TNF-related apoptosis inducing ligand (TRAIL) and its receptors in tumor surveillance and cancer therapy

TNF-related apoptosis-inducing ligand (TRAIL/APO-2L) is a typical member of the TNF ligand family that induces apoptosis by activating the death receptors TRAIL-R1 and TRAIL-R2. TRAIL has attracted great attention in recent years as a promising anti cancer reagent because recombinant soluble TRAIL derivatives induce apoptosis in a broad range of tumor cells but not or only rarely in non-transformed cells. In this review we will address the putative role of TRAIL in cancer treatment in the light of the emerging importance of TRAIL in tumor surveillance and discuss the molecular basis of the cooperation of TRAIL and chemotherapeutic drugs. In particular, we debate controversial data in the literature concerning the cytotoxicity of different TRAIL derivatives on primary human cells.     

Helicobacter pylori sensitizes TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric epithelial cells through regulation of FLIP

Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcer and gastric cancer. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Here we show that human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death receptor signaling. Human gastric epithelial cells are intrinsically resistant to TRAIL-mediated apoptosis. The induction of TRAIL sensitivity by H. pylori is dependent on the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex (DISC) through downregulation of cellular FLICE-inhibitory protein (FLIP). Overexpression of FLIP abolished the H. pylori-induced TRAIL sensitivity in human gastric epithelial cells. Our study thus demonstrates that H. pylori induces sensitivity to TRAIL apoptosis by regulation of FLIP and assembly of DISC, which initiates caspase activation, resulting in the breakdown of resistance to apoptosis, and provides insight into the pathogenesis of gastric damage in Helicobacter infection. Modulation of host apoptosis signaling by bacterial interaction adds a new dimension to the pathogenesis of Helicobacter.     

Cellular inhibitor of apoptosis 1 (cIAP-1) degradation by caspase 8 during TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

TNF-related apoptosis-inducing ligand (TRAIL) is a potential chemotherapeutic agent with high selectivity for malignant cells. Many tumors, however, are resistant to TRAIL cytotoxicity. Although cellular inhibitors of apoptosis 1 and 2 (cIAP-1 and -2) are often over-expressed in cancers, their role in mediating TRAIL resistance remains unclear. Here, we demonstrate that TRAIL-induced apoptosis of liver cancer cells is associated with degradation of cIAP-1 and X-linked IAP (XIAP), whereas cIAP-2 remains unchanged. Lower concentrations of TRAIL causing minimal or no apoptosis do not alter cIAP-1 or XIAP protein levels. Silencing of cIAP-1 expression, but not XIAP or cIAP-2, as well as co-treatment with a second mitochondrial activator of caspases (SMAC) mimetic (which results in rapid depletion of cIAP-1), sensitizes the cells to TRAIL. TRAIL-induced loss of cIAP-1 and XIAP requires caspase activity. In particular, caspase 8 knockdown stabilizes both cIAP-1 and XIAP, while caspase 9 knockdown prevents XIAP, but not cIAP-1 degradation. Cell-free experiments confirmed cIAP-1 is a substrate for caspase 8, with likely multiple cleavage sites. These results suggest that TRAIL-mediated apoptosis proceeds through caspase 8-dependent degradation of cIAP-1. Targeted depletion of cIAP-1 by SMAC mimetics in conjunction with TRAIL may be beneficial for the treatment of human hepatobiliary malignancies.     

Hypoxia inhibition of apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Hypoxia is a common environmental stress. Particularly, the center of rapidly growing solid tumors is easily exposed to hypoxic conditions. Thus, tumor cell response to hypoxia plays an important role in tumor progression as well as tumor therapy. However, little is known about hypoxic effect on apoptotic cell death. To examine the effects of hypoxia on TRAIL-induced apoptosis, human lung carcinoma A549 cells were exposed to hypoxia and treated with TRAIL protein. Hypoxia significantly protected A549 cells from apoptosis induced by TRAIL. Western blotting analysis demonstrated that hypoxia increased expression of antiapoptotic proteins such as Bcl-2, Bcl-XL, and IAP family members. The increase of these antiapoptotic molecules is believed to play an hypoxia-mediated protective role in TRAIL-induced apoptosis. Our findings suggest that an increase of antiapoptotic proteins induced by hypoxia may regulate the therapeutic activity of TRAIL protein in cancer therapy.     

Combining proteasome inhibition with TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) for cancer therapy

Apoptosis has an essential role in embryogenesis, adult tissue homeostasis and cellular responses to stressful stimuli. Therefore, increased apoptosis is involved in the pathogenesis of various ischaemic, degenerative and immune disorders. Conversely, genetic aberration that results in a reduction or abolition of apoptosis can promote tumorigenesis and underlie the resistance of cancer cells to various genotoxic anticancer agents. Therefore, a detailed knowledge of the control of apoptotic pathways could aid in the rational design of effective therapeutics for a variety of human diseases including cancer. One major way to promote apoptosis involves signaling through members of the tumor necrosis factor (TNF) superfamily. On binding to their appropriate receptors, some TNF family members can promote caspase activation and apoptosis. Early studies on TNF indicated that a limited number of tumor cell lines could be induced to undergo apoptosis on exposure to TNF. Another member of the TNF family Fas ligand (FasL) is also known to induce apoptosis in a variety of tumor cells. Although TNF and FasL can efficiently induce apoptosis in a limited number of tumor cells, administration of either of these agents is associated with extreme toxicity. This toxicity has precluded further development of either TNF or FasL for cancer therapy. However, within the last 8 years another member of the TNF family, TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) has been characterized, which induces apoptosis of a wider range of cancer cells than either TNF or FasL. Surprisingly, most normal non-transformed cells are quite resistant to the apoptotic effects of Apo2L/TRAIL. This selective toxicity for cancer cells is the basis for the current enthusiasm for Apo2L/TRAIL as a potential novel anticancer therapy. In this symposium report, we provide a brief overview of Apo2L/TRAIL, its receptors and their signaling pathways. We discuss findings on the antitumor effects of Apo2L/TRAIL alone or in combination with radiotherapy or chemotherapy. In addition, we present recent information from our groups concerning the possible therapeutic benefits of combining Apo2L/TRAIL with the proteasome inhibitor bortezomib. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004     

Alizarin,a nature compound,inhibits the growth of pancreatic cancer cells by abrogating NF-κB activation

The current performance of nature compounds in antitumor field is gradually attracted more and more attention, we discovered a nature active ingredient alizarin possess potent natural reductive NF-κB activity to against pancreatic cancer. However, the preclinical pharmacology and therapeutic effect, and the underlying mechanisms of alizarin in inhibiting pancreatic cancer are still unclear. After high-throughput screening, this is the first report that alizarin can induce a potent inhibitory effect against pancreatic cancer cells. Alizarin induced cell cycle arrest and promoted cell apoptosis by inhibiting TNF-α-stimulated NF-κB activity and nuclear translocation, and inactivated its related TNF-α-TAK1-NF-κB signaling cascade followed by downregulation of NF-κB target genes involved in cell apoptosis (Bcl-2, Bcl-xL, XIAP) and in the cell cycle and growth (cyclin D, c-myc). Due to the abrogation of NF-κB activity, combination of alizarin and gemcitabine exerted a better inhibitory effect on pancreatic cancer. In summary, natural component alizarin, inhibited cell proliferation and induced apoptosis in vitro and in vivo through targeting of the NF-κB signaling cascade with minimal toxicity, which combine with gemcitabine, can significantly enhance the antitumor capability, playing a synergistic effect. Therefore, alizarin may play a role in reversing gemcitabine resistance caused by overactivated NF-κB in clinical application in the future.     

Engagement of Nucleotide-binding Oligomerization Domain-containing Protein 1 (NOD1) by Receptor-interacting Protein 2 (RIP2) Is Insufficient for Signal Transduction

Following activation, the cytoplasmic pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) interacts with its adaptor protein receptor-interacting protein 2 (RIP2) to propagate immune signaling and initiate a proinflammatory immune response. This interaction is mediated by the caspase recruitment domain (CARD) of both proteins. Polymorphisms in immune proteins can affect receptor function and predispose individuals to specific autoinflammatory disorders. In this report, we show that mutations in helix 2 of the CARD of NOD1 disrupted receptor function but did not interfere with RIP2 interaction. In particular, N43S, a rare polymorphism, resulted in receptor dysfunction despite retaining normal cellular localization, protein folding, and an ability to interact with RIP2. Mutation of Asn-43 resulted in an increased tendency to form dimers, which we propose is the source of this dysfunction. We also demonstrate that mutation of Lys-443 and Tyr-474 in RIP2 disrupted the interaction with NOD1. Mapping the key residues involved in the interaction between NOD1 and RIP2 to the known structures of CARD complexes revealed the likely involvement of both type I and type III interfaces in the NOD1·RIP2 complex. Overall we demonstrate that the NOD1-RIP2 signaling axis is more complex than previously assumed, that simple engagement of RIP2 is insufficient to mediate signaling, and that the interaction between NOD1 and RIP2 constitutes multiple CARD-CARD interfaces.     

VRK2 activates TNFα/NF-κB signaling by phosphorylating IKKβ in pancreatic cancer

NF-κB signaling is active in more than 50% of patients with pancreatic cancer and plays an important role in promoting the progression of pancreatic cancer. Revealing the activation mechanism of NF-κB signaling is important for the treatment of pancreatic cancer. In this study, the regulation of TNFα/NF-κB signaling by VRK2 (vaccinia-related kinase 2) was investigated. The levels of VRK2 protein were examined by immunohistochemistry (IHC). The functions of VRK2 in the progression of pancreatic cancer were examined using CCK8 assay, anchorage-independent assay, EdU assay and tumorigenesis assay. The regulation of VRK2 on the NF-κB signaling was investigated by immunoprecipitation and invitro kinase assay. It was discovered in this study that the expression of VRK2 was upregulated in pancreatic cancer and that the VRK2 expression level was significantly correlated with the pathological characteristics and the survival time of patients. VRK2 promoted the growth, sphere formation and subcutaneous tumorigenesis of pancreatic carcinoma cells as well as the organoid growth derived from the pancreatic cancer mouse model. Investigation of the molecular mechanism indicated that VRK2 interacts with IKKβ, phosphorylating its Ser177 and Ser181 residues and thus activating the TNFα/NF-κB signaling pathway. An IKKβ inhibitors abolished the promotive effect of VRK2 on the growth of organoids. The findings of this study indicate that VRK2 promotes the progression of pancreatic cancer by activating the TNFα/NF-κB signaling pathway, suggesting that VRK2 is a potential therapeutic target for pancreatic cancer.     

Time sequence of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cisplatin treatment is responsible for a complex pattern of synergistic cytotoxicity

The combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cisplatin resulted in a greater cytotoxicity than could be accounted for by the addition of the cytotoxic effects of the agents alone. In this study, we hypothesized that the synergistic interaction between the two modalities can be changed when both the sequence and the time interval between the two treatments are varied. To test the hypothesis, human head-and-neck squamous-cell carcinoma (HNSCC)-6 cells were either pretreated with 0.01-0.5 microg/ml TRAIL for various times (0-24 h) followed by treatment with 5 microg/ml cisplatin or pretreated with 5 microg/ml cisplatin for various times (0-24 h) followed by treatment with 0.5 microg/ml TRAIL. In latter case, the synergistic effect was gradually increased when the time interval between the two treatments was increased. In former case, a maximal synergy occurred within 0-4 h of pretreatment with TRAIL. However, the synergistic effect was gradually decreased when the time interval between the two treatments was increased. Data from immunoblotting analysis reveal that a similar pattern emerged for the PARP cleavage and caspase activation. The synergistic effect is not associated with DR4, DR5, FADD, and FLIP(L). Interestingly, a complex pattern of synergistic interaction between TRAIL and cisplatin is related to the cleavage of FLIP(S). Although overexpression of FLIP(S) protected cells from FLIP(S) cleavage and apoptotic death, blockage of FLIP(S) cleavage by replacing Asp(39) and Asp(42) residues with alanine did not further enhance FLIP(S)-mediated protection. Taken together, FLIP(S) cleavage reflects apoptotic damage, but it does not cause apoptosis.     

Radiation-inducible human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy: a novel treatment for radioresistant uveal melanoma

Uveal melanoma (UM) is one of the most therapy-resistant cancers. Radiotherapy is the preferred treatment for most cases of UM. However, some UM cells, such as the SP6.5 or OM431 cell lines, are relatively radioresistant. In this study, we attempted to improve the current UM therapy using an adenovirus radio-inducible gene therapy system. The antitumor adenovirus was constructed by inclusion of the radiation-inducible early growth response gene 1 (EGR1) promoter and the anticancer tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene. We demonstrated that the UM SP6.5 and OM431 cell lines were susceptible to the TRAIL-induced antitumor effect. TRAIL expression was enhanced in the adenovirus containing EGR1/TRAIL (Ad-ET) treatment group by radiotherapy, whereas Ad-ET significantly increased cell death and apoptosis caused by radiotherapy. In mice bearing xenograft tumors, apoptotic cells were detected in pathological tumor sections. Adenovirus Ad-ET combined with radiation therapy significantly inhibited tumor growth compared with the other treatment groups (P < 0.01). Our findings indicate that radioresponsive gene therapy has the potential to be a more effective and specific therapy for UM because the therapeutic gene can be spatially or temporally controlled by exogenous radiation.     

Dynamic Imaging of Pancreatic Nuclear Factor κB (NF-κB) Activation in Live Mice Using Adeno-associated Virus (AAV) Infusion and Bioluminescence

Nuclear factor κB (NF-κB) is an important signaling molecule that plays a critical role in the development of acute pancreatitis. Current methods for examining NF-κB activation involve infection of an adenoviral NF-κB-luciferase reporter into cell lines or electrophoretic mobility shift assay of lysate. The use of adeno-associated viruses (AAVs) has proven to be an effective method of transfecting whole organs in live animals. We examined whether intrapancreatic duct infusion of AAV containing an NF-κB-luciferase reporter (AAV-NF-κB-luciferase) can reliably measure pancreatic NF-κB activation. We confirmed the infectivity of the AAV-NF-κB-luciferase reporter in HEK293 cells using a traditional luciferase readout. Mice were infused with AAV-NF-κB-luciferase 5 weeks before induction of pancreatitis (caerulein, 50 μg/kg). Unlike transgenic mice that globally express NF-κB-luciferase, AAV-infused mice showed a 15-fold increase in pancreas-specific NF-κB bioluminescence following 12 h of caerulein compared with baseline luminescence (p < 0.05). The specificity of the NF-κB-luciferase signal to the pancreas was confirmed by isolating the pancreas and adjacent organs and observing a predominant bioluminescent signal in the pancreas compared with liver, spleen, and stomach. A complementary mouse model of post-ERCP-pancreatitis also induced pancreatic NF-κB signals. Taken together these data provide the first demonstration that NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. We believe this technique offers a valuable tool to study real-time activation of NF-κB in vivo.     

Study on the Mechanism of BMSCs in Regulating NF-κB Signal Pathway by Targeting miR-449a to Improve the Inflammatory Response to Peripheral Nerve Injury

Objective:To evaluate the mechanism of Bone Marrow Mesenchymal Stem Cells (BMSCs) in regulating NF-κB signal pathway by targeting miR-449a.Methods:Stem cells were transfected by over-expressing and inhibiting miR-449a to detect the levels and viability of miR-449a in stem cells after transfection. Stem cells and neurons were co-cultured in vitro to evaluate the in vitro mechanism of stem cells over-expressing miR-449a on neurons.Results:After the addition of neurons, the neuronal activity of miR-449a over-expression group increased significantly, the expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, and the inflammatory cytokines (TNF-α and IL-1β) decreased significantly (P<0.05). In vivo experiments in rats also showed that rats were unresponsive, did not chirp or elude after being stimulated. After stem cell therapy, the weight and response of rats gradually returned to normal levels. miR-449a expression significantly increased in the stem cell + miR-449a over-expression group, expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, inflammatory cytokines (TNF-α and IL-1β) significantly decreased, and cell activity significantly increased (P<0.05).Conclusions:BMSCs can modulate NF-κB signaling pathway by targeting miR-449a, so as to reduce the inflammatory response to peripheral nerve injury and repair nerve injury.     

Generation of a novel proform of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein that can be reactivated by matrix metalloproteinases

TRAIL has been suggested to induce the cell death in various tumor cells but not in normal cells; however, several studies have provided the evidence that TRAIL can induce the cell death in some normal cells including human normal hepatocytes, suggesting that TRAIL may show hepatic toxicity in human. In this study, we designed a pro-form of TRAIL (sTRAIL:IL-18) in that soluble TRAIL (sTRAIL) is fused to IL-18, and a matrix metalloproteinases (MMPs) cleavage site is introduced at the connecting site. We showed that sTRAIL:IL-18 has significantly diminished the killing activity in HeLa cells but regains the activity by releasing the free sTRAIL through MMP-2-mediated cleavage. In addition, the killing activity of sTRAIL:IL-18 was significantly increased in HeLa cells when active MMP-2 was produced by TNF-alpha. Taken together, the data suggested that the sTRAIL:IL-18 can be reactivated at the specialized areas where MMPs are pathologically produced.     

Anti-Inflammatory Effect of Pineapple Rhizome Bromelain through Downregulation of the NF-κB- and MAPKs-Signaling Pathways in Lipopolysaccharide (LPS)-Stimulated RAW264.7 Cells

Bromelain is a mixture of proteolytic enzymes derived from pineapple (Ananas comosus) fruit and stem possessing several beneficial properties, particularly anti-inflammatory activity. However, the molecular mechanisms underlying the anti-inflammatory effects of bromelain are unclear. This study investigated the anti-inflammatory effects and inhibitory molecular mechanisms of crude and purified rhizome bromelains on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. RAW264.7 cells were pre-treated with various concentrations of crude bromelain (CB) or purified bromelain (PB), and then treated with LPS. The production levels of pro-inflammatory cytokines and mediators, including nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α were determined by Griess and ELISA assays. The expressions of inducible nitric oxide synthetase (iNOS), cyclooxygenase (COX)-2, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinases (MAPKs)-signaling pathway-related proteins were examined by western blot analysis. The pre-treatment of bromelain dose-dependently reduced LPS-induced pro-inflammatory cytokines and mediators, which correlated with downregulation of iNOS and COX-2 expressions. The inhibitory potency of PB was stronger than that of CB. PB also suppressed phosphorylated NF-κB (p65), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, extracellular signal-regulated kinases, c-Jun amino-terminal kinases, and p38 proteins in LPS-treated cells. PB then exhibited potent anti-inflammatory effects on LPS-induced inflammatory responses in RAW264.7 cells by inhibiting the NF-κB and MAPKs-signaling pathways.     

Trifluoromethyl-substituted 3,5-bis(arylidene)-4-piperidones as potential anti-hepatoma and anti-inflammation agents by inhibiting NF-кB activation

Some methoxy-, hydroxyl-, pyridyl-, or fluoro-substituted 3,5-bis(arylidene)-4-piperidones (BAPs) could reduce inflammation and promote hepatoma cell apoptosis by inhibiting activation of NF-κB, especially after introduction of trifluoromethyl. Herein, a series of trifluoromethyl-substituted BAPs (4-30) were synthesised and the biological activities were evaluated. We successfully found the most potential 16, which contains three trifluoromethyl substituents and exhibits the best anti-tumour and anti-inflammatory activities. Preliminary mechanism research revealed that 16 could promote HepG2 cell apoptosis in a dose-dependent manner by down-regulating the expression of Bcl-2 and up-regulating the expression of Bax, C-caspase-3. Meanwhile, 16 inhibited activation of NF-κB by directly inhibiting the phosphorylation of p65 and IκBα induced by LPS, together with indirectly inhibiting MAPK pathway, thereby exhibiting both anti-hepatoma and anti-inflammatory activities. Molecular docking confirmed that 16 could bind to the active sites of Bcl-2, p65, and p38 reasonably. The above results suggested that 16 has enormous potential to be developed as a multifunctional agent for the clinical treatment of liver cancers and inflammatory diseases.     

Diverse Ras-related GTPase DIRAS2, downregulated by PSMD2 in a proteasome-mediated way,inhibits colorectal cancer proliferation by blocking NF-κB signaling

Colorectal cancer (CRC) is the most common gastrointestinal cancer, with a high mortality rate but limited therapeutic targets. DIRAS family GTPase 2 (DIRAS2) is a member of the Ras-related small G-protein family whose biological functions and underlying mechanism in CRC remain poorly understood. In this study, we identified the crucial roles of DIRAS2 in CRC. DIRAS2 expression was downregulated in CRC and closely correlated with poor prognosis. Functionally, DIRAS2 inhibited CRC cell proliferation and affected cell-cycle protein expression. Mechanistically, DIRAS2 blocked nuclear factor kappa light-chain enhancer of activated B-cell signaling pathways, inducing G0/G1 arrest. Moreover, DIRAS2 interacted with 26S proteasome non-ATPase regulatory subunit 2, which facilitates the degradation of DIRAS2 in a proteasome-mediated way. Together, these results demonstrate potential functions of DIRAS2 as a tumor-suppressor gene in CRC and reveal a distinct mechanism of DIRAS2 in CRC tumorigenesis, indicating its role as a potential biomarker and target for CRC therapy.