Differential modulation of the TRAIL receptors and the CD95 receptor in colon carcinoma cell lines

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 ligand (CD95L) are potent inducers of apoptosis in various tumour cell types. Death receptors DR4 and DR5 can induce and decoy receptors DcR1 and DcR2 can inhibit TRAIL-mediated apoptosis. The study aim was to investigate whether anticancer agents can modulate similarly TRAIL-receptor and CD95 membrane expression and TRAIL and CD95L sensitivity.Three colon carcinoma cell lines (Caco-2, Colo320 and SW948) were treated with 5-fluorouracil (5-FU), cisplatin or interferon-γ. TRAIL-receptor and CD95 membrane expression was determined flow cytometrically. Sensitivity to TRAIL or CD95L agonistic anti-CD95 antibody was determined with cytotoxicity and apoptosis assays. SW948 showed highest TRAIL sensitivity. The protein synthesis inhibitor cycloheximide decreased FLICE-like inhibitory protein levels in all cell lines, and the TRAIL-resistant cell lines Caco-2 and Colo320 became sensitive for TRAIL. Exposure of the cell lines to 5-FU, cisplatin and interferon-γ left TRAIL-receptor membrane expression and TRAIL sensitivity unaffected. CD95 membrane expression and anti-CD95 sensitivity was, however, modulated by the same drugs in all lines. Cisplatin and interferon-γ raised CD95 membrane levels 6–8-fold, interferon-γ also increased anti-CD95 sensitivity. These results indicate that the CD95 and TRAIL pathways use different mechanisms to respond to various anticancer agents. Induced CD95 membrane upregulation was associated with increased anti-CD95 sensitivity, whereas no upregulation of TRAIL-receptor membrane expression or TRAIL sensitisation could be established. For optimal use of TRAIL-mediated apoptosis for cancer therapy in certain tumours, downregulation of intracellular inhibiting factors may be required

β-Elemene Piperazine Derivatives Induce Apoptosis in Human Leukemia Cells through Downregulation of c-FLIP and Generation of ROS

β-Elemene is an active component of the herb medicine Curcuma Wenyujin with reported antitumor activity. To improve its antitumor ability, five novel piperazine derivatives of β-elemene, 13-(3-methyl-1-piperazinyl)-β-elemene (DX1), 13-(cis-3,5-dimethyl-1-piperazinyl)-β-elemene (DX2), 13-(4-ethyl-1-piperazinyl)-β-elemene (DX3), 13-(4-isopropyl-1-piperazinyl)-β-elemene (DX4) and 13-piperazinyl-β-elemene (DX5), were synthesized. The antiproliferative and apoptotic effects of these derivatives were determined in human leukemia HL-60, NB4, K562 and HP100-1 cells. DX1, DX2 and DX5, which contain a secondary amino moiety, were more active in inhibiting cell growth and in inducing apoptosis than DX3 and DX4. The apoptosis induction ability of DX1 was associated with the generation of hydrogen peroxide (H2O2), a decrease of mitochondrial membrane potential (MMP), and the activation of caspase-8. Pretreatment with the antioxidants N-acetylcysteine and catalase completely blocked DX1-induced H2O2 production, but only partially its activation of caspase-8 and induction of apoptosis. HL-60 cells were more sensitive than its H2O2-resistant subclone HP100-1 cells to DX1-induced apoptosis. The activation of caspase-8 by these compounds was correlated with the decrease in the levels of cellular FLICE-inhibitory protein (c-FLIP). The proteasome inhibitor MG-132 augmented the decrease in c-FLIP levels and apoptosis induced by these derivatives. FADD- and caspase-8-deficient Jurkat subclones have a decreased response to DX1-induced apoptosis. Our data indicate that these novel β-elemene piperazine derivatives induce apoptosis through the decrease in c-FLIP levels and the production of H2O2 which leads to activation of both death receptor- and mitochondrial-mediated apoptotic pathways

Histone deacetylase inhibitors strongly sensitise neuroblastoma cells to TRAIL-induced apoptosis by a caspases-dependent increase of the pro- to anti-apoptotic proteins ratio

BACKGROUND: Neuroblastoma (NB) is the second most common solid childhood tumour, an aggressive disease for which new therapeutic strategies are strongly needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumour cells, but not in normal tissues and therefore represents a valuable candidate in apoptosis-inducing therapies. Caspase-8 is silenced in a subset of highly malignant NB cells, which results in full TRAIL resistance. In addition, despite constitutive caspase-8 expression, or its possible restoration by different strategies, NB cells remain weakly sensitive to TRAIL indicating a need to develop strategies to sensitise NB cells to TRAIL. Histone deacetylase inhibitors (HDACIs) are a new class of anti-cancer agent inducing apoptosis or cell cycle arrest in tumour cells with very low toxicity toward normal cells. Although HDACIs were recently shown to increase death induced by TRAIL in weakly TRAIL-sensitive tumour cells, the precise involved sensitisation mechanisms have not been fully identified. METHODS: NB cell lines were treated with various doses of HDACIs and TRAIL, then cytotoxicity was analysed by MTS/PMS proliferation assays, apoptosis was measured by the Propidium staining method, caspases activity by colorimetric protease assays, and (in)activation of apoptotic proteins by immunoblotting. RESULTS: Sub-toxic doses of HDACIs strongly sensitised caspase-8 positive NB cell lines to TRAIL induced apoptosis in a caspases dependent manner. Combined treatments increased the activation of caspases and Bid, and the inactivation of the anti-apoptotic proteins XIAP, Bcl-x, RIP, and survivin, thereby increasing the pro- to anti-apoptotic protein ratio. It also enhanced the activation of the mitochondrial pathway. Interestingly, the kinetics of caspases activation and inactivation of anti-apoptotic proteins is accelerated by combined treatment with TRAIL and HDACIs compared to TRAIL alone. In contrast, cell surface expression of TRAIL-receptors or TRAIL is not affected by sub-toxic doses of HDACIs. CONCLUSION: HDACIs were shown to activate the mitochondrial pathway and to sensitise NB cells to TRAIL by enhancing the amplitude of the apoptotic cascade and by restoring an apoptosis-prone ratio of pro- to anti-apoptotic proteins. Combining HDACIs and TRAIL could therefore represent a weakly toxic and promising strategy to target TRAIL-resistant tumours such as neuroblastomas

Decitabine immunosensitizes human gliomas to NY-ESO-1 specific T lymphocyte targeting through the Fas/Fas Ligand pathway

<p>Abstract</p> <p>Background</p> <p>The lack of effective treatments for gliomas makes them a significant health problem and highlights the need for the development of novel and innovative treatment approaches. Immunotherapy is an appealing strategy because of the potential ability for immune cells to traffic to and destroy infiltrating tumor cells. However, the absence of well-characterized, highly immunogenic tumor-rejection antigens (TRA) in gliomas has limited the implementation of targeted immune-based therapies.</p> <p>Methods</p> <p>We hypothesized that treatment with the demethylating agent, decitabine, would upregulate the expression of TRA on tumor cells, thereby facilitating enhanced surveillance by TRA-specific T cells.</p> <p>Results and Discussion</p> <p>Treatment of human glioma cells with decitabine increased the expression of NY-ESO-1 and other well characterized cancer testes antigens. The upregulation of NY-ESO-1 made these tumors susceptible to NY-ESO-1-specific T-cell recognition and lysis. Interestingly, decitabine treatment of T98 glioma cells also sensitized them to Fas-dependent apoptosis with an agonistic antibody, while a Fas blocking antibody could largely prevent the enhanced functional recognition by NY-ESO-1 specific T cells. Thus, decitabine treatment transformed a non-immunogenic glioma cell into an immunogenic target that was efficiently recognized by NY-ESO-1--specific T cells.</p> <p>Conclusions</p> <p>Such data supports the hypothesis that agents which alter epigenetic cellular processes may "immunosensitize" tumor cells to tumor-specific T cell-mediated lysis.</p

TLR2 Signaling Contributes to Rapid Inflammasome Activation during F. novicida Infection

Early detection of microorganisms by the innate immune system is provided by surface-expressed and endosomal pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Detection of microbial components by TLRs initiates a signaling cascade leading to the expression of proinflammatory cytokines including IL-6 and IL-1β. Some intracellular bacteria subvert the TLR response by rapidly escaping the phagosome and entering the cytosol. However, these bacteria may be recognized by the inflammasome, a multi-protein complex comprised of a sensor protein, ASC and the cysteine protease caspase-1. Inflammasome activation leads to release of the proinflammatory cytokines IL-1β and IL-18 and death of the infected cell, an important host defense that eliminates the pathogen's replicative niche. While TLRs and inflammasomes are critical for controlling bacterial infections, it is unknown whether these distinct host pathways cooperate to activate defenses against intracellular bacteria.Using the intracellular bacterium Francisella novicida as a model, we show that TLR2(-/-) macrophages exhibited delayed inflammasome activation compared to wild-type macrophages as measured by inflammasome assembly, caspase-1 activation, cell death and IL-18 release. TLR2 also contributed to inflammasome activation in response to infection by the cytosolic bacterium Listeria monocytogenes. Components of the TLR2 signaling pathway, MyD88 and NF-κB, were required for rapid inflammasome activation. Furthermore, TLR2(-/-) mice exhibited lower levels of cell death, caspase-1 activation, and IL-18 production than wild-type mice upon F. novicida infection.These results show that TLR2 is required for rapid inflammasome activation in response to infection by cytosolic bacterial pathogens. In addition to further characterizing the role of TLR2 in host defense, these findings broaden our understanding of how the host integrates signals from spatiotemporally separated PRRs to coordinate an innate response against intracellular bacteria

XIAP Regulates Cytosol-Specific Innate Immunity to Listeria Infection

The inhibitor of apoptosis protein (IAP) family has been implicated in immune regulation, but the mechanisms by which IAP proteins contribute to immunity are incompletely understood. We show here that X-linked IAP (XIAP) is required for innate immune control of Listeria monocytogenes infection. Mice deficient in XIAP had a higher bacterial burden 48 h after infection than wild-type littermates, and exhibited substantially decreased survival. XIAP enhanced NF-κB activation upon L. monocytogenes infection of activated macrophages, and prolonged phosphorylation of Jun N-terminal kinase (JNK) specifically in response to cytosolic bacteria. Additionally, XIAP promoted maximal production of pro-inflammatory cytokines upon bacterial infection in vitro or in vivo, or in response to combined treatment with NOD2 and TLR2 ligands. Together, our data suggest that XIAP regulates innate immune responses to L. monocytogenes infection by potentiating synergy between Toll-like receptors (TLRs) and Nod-like receptors (NLRs) through activation of JNK- and NF-κB–dependent signaling

HGS-ETR1, a fully human TRAIL-receptor 1 monoclonal antibody, induces cell death in multiple tumour types in vitro and in vivo

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumour cells through activation of TRAIL-R1 and TRAIL-R2 death signalling receptors. Here, we describe the characterisation and activity of HGS-ETR1, the first fully human, agonistic TRAIL-R1 mAb that is being developed as an antitumour therapeutic agent. HGS-ETR1 showed specific binding to TRAIL-R1 receptor. HGS-ETR1 reduced the viability of multiple types of tumour cells in vitro, and induced activation of caspase 8, Bid, caspase 9, caspase 3, and cleavage of PARP, indicating activation of TRAIL-R1 alone was sufficient to induce both extrinsic and intrinsic apoptotic pathways. Treatment of cell lines in vitro with HGS-ETR1 enhanced the cytotoxicity of chemotherapeutic agents (camptothecin, cisplatin, carboplatin, or 5-fluorouracil) even in tumour cell lines that were not sensitive to HGS-ETR1 alone. In vivo administration of HGS-ETR1 resulted in rapid tumour regression or repression of tumour growth in pre-established colon, non-small-cell lung, and renal tumours in xenograft models. Combination of HGS-ETR1 with chemotherapeutic agents (topotecan, 5-fluorouracil, and irinotecan) in three independent colon cancer xenograft models resulted in an enhanced antitumour efficacy compared to either agent alone. Pharmacokinetic studies in the mouse following intravenous injection showed that HGS-ETR1 serum concentrations were biphasic with a terminal half-life of 6.9–8.7 days and a steady-state volume of distribution of approximately 60 ml kg−1. Clearance was 3.6–5.7 ml−1 day−1 kg−1. These data suggest that HGS-ETR1 is a specific and potent antitumour agent with favourable pharmacokinetic characteristics and the potential to provide therapeutic benefit for a broad range of human malignancies

IL-1β Processing in Host Defense: Beyond the Inflammasomes

Stimulation and release of proinflammatory cytokines is an essential step for the activation of an effective innate host defense, and subsequently for the modulation of adaptive immune responses. Interleukin-1β (IL-1β) and IL-18 are important proinflammatory cytokines that on the one hand activate monocytes, macropages, and neutrophils, and on the other hand induce Th1 and Th17 adaptive cellular responses. They are secreted as inactive precursors, and the processing of pro-IL-1β and pro-IL-18 depends on cleavage by proteases. One of the most important of these enzymes is caspase-1, which in turn is activated by several protein platforms called the inflammasomes. Inflammasome activation differs in various cell types, and knock-out mice defective in either caspase-1 or inflammasome components have an increased susceptibility to several types of infections. However, in other infections and in models of sterile inflammation, caspase-1 seems to be less important, and alternative mechanisms such as neutrophil-derived serine proteases or proteases released from microbial pathogens can process and activate IL-1β. In conclusion, IL-1β/IL-18 processing during infection is a complex process in which the inflammasomes are only one of several activation mechanisms