Structural determinants of DISC function: New insights into death receptor-mediated apoptosis signalling

Death receptors are members of the tumour necrosis factor (TNF) receptor superfamily characterised by an ~ 80 amino acid long alpha-helical fold, termed the death domain (DD). Death receptors diversified during early vertebrate evolution indicating that the DD fold has plasticity and specificity that can be easily adjusted to attain additional functions. Eight members of the death receptor family have been identified in humans, which can be divided into four structurally homologous groups or clades, namely: the p75^NTR clade (consisting of ectodysplasin A receptor, death receptor 6 (DR6) and p75 neurotrophin (NTR) receptor); the tumour necrosis factor receptor 1 clade (TNFR1 and DR3), the CD95 clade (CD95/FAS) and the TNF-related apoptosis-inducing ligand receptor (TRAILR) clade (TRAILR1 and TRAILR2). Receptors in the same clade participate in similar processes indicating that structural diversification enabled functional specialisation. On the surface of nearly all human cells multiple death receptors are expressed, enabling the cell to respond to a plethora of external signals. Activation of different death receptors converges on the activation of three main signal transduction pathways: nuclear factor-κB-mediated differentiation or inflammation, mitogen-associated protein kinase-mediated stress response and caspase-mediated apoptosis. While the ability to induce cell death is true for nearly all DRs, the FAS and TRAILR clades have specialised in inducing cell death. Here we summarise recent discoveries about the molecular regulation and structural requirements of apoptosis induction by death receptors and discuss how this information can be used to better explain the biological functions, similarities and distinguishing features of death receptors.     

表没食子儿茶素没食子酸酯联合TRAIL对黑素瘤A375细胞的凋亡作用

为探讨肿瘤坏死因子相关诱导凋亡配体(TRAIL)与表没食子儿茶素没食子酸酯(EGCG)联合应用对人恶性黑色素瘤A375细胞的抑制作用,采用以甲基噻唑基四唑(MTT)比色法检测TRAIL和EGCG作用组的抑制率,并以流式细胞仪检测各组凋亡率,流式检测EGCG单独作用后A375细胞表面死亡受体DR4、DR5的变化.亚毒剂量的TRAIL、EGCG及联合作用组致恶性黑素瘤细胞A375细胞凋亡率分别为12%、5.1%、60%.Caspase-3活性明显增强.单独应用组与联合应用组之间存在显著性差异(P<0.01).EGCG单独作用于A375细胞24 h后,死亡受体TRAIL-R1/DR4在细胞的表达明显(P<0.05).TRAIL-R2/DR5表达无明显变化.TRAIL和EGCG联合应用对A375细胞具有明显的协同杀伤效果,这一作用可能与EGCG上调DR4的表达及增强Caspase-3活性有关.     

Synthesis,characterization, and in vitro evaluation of TRAIL-modified,cabazitaxel -loaded polymeric micelles for achieving synergistic anticancer therapy

Abstract

Combination therapy of two or more drugs has gradually become of outmost importance in cancer treatment. Cabazitaxel (CTX) is a taxoid drug and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of TNF superfamily. In this study, we prepared TRAIL-modified and CTX-loaded polymer micelle (TRAIL-M-CTX). This nanoparticle was self-assembled from biodegradable amphiphilic copolymers, monomethoxyl poly(ethylene glycol)–b-poly(DL-lactide) (mPEG-PLA) and COOH-PEG-PLA, via a nanoprecipitation method and were modified with the TRAIL protein, resulting in a particle size of 39.75 ± 0.17 nm in diameter and a drug encapsulation efficiency of 95.52 ± 1.69%. The successful coupling was confirmed by ¹H NMR, FTIR spectroscopy, and DLS article size measurement. Pharmacodynamic analysis in two human cancer cell lines with different TRAIL sensitivities showed that TRAIL-M-CTX has a significantly better anticancer efficacy than the individual CTX and TRAIL protein. Importantly, TRAIL-M-CTX showed synergistic effects against TRAIL-insensitive cells (MCF-7). A study of cellular uptake implied that the modified micelles were internalized into MCF-7 cells more effectively than unmodified micelles, owing to the coupled TRAIL protein. A cell cycle assay of MCF-7 cells revealed that TRAIL-M-CTX significantly increased the sub-G1 population compared with CTX or TRIAL, thus, facilitating cancer cell apoptosis. These results suggest that TRAIL-M-CTX micelles have potential as a cancer chemotherapy formulation.     

Heat Shock Protein translocation induced by membrane fluidization increases tumor-cell sensitivity to chemotherapeutic drugs

Treatment of chronic lymphocytic leukemia (CLL) remains a challenge due to the frequency of drug resistance amongst patients. Improving the delivery of chemotherapeutic agents while reducing the expression of anti-apoptotic Heat Shock Proteins (HSPs) within the cancer cells may facilitate in overcoming this drug resistance. We demonstrate for the first time that sub-lethal doses of chemotherapeutic agents can be combined with membrane fluidizing treatments to produce a significant increase in drug efficacy and apoptosis in vitro. We show that fluidizers result in a transient decrease in intracellular HSPs, resulting in increased tumor-cell sensitivity and a membrane-associated induction of HSP gene expression.     

Melatonin sensitizes human malignant glioma cells against TRAIL-induced cell death

Despite the common expression of death receptors, many types of cancer including gliomas are resistant to the death receptor ligand (TRAIL). Melatonin antitumoral actions have been extensively described, including oncostatic properties on several tumor types and improvement of chemotherapeutic regimens. Here, we found that melatonin effectively increase cell sensitivity to TRAIL-induced cell apoptosis in A172 and U87 human glioma cells. The effect seems to be related to a modulation of PKC activity which in turns decreases Akt activation leading to an increase in death receptor 5 (DR5) levels and a decrease in the antiapoptotic proteins survivin and bcl-2 levels.     

Micellar formulations of pro-apoptotic DM-PIT-1 analogs and TRAIL in vitro and in vivo

Abstract

We have developed and characterized micellar formulations of analogs to the recently developed inhibitor of the phosphatidylinositol-3-kinase (PI3K) pathway (N-[(2-hydroxy-5-nitrophenyl)amino]carbonothioyl-3,5-dimethylbenzamide (DM-PIT-1)) for their physicochemical, loading and cytotoxic properties. The first generation inhibitor DM-PIT-1 is a non-lipid, small molecule inhibitor of phosphatidylinositol-3,4,5-triphosphate/Pleckstrin homology (PIP₃/PH) binding capable of inhibiting the growth of tumor cells both in vitro and in vivo. A second generation of improved and druggable analogs has been developed. All compounds were successfully loaded (>70%) in PEG₂₀₀₀-PE micelles of 16–20?nm in size with several analogs demonstrating favorable cytotoxic activity against A2780 ovarian carcinoma. These compounds were also successfully incorporated into polyethylene glycol-phosphatidylethanolamine (PEG-PE) micelles combined with surface-bound tumor necrosis factor related apoptosis inducing ligand (TRAIL). The resulting multifunctional combination micelles were able to significantly enhance cytotoxic activity in the TRAIL-resistant A2780 cell line. Additionally, analogs NCL-176 and NCL-240 were effective in inhibiting tumor growth in an in vivo subcutaneous tumor model of A2780. These results indicate the utility of delivering TRAIL and PI3K pathway inhibitors in a combined micellar preparation.     

Sarcomas as a mise en abyme of mesenchymal stem cells: Exploiting interrelationships for cell mediated anticancer therapy

Mise en abyme meaning “placed into abyss or infinite recurrence” is an apt paradigm for the relentless growth of sarcoma cells. Its alternative meaning, “self-reflexive embedding” fits the central role attributed to cancer stem cells (CSCs). Diversely sourced and defined, mesenchymal stem cells (MSCs) may be the cells of sarcoma origin, evolve a CSC phenotype and/or contribute to tumor growth through inherent qualities for homing, neovascularization, paracrine cross-feeding, microvesicle secretion, cell fusion, entosis and immune modulation. Exploiting these qualities, MSC expressing modified forms of the TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) are being developed to complement more conventional radiation and chemotherapy.     

AMPK-independent down-regulation of cFLIP and sensitization to TRAIL-induced apoptosis by AMPK activators

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a TNF superfamily member that is being considered as a new strategy in anticancer therapy because of its ability to induce apoptosis, alone or in combination with other stimuli, in many cancer cells. AMP-activated protein kinase (AMPK) is an evolutionarily conserved key regulator of cellular energy homeostasis that protects the cell from energy depletion and stress by activating several biochemical pathways that lead to the conservation, as well as generation, of ATP. Here we report that a number of AMPK activators, including the small molecule activator A-769662, markedly sensitize TRAIL-resistant breast cancer cells to TRAIL-induced apoptosis. However, silencing AMPKα1 expression with siRNA or over-expression of DN-AMPKα1 does not inhibit AICAR, glucose deprivation, phenformin or A-769662-induced sensitization to TRAIL. Furthermore, the expression of constitutively active AMPK subunits does not sensitize resistant breast cancer cells to TRAIL-induced apoptosis. The cellular FLICE-inhibitory proteins (cFLIP_L and cFLIP_S) were significantly down-regulated following exposure to AMPK activators through an AMPK-independent mechanism. Furthermore, in cells over-expressing cFLIP_L, sensitization to TRAIL by AMPK activators was markedly reduced. In summary, our results indicate that AMPK activators facilitate the activation by TRAIL of an apoptotic cell death program through a mechanism independent of AMPK and dependent on the down-regulation of cFLIP levels.