的JNK磷酸化：卡铂作用T47D细胞可诱导JNK一过性磷酸化；然而,转染tmTNF-α可明显抑制卡铂诱导JNK的磷酸化。提示tmTNF-α反向信号抑制JNK磷酸化,从而抵抗卡铂的胞毒效应。
5. tmTNF-α反向信号导致PI3K组成性表达：卡铂作用T47D细胞可诱导PI3K亚单位p85微量表达；转染tmTNF-α则导致T47D细胞PI3K高水平组成性表达,但卡铂对其无明显影响。提示tmTNF-α反向信号诱导PI3K组成性活化,可能通过促进抗凋亡因子表达而抵抗卡铂的胞毒效应。
6. tmTNF-α反向信号诱导p53组成性表达：卡铂作用T47D细胞可诱导p53高表达,而转染tmTNF-α则导致T47D细胞p53高水平组成性表达,卡铂对之无明显影响。提示tmTNF-α反向信号诱导p53组成性高表达,可能通过增强DNA修复,抵抗卡铂造成DNA损伤而导致的细胞凋亡。
综上所述,本工作阐明了tmTNF-α反向信号复合物成员的相互作用,即通过其胞浆段脱磷酸化,募集TRAF1,后者将IKK-α募集到tmTNF-α反向信号复合物中,活化NF-κB旁路,通过上调抗凋亡基因表达,导致肿瘤细胞抵抗sTNF-α和化疗药物的杀伤。此外,tmTNF-α反向信号还通过促进肿瘤细胞PI3K和p53组成性表达,诱导ERK组成性磷酸化,抑制卡铂诱导的JNK磷酸化而导致肿瘤细胞耐药。该研究通过深入探索tmTNF-α反向信号通路及其保护肿瘤抵抗凋亡的分子机制,为临床干预肿瘤耐药提供新的线索和分子靶点。
transmembrane TNF-a(tmTNF-a) can not only interact directly with TNFR and transduce forward signaling into TNFR bearing cells to play effects, this signaling pathway is also called forward signaling pathway. Meanwhile, tmTNF-a can also work as receptors to accept membrane bound TNFR or soluble TNFR stimulation, and transdues signaling into tmTNF-a bearing cells, this signaling pathway is called reverse signaling pathway, which mediate several biological effects. The reverse signaling of tmTNF-a can upregulate activated T cells to secret IFN-y and downregulate generation of IL-4; it can also inhibit induced monocytes by LPS to secret proapoptotic factors and proinflammation factors, such as TNF-α、IL-1、IL-6 generation; but it upregulate IL-12 secretion. Furthermore, reverse signaling pathway induced by tmTNF-a can induce HTLV infected T cells or PHA activated CD4+T cells expression of E-selectin. We previously found that tumor cells with high expression of tmTNF-a can protect tumor cells from secretory TNF-a (sTNF-a) cytotoxicity. However, the reverse signaling pathway is still unknown.
We previously prepared a specific monoclonal tmTNF-a antibody, and we used this specific antibody to immunoprecipitate, and we had got IKKα、TRAF1 and NF-κBp52 to interact with the intracellular domain of tmTNF-a. This research is mainly studying the relationship of the member of the reverse signaling pathway of tmTNF-a, and their relationship with mitochondria. We also worked on the relationship between tmTNF-a expression on tumor cells and chemoresistance, and we also further study into the molecular mechanism of the chemoresistance induced by tmTNF-a, so as to provide new target molecular and theory basis for clinical intervention. Main results as below:
I. Construction and identification of prokaryotic、eukaryotic and fluorescence fusion expression vectors of TNF-LS、IKKαand TRAF1
1. Construction and identification of prokaryotic、eukaryotic and fluorescence fusion expression vectors of TNF-LS、IKKαand TRAF1:
PCR and molecular cloning techniques were used to construct prokaryotic fusion vectors of pTriEx-4C-TNF-LS、pTriEx-4C-TRAF1、pTrieX-4C-IKKα、pET28a-his-TRAF1 and pET28a-his-IKKa; fluorescence expression vectors of pEGFPNl-TNF-LS、pDsRedN1-TNF-LS、pCFPN1-TNF-LS and pEGFPC1-IKKa; eukaryotic fusion vectors of pTriEx-4C-TNF-LS、pTriEx-4C-TRAF、pTriEx-4C-IKKa and pcDNA3.1-IKKa. And they were successfully constructed by sequencing and blasting.
2. Construction and identification of the mutated TNF-LS in eukaryotic and fluorescence fusion expression vectors
pcDNA3.1/V5-His-TOPO-TNF-LS was taken as a template, PCR amplification of a fragment about 208 bp, restriction to connect to pcDNA3.1, transformation of the ligated product into competent DH5a bacteria, choosing positive clones, restriction enzyme digestion and sequencing. And then the correct sequenced pcDNA3.1-△-75--69-TNF-LS and pcDNA3.1-△-68～-62-TNF-LS digested, recycling the fragment, after digestion, connected to digested pEGFPNl, the identification of the positive clones by digesttion.
II. Identification of the major members of the reverse signaling pathway of tmTNF-αTRAFl and IKKa and the relationship among them
1. IKKα、TRAF-1 and NF-κBp52 can be immunoprecipitated by tmTNF-αmonoclonal antibody
In order to investigate into the reverse signaling pathway of tmTNF-α, specific tmTNF-αmonoclonal antibody was used to IP-Wetern. The results show that TRAFl, IKK-αand NF-κBp52 can be sedimentated by tmTNF-αantibody. However, TNFR1/2, TRAF2/3, NIK, SODD, FADD and NF-κBp65 can not be immunoprecipitated. Conversely, using TRAF1, IKKa and NF-κBp52 antibody to re-IP, all of them can immunoprecipitated tmTNF-α. The results imply that TRAFl and IKKa might participitate in the formation of reverse signaling complexes of tmTNF-αso as to activate NF-κB.
2. TNF-LS directly interact with TRAFl
First of all, we intend to explore the relationship between TNF-LS and TRAFl, direct or indirect interactions. Prokaryotic expression with his label with TRAF1 and prokaryotic expression of the non-his tagged TNF-LS in vitro Pull down, we confirmed that TNF-LS and TRAFlinteract directly. In order to further confirm this direct interaction, we transfected TRAF1 and TNF-LS plasmid alone or co-transfected into 293T cells to overexpress, and then by IP-Western to verify their interaction in vivo and found that tmTNF-αwith monoclonal antibodies can pull down significantly higher TRAFl in cells cotransfected with TNF-LS and TRAF1 than that of the single-TRAFl transgenic group or the blank control group. Further, we build TRAFl and TNF-LS fluorescence fusion vectors with different corresponding fluorescence to study their cellular localization, found that over-expression of TRAFl in 293T cells showed the limited point-like distribution, and TNF-LS distributed near the membrane. Co-transfected TRAF1 and TNF-LS resulted in gathering of TNF-LS to TRAF1 localization, the confocal results showed that the fluoresence of the TRAF1 and the fluorescence of the TNF-LS merged. Direct interaction of TNF-LS with TRAF1 confirmed in Three-pronged approach.
3.-75～-69 amino acids of the N terminal of TNF-LS determined the direct interaction of TNF-LS and TRAFl
The intracellular domain of TNF-LS has only 30 amino acids, and we produced mutation of TNF-LS deletion of the N-terminal -75～-69 or -68～-61 amino acids. Mutants or wild type tmTNF-αor TNF-LS together with TRAF1 were co-transfected into 293T cells and found that the TNF-LS deletion of the N-terminal of -75～-69 amino acids influenced greatly on the interaction of TRAF1 and TNF-LS, suggesting that the N-terminal -75～-69 amino acids of TNF-LS plays a decisive role in TRAF1 and TNF-LS interaction.
4. Dephosphorylation of the intracellular domain of TNF-LS caused the recruitment of the reverse signaling complex, leading to NF-κB activation, resulting in the resistance of tmTNF-αhigh expression tumors resistance to ADM and sTNF-α
The N-terminal-75 to -69 amino acids decides TRAFl and TNF-LS interaction, we found that the N-terminal-75 to -69 amino acids of tmTNF-αis the site for CKI phosphorylation. Using specific inhibitors of CKI inhibitor D4476 to inhibit the activity of CKI, and then tmTNF-a monoclonal antibody was used to IP, compared with untreated Raji cell group, treatment group IP to TRAF1, IKKa and NF-κBp52 significantly more than untreated group. The results implies that phosphorylation of tmTNF-a by CKI inhibited the reverse transmission signal pathway. CKI inhibitor with Raji cells inhibited the activity of CKI can lead to NF-κB activation and further lead to Raji cells resistant to sTNF-a and chemotherapy drug doxorubicin. Suggesting that tmTNF-a may be involved in a reverse signal of NF-κB activation and resistance to chemotherapy drugs.
5. TRAFl and IKKa interacted directly
We also confirmed direct interaction of TRAF1 and IKKa from three-pronged approaches. First of all, we prokaryotically expressed TRAF1 and IKKa, Pull down assay was done in vitro and confirmed that TRAF1 directly interacted with IKKa. In addition, we use IP in the body to further give evidence that TRAF1 directly interacted with IKKa. Finally, TRAF1 and IKKa with different color fluorescent protein fusion expressed in 293T cells, after co-transfection of TRAF1 and IKKa, TRAF1 resulted in partial aggregation of IKKa protein and the fluorescence of TRAFl and IKKa merged.
6. TNF-LS can not interact directly with IKKa
Prokaryotically expressed TNF-LS and IKKa, and in vitro pull down assay was done, we found that TNF-LS can not directly interact with IKKa
III. The close relationship of mitochondrial with the reverse signaling complex molecular TNF-LS、TRAF1 and IKKα
1. TRAFl co-localized with mitochondria
In 293T cells, mitochondria showed the mesh point distribution, after transfection of TRAF1, the distribution of mitochondria changed into point-like distribution, and co-localized with TRAF1.
2. Members of the reverse signaling of tmTNF-a all exist in the mitochondria
mitochondira was extracted, and western blot was taken to indentify if there was any relationship between members of the reverse signaling complex members of TRAF1、 IKKa and NF-κBp52 and mitochondria. We found that TRAF1、IKKαand NF-κB p52 all could localize in the mitochondria. Co-transfection of 293T cells with TNF-LS and TRAF1 could induce mitochondria aggregation, and TRAF1 changed the TNF-LS distribution on the membrane which assembled to the position of TRAF1. IP was further taken to immunoprecipitated mitochondria proteins with monoclonal tmTNF-αantibody and NF-KBp52 can be precipitated, implying that the reverse signaling pathway might occur in the mitochondria.
3. tmTNF-αand related reverse signaling complex members can be found in the nucleus
nuclear proteins were prepared, and western blot was used and we identified the nuclear localization of tmTNF-a and related molecular TRAF1、IKKa and NF-κB p52, but there is no report about their relationship until now.
IV. TNFLS stably high expression in MCF-7 induced the tolerance of such cells to sTNF-α
1. High expression of tmTNF-a protects cells from the sTNF-a induced cytotoxicity
Sources of the same breast cancer tumor, some sensitive to sTNF-a induced cytotoxicity,some resistant, we found that high expression of tmTNF-a in the MDA-MB-231 induced sTNF-αtolerance, while low tmTNF-a expression in the MCF-7 cells were sensitive to sTNF-α. Prompt, tmTNF-a expression may be the protection effect from sTNF-αinduced cytotoxicity.
2. Down-regulation of tmTNF-a in MDA-MB-231 cells reduced NF-κB activity with a reversal of the cells on the sensitivity to sTNF-α
Interestingly, down-regulation of tmTNF-a expression in MDA-MB-231 cells with TNF siRNA resulted in the recovery of MDA-MB-231 cells sensitive to sTNF-αto some extent.
3. TNF-LS expressed on the membrane of MCF-7 cells
After transfection of pIRES2-EGFP/TNF-LS into MCF-7 cells, the expression of EGFP and TNF-LS were separate. tmTNF-a monoclonal antibody were used for staining, and then adding the second TRITC labeled antibody. Indirect fluorescence under the microscope shows that the diffuse cytoplasmic green fluorescence with TNF-LS distribution in the cell membrane, which shows a red aperture under the fluorescence microscope.
4. High expression of TNFLS in MCF-7 cells caused the cells tolerant to sTNF-αand persistent NF-κB activation
Establishment of TNF-LS stably expressed in MCF-7 cells, we found that TNF-LS/MCF-7 cells are tolerant to sTNF-a and persistent NF-κB activation, showing the same features like another breast cancer cell line MDA-MB-231 cell with high tmTNF-a expression. Prompt, TNF-LS has some tmTNF-a properties.We speculated that the reverse signaling pathway of tmTNF-a may determined by TNF-LS.
5. NF-κB inhibitor PDTC inhibited stably transfected MCF-7 cells with TNF-LS NF-κB activity, resulting in reversal of the stable cell lines TNF-LS/MCF-7 sensitive to sTNF-α
PDTC can selectivly inhibited the activity of NF-κB. Selective inhibition of NF-κB activity in TNF-LS/MCF-7 cells and found that the sensitivity of TNF-LS/MCF-7 cells to sTNF-a increased, suggesting that TNF-LS lead to the stability of the TNF-LS/MCF-7 cells to sTNF-a for NF-κB activation.
6. Anti-apoptotic gene expression leading to TNF-LS/MCF-7 cells resistant to sTNF-a induced cytotoxicity
NF-κB is an important factor in determining cell death and survival, and cIAPl is one of the target gene of NF-κB which is an antiapoptotic factor. comparing before and after STNF-a stimulation, the expression of cIAPl was significantly higher in MCF-7 cells stably transfected with TNF-LS than MCF-7 cells untranfected or transfected empty vectors.sTNF-a stimulation led to further cIAPl gene expression. The results suggest that regulation of antiapoptotic gene expression by NF-κB activation may be the reason for TNF-LS/MCF-7 cells resistant to sTNF-αinduced cytotoxicity.
7. The intracellualr domain of tmTNF-a induced durable NF-κB activation in MCF-7 cells
High tmTNF-a expression in breast cancer cell line MDA-MB-231 cells can lead to NF-κB activation,while, TNF-LS stabily expressed on MCF-7 cells also results in a persistent activation of NF-κB. Comparison transfection of Acs-tmTNF-α、TNF-LS and tmTNF-a into MCF-7 cell lines, we only could find durable NF-κB activation in cells instant transfection of tmTNF-a and TNF-LS. Cells transfected with Acs-tmTNF-a could not activate NF-κB when compared with untransfected or transfected empty vector groups. Our results implied that high tmTNF-a expression in breast cancer cells induced durable NF-κB activation possibly by TNF-LS.
8. Endocytosis inhibitor MDC pretreatment TNF-LS/MCF-7 cellS induced surface expression of TNF-LS
Inhibition of TNF-LS endocytosis, TNF-LS/MCF-7 increased cell surface expression of TNF-LS may be a further increase in NF-κB activity in TNF-LS/MCF-7 cells.
9. Signal peptide peptidase inhibitor ZLL pretreatment increased TNF-LS/MCF-7 cell surface TNF-LS expression, resulting in further NF-κB B activation
Inhibition TNF-LS shear from membrane by signal peptide peptidase inhibitor increased TNF-LS/MCF-7 cell surface TNF-LS expression, and this may be a reason for further increase in NF-κB activity in TNF-LS/MCF-7 cells.
V. Reverse signaling of tmTNF-a induced the resistance of breast cancer cells to chemotherapy and related molecular mechanisms
1. High expression of tmTNF-a on breast cancers induced carboplatin resistance
Comparing the sensitivity difference of breast cancer cells with different tmTNF-αexpression to carboplatin, we found that MDA-MB-435 cells with high level of tmTNF-αexpression were much more resistant to carboplatin than T47D cells with low level of tmTNF-αexpression did, and found that MDA-MB-435 cells with high tmTNF-αexpression showed durable NF-κB activation, while no obvious NF-κB activity was observed in T47D cells with low tmTNF-αexpression.
2. Upregulation of tmTNF-αexpression on T47D cells induced their resistance to carboplatin with the phenomenon that the cleavage of caspase-9 decreased, and NF-κB inhibitor PDTC can reverse the resistance
After transfection of T47D with tmTNF-α, the cells are resistant to carboplatin cytotoxicity, the cleavage of caspase-9 decreased. NF-κB inhibitor can reverse the resistance to carboplatin at a certain level.
3. MCF-7 cells stably transfected with TNF-LS were resistant to carboplatin cytotoxicity
It has been confirmed that stable transfection of TNF-LS into MCF-7 cells resulted in durable NF-κB activation and tolerance to sTNF-α. In this, we have further found that TNF-LS/MCF-7 cells are more resistant to carboplatin chemotherapy treatment than untransfected or transfected with empty vectors.
4. MCF-7 cells with TNF-LS inhibited the sensitivity of MCF-7 cells to carboplatin
Treatment of transfected and untransfected MCF-7 cells with different concentration of carboplatin, we found that the IC50 of carboplatin to untransfected MCF-7 cells was just 231μg/ml, however, the IC50 of transfected MCF-7 cells were much higher than 400μg/ml.
5. The proliferation of MCF-7 cells with high expression of tmTNF-αis quicker than MCF-7 cells with low tmTNF-αexpression after treatment of MCF-7 cells with carboplatin
We got two lines of MCF-7 cells with high tmTNF-αexpression and low tmTNF-αexpression, and we compared these two lines sensitivity to carboplatin. We found that high expression of tmTNF-αin MCF-7 cells proliferated significantly faster than the speed of low tmTNF-αexpression MCF-7 cells treated with carboplatin. And extended over time, the inhibitory effect of carboplatin to the MCF-7 cells with high tmTNF-αexpression becomes weaker and weaker.
6. Inhibition NF-κB activation in MCF-7 cell line 2-E8 stably transfected with TNF-LS could reverse sensitivity of 2-E8 to carboplatin at a certain extent
As has reported in part four that stably transfected MCF-7 cells with TNF-LS induced durable NF-κB activation in these cell lines, however, inhibiting the NF-κB activity in these cells can reverse the sensitivity of these cells to carboplatin.
7. Comparing the ERK phosphorylation status of T47D cells untransfected or transfected with tmTNF-a after treatment with carboplatin
ERK phosphorylation was not very clear for untransfected T47D cells.After carboplatin treatment, as early as five minutes after treatment with carboplatin can resulted in ERK phosphorylation, and the ERK phosphorylation level reached peak at 10 minutes with significant decrease at 30 minutes, suggesting that stimulation of carboplatin resulted in a transient ERK phosphorylation. Instant transfection of tmTNF-a into MCF-7 cells resulted in durable ERK phosphorylation to certain degree, and activation was not affected by treatment with carboplatin.
8. Comparing the JNK phosphorylation status of T47D cells untransfected or transfected with tmTNF-αafter treatment with carboplatin
Both transfected and untransfected T47D cells have no JNK phosphorylation without stimulation. After treatment with carboplatin, the JNK phosphorylation level of untransfected T47D cells were significantly higher than T47D cells transfected with tmTNF-α, and both JNK phosphorylation status reduced after 30 min stimulation.
9. Comparing the PI3K expression status of T47D cells untransfected or transfected with tmTNF-αafter treatment with carboplatin
T47D cells transfected with tm TNF-α, we can see continuous PI3K p85 expression, and carboplatin treatment has little effect on the expression level of PI3K. However, no observed PI3K p85 expression could be found in non-transfected T47D cells in the resting state. Carboplatin treatment of untransfected T47D cells led to a small amount of PI3K p85 expression, suggesting that PI3K may be involved in a high tmTNF-a expression in tumor cells resistant to chemotherapeutic drugs.
lO.Comparing the expression of p53 in T47D cells untransfected or transfected with tmTNF-a after treatment with carboplatin
Comparing the expression status of p53, we found a very interesting results. We found that after treatment of untransfected T47D cells with carboplatin, the expression of p53 in cells gradually upregulated as time went by. While, we could see p53 expression even after transfection of cells with tmTNF-αwithout treatment of carboplatin, and expression of p53 in T47D cells transfected with tmTNF-awas independent on carboplatin treatment.
本实验室前期用自己制备的特异性针对tmTNF-α抗体进行免疫共沉淀,沉淀到与tmTNF-α胞内段发生作用的IKKa、TRAF1和NF-κBp52。本研究主要探讨tmTNF-α反向信号复合物成员之间的相互作用,该复合物与线粒体之间的联系,以及tmTNF-α与肿瘤耐药的关系并深入探索其分子机制,为临床干预肿瘤耐药提供新的分子靶点和理论依据。
主要实验结果如下：
一、TNF-LS、IKKα和TRAF1原核表达、真核表达和荧光融合表达载体的构建和鉴定
1. TNF-LS、IKKα和TRAF1原核表达、真核表达和荧光融合表达载体的构建和鉴定：用PCR和分子克隆技术成功构建pTriEx-4C-TNF-LS、pTriEx-4C-TRAF1、pTrieX-4C-IKKα、pET28a-his-TRAF1和pET28α-his-IKKα原核表达重组体；pEGFPN1-TNF-LS、pDsRedN1-TNF-LS、pCFPN1-TNF-LS和pEGFPC1-IKKα等荧光融合表达载体；pTriEx-4C-TNF-LS、pTriEx-4C-TRAF1、pTriEx-4C-IKKα、pcDNA3.1-IKKα等真核表达载体。
2. TNF-LS缺失突变体的构建和鉴定：用重组PCR定位突变技术成功构建了△-75～-69-TNFLS和△-68～-62-TNFLS突变体,并分别连接至pcDNA3.1和pEGFPN1载体上。
二、tmTNF-α反向信号复合物主要成员TNF-LS、TRAF1与IKKα的细胞亚定位和相互关系的确定
1. tmTNF-α与IKK-α, TRAF-1和NF-κBp52发生共沉淀：采用仅识别tmTNF-α的单克隆抗体进行IP-Wetern,结果可共沉淀TRAF1、IKKα和NF-κBp52,但不能与TNFR1/2、TRAF2/3、NIK、SODD、FADD和NF-κBp65等分子共沉淀。反之,分别用TRAF1、IKKα和NF-κBp52抗体可与tmTNF-α共沉淀。提示TRAF-1、IKKα和NF-κBp52可能与tmTNF-α胞浆段形成信号复合物参与NF-κB的活化。
2. TNF-LS与TRAF1直接相互作用：用体外Pull down证实了TRAF1和TNF-LS可直接相互作用；共转染TRAF1和tmTNF-α或TNF-LS可共沉淀到大量TRAF1,当TRAF1与缺失胞浆段的tmTNF-α共转,则不能募集TRAF1;此外,共转TRAF1和TNF-LS可使在原本胞膜周围弥散性分布的TNF-LS改变为与TRAF1共定位,且呈局限性聚集。
3. TNF-LS N端的-75～-69位氨基酸决定了TRAF1和TNF-LS的相互作用：共转△-75～-69-TNF-LS和TRAF1,并不能导致共沉淀的TRAF1增加,提示该位点可能为募集TRAF1所必需。
4. TRAF1与IKKα直接相互作用：用体外Pull down证实TRAF1可与IKKα直接相互作用。共转TRAF1和IKKα使共沉淀的IKKα量明显高于单转TRAF1或者IKKα,并使胞浆内弥散性分布的IKKα向TRAF1分布的地方聚集。
5. TNF-LS不能与IKKα直接相互作用：用体外Pull down实验证实TNF-LS和IKKα之间无直接相互作用。
6.抑制CKI对tmTNF-α胞内段磷酸化导致反向信号复合物募集,NF-κB活化,并减低对ADM和sTNF-α杀伤的敏感性：用CKI抑制剂D4476抑制CKI对tmTNF-α-75～-69位点的磷酸化,促进tmTNF-α与TRAF1、IKKα和NF-κBp52形成复合物增多,NF-κB进一步活化,并保护肿瘤细胞抵抗sTNF-α和阿霉素诱导的凋亡。
三、tmTNF-α反向信号复合物与线粒体的关系
1. TRAF1诱导线粒体聚集,并和线粒体共定位：293T细胞线粒体呈点网状分布,转染TRAF1后,线粒体呈局限性点状分布,且和TRAF1共定位。
2. tmTNF-α反向信号复合物存在于线粒体：提取线粒体蛋白,用Western blot证实在线粒体中可检测到tmTNF-α及其反向信号复合物成员TRAF1、IKKα和NF-κBp52；共转293T细胞TNF-LS和TRAF1可导致线粒体聚集,且聚集的线粒体与TNF-LS和TRAF1发生共定位；用IP证实tmTNF-α和NF-KBp52可在线粒体中发生免疫共沉淀,提示tmTNF-α反向信号复合物可能存在于线粒体中。
3. tmTNF-α及其反向信号复合物成员可在胞核中检出：提取核蛋白,用Western blot在核蛋白中检出完整的tmTNF-α分子及其反向信号复合物成员TRAF1、IKKα和NF-κBp52,但是,它们与tmTNF-α之间的关系尚待研究。
四、高表达TNF-LS诱导MCF-7细胞抵抗sTNF-α的胞毒作用
1.高表达tmTNF-α保护乳腺癌细胞抵抗sTNF-α的胞毒效应：高表达tmTNF-α的MDA-MB-231乳腺癌细胞NF-κB持续性活化,对sTNF-α导致的胞毒效应发生抵抗；而低表达tmTNF-α的MCF-7乳腺癌细胞NF-κB无激活,且对sTNF-α胞毒效应敏感。
2.下调tmTNF-α可逆转MDA-MB-231细胞对sTNF-α的敏感性：用siRNA下调MDA-MB-231细胞tmTNF-α的基因表达,可抑制NF-κB活化,逆转其对sTNF-α杀伤的敏感性。
3.稳转TNF-LS使MCF-7细胞由sTNF-α敏感株变为耐受株：稳转TNF-LS的MCF-7细胞高表达TNF-LS,且TNF-LS主要分布在胞膜上；稳转TNF-LS使MCF-7细胞NF-κB持续性活化,并由sTNF-α敏感细胞株转变为耐受株。用PDTC抑制NF-κB活化,可抑制抗凋亡分子cIAP1的基因表达,并逆转稳转TNF-LS的MCF-7细胞对sTNF-α的敏感性。
4. TNF-LS介导tmTNF-α的反向信号：瞬转TNF-LS和wt-tmTNF-α均可导致NF-κB活化,对sTNF-α胞毒效应耐受；而瞬转缺失胞浆段的△cs-tmTNF-α则几乎不引起NF-κB活化,并保留亲本细胞对sTNF-α的敏感性。提示TNF-LS介导tmTNF-α的反向信号。
5.抑制TNF-LS内吞促进MCF-7细胞对sTNF-α的耐受：用MDC抑制内吞可增加细胞表面TNF-LS的表达,导致NF-κB活性进一步增加,使MCF-7细胞对sTNF-α的耐受增强。提示tmTNF-α的反向信号不需要该分子内化。
6.抑制TNF-LS胞浆段剪切促进MCF-7细胞对sTNF-α的耐受：用ZLL抑制信号肽肽酶对TNF-LS胞浆段的剪切,也可进一步增加TNF-LS在细胞表面的表达,进而促进NF-κB活化,增强MCF-7细胞对sTNF-α的耐受。提示tmTNF-α的反向信号依赖其跨膜性。
五、tmTNF-α反向信号诱导乳腺癌细胞抵抗化疗及其分子机制
1.高表达tmTNF-α导致乳腺癌细胞卡铂耐药：用胞毒实验证实高表达tmTNF-α乳腺癌细胞系MDA-MB-435抵抗卡铂杀伤效应,其NF-κB组成性活化；而低表达tmTNF-α乳腺癌细胞系T47D的NF-κB无活化,对卡铂杀伤则敏感。瞬转tmTNF-α可明显抑制T47D细胞对卡铂的敏感性,用PDTC抑制NF-κB活性则能在一定程度上逆转该转染细胞对卡铂的敏感性。
2. tmTNF-α反向信号与乳腺癌细胞耐药相关：比较3株稳转缺失胞外段的TNF-LS的MCF-7细胞,因TNF-LS表达量不同而对卡铂的敏感性不同,即TNF-LS表达量越高的MCF-7细胞对卡铂的敏感性越差(IC50>400μg/ml),而亲本MCF-7细胞的IC50则为231μg/ml。用PDTC特异性抑制NF-κB的活性,可以在一定程度上逆转高表达TNF-LS的MCF-7细胞对卡铂的敏感性。提示tmTNF-α反向信号与乳腺癌细胞耐药相关。
3. tmTNF-α反向信号导致ERK组成性磷酸化：转染tmTNF-α可导致T47D细胞ERK组成性磷酸化增加,卡铂对其无明显影响；但是卡铂可明显诱导未转染T47D细胞ERK的磷酸化。
4. tmTNF-α反向信号抑制卡铂诱导的JNK磷酸化：卡铂作用T47D细胞可诱导JNK一过性磷酸化；然而,转染tmTNF-α可明显抑制卡铂诱导JNK的磷酸化。提示tmTNF-α反向信号抑制JNK磷酸化,从而抵抗卡铂的胞毒效应。
5. tmTNF-α反向信号导致PI3K组成性表达：卡铂作用T47D细胞可诱导PI3K亚单位p85微量表达；转染tmTNF-α则导致T47D细胞PI3K高水平组成性表达,但卡铂对其无明显影响。提示tmTNF-α反向信号诱导PI3K组成性活化,可能通过促进抗凋亡因子表达而抵抗卡铂的胞毒效应。
6. tmTNF-α反向信号诱导p53组成性表达：卡铂作用T47D细胞可诱导p53高表达,而转染tmTNF-α则导致T47D细胞p53高水平组成性表达,卡铂对之无明显影响。提示tmTNF-α反向信号诱导p53组成性高表达,可能通过增强DNA修复,抵抗卡铂造成DNA损伤而导致的细胞凋亡。
综上所述,本工作阐明了tmTNF-α反向信号复合物成员的相互作用,即通过其胞浆段脱磷酸化,募集TRAF1,后者将IKK-α募集到tmTNF-α反向信号复合物中,活化NF-κB旁路,通过上调抗凋亡基因表达,导致肿瘤细胞抵抗sTNF-α和化疗药物的杀伤。此外,tmTNF-α反向信号还通过促进肿瘤细胞PI3K和p53组成性表达,诱导ERK组成性磷酸化,抑制卡铂诱导的JNK磷酸化而导致肿瘤细胞耐药。该研究通过深入探索tmTNF-α反向信号通路及其保护肿瘤抵抗凋亡的分子机制,为临床干预肿瘤耐药提供新的线索和分子靶点。
transmembrane TNF-a(tmTNF-a) can not only interact directly with TNFR and transduce forward signaling into TNFR bearing cells to play effects, this signaling pathway is also called forward signaling pathway. Meanwhile, tmTNF-a can also work as receptors to accept membrane bound TNFR or soluble TNFR stimulation, and transdues signaling into tmTNF-a bearing cells, this signaling pathway is called reverse signaling pathway, which mediate several biological effects. The reverse signaling of tmTNF-a can upregulate activated T cells to secret IFN-y and downregulate generation of IL-4; it can also inhibit induced monocytes by LPS to secret proapoptotic factors and proinflammation factors, such as TNF-α、IL-1、IL-6 generation; but it upregulate IL-12 secretion. Furthermore, reverse signaling pathway induced by tmTNF-a can induce HTLV infected T cells or PHA activated CD4+T cells expression of E-selectin. We previously found that tumor cells with high expression of tmTNF-a can protect tumor cells from secretory TNF-a (sTNF-a) cytotoxicity. However, the reverse signaling pathway is still unknown.
We previously prepared a specific monoclonal tmTNF-a antibody, and we used this specific antibody to immunoprecipitate, and we had got IKKα、TRAF1 and NF-κBp52 to interact with the intracellular domain of tmTNF-a. This research is mainly studying the relationship of the member of the reverse signaling pathway of tmTNF-a, and their relationship with mitochondria. We also worked on the relationship between tmTNF-a expression on tumor cells and chemoresistance, and we also further study into the molecular mechanism of the chemoresistance induced by tmTNF-a, so as to provide new target molecular and theory basis for clinical intervention. Main results as below:
I. Construction and identification of prokaryotic、eukaryotic and fluorescence fusion expression vectors of TNF-LS、IKKαand TRAF1
1. Construction and identification of prokaryotic、eukaryotic and fluorescence fusion expression vectors of TNF-LS、IKKαand TRAF1:
PCR and molecular cloning techniques were used to construct prokaryotic fusion vectors of pTriEx-4C-TNF-LS、pTriEx-4C-TRAF1、pTrieX-4C-IKKα、pET28a-his-TRAF1 and pET28a-his-IKKa; fluorescence expression vectors of pEGFPNl-TNF-LS、pDsRedN1-TNF-LS、pCFPN1-TNF-LS and pEGFPC1-IKKa; eukaryotic fusion vectors of pTriEx-4C-TNF-LS、pTriEx-4C-TRAF、pTriEx-4C-IKKa and pcDNA3.1-IKKa. And they were successfully constructed by sequencing and blasting.
2. Construction and identification of the mutated TNF-LS in eukaryotic and fluorescence fusion expression vectors
pcDNA3.1/V5-His-TOPO-TNF-LS was taken as a template, PCR amplification of a fragment about 208 bp, restriction to connect to pcDNA3.1, transformation of the ligated product into competent DH5a bacteria, choosing positive clones, restriction enzyme digestion and sequencing. And then the correct sequenced pcDNA3.1-△-75--69-TNF-LS and pcDNA3.1-△-68～-62-TNF-LS digested, recycling the fragment, after digestion, connected to digested pEGFPNl, the identification of the positive clones by digesttion.
II. Identification of the major members of the reverse signaling pathway of tmTNF-αTRAFl and IKKa and the relationship among them
1. IKKα、TRAF-1 and NF-κBp52 can be immunoprecipitated by tmTNF-αmonoclonal antibody
In order to investigate into the reverse signaling pathway of tmTNF-α, specific tmTNF-αmonoclonal antibody was used to IP-Wetern. The results show that TRAFl, IKK-αand NF-κBp52 can be sedimentated by tmTNF-αantibody. However, TNFR1/2, TRAF2/3, NIK, SODD, FADD and NF-κBp65 can not be immunoprecipitated. Conversely, using TRAF1, IKKa and NF-κBp52 antibody to re-IP, all of them can immunoprecipitated tmTNF-α. The results imply that TRAFl and IKKa might participitate in the formation of reverse signaling complexes of tmTNF-αso as to activate NF-κB.
2. TNF-LS directly interact with TRAFl
First of all, we intend to explore the relationship between TNF-LS and TRAFl, direct or indirect interactions. Prokaryotic expression with his label with TRAF1 and prokaryotic expression of the non-his tagged TNF-LS in vitro Pull down, we confirmed that TNF-LS and TRAFlinteract directly. In order to further confirm this direct interaction, we transfected TRAF1 and TNF-LS plasmid alone or co-transfected into 293T cells to overexpress, and then by IP-Western to verify their interaction in vivo and found that tmTNF-αwith monoclonal antibodies can pull down significantly higher TRAFl in cells cotransfected with TNF-LS and TRAF1 than that of the single-TRAFl transgenic group or the blank control group. Further, we build TRAFl and TNF-LS fluorescence fusion vectors with different corresponding fluorescence to study their cellular localization, found that over-expression of TRAFl in 293T cells showed the limited point-like distribution, and TNF-LS distributed near the membrane. Co-transfected TRAF1 and TNF-LS resulted in gathering of TNF-LS to TRAF1 localization, the confocal results showed that the fluoresence of the TRAF1 and the fluorescence of the TNF-LS merged. Direct interaction of TNF-LS with TRAF1 confirmed in Three-pronged approach.
3.-75～-69 amino acids of the N terminal of TNF-LS determined the direct interaction of TNF-LS and TRAFl
The intracellular domain of TNF-LS has only 30 amino acids, and we produced mutation of TNF-LS deletion of the N-terminal -75～-69 or -68～-61 amino acids. Mutants or wild type tmTNF-αor TNF-LS together with TRAF1 were co-transfected into 293T cells and found that the TNF-LS deletion of the N-terminal of -75～-69 amino acids influenced greatly on the interaction of TRAF1 and TNF-LS, suggesting that the N-terminal -75～-69 amino acids of TNF-LS plays a decisive role in TRAF1 and TNF-LS interaction.
4. Dephosphorylation of the intracellular domain of TNF-LS caused the recruitment of the reverse signaling complex, leading to NF-κB activation, resulting in the resistance of tmTNF-αhigh expression tumors resistance to ADM and sTNF-α
The N-terminal-75 to -69 amino acids decides TRAFl and TNF-LS interaction, we found that the N-terminal-75 to -69 amino acids of tmTNF-αis the site for CKI phosphorylation. Using specific inhibitors of CKI inhibitor D4476 to inhibit the activity of CKI, and then tmTNF-a monoclonal antibody was used to IP, compared with untreated Raji cell group, treatment group IP to TRAF1, IKKa and NF-κBp52 significantly more than untreated group. The results implies that phosphorylation of tmTNF-a by CKI inhibited the reverse transmission signal pathway. CKI inhibitor with Raji cells inhibited the activity of CKI can lead to NF-κB activation and further lead to Raji cells resistant to sTNF-a and chemotherapy drug doxorubicin. Suggesting that tmTNF-a may be involved in a reverse signal of NF-κB activation and resistance to chemotherapy drugs.
5. TRAFl and IKKa interacted directly
We also confirmed direct interaction of TRAF1 and IKKa from three-pronged approaches. First of all, we prokaryotically expressed TRAF1 and IKKa, Pull down assay was done in vitro and confirmed that TRAF1 directly interacted with IKKa. In addition, we use IP in the body to further give evidence that TRAF1 directly interacted with IKKa. Finally, TRAF1 and IKKa with different color fluorescent protein fusion expressed in 293T cells, after co-transfection of TRAF1 and IKKa, TRAF1 resulted in partial aggregation of IKKa protein and the fluorescence of TRAFl and IKKa merged.
6. TNF-LS can not interact directly with IKKa
Prokaryotically expressed TNF-LS and IKKa, and in vitro pull down assay was done, we found that TNF-LS can not directly interact with IKKa
III. The close relationship of mitochondrial with the reverse signaling complex molecular TNF-LS、TRAF1 and IKKα
1. TRAFl co-localized with mitochondria
In 293T cells, mitochondria showed the mesh point distribution, after transfection of TRAF1, the distribution of mitochondria changed into point-like distribution, and co-localized with TRAF1.
2. Members of the reverse signaling of tmTNF-a all exist in the mitochondria
mitochondira was extracted, and western blot was taken to indentify if there was any relationship between members of the reverse signaling complex members of TRAF1、 IKKa and NF-κBp52 and mitochondria. We found that TRAF1、IKKαand NF-κB p52 all could localize in the mitochondria. Co-transfection of 293T cells with TNF-LS and TRAF1 could induce mitochondria aggregation, and TRAF1 changed the TNF-LS distribution on the membrane which assembled to the position of TRAF1. IP was further taken to immunoprecipitated mitochondria proteins with monoclonal tmTNF-αantibody and NF-KBp52 can be precipitated, implying that the reverse signaling pathway might occur in the mitochondria.
3. tmTNF-αand related reverse signaling complex members can be found in the nucleus
nuclear proteins were prepared, and western blot was used and we identified the nuclear localization of tmTNF-a and related molecular TRAF1、IKKa and NF-κB p52, but there is no report about their relationship until now.
IV. TNFLS stably high expression in MCF-7 induced the tolerance of such cells to sTNF-α
1. High expression of tmTNF-a protects cells from the sTNF-a induced cytotoxicity
Sources of the same breast cancer tumor, some sensitive to sTNF-a induced cytotoxicity,some resistant, we found that high expression of tmTNF-a in the MDA-MB-231 induced sTNF-αtolerance, while low tmTNF-a expression in the MCF-7 cells were sensitive to sTNF-α. Prompt, tmTNF-a expression may be the protection effect from sTNF-αinduced cytotoxicity.
2. Down-regulation of tmTNF-a in MDA-MB-231 cells reduced NF-κB activity with a reversal of the cells on the sensitivity to sTNF-α
Interestingly, down-regulation of tmTNF-a expression in MDA-MB-231 cells with TNF siRNA resulted in the recovery of MDA-MB-231 cells sensitive to sTNF-αto some extent.
3. TNF-LS expressed on the membrane of MCF-7 cells
After transfection of pIRES2-EGFP/TNF-LS into MCF-7 cells, the expression of EGFP and TNF-LS were separate. tmTNF-a monoclonal antibody were used for staining, and then adding the second TRITC labeled antibody. Indirect fluorescence under the microscope shows that the diffuse cytoplasmic green fluorescence with TNF-LS distribution in the cell membrane, which shows a red aperture under the fluorescence microscope.
4. High expression of TNFLS in MCF-7 cells caused the cells tolerant to sTNF-αand persistent NF-κB activation
Establishment of TNF-LS stably expressed in MCF-7 cells, we found that TNF-LS/MCF-7 cells are tolerant to sTNF-a and persistent NF-κB activation, showing the same features like another breast cancer cell line MDA-MB-231 cell with high tmTNF-a expression. Prompt, TNF-LS has some tmTNF-a properties.We speculated that the reverse signaling pathway of tmTNF-a may determined by TNF-LS.
5. NF-κB inhibitor PDTC inhibited stably transfected MCF-7 cells with TNF-LS NF-κB activity, resulting in reversal of the stable cell lines TNF-LS/MCF-7 sensitive to sTNF-α
PDTC can selectivly inhibited the activity of NF-κB. Selective inhibition of NF-κB activity in TNF-LS/MCF-7 cells and found that the sensitivity of TNF-LS/MCF-7 cells to sTNF-a increased, suggesting that TNF-LS lead to the stability of the TNF-LS/MCF-7 cells to sTNF-a for NF-κB activation.
6. Anti-apoptotic gene expression leading to TNF-LS/MCF-7 cells resistant to sTNF-a induced cytotoxicity
NF-κB is an important factor in determining cell death and survival, and cIAPl is one of the target gene of NF-κB which is an antiapoptotic factor. comparing before and after STNF-a stimulation, the expression of cIAPl was significantly higher in MCF-7 cells stably transfected with TNF-LS than MCF-7 cells untranfected or transfected empty vectors.sTNF-a stimulation led to further cIAPl gene expression. The results suggest that regulation of antiapoptotic gene expression by NF-κB activation may be the reason for TNF-LS/MCF-7 cells resistant to sTNF-αinduced cytotoxicity.
7. The intracellualr domain of tmTNF-a induced durable NF-κB activation in MCF-7 cells
High tmTNF-a expression in breast cancer cell line MDA-MB-231 cells can lead to NF-κB activation,while, TNF-LS stabily expressed on MCF-7 cells also results in a persistent activation of NF-κB. Comparison transfection of Acs-tmTNF-α、TNF-LS and tmTNF-a into MCF-7 cell lines, we only could find durable NF-κB activation in cells instant transfection of tmTNF-a and TNF-LS. Cells transfected with Acs-tmTNF-a could not activate NF-κB when compared with untransfected or transfected empty vector groups. Our results implied that high tmTNF-a expression in breast cancer cells induced durable NF-κB activation possibly by TNF-LS.
8. Endocytosis inhibitor MDC pretreatment TNF-LS/MCF-7 cellS induced surface expression of TNF-LS
Inhibition of TNF-LS endocytosis, TNF-LS/MCF-7 increased cell surface expression of TNF-LS may be a further increase in NF-κB activity in TNF-LS/MCF-7 cells.
9. Signal peptide peptidase inhibitor ZLL pretreatment increased TNF-LS/MCF-7 cell surface TNF-LS expression, resulting in further NF-κB B activation
Inhibition TNF-LS shear from membrane by signal peptide peptidase inhibitor increased TNF-LS/MCF-7 cell surface TNF-LS expression, and this may be a reason for further increase in NF-κB activity in TNF-LS/MCF-7 cells.
V. Reverse signaling of tmTNF-a induced the resistance of breast cancer cells to chemotherapy and related molecular mechanisms
1. High expression of tmTNF-a on breast cancers induced carboplatin resistance
Comparing the sensitivity difference of breast cancer cells with different tmTNF-αexpression to carboplatin, we found that MDA-MB-435 cells with high level of tmTNF-αexpression were much more resistant to carboplatin than T47D cells with low level of tmTNF-αexpression did, and found that MDA-MB-435 cells with high tmTNF-αexpression showed durable NF-κB activation, while no obvious NF-κB activity was observed in T47D cells with low tmTNF-αexpression.
2. Upregulation of tmTNF-αexpression on T47D cells induced their resistance to carboplatin with the phenomenon that the cleavage of caspase-9 decreased, and NF-κB inhibitor PDTC can reverse the resistance
After transfection of T47D with tmTNF-α, the cells are resistant to carboplatin cytotoxicity, the cleavage of caspase-9 decreased. NF-κB inhibitor can reverse the resistance to carboplatin at a certain level.
3. MCF-7 cells stably transfected with TNF-LS were resistant to carboplatin cytotoxicity
It has been confirmed that stable transfection of TNF-LS into MCF-7 cells resulted in durable NF-κB activation and tolerance to sTNF-α. In this, we have further found that TNF-LS/MCF-7 cells are more resistant to carboplatin chemotherapy treatment than untransfected or transfected with empty vectors.
4. MCF-7 cells with TNF-LS inhibited the sensitivity of MCF-7 cells to carboplatin
Treatment of transfected and untransfected MCF-7 cells with different concentration of carboplatin, we found that the IC50 of carboplatin to untransfected MCF-7 cells was just 231μg/ml, however, the IC50 of transfected MCF-7 cells were much higher than 400μg/ml.
5. The proliferation of MCF-7 cells with high expression of tmTNF-αis quicker than MCF-7 cells with low tmTNF-αexpression after treatment of MCF-7 cells with carboplatin
We got two lines of MCF-7 cells with high tmTNF-αexpression and low tmTNF-αexpression, and we compared these two lines sensitivity to carboplatin. We found that high expression of tmTNF-αin MCF-7 cells proliferated significantly faster than the speed of low tmTNF-αexpression MCF-7 cells treated with carboplatin. And extended over time, the inhibitory effect of carboplatin to the MCF-7 cells with high tmTNF-αexpression becomes weaker and weaker.
6. Inhibition NF-κB activation in MCF-7 cell line 2-E8 stably transfected with TNF-LS could reverse sensitivity of 2-E8 to carboplatin at a certain extent
As has reported in part four that stably transfected MCF-7 cells with TNF-LS induced durable NF-κB activation in these cell lines, however, inhibiting the NF-κB activity in these cells can reverse the sensitivity of these cells to carboplatin.
7. Comparing the ERK phosphorylation status of T47D cells untransfected or transfected with tmTNF-a after treatment with carboplatin
ERK phosphorylation was not very clear for untransfected T47D cells.After carboplatin treatment, as early as five minutes after treatment with carboplatin can resulted in ERK phosphorylation, and the ERK phosphorylation level reached peak at 10 minutes with significant decrease at 30 minutes, suggesting that stimulation of carboplatin resulted in a transient ERK phosphorylation. Instant transfection of tmTNF-a into MCF-7 cells resulted in durable ERK phosphorylation to certain degree, and activation was not affected by treatment with carboplatin.
8. Comparing the JNK phosphorylation status of T47D cells untransfected or transfected with tmTNF-αafter treatment with carboplatin
Both transfected and untransfected T47D cells have no JNK phosphorylation without stimulation. After treatment with carboplatin, the JNK phosphorylation level of untransfected T47D cells were significantly higher than T47D cells transfected with tmTNF-α, and both JNK phosphorylation status reduced after 30 min stimulation.
9. Comparing the PI3K expression status of T47D cells untransfected or transfected with tmTNF-αafter treatment with carboplatin
T47D cells transfected with tm TNF-α, we can see continuous PI3K p85 expression, and carboplatin treatment has little effect on the expression level of PI3K. However, no observed PI3K p85 expression could be found in non-transfected T47D cells in the resting state. Carboplatin treatment of untransfected T47D cells led to a small amount of PI3K p85 expression, suggesting that PI3K may be involved in a high tmTNF-a expression in tumor cells resistant to chemotherapeutic drugs.
lO.Comparing the expression of p53 in T47D cells untransfected or transfected with tmTNF-a after treatment with carboplatin
Comparing the expression status of p53, we found a very interesting results. We found that after treatment of untransfected T47D cells with carboplatin, the expression of p53 in cells gradually upregulated as time went by. While, we could see p53 expression even after transfection of cells with tmTNF-αwithout treatment of carboplatin, and expression of p53 in T47D cells transfected with tmTNF-awas independent on carboplatin treatment.
引文
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