曲古抑菌素A增加人肝癌Bel7402细胞对TRAIL敏感度的实验

目的 探讨曲古抑菌素A(Trichostatin A,TSA)联合肿瘤坏死因子(tumor necrosis factor,TNF)相关凋亡诱导配体(TNF related apoptosis inducing ligand,TRAIL)对肝癌细胞Bel7402增殖凋亡的影响及其机制.方法 采用四甲基偶氮唑盐(MTT)染色法分别检测TSA、TRAIL及低浓度TSA联合TRAIL处理Bel7402细胞的生长抑制率;4,6-二脒基-2-苯基吲哚二盐酸盐(DAPI)染色法对药物联合处理后的细胞进行凋亡形态学观察;免疫细胞化学和Western blot技术观察药物联合作用后p65蛋白在细胞中表达和定位的变化.结果 不同浓度TSA作用6、12和24 h对人肝癌Bel7402细胞的增殖没有明显抑制作用,而作用48 h后的细胞增殖抑制率明显升高,和对照组比较差异有统计学意义(P＜0.05);不同浓度TRAIL处理Bel7402细胞存活率没有明显改变;低浓度TSA(20 ng/ml)预处理能够增加Bel7402细胞对TRAIL治疗的敏感度,TSA预处理联合TRAIL(100ng/ml)作用细胞24 h后,细胞生存率为(57.1±5.4)％,和单独药物处理组及对照组比较差异有统计学意义(P＜0.05);DAPI染色显示TSA和TRAIL联合作用后Bel7402细胞有核凋亡出现.荧光显微镜观察证明单独应用TSA(200 ng/ml)或TRAIL(100 ng/ml)处理的细胞p65蛋白有部分核内移位,而两种药物联合应用导致p65蛋白表达量降低,并且发生明显的核内转移和集聚.结论 低浓度TSA能够增加肝癌Bel7402细胞对TRAIL的敏感度;其机制可能是两种药物联合应用降低p65的表达和活性,从而诱导Bel7402细胞凋亡.     

Four-arm PEG cross-linked hyaluronic acid hydrogels containing PEGylated apoptotic TRAIL protein for treating pancreatic cancer

Four-arm polyethylene glycol (PEG) cross-linked hyaluronic acid (HA) hydrogels containing PEGylated tumor necrosis factor-related apoptosis-inducing ligand (PEG-TRAIL) were fabricated, and their antitumor effects were evaluated in pancreatic cell (Mia Paca-2)-xenografted mice. HA was conjugated with 4-arm PEG_10k-amine (a cross-linker) at ratios of 100:1 and 100:2 using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride as a cross-linker, and TRAIL or PEG-TRAIL was incorporated into these HA hydrogels. HA hydrogels at a 100:1 ratio were prepared in good yields (>88%), were moderately stiff, and gradually released PEG-TRAIL over ∼14 days in vitro and over ∼7 days in vivo (as determined by high-pressure liquid chromatography and infrared imaging). The released PEG-TRAIL was found to have obvious apoptotic activity in Mia Paca-2 cells. PEG-TRAIL HA hydrogels displayed remarkably more antitumor efficacy than TRAIL HA hydrogels in Mia Paca-2 cell-xenografted mice in terms of tumor volumes (size) and weights (453.2 mm³ and 1.03 g vs. 867.5 mm³ and 1.86 g). Furthermore, this improved antitumor efficacy was found to be due to the apoptotic activity of PEG-TRAIL in vivo (determined by a TUNEL assay) despite its substantially lower cytotoxicity than native TRAIL (IC₅₀ values: 71.8 and 202.5 ng ml⁻¹, respectively). This overall enhanced antitumor effect of PEG-TRAIL HA hydrogels appeared to be due to the increased stability of PEGylated TRAIL in HA hydrogels. These findings indicate that this HA hydrogel system combined with PEG-TRAIL should be considered a potential candidate for the treatment of pancreatic cancer.     

Alveolar epithelial cells in idiopathic pulmonary fibrosis display upregulation of TRAIL,DR4 and DR5 expression with simultaneous preferential over-expression of pro-apoptotic marker p53

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating, and fatal lung disease of unknown aetiology with no current cure. The pathogenesis of IPF remains unclear but repeated alveolar epithelial cell (AEC) injuries and subsequent apoptosis are believed to be among the initiating/ongoing triggers. However, the precise mechanism of apoptotic induction is hitherto elusive. In this study, we investigated expression of a panel of pro-apoptotic and cell cycle regulatory proteins in 21 IPF and 19 control lung tissue samples. We reveal significant upregulation of the apoptosis-inducing ligand TRAIL and its cognate receptors DR4 and DR5 in AEC within active lesions of IPF lungs. This upregulation was accompanied by pro-apoptotic protein p53 overexpression. In contrast, myofibroblasts within the fibroblastic foci of IPF lungs exhibited high TRAIL, DR4 and DR5 expression but negligible p53 expression. Similarly, p53 expression was absent or negligible in IPF and control alveolar macrophages and lymphocytes. No significant differences in TRAIL expression were noted in these cell types between IPF and control lungs. However, DR4 and DR5 upregulation was detected in IPF alveolar macrophages and lymphocytes. The marker of cellular senescence p21^WAF1 was upregulated within affected AEC in IPF lungs. Cell cycle regulatory proteins Cyclin D1 and SOCS3 were significantly enhanced in AEC within the remodelled fibrotic areas of IPF lungs but expression was negligible in myofibroblasts. Taken together these findings suggest that, within the remodelled fibrotic areas of IPF, AEC can display markers associated with proliferation, senescence, and apoptotosis, where TRAIL could drive the apoptotic response. Clear understanding of disease processes and identification of therapeutic targets will direct us to develop effective therapies for IPF.     

Quercetin-mediated Mcl-1 and survivin downregulation restores TRAIL-induced apoptosis in non-Hodgkin's lymphoma B cells

Background

Non-Hodgkin''s B-cell lymphomas account for approximately 70% of B-cell lymphomas. While its incidence is dramatically increasing worldwide, the disease is still associated with high morbidity due to ineffectiveness of conventional therapies, creating an urgent need for novel therapeutic approaches. Unconventional compounds, including polyphenols and the cytokine TRAIL, are being extensively studied for their capacity to restore apoptosis in a large number of tumors, including lymphomas.

Design and Methods

Molecular mechanisms of TRAIL-resistance and reactivation of the apoptotic machinery by quercetin in non-Hodgkin’s lymphoma cell lines were determined by Hoescht, flow cytometry, Western blot, qPCR, by use of siRNA or pharmacological inhibitors of the mitochondrial pathway and by immunoprecipitation followed by post-translational modification analysis.

Results

Results demonstrate that quercetin, a natural flavonoid, restores TRAIL-induced cell death in resistant transformed follicular lymphoma B-cell lines, despite high Bcl-2 expression levels due to the chromosomal translocation t(14;18). Quercetin rescues mitochondrial activation by inducing the proteasomal degradation of Mcl-1 and by inhibiting survivin expression at the mRNA level, irrespective of p53. Restoration of the TRAIL pathway requires Bax and Bak but is independent of enhanced TRAIL DISC formation.

Conclusions

We demonstrate that inactivation of survivin and Mcl-1 expression by quercetin is sufficient to restore TRAIL sensitivity in resistant non–Hodgkin’s lymphoma B cells. Our results suggest, therefore, that combining quercetin with TRAIL treatments may be useful in the treatment of non–Hodgkin’s lymphoma.     

人TRAIL胞外区原核可溶性表达及活性鉴定

目的获得具有生物活性的肿瘤坏死因子相关凋亡诱导配体(TRAIL)胞外段蛋白。方法根据大肠杆菌密码子偏爱性要求,设计合成编码TRAIL胞外段的DNA序列,构建成pET30a-TRAIL胞内融合表达质粒,重组质粒转化表达宿主E.coli BL21。在不同温度、不同浓度的IPTG条件下诱导表达TRAIL,SDS-PAGE分析表达产物,MTT法检测产物活性。结果构建的工程菌株表达29KD的可溶性TRAIL融合蛋白,能诱导Jurkat细胞凋亡。结论成功表达了人TRAIL胞外段蛋白,为进一步研究提供基础。     

p53-dependent and p53-independent anticancer effects of different histone deacetylase inhibitors

Background:

Histone deacetylase inhibitors (HDACi) are promising antineoplastic agents, but their precise mechanisms of actions are not well understood. In particular, the relevance of p53 for HDACi-induced effects has not been fully elucidated. We investigated the anticancer effects of four structurally distinct HDACi, vorinostat, entinostat, apicidin and valproic acid, using isogenic HCT-116 colon cancer cell lines differing in p53 status.

Methods:

Effects were assessed by MTT assay, flow-cytometric analyses of propidium iodide uptake, mitochondrial depolarisation and cell-cycle distribution, as well as by gene expression profiling.

Results:

Vorinostat was equally effective in p53 wild-type and null cells, whereas entinostat was less effective in p53 null cells. Histone deacetylase inhibitors treatment suppressed the expression of MDM2 and increased the abundance of p53. Combination treatments showed that vorinostat enhanced the cytotoxic activity of TRAIL and bortezomib, independent of the cellular p53 status. Investigations into the effects of an inhibitor of the sirtuin class of HDAC, tenovin-1, revealed that tenovin-1-mediated cell death hinged on p53.

Conclusion:

These results demonstrate that vorinostat activates p53, but does not require p53 for inducing its anticancer action. Yet they also demonstrate that entinostat-induced cytotoxic effects partially depend on p53, indicating that different HDACi have a different requirement for p53.     

Cell death pathways – potential therapeutic targets

Programmed cell death and its morphological manifestation termed apoptosis is a conserved pathway that appears to operate in all multicellular organisms. During embryonic development, cell death is essential for successful organogenesis, and apoptosis also operates in adult organisms to maintain normal cellular homeostasis. The removal of disordered cells by a controlled cellular mechanism is especially critical in long-lived mammals that must integrate multiple physiological as well as pathological death signals. Gain- and loss-of-function models of genes in the core apoptotic pathway suggest that perturbation of cellular homeostasis can be a primary pathogenic event that results in disease. Indeed, there is now compelling evidence that insufficient apoptosis can manifest as cancer or autoimmunity, whereas accelerated cell death is evident in acute and chronic degenerative diseases, further highlighting the fact that deregulation of cell death pathways has major health implications. Not surprisingly, during the past 25 years a huge endeavour aimed at unravelling this fundamental biological process has led to major advances in our understanding of cell death pathways. Therapeutic strategies to manipulate apoptosis have immense potential and this review highlights several potentially viable drug targets for modulating cell death that have been discovered from the elegant work of many scientists in elucidating the protein components and key regulators of apoptosis signalling pathways.     

siRNA腺病毒载体沉默MEKK3基因促进TRAIL诱导肝癌HCC-9204细胞凋亡

目的研究抑制丝裂原活化蛋白激酶/细胞外信号调节激酶-激酶3（MEKK3）基因表达促进TRAIL诱导人肝癌HCC-9204细胞凋亡的作用。方法构建人MEKK3基因的siRNA重组腺病毒载体,转导人肝癌HCC-9204细胞,以RTPCR和印迹法检测MEKK3 mRNA和蛋白的表达,筛选并建立稳定沉默MEKK3基因表达的人肝癌HCC-9204细胞株,以流式细胞仪检测TRAIL诱导细胞凋亡的情况。结果①成功构建并鉴定表达人MEKK3基因的siRNA重组腺病毒载体。②建立MEKK3基因有效且稳定沉默的人肝癌HCC-9204细胞株。③500 g/LTRAIL处理MEKK3基因沉默的HCC-9204细胞株,细胞凋亡率较野生HCC-9204细胞株显著提高。结论本研究成功建立有效且稳定沉默MEKK3基因表达的人肝癌HCC-9204细胞株模型,初步证实MEKK3基因表达与TRAIL诱导肝癌细胞凋亡的敏感性相关。     

Synergistic induction of TRAIL-mediated apoptosis by anisomycin in human hepatoma cells via the BH3-only protein Bid and c-Jun/AP-1 signaling pathway

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF super-family, and it has been shown that many human cancer cell lines are refractory to TRAIL-induced cell death. However, the molecular mechanisms underlying resistance are unclear. In the present study, we show that TRAIL-resistance is reversed in human hepatoma cells by anisomycin, which is known to inhibit protein synthesis and induce ribotoxic stress. Synergistic induction of apoptosis in cells treated with anisomycin plus TRAIL was associated with activation of caspases and cleavage of Bid, a pro-apoptotic BH3-only protein. Silencing of Bid expression by small interfering RNA (siRNA) significantly attenuated the loss of mitochondrial membrane potential (MMP, Δψ_m) and significantly increased induction of apoptosis in cells treated with anisomycin and TRAIL, confirming that Bid cleavage is required for the response. In addition, c-Jun/AP-1 was rapidly activated upon stimulation with anisomycin; however, the knockdown of c-Jun/AP-1 expression by c-Jun siRNA markedly reduced anisomycin plus TRAIL-induced loss of MMP and apoptosis. Taken together, the findings show that anisomycin sensitizes TRAIL-mediated hepatoma cell apoptosis via the mitochondria-associated pathway, involving the cleavage of Bid and activation of the c-Jun/AP-1 pathway, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.