Lipopolysaccharide initiates a bypass feedback loop of epidermal growth factor receptor signaling by HPS70-induced COX-2 in H22 hepatocarcinoma cells

LPS can induce TACE upregulation via signaling from TLR4-derived EGFR activation in tumor cells. The regulation and activity of TACE have been investigated with the observation that gene expression is upregulated in response to LPS followed by EGFR activation, however, the process remains poorly understood. In this study, we examined the effects of LPS on H22 hepatocarcinoma cells that displayed constitutively active TLR4 expression. Upon TLR4 shRNA transfection into H22 cells, HSP70 expression significantly increased. However, LPS induced early phosphorylation of EGFR in H22 cells, which reached maximum levels within 30?min. Inhibition of TLR4 in H22 cells resulted in a significant rise in both EGFR phosphorylation and TACE upregulation 24?h after exposure to LPS. Exogenous HSP70 also induced rapid phosphorylation of EGFR, upregulated the expression of COX-2 via a signaling pathway that involved TACE-dependent TGF-α release. Furthermore, inhibition of EGFR activation and reduction of COX-2 expression by COX-2 inhibitor prevented HSP70-induced cell invasion in vitro. These findings demonstrate that the biological importance of HSP70/COX-2 is crucial to the second, but not the first, phase of EGFR phosphorylation in tumor cells. The growth of tumor cells by inserting shRNA plasmid TLR4 combination with COX-2 inhibitor could be effectively reduced in LPS stimulation. We concluded that LPS triggered a bypass feedback loop of EGFR activation and involved HSP70/COX-2 in H22 cells by inhibition of TLR4 and that EGFR phosphorylation is implicated in tumor growth by LPS stimulation.     

同种肿瘤来源的HSP70-PC对原位的大鼠膀胱肿瘤的治疗研究

 目的 研究用来自亚高温盆腔区域热疗后原位大鼠膀胱肿瘤的热休克蛋白70-肽复合物（HSP70-PC）对原位大鼠膀胱肿瘤的治疗作用机制。方法 经尿道膀胱灌注MNU制备出大鼠膀胱肿瘤动物模型。将动物随机分为A（肿瘤对照）组、B（HSP70-PC治疗）组、C（肿瘤细胞裂解液治疗）组，每组5只。皮下注射治疗1个月后检测膀胱及肿瘤湿重，肿瘤分期分级，TUNEL法检测肿瘤细胞凋亡指数（AI），免疫组化检测肿瘤细胞增殖细胞核抗原指数（PCNA LI）、肿瘤组织中S-100蛋白阳性细胞率及CD8阳性细胞率、膀胱引流区域髂动脉及腹主动脉旁淋巴结中S-100蛋白阳性细胞率、脾脏S-100蛋白阳性细胞率及CD8阳性细胞率，ELISA法检测血清Rat IFN-γ含量。结果 膀胱及肿瘤湿重B组低于A、C组（P＜0.01）。A、C组间差异无显著性意义（P = 0.610）。B组肿瘤分期低于A、C组（P＜0.01）；A、C组间差异无显著性意义（P =1.00）。肿瘤分级各组间差异均无显著性意义（P =1.00）。B组肿瘤细胞AI、膀胱肿瘤组织中CD8阳性细胞率、血清Rat IFN-γ含量均高于A、C组（P＜0.01）；A、C组间差异均无显著性意义（P =0.870，0.597，0.979）。脾脏S-100蛋白阳性细胞率及CD8阳性细胞率由高到低依次为B、C、A组（P＜0.01）。肿瘤细胞PCNA LI、膀胱肿瘤组织中S-100蛋白阳性细胞率、淋巴结中S-100蛋白阳性细胞率三组之间差异均无显著性意义（P =0.808，0.718，0.847）。结论 皮下注射HSP70-PC可以上调原位的膀胱肿瘤大鼠的细胞免疫状态，促进肿瘤凋亡，但对肿瘤细胞增生无影响。     

HIFU治疗后肿瘤抗原对树突状细胞的活化及其抗肿瘤效应

目的：探讨HIFU治疗小鼠H22抑制性肝癌后产生的肿瘤抗原对机体抗肿瘤免疫功能增强的机制。方法：正常小鼠骨髓中提取骨髓细胞,在rmIL-4、rmGM-CSF奈件下培养7d,制备小鼠骨髓树突状细胞,用HIFU治疗小鼠移植性肝癌后产生的肿瘤抗原活化树突状细胞,再用活化后的树突状细胞激活T淋巴细胞为细胞毒性T细胞,用MTT法检测CTL在体外特异性杀伤肿瘤靶细胞的能力。结果：B16肿瘤HSP70-肽复合物组和H22肿瘤HSP70-肽复合物组的脾淋巴细胞的增殖率均高于阴性对照组、H22肿瘤粗提物组和HIFU后H22肿瘤粗提物组,P〈0．001;但两组之间差异无统计学意义,P〉0．05。H22肿瘤HSP70-肽复合物组CTL对H22肿瘤细胞的杀伤率为70．0％,明显高于阴性对照组、H22肿瘤粗提物组和HIFU后H22肿瘤粗提物组（P〈0．001）,但对非靶细胞B16肿瘤的杀伤率与上述各组的差异无统计学意义;B16肿瘤HSP70-肽复合物组CTL对B16肿瘤细胞的杀伤率为78．5％,对H22细胞的杀伤率为21．4％,表明CTL对肿瘤细胞的杀伤作用具有特异性。结论：HIFU治疗后坏死肿瘤组织中的HSP70-肽复合物作为肿瘤疫苗,通过活化DC和刺激T淋巴细胞增殖为CTL,发挥特异性抗肿瘤免疫功能。     

TFF1和HSP70在结肠癌中的表达及临床意义

目的 探讨TFF1和HSP70在结肠癌中表达及临床意义。方法 用免疫组化S-P法检测71例结肠癌术后组织和15例正常组织TFF1和HSP70表达。结果 结肠癌组织TFF1和HSP70表达率分别为57．7％和70．4％，高于正常组织（26．7％和40％；P=0．03；0．04）。TFF1表达与Dukes分期和分化程度有关（P=0．025；0．005）。HSP70表达与Dllkes分期和分化程度有关（P=0．032；0．031）。TFF1和HSP70均与性别、年龄、肿瘤浸润深度、生长方式、淋巴结转移和预后无关。TFF1和HSP70在结肠癌中表达呈正相关（r=0．306，P=0．1309）。结论 本实验首次研究TFF1与HSP70在结肠癌中的表达相关性，提示两者在肿瘤发生中可能存在相互作用。TFF1和HSP70在结肠癌高表达，可能参与肿瘤的发生发展过程。     

TAK-242对人多发性骨髓瘤细胞增殖及凋亡的影响

 目的 探讨Toll样受体4（TLR4）特异性抑制剂TAK-242对人多发性骨髓瘤细胞株RPMI8226增殖及凋亡的影响。方法 取对数期生长的人多发性骨髓瘤细胞株RPMI8226分为A、B、C组及对照组（Control），A、B、C组分别加入终浓度为20、40、80 μmol/L的TAK-242，对照组不加抑制剂，培养24 h后，采用CCK-8法检测细胞增殖抑制率；Annexin-V/PI检测细胞凋亡率；RT-PCR检测细胞TLR4、Myd88 mRNA的表达水平；Western blot检测细胞Myd88、NF-κB的蛋白表达情况。结果 A、B、C组细胞增殖抑制率、凋亡率升高，TLR4、Myd88 mRNA表达水平及Myd88、NF-κB的蛋白表达水平降低，各浓度组之间的差异均有统计学意义（P<0.05）。结论 TAK-242可抑制人多发性骨髓瘤细胞RPMI8226的增殖，促进细胞凋亡，其机制可能为NF-κB信号通路功能被抑制。     

Anti-Tumor Effect of Heat Shock Protein 70-Peptide Complexes on A-549 Cells

Objective: To investigate the anti-tumor immunity in vitro of heat shock protein 70-peptide complexes (HSP70-PC) from human lung cancer tissue. Methods: HSP70-PC was purified from lung tumor tissues and corresponding non-tumor lung samples with the methods of ADP-affinity chromatography, DEAE ion-exchange chromatography and Western-blot. The activation and proliferation of PBMC induced by different HSP70-PC and tumor cytotoxic reactivity to A549 cells in vitro were measured by the MTT cell proliferation assay. Results: The purified HSP70-PC had a very high purity found by SDS-PAGE and Western-blot. Human lymphocytes were sensitized efficiently by HSP70 preparation purified from lung cancer tissues and a definite cytotoxicity to A-549 cells was observed. There was significant difference with HSP70-PC purified from lung cancer, compared with the control group (P<0.001). Conclusion: High purity of HSP70-PC could be achieved from tumor tissues in this study. HSP70-PC purified from human tumor tissues can induce anti-tumor immunity in vitro mainly implemented by eliciting CTL immunity.     

紫铆因抑制膀胱癌细胞增殖的体内外研究

目的 观察紫铆因对膀胱移行细胞癌细胞BLS增殖的影响以及对膀胱癌裸鼠移植瘤生长的 影响。方法 不同浓度紫铆因处理细胞,四甲基偶氮唑蓝比色实验（MTT）及平板克隆形成实验观 察细胞增殖能力,流式细胞仪分析细胞周期,蛋白印迹检测核转录因子κB (NF-κB)p65核内表达及 细胞外信号调节激酶1/2（ERK1/2）的磷酸化程度,并检测NF-κB下游靶基因细胞周期素D1（Cyclin D1）及环氧合酶-2（COX2）的表达。建立膀胱癌裸鼠皮下移植瘤,采用腹腔注射的给药途径,测 量移植瘤的体积和重量。结果 紫铆因抑制了膀胱癌细胞增殖并诱导G2/M期细胞周期阻滞。紫铆因 处理后NF-κB p65的核内表达及ERK1/2的磷酸化程度下降（P<0.05）,Cyclin D1及COX2基因表达下 调（P<0.05）,体内实验发现紫铆因治疗组较对照组皮下移植瘤的生长明显受抑制（P<0.05）。结 论 紫铆因具有抗膀胱癌细胞增殖作用,可能与其抑制ERK及NF-κB信号激活有关。     

AAV–sBTLA facilitates HSP70 vaccine-triggered prophylactic antitumor immunity against a murine melanoma pulmonary metastasis model in vivo

Activation of the BTLA–HVEM co-inhibitory signaling pathway impairs antitumor immunity. Our previous study demonstrated that the extracellular domain of murine BTLA (the soluble form of BTLA) can facilitate HSP70 vaccine-triggered antitumor immunity by blocking BTLA–HVEM interactions in a murine TC-1 non-metastatic tumor model. However, it is unknown whether this strategy has beneficial effects on highly malignant metastatic tumors, such as melanoma. To address this question, we expressed the soluble form of BTLA (sBTLA) in combination with HSP70 vaccine and examined the resulting antitumor activity in a melanoma pulmonary metastasis model. A recombinant adeno-associated virus (AAV) vector was used for the sBTLA gene delivery because of its high transfection efficiency and low toxicity. In vitro expression of AAV–sBTLA enhanced lymphocyte activation and induced specific cytotoxicity against B16F1 murine melanoma cells, while in vivo administration of AAV–sBTLA plus HSP70 vaccine by tail vein injection exerted a limited, late-stage antitumor effect against the existing B16F1 cells. However, the combination treatment generated a potent prophylactic antitumor response in the melanoma lung metastasis model in B6 mice. In this case, most of the metastatic foci were inhibited, and mouse survival was prolonged. Furthermore, the Th1 cytokines IL-2 and IFN-γ were up-regulated, while the negative regulatory molecules IL-10 and TGF-β were down-regulated. The number of regulatory T cells also decreased in the tumor environment. Therefore, AAV–sBTLA plus HSP70 vaccine may have therapeutic potential for the prevention of metastatic melanoma.     

TLR4基因多态性对肝癌细胞HepG2增殖、迁移及凋亡的影响

目的:研究TLR4基因多态性对人肝癌细胞HepG2细胞增殖、迁移及凋亡的影响,并探讨其相关分子机制.方法:构建TLR4野生型(WT)、1196(C/T)位点及896(A/G)位点突变型质粒并将其稳转HepG2细胞株中;CCK-8法检测、Annexin V-PE/7-AAD流式细胞仪及Transwell小室实验检测TLR4基因多态性对HepG2细胞增殖、迁移及凋亡的影响;Western blot检测NF-κB p65(nuclear factorκB p65)表达水平的差异.结果:Western blot结果表明三组TLR4过表达细胞株中TLR4的含量显著高于正常组.与正常HepG2细胞相比,三组TLR4过表达细胞株增殖能力、迁移能力及p65表达量均增加;与TLR4野生型组相比,两组突变型细胞株增殖能力、迁移能力及p65表达量均减弱,且凋亡率增加,差异具有统计学意义(P<0.05).结论:TLR4可能通过影响NF-κB p65相关蛋白的表达促进肝癌细胞HepG2的增殖及迁移,其基因1196(C/T)位点及896(A/G)位点的突变会减弱肝癌细胞的增殖及迁移能力.     

Regulation of HSP70 on activating macrophages using PDT‐induced apoptotic cells

Although anti‐tumor immunological responses have been mainly associated with necrosis, apoptosis‐associated immune responses have been recently suggested as well. In this study, we investigated anti‐tumor immune responses and regulatory mechanisms of HSP70 using apoptotic cells induced by photodynamic therapy (PDT). The relationships between HSP70 release, HSP70 translocation and macrophage responses were studied using confocal fluorescence microscopy, FACS and ELISA. Macrophages incubated with apoptotic cells as well as necrotic tumor cells showed a high level of TNFα secretion. Apoptotic cells but not the apoptotic cell supernatants induced TNFα secretion. During both necrosis and apoptosis processes, the TNFα production was diminished drastically when HSP70 or TLR‐2 was inhibited. After the PDT treatment, cytoplasmic HSP70 was released from the necrotic cells, while HSP70 rapidly translocated to the surface of the apoptotic cells. Furthermore, the TNFα secretion and the tumor cytotoxicity of splenocytes from mice immunized with apoptotic cells appeared similar to that of splenocytes immunized with necrotic cells. Our in vitro and in vivo results show that apoptosis can potentially have higher impact in inducing immunological responses, hence clarifying the immunological regulatory mechanisms of HSP70 under cell apoptosis and necrosis induced by PDT treatment. These findings could lead to an optimal PDT treatment based on immunological responses. © 2009 UICC     

转录核因子-κB信号传导在消化道肿瘤中的研究进展

具有多向转录调节功能的转录核因子-κB(NF-κB)与胃、肠、肝等消化道组织细胞增殖和凋亡的失衡有关.其活化后产生的信号传导广泛参与了这些消化道肿瘤的癌前病变、肿瘤发生以及浸润、转移等过程.针对NF-κB活化途径的治疗,可以促进肿瘤细胞凋亡,削弱肿瘤对放化疗的抵抗能力并减轻肿瘤引起的恶病质.     

A small molecule inhibitor of NF-κB,dehydroxymethylepoxyquinomicin (DHMEQ), suppresses growth and invasion of nasopharyngeal carcinoma (NPC) cells

Despite the demonstrated constitutive activation of NF-κB in nasopharyngeal carcinoma (NPC), the therapeutic potential of targeting this pathway has not been investigated. Here, we employed a small molecule inhibitor of NF-κB, DHMEQ (which mainly blocks nuclear translocation of activated NF-κB) and demonstrated significant inhibition of NPC cell proliferation, migration, invasion, as well as anchorage-independent growth. These antitumor effects were associated with induction of G₂/M cell cycle arrest and apoptosis, and downregulation of NF-κB target genes (EGFR, cyclin D1 and survivin). This first demonstration of therapeutic benefits of NF-κB targeting in NPC implicates the importance of targeting this pathway in NPC.     

miR-485-3p通过靶向TLR1/NF-κB信号通路调节胃癌细胞放射敏感性

目的 研究miR-485-3p是否通过靶向TLR1对胃癌细胞放射敏感性起作用。方法分别用qRT-PCR和蛋白印迹法检测miR-485-3p和TLR1的表达变化,DIANA、TargetScan和miRanda软件预测和双荧光素酶报告实验验证miR-485-3p对TLR1的靶向作用。将miR-485-3p mimic或TLR1 siRNA转染到胃癌MGC803细胞中,放射处理细胞,凋亡实验、克隆形成实验和MTT检测细胞放射敏感性的变化。双荧光素酶报告实验检测miR-485-3p上调和TLR1沉默对NF-κB活性的影响。蛋白免疫印迹实验探究miR-485-3p上调和TLR1沉默对NF-κB靶基因的影响。结果 放射处理后胃癌细胞miR-485-3p表达下调,TLR1表达上调。靶基因预测软件发现TLR1可能是miR-485-3p的靶基因,双荧光素酶报告实验进一步验证了TLR1是miR-485-3p的直接靶点。miR-485-3p负调控TLR1的表达。miR-485-3p的过表达提高了细胞凋亡率,降低了细胞克隆形成和细胞群体增殖能力,增强了细胞的放射敏感性,且TLR1的沉默也具有相同作用。miR-485-3p上调和TLR1沉默均降低了NF-κB的活性,下调了NF-κB多个靶基因的表达。结论 miR-485-3p可能通过靶向TLR1调控NF-κB信号通路增强胃癌细胞的放射敏感性。