采用组织芯片技术研究c-FLIP在胃癌中的表达及其意义

目的 检测C-FLIP(细胞型fas相关死亡区域蛋白样白介素1-β转化酶抑制蛋白,FADD-like interleukin-1-β converting enzyme inhibitory protein,c-FLIP)在胃癌中的表达并提示其在胃癌发生、发展中的意义.方法 收集60例胃癌和27例正常胃黏膜标本,用组织芯片(tissue micro-array)和免疫组织化学法检测c-FLIP基因的表达.结果 c-FLIP在60例胃癌组织中有59例呈阳性表达(98.03%),在27例正常胃黏膜组织中有1例呈阳性表达(3.7%),胃癌组织中c-FILP基因表达显著高于正常胃黏膜(P<0.05).结论 c-FLIP基因高表达于胃癌组织,这为揭示胃癌的发生、发展提供了新的理论依据,并为胃癌的有效治疗提供了新思路.应用组织芯片大规模高效检测临床样本是可行的,具有快速、准确、方便、经济的特点.     

Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.     

凋亡相关蛋白死亡受体5、细胞内Fas相关死亡域样白介素1-β转换酶抑制蛋白在胃组织中的表达及临床意义

目的探讨不同胃组织中凋亡相关蛋白死亡受体(DR5)、Fas相关死亡域样白介素1-β转换酶抑制蛋白(c-FLIP)表达的临床意义。方法检测胃癌、正常胃黏膜及胃炎组织中DR5和c-FLIP蛋白的表达,以及不同分化程度的胃癌组织中的表达差异,对两者进行相关性分析。结果 DR5在胃癌、慢性胃炎、正常胃黏膜中阳性表达率分别为76.67%、53.85%和28.13%;c-FLIP蛋白阳性率分别为85%、30.77%和18.75%,各组比较差异有显著性意义(P<0.05)。DR5蛋白在高、中、低分化腺癌中的表达率分别为38.1%、55%、78.95%,c-FLIP分别为47.62%、70%、94.74%,各组比较差异有显著性意义(P<0.05)。结论 DR5与c-FLIP蛋白在胃癌中均高表达,两者均参与了胃癌的发生。     

Targeting cellular FLICE-like inhibitory protein as a novel approach to the treatment of Hodgkin’s lymphoma

Hodgkin’s lymphoma is one of the most common lymphoid cancers, particularly among young adults. Although there have been dramatic improvements in the treatment of Hodgkin’s lymphoma, leading to high cure rates in some groups, current combination chemotherapy regimes are associated with significant secondary complications in long-term survivors. Furthermore, although a proportion of patients with Hodgkin’s lymphoma will be cured, there still remains a significant rate of relapse and also a smaller proportion of poor responders who will go on to die of their disease. Therefore, developments in the treatment of Hodgkin’s lymphoma must be directed at improving cure rates and reducing the burden of secondary complications. In recent years, the underlying pathogenesis of Hodgkin’s lymphoma has become better understood. In particular, it is emerging that a key pathogenic event in Hodgkin’s lymphoma is protection from Fas-induced cell death. Recent studies by the authors’ group, and others, have demonstrated that this is, in part, due to the expression by Hodgkin/Reed–Sternberg cells of the cellular Fas-associated death domain-like IL-1 converting enzyme (FLICE)-like inhibitory protein molecule, a potent inhibitor of Fas-induced death. In this review, the role of cellular FLICE-like inhibitory protein in the pathogenesis of Hodgkin’s lymphoma will be explored and also the possibility of targeting this molecule in order to provide an alternative and potentially safe approach to the treatment of Hodgkin’s lymphoma will be investigated.     

Pentoxifylline augments TRAIL/Apo2L mediated apoptosis in cutaneous T cell lymphoma (HuT-78 and MyLa) by modulating the expression of antiapoptotic proteins and death receptors

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising anticancer agent but cutaneous T lymphoma cells (CTCL) are less sensitive to TRAIL-induced apoptosis. Here, we report that pentoxifylline (PTX), a phosphodiesterase inhibitor, augments TRAIL-mediated apoptosis in HuT-78 and MyLa cells through modulating extrinsic death receptors and intrinsic mitochondria dependent pathways. Our results clearly show that PTX augments TRAIL-mediated activation of caspase-8 and induces cleavage of Bid, although PTX alone cannot activate caspase-8. This is followed by cytochrome c release and subsequent, activation of caspase-9 and caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). Combined treatment downregulates the expression of various antiapoptotic proteins including c-FLIP, Bcl-xl, cIAP-1, cIAP-2 and XIAP. PTX induces the expression of death receptors DR4 and DR5 on cell surface of both the cell types where c-Jun NH2-terminal kinase (JNK) pathway plays an important role. Moreover, combined silencing of DR4 and DR5 by small interfering RNA abrogates the ability of PTX to induce TRAIL-mediated apoptosis. Thus, this is the first demonstration that PTX can potentiate TRAIL-mediated apoptosis through downregulation of cell survival gene products and upregulation of death receptors.     

c-FLIP与细胞凋亡及肿瘤靶向治疗

细胞型FLIP(c-FLIP)是近年来发现的细胞凋亡过程中的抑制蛋白.凋亡过程是通过结合死亡受体(DR)而触发的,例如Fas或TRAIL.通过征募结合DR复合体阻止caspase的活化,从而抑制细胞凋亡.已明确c-FLIP的过表达与多种肿瘤的发生、发展有密切关系.     

c-FLIP反义寡核苷酸诱导胚胎型横纹肌肉瘤细胞凋亡的研究

目的探讨c-FLIP反义寡核苷酸(c-FLIPantisenseoligodeoxynucleotide,c-FLIP-ASODN)对c-FLIP的表达调控及诱导人胚胎型横纹肌肉瘤(rhabdomyosarcoma,RD)细胞凋亡的作用。方法以脂质体为载体,将c-FLIP-ASODN及对照序列分别转染RD细胞,分别通过WesternBlotting、RT-PCR方法检测ASODN对c-FLIP蛋白和mRNA表达量的调控,用流式细胞术测定ASODN干预前后RD细胞凋亡率的变化。结果与对照序列相比,Western印迹显示,转染了c-FLIP-ASODN的RD细胞其FLIPS和FLIPL的表达量分别下降为空白对照组的51%和57%;RT-PCR检测表明c-FLIP-ASODN能抑制c-FLIPmRNA的表达;诱导凋亡48h后,转染c-FLIP-ASODN的RD细胞凋亡率升高(29.3±6·3)%.结论c-FLIP-ASODN可有效转染RD细胞,下调c-FLIPmRNA表达,从而诱导RD细胞的凋亡,成为眼眶RD基因治疗的新靶向。     

Inhibition of c-FLIP by RNAi Enhances Sensitivity of the Human Osteogenic Sarcoma Cell Line U2OS to TRAILInduced Apoptosis

To study effects of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP)inhibition by RNA interference (RNAi) on sensitivity of U2OS cells to tumor necrosis factor (TNF)-relatedapoptosis-inducing ligand (TRAIL)-induced apoptosis, plasmid pSUPER-c-FLIP-siRNA was constructed andthen transfected into U2OS cells. A stable transfection cell clone U2OS/pSUPER-c-FLIP-siRNA was screenedfrom the c-FLIP-siRNA transfected cells. RT-PCR and Western blotting were applied to measure the expressionof c-FLIP at the levels of mRNA and protein. The results indicated that the expression of c-FLIP was significantlysuppressed by the c-FLIP-siRNA in the cloned U2OS/pSUPER-c-FLIP-siRNA as compared with the controlcells of U2OS/pSUPER. The cloned cell line of U2OS/pSUPER-c-FLIP-siRNA was further examined for TRAILinducedcell death and apoptosis in the presence of a pan-antagonist of inhibitor of apoptosis proteins (IAPs)AT406, with or without 4 hrs pretreatment with rocaglamide, an inhibitor of c-FLIP biosynthesis, for 24 hrs.Cell death effects and apoptosis were measured by the methods of MTT assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry, respectively. The results indicated that TRAIL-inducedcell death in U2OS/pSUPER-c-FLIP-siRNA was increased compared with control cells U2OS/pSUPER in thepresence or absence of AT406. Flow cytometry indicated that TRAIL-induced cell death effects proceeded throughcell apoptosis pathway. However, in the presence of rocaglamide, cell death or apoptotic effects of TRAIL weresimilar and profound in both cell lines, suggesting that the mechanism of action for both c-FLIP-siRNA androcaglamide was identical. We conclude that the inhibition of c-FLIP by either c-FLIP-siRNA or rocaglamidecan enhance the sensitivity of U2OS to TRAIL-induced apopotosis, suggesting that inhibition of c-FLIP is a goodtarget for anti-cancer therapy.