Augmentation of tumor necrosis factor family-induced apoptosis by E3330 in human hepatocellular carcinoma cell lines via inhibition of NFκB

AIM: To investigate the reduction of cell viability in human hepatocellular carcinoma (HCC) cell lines induced by inhibition of nuclear factor κB (NFκB).METHODS: HLE, SKHep1, and HepG2 were incubated and E3330 was used to compare the stimulation of some chemotherapeutic drugs with that of TNF family, Fas ligand, TNFα and TNF-related apoptosis-inducing ligand (TRAIL) at the point of the reduction of cell viability by inhibiting NFκB.RESULTS: E3330 decreased NFκB levels in HLE cells stimulated by TNF and TRAIL. The cytotoxicity of the combination of TRAIL, TNFα, Fas ligand, and E3330increased synergistically in a dose-dependent manner compared to either E3330 alone in all HCC cell lines by MTT assay. However, the combination of some chemotherapeutic drugs and E3330 did not decrease the cell viability.CONCLUSION: Inhibition of NFκB sensitizes human HCC cell lines to TNF-mediated apoptosis including TRAIL, and TRAIL-based tumor therapy might be a powerful potential therapeutic tool in the treatment of human HCC.     

TRAIL蛋白原核表达载体的构建及表达研究

目的 利用蛋白自剪切功能原核表达系统构建肿瘤坏死因子相关凋亡诱导配体（TRAIL）胞膜外段融合蛋白表达载体,并进行表达条件优化和初步纯化。方法 设计基因拼接引物,合成TRAIL基因胞膜外段双链cDNA序列,将其连接于克隆载体pMD18-T中;将酶切、纯化的TRAIL基因与经相同处理的载体pTWIN1相连接,转化感受态大肠杆菌JM109,筛选阳性重组子。将鉴定（酶切及序列分析）阳性的重组子质粒转人大肠埃希菌ER2566表达菌中,采用蛋白质SDS—PAGE电泳方法分析不同浓度诱导剂异丙基硫代-β-D-半乳糖苷（IPTG）、不同诱导温度、不同诱导时间下目的蛋白表达情况。结果 成功获得了包含TRAIL基因胞膜外段的双链cDNA序列,酶切及序列分析证实成功构建了TRAIL胞膜外段蛋白自剪切功能原核表达载体。采用0．3mmol／LIPTG、15℃诱导过夜（14～16h）,目的蛋白获得较高表达量,且大部分为可溶性蛋白。结论 采用大肠埃希氏菌ER2566,TRAIL胞膜外段融合蛋白获得高表达,经简单后续处理即可获得不含任何额外氨基酸的可溶性目的蛋白。     

Novel engineered TRAIL-based chimeric protein strongly inhibits tumor growth and bypasses TRAIL resistance

Targeting of the TRAIL-DR4/5 pathway was proposed as a promising approach for specific induction of apoptosis in cancer cells. Clinical trials, however, showed inadequate efficiency of TRAIL as a monotherapy. It is a widely held view that the application of multifunctional molecules or combination therapy may lead to substantial improvement. Here, we demonstrate the effectiveness and safety of a novel chimeric protein, AD-O51.4, which is a TRAIL equipped with positively charged VEGFA-derived effector peptides. The study was performed in multiple cancer cell line- and patient-derived xenografts. A pharmacokinetic profile was established in monkeys. AD-O51.4 strongly inhibits tumor growth, even leading to complete long-term tumor remission. Neither mice nor monkeys treated with AD-O51.4 demonstrate symptoms of drug toxicity. AD-O51.4 exhibits a satisfactory half-life in plasma and accumulates preferentially in tumors. The cellular mechanism of AD-O51.4 activity involves both cytotoxic effects in tumor cells and antiangiogenic effects on the endothelium. The presence of DRs in cancer cells is crucial for AD-O51.4-driven apoptosis execution. The TRAIL component of the fusion molecule serves as an apoptosis inducer and a cellular anchor for the effector peptides in TRAIL-sensitive and TRAIL-resistant cancer cells, respectively. The FADD-dependent pathway, however, seems to be not indispensable in death signal transduction; thus, AD-O51.4 is capable of bypassing the refractoriness of TRAIL. AD-O51.4-driven cell death, which exceeds TRAIL activity, is achieved due to the N-terminally fused polypeptide, containing VEGFA-derived effector peptides. The high anticancer efficiency of AD-O51.4 combined with its safety has led to the entry of AD-O51.4 into toxicological studies.     

顺铂在TRAIL/APO-2L致胶质瘤细胞凋亡作用中影响机制的研究

目的探讨TRAIL对胶质瘤细胞凋亡的影响,并进一步研究DNA破坏药物顺铂(CDDP)在与其联合作用诱导细胞凋亡中影响作用的机制。方法行人脑胶质瘤U251细胞株的培养,应用不同浓度TRAIL和CDDP作用于胶质瘤U251细胞,MTT法检测TRAIL和CDDP分别及联合作用于U251细胞后的存活率,并以亚毒剂量的TRAIL和CDDP联合作用后,流式细胞仪检测其凋亡率。Westernblot方法检测CDDP作用前后TRAIL受体DR5表达量的变化。结果MTT结果:在一定时间范围内,随着药物浓度增加,TRAIL和CDDP分别及联合作用皆使胶质瘤U251细胞存活率逐渐降低,其中两者的联合作用效果明显,与分别作用组差异有统计学意义(P<0.01)。亚毒剂量的TRAIL(50ng·ml-1)、CDDP(4μg·ml-1)分别及联合作用人脑胶质瘤U251细胞24h后,存活率分别为:72.43%,75.56%,23.45%。亚毒剂量的TRAIL(50ng·ml-1)、CDDP(4μg·ml-1)单独或联合作用组致胶质瘤细胞凋亡率分别为25.61%、20.59%、62.33%。单独应用组与联合组之间差异有统计学意义(P<0.01)。Westernblot法检测显示:CDDP作用后较作用前DR5表达有明显增高,随CDDP剂量增多,DR5表达量逐渐增加。结论TRAIL和CDDP联合作用能显著增强其对人U251胶质瘤细胞凋亡的诱导,其机制可能与CDDP能上调TRAIL死亡受体DR5的表达有关。     

TRAIL参与氧合血红蛋白诱导的脑血管内皮细胞凋亡

目的目前对自发性蛛网膜下腔出血（SAH）后脑血管痉挛（CVS）的机制尚不十分清楚,研究认为脑血管内皮细胞的凋亡发挥了重要作用。本实验探讨了肿瘤坏死因子相关凋亡诱导配体（TRAIL）及caspase3在血管内皮细胞凋亡中作用。方法在离体培养的脑血管内皮细胞中,观察给予氧合血红蛋白（OxyHb）处理以及给予caspase3拮抗剂或TRAIL后,运用实时荧光定量聚合酶链反应（PCR）和Western blot检测两者在mRNA和蛋白水平表达的改变。结果和生理盐水阴性对照相比,单纯OxyHb处理以及OxyHb＋TRAIL处理后,血管内皮caspase3表达均显著升高（P〈0．05）。TRAIL能够上调OxyHb刺激诱导的caspase3表达（P〈0．05）。和生理盐水对照组相比,OxyHb处理后,血管内皮TRAIL表达显著上调（P〈0．05）。但是,和OxyHb处理相比,caspase3拮抗剂Ac—DEVD—CHO（ADC）不能明显改变OxyHb刺激诱导的TRAIL蛋白的表达上调（P〉0．05）。结论TRAIL能够促进SAH后OxyHb刺激导致的内皮细胞凋亡。在凋亡的级联反应过程中,TRAIL能够活化下游caspase3,从而启动凋亡过程。因此,TRAIL及其受体在SAH后CVS的发生、发展过程中可能有着重要作用。     

绒毛中TRAIL及其受体的表达与不明原因反复自然流产的相关性的探讨

目的探讨肿瘤坏死因子相关凋亡诱导配体TRAIL及其受体在不明原因早期反复自然流产患者(URSA)绒毛中的表达及二者的相关性。方法选择实验组妊娠45～60 d的URSA患者15例,对照组健康人工流产15例。两组分别取绒毛组织采用免疫组化法检测TRAIL及其受体的表达,进行方差分析和t检验比较两组绒毛组织中TRAIL及其受体蛋白表达水平的差异。结果实验组绒毛组织中TRAIL及其受体DR4、DR5蛋白表达较强,而诱骗受体DcR1及DcR2蛋白与正常对照相比表达较少,P值均小于0.05。结论TRAIL及其受体可能是造成自然流产的机制之一。     

TRAIL在原发免疫性血小板减少症患者外周血单个核细胞中的表达及临床意义

目的：探讨原发免疫性血小板减少症（primary immune thrombocytopenia,ITP）中TRAIL的表达及其在发病机制中的作用。方法使用Ficoll密度梯度离心法,收集28例ITP患者和30例健康对照的外周血单个核细胞（PBMC）。采用实时荧光定量PCR检测TRAIL基因的表达,采用 ELISA方法检测血清中细胞因子TNF-α,IFN-γ,IL-4和 IL-10的量,并分析TRAIL与患者TNF-α,INF-γ,IL-10和血小板计数的相关性。结果与健康对照组相比,ITP患者 PBMC中 TRAIL表达显著增高（2.80±0.43 vs 1.00±0.24,t＝19.72,P＜0.001）。与健康对照组相比,ITP患者血清中IFN-γ和TNF-α升高（52.43±11.04 pg/ml vs 23.26±8.15 pg/ml;69.14±10.89 pg/ml vs 36.14±13.17 pg/ml,t＝10.36～11.50,P值均＜0.001）,IL-10降低（11.18±6.13 pg/ml vs 33.28±9.85 pg/ml,t＝10.17,P＜0.001）,IL-4的变化差异无统计学意义（35.75±12.52 pg/ml vs 40.16±14.26 pg/ml,t＝1.25,P＞0.05）。且ITP患者TRAIL的表达与血清中IFN-γ,TNF-α正相关（r＝0.432,0.541,P 值均＜0.05）,与 IL-10,血小板计数负相关（r＝-0.424,-0.553,P 值均＜0.05）。结论TRAIL可能通过介导细胞因子免疫紊乱,参与了 ITP的发生与发展,这为进一步研究 ITP的免疫干预治疗提供了新的线索和思路。     

膀胱癌中DR4和DR5的表达水平与患者术后化疗及患者预后的关系

目的：在临床前期实验中,肿瘤坏死因子相关凋亡诱导配体（TRAIL）与化疗药物在抗癌中存在协同作用。表阿霉素广泛应用于膀胱癌的辅助化疗中。在这里,我们假设膀胱癌中死亡受体 DR4和 DR5表达水平与膀胱癌患者的术后化疗及预后相关。方法采用免疫组化法检测229例经尿道切除术后的膀胱癌患者及癌旁正常组织中的DR4和DR5的表达。结果膀胱癌患者细胞质中 DR4和 DR5的表达率分别为75.1％和74．2％。DR4与 DR5均高表达的患者术后10年随访期间的无复发生存率明显好于低表达者。多变量分析显示 DR4（P＜0．001）、DR5（P＜0．001）的表达及表阿霉素的治疗（P＝0．034）是膀胱癌患者预后的独立指标,而且表阿霉素治疗能够明显提高DR4（P＝0．006）或DR5（P＝0．042）高表达的膀胱癌患者的无复发生存率。结论 DR4和DR5均高表达的患者可能更适合接受表阿霉素治疗。     

白细胞介素12通过抑制survivin表达加强肿瘤坏死因子相关凋亡诱导配体诱导的肝癌细胞凋亡

目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)对肝细胞癌(HCC)的治疗作用,以及TRAIL耐药的可能机制和逆转耐药方案。方法 采用原位杂交方法观察HCC与正常肝组织中TRAIL的表达差异。采用不同浓度的sTRAIL(可溶性)处理HCC细胞株及真核表达质粒pIRES-EGFP-sTRAIL转染HCC细胞株,观察sTRAIL的抑癌疗效。建立裸鼠肝癌模型,观察sTRAIL的体内抑癌作用。进一步,检测HCC中survivin的表达并采用反义寡核苷酸封闭治疗。最后,观测sTRAIL和IL-12联合抗癌效果。结果 肝癌组织DR表达量显著强于正常肝组织DR表达量。60例肝癌组织中54例不表达诱捕受体DcRl,25例不表达DcR2,而20例正常肝组织均表达DcR。两种．HCC细胞株中DcR1表达缺失。经sTRAIL(100ng/ml)处理24h,HCC细胞凋亡发生率约10％,而Jurkat细胞凋亡率达70％以上。体外pIRES-EGFP-sTRAIL转染对肝癌细胞杀伤作用不敏感。体内直接瘤体注射pIRES-EGFP-sTRAIL可对裸鼠肝癌无明显抑制作用。HCC高表达survivin,反义寡核苷酸封闭可部分逆转TRAIL耐药。IL-12使survivin表达明显下调,显著加强TRAIL对HCC细胞的杀伤作用。结论 HCC对TRAIL诱导的凋亡有耐药现象。survivin参与HCC对TRAIL的耐药机制,反义寡核苷酸封闭可部分逆转TRAIL耐药。IL-12可通过抑制siurvivin表达增强TRAIL对HCC杀癌作用。联合基因治疗(如TRAIL和IL-12基因)可能成为一种有前途HCC治疗方案。     

Effects of sequential treatments with chemotherapeutic drugs followed by TRAIL on prostate cancer in vitro and in vivo

BACKGROUND: Tumor necrosis factor related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo-2L) is a novel anticancer agent, capable of inducing apoptosis preferentially in tumor and transformed cells. TRAIL-R1/death receptor (DR)4 and TRAIL-R2/DR5 are members of the tumor necrosis factor (TNF) receptor family, and can be activated by the TRAIL. We examined the clinical potential of chemotherapeutic drugs and TRAIL for the treatment of prostate cancer. METHODS: Prostate and bladder cancer cells were exposed to chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, doxorubicin, and camptothecin) and TRAIL. Cell viability was measured by sodium 3'[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) assay; expressions of death receptors and Bcl-2 family members were measured by Western blotting, ELISA and ribonuclease protection assay. PC-3 tumor cells xenografted athymic nude mice were exposed to chemotherapeutic drugs and TRAIL, either alone or in combination, to measure tumor growth and survival of mice. Apoptosis was measured by annexin V-FITC/propidium iodide staining, and terminal deoxynucleotidyltransferase-mediated nick end labeling assay. Caspase-3 activity was measured by the Western blotting and immunohistochemistry. RESULTS: TRAIL induced apoptosis with varying sensitivity. Chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, doxorubicin, and camptothecin) significantly augmented TRAIL-induced apoptosis in cancer cells through up-regulation of DR4, DR5, Bax, and Bak, and induction of caspase activation. Mitochondrial pathway enhanced the synergistic interactions between drugs and TRAIL. The sequential treatment of mice with chemotherapeutic drugs followed by TRAIL induced caspase-3 activity, and apoptosis, inhibited angiogenesis, completely eradicated the established tumors, and enhanced survival of mice. CONCLUSIONS: Chemotherapeutic drugs can be used to enhance the therapeutic potential of TRAIL in prostate cancer.