Targeting Death Receptor Research Articles

Targeting Death Receptor 5 (DR5) for the imaging and treatment of primary bone and soft tissue tumors: an update of the literature.

Death Receptor 5 (DR5) is expressed on the surface of primary bone and soft tissue sarcoma cells, and its activation induces cell death primarily through apoptosis. The combination of DR5 agonists and commonly used chemotherapeutic agents, such as doxorubicin, can promote cell death. Currently, clinical trials are investigating the effectiveness of DR5 activation using new biological agents, such as bi-specific or tetravalent antibodies, in improving the survival of patients with relapsed or refractory cancers. Furthermore, investigations continue into the use of novel combination therapies to enhance DR5 response, for example, with inhibitor of apoptosis protein (IAP) antagonist agents [such as the second mitochondria-derived activator of caspase (SMAC) mimetics] and with immune checkpoint inhibitor anti-programmed death-ligand 1 (anti-PD-L1) or anti-programmed cell death-1 (anti-PD-1) antibodies. Other therapies include nanoparticle-mediated delivery of TRAIL plasmid DNA or TRAIL mRNA and stem cells as a vehicle for the targeted delivery of anti-cancer agents, such as TRAIL, to the tumor. Scoping review of the literature from November 2017 to March 2024, utilizing PubMed and Google Scholar. New agents under investigation include nanoTRAIL, anti-Kv10.1, multimeric IgM, and humanized tetravalent antibodies. Developments have been made to test novel agents, and imaging has been used to detect DR5 in preclinical models and patients. The models include 3D spheroids, genetically modified mouse models, a novel jaw osteosarcoma model, and patient-derived xenograft (PDX) animal models. There are currently two ongoing clinical trials focusing on the activation of DR5, namely, IGM-8444 and INBRX-109, which have progressed to phase 2. Further modifications of TRAIL delivery with fusion to single-chain variable fragments (scFv-TRAIL), directed against tumor-associated antigens (TAAs), and in the use of stem cells focus on targeted TRAIL delivery to cancer cells using bi-functional strategies. In vitro, in vivo, and clinical trials, as well as advances in imaging and theranostics, indicate that targeting DR5 remains a valid strategy in the treatment of some relapsed and refractory cancers.

MicroRNA-148a Inhibits Hepatocellular Carcinoma Cell Growth via Epithelial-to-Mesenchymal Transition and PI3K/AKT Signaling Pathways by Targeting Death Receptor-5.

The purpose of this study was to investigate the role of microRNA-148a (miR-148a) in hepatocellular carcinoma (HCC) metastasis and explore its potential mechanism in HCC cells. Expression levels of miR-148a were measured using qRT-PCR in 120 HCC tissue samples and two HCC cell lines. Migration and invasion assays were used to determine the role of miR-148a in HCC cells. Flow cytometry was used to access the effect of miR-148a on cell cycle of HCC cells. Western blot was performed to analyze the effect of miR-148a on epithelial-to-mesenchymal transition (EMT) and PI3K/AKT signaling pathways in HCC cells. Luciferase reporter assay was conducted to explore the downstream targets and biological function of miR-148a in HCC cells. The results showed that level of miR-148a was significantly downregulated in both HCC tissue and plasma samples in HCC patients. A higher level of miR-148a was positively correlated with better survival time and prognosis of HCC patients. Transfection of miR-148a inhibited the proliferation, migration and invasion of HCC cell lines. Transfection of miR-148a arrested HCC cells at S phase and promoted apoptosis of HCC cells. Death receptor-5 (DR-5) was identified as a direct target of miR-148a in HCC cell lines. Western blot and qRT-PCR analyses showed that miR-148a upregulated EMT and downregulated PI3K/AKT signaling pathways in HCC cell lines. In conclusion, data in the current study indicate that miR-148a inhibits HCC cells growth via downregulation of EMT and PI3K/AKT signaling pathways by targeting death receptor. These data suggest that miR-148a may serve as a therapeutic target for HCC cancer therapy in the future.

TRAIL sensitivity of nasopharyngeal cancer cells involves redox dependent upregulation of TMTC2 and its interaction with membrane caspase-3

AimsPreferential expression of receptors for TNF-family related apoptosis inducing ligand (TRAIL), DR4 and DR5 makes TRAIL an attractive anti-cancer therapeutic. However, the efficacy of targeting death receptors has not been extensively studied in nasopharyngeal cancer (NPC). Here we investigated TRAIL sensitivity and its underlying mechanism in NPC cell lines, and assessed the potential of TRAIL as a therapeutic option against NPC. ResultsUsing two established NPC cell lines, we report the expression of DR4 and DR5, which respond to TRAIL ligation by triggering efficient Type II apoptosis. Mechanistically, early activation of caspase-3 and its membrane recruitment is identified in NPC cell lines, which is associated with, hitherto unreported, interaction with transmembrane and tetratricopeptide repeat containing 2 (TMTC2) in the lipid raft domains. TMTC2 expression is induced upon exposure to TRAIL and involves intracellular increase in peroxynitrite (ONOO−) production. While ONOO− increase is downstream of caspase-8 activation, it is involved in the upregulation of TMTC2, gene knockdown of which abrogated TRAIL-induced apoptotic execution. Bioinformatics analyses also provide evidence for a strong correlation between TMTC2 and DR4 or caspase-3 as well as a significantly better disease-free survival in patients with high TMTC2 expression. Innovation and conclusionCollectively, redox-dependent execution of NPC cells upon ligation of TRAIL receptors reintroduces the possible therapeutic use of TRAIL in NPC as well as underscores the potential of using TMTC2 as a biomarker of TRAIL sensitivity.

P-192: Circularly permuted TRAIL (CPT) combined with Thalidomide and Dexamethasone in patients with relapsed/refractory Multiple Myeloma: a randomized, double-blind, placebo-controlled phase 3 study

<h3>Background</h3> CPT, a recombinant permuted human TNF-related apoptosis-inducing ligand (TRAIL), is a 1rst-in-human anti-myeloma drug targeting death receptor 4/5. In order to verify the effectiveness and safety of CPT, we have conducted this multi-center, double-blind, placebo-controlled phase 3 study. <h3>Methods</h3> In this study, 417 RRMM patients previously treated with two or more lines of therapies were randomly assigned (2:1) to receive CPT+TD (CPT group) vs. placebo+TD (control group). CPT or placebo at a dose of 10 mg/kg was intravenously administered on days 1 to 5, both groups receive dexamethasone 40 mg orally on days 1 to 4, and thalidomide 150mg daily, 28 days per cycle until to disease progressions or unacceptable toxicity. The primary endpoint was progression-free survival (PFS), and the key secondary endpoints overall survival (OS), overall response rate (ORR), and safety. <h3>Results</h3> Of the 417 patients enrolled, 415 patients received CPT+TD (n=276) or placebo+TD (n=139) treatment. The demographic, baseline disease and clinical characteristic of the two groups were comparable. The median age of the patients was 59 years, and the median time from the initial diagnosis of multiple myeloma (MM) to participating this trial was 2.6 years. The median number of lines of prior therapies was 3. Proteasome inhibitors (PI) and immunomodulators (IMiD) were previously used in 74% and 86.5% of patients, respectively. At date cutoff, the primary endpoint of PFS was significantly longer in the CPT group than in the control group (median 5.5 vs. 3.1 months; hazard ratio [HR] 0.619; P3 lines of prior therapies. PFS and ORR were also significantly improved in the patients with PI and IMiD double-refractory MM. The serious adverse events (SAE) rates were similar in the CPT group and the control group (40.6% vs. 37.4%), as were the death rates during the treatments (7.6% vs. 8.6%). Treatment-emergent adverse events (TEAEs) with at least a 10% greater frequency in the CPT group vs. the control group were elevated alanine transaminase (ALT), elevated aspartate transaminase (AST), elevated lactate dehydrogenase (LDH), increased monocyte counts, hypocalcaemia and upper respiratory tract infections. <h3>Conclusions</h3> The combination of CPT with TD significantly prolonged the PFS and OS, increased the ORR. CPT combined with TD was well tolerated, the adverse events were mild, transient and reversible.

JDP2 is directly regulated by ATF4 and modulates TRAIL sensitivity by suppressing the ATF4-DR5 axis.

Jun dimerization protein 2 (JDP2) is a bZip-type transcription factor, which acts as a repressor or activator of several cellular processes, including cell differentiation and chromatin remodeling. Previously, we found that a stress-responsive transcription factor, known as activating transcription factor 4 (ATF4), enhances JDP2 gene expression in human astrocytoma U373MG and cervical cancer HeLa cells; however, the role of JDP2 in the ATF4-mediated stress response remained unclear. Here, we reported that siRNA-mediated JDP2 knockdown enhances the expression of several ATF4 target genes, including ASNS, and death receptors 4 and 5 (DR4 and DR5) in HeLa cells. In addition, the results of a transient reporter assay indicate that JDP2 overexpression represses ER stress-mediated DR5 promoter activation suggesting that JDP2 negatively regulates ATF4-mediated gene expression. Curiously, knockdown of JDP2 increases the sensitivity of cells to TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in cancer cells through DR4 and DR5. These results indicate that JDP2 functions as a negative feedback regulator of the ATF4 pathway and contributes to TRAIL resistance in cancer cells.

The Salmonella Effector SseK3 Targets Small Rab GTPases.

During infection, Salmonella species inject multiple type III secretion system (T3SS) effector proteins into host cells that mediate invasion and subsequent intracellular replication. At early stages of infection, Salmonella exploits key regulators of host intracellular vesicle transport, including the small GTPases Rab5 and Rab7, to subvert host endocytic vesicle trafficking and establish the Salmonella-containing vacuole (SCV). At later stages of intracellular replication, interactions of the SCV with Rab GTPases are less well defined. Here we report that Rab1, Rab5, and Rab11 are modified at later stages of Salmonella infection by SseK3, an arginine N-acetylglucosamine (GlcNAc) transferase effector translocated via the Salmonella pathogenicity island 2 (SPI-2) type III secretion system. SseK3 modified arginines at positions 74, 82, and 111 within Rab1 and this modification occurred independently of Rab1 nucleotide binding. SseK3 exhibited Golgi localization that was independent of its glycosyltransferase activity but Arg-GlcNAc transferase activity was required for inhibition of alkaline phosphatase secretion in transfected cells. While SseK3 had a modest effect on SEAP secretion during infection of HeLa229 cells, inhibition of IL-1 and GM-CSF cytokine secretion was only observed upon over-expression of SseK3 during infection of RAW264.7 cells. Our results suggest that, in addition to targeting death receptor signaling, SseK3 may contribute to Salmonella infection by interfering with the activity of key Rab GTPases.

Abstract 5403: Targeting death-receptors in therapy resistant progressive neuroblastoma: A preliminary insight

Abstract Neuroblastoma is the most common cancer in infants and accounts for 9.1% of childhood cancer-related deaths. Despite the current standard of care (SOC), drug-resistant neuroblastoma significantly compromises patient survival. Hence it is critical to identify new and less toxic therapeutic approaches for better patient outcomes. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which mediates its effect through the activation of the death receptor (DR) 4 and 5 are found on many cell types and are the main pathway for apoptosis. These can death receptors can kill most tumor cell types expressing TRAIL-receptor 1. Mining RNA-Seq data across multiple cohorts of neuroblastoma patients indicated that high levels of DR4 and DR5 significantly associated with poor clinical outcomes (OS, EFS, RFS). Further, examining the surface expression of DR4 and DR5 by immunohistochemistry using anti-human rabbit monoclonal antibodies on custom-archived TMAs for 100 NB patients revealed profound correlation with poor OS and RFS. Immunoblot analysis for DR4 and DR5 level in a panel of stage 4 patient-derived cell-lines during diagnosis and from progressive disease after therapy displayed high-levels of death receptor expression in progressive disease. Preliminary co-culture investigations with genetically engineered mesenchymal stem cells (MSCs) expressing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) mimicking targeted delivery showed heightened differentiation and death (limited dilution tumorosphere assay) of therapy resistant cells. Further these studies exhibited inhibition of tumor cell migration and invasion. The results demonstrate the benefit of targeted TRAIL delivery in in the treatment of therapy resistant neuroblastoma and further imply that the genetically engineered MSCs could serve as a clinically valuable tool for improved therapeutic strategy in this setting. Citation Format: Dinesh Babu Somasundaram, Andrew Maher, Sheeja Aravindan, Terence S. Herman, Natarajan Aravindan. Targeting death-receptors in therapy resistant progressive neuroblastoma: A preliminary insight [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5403.

Cell death induced by the ER stressor thapsigargin involves death receptor 5, a non-autophagic function of MAP1LC3B, and distinct contributions from unfolded protein response components

BackgroundCell death triggered by unmitigated endoplasmic reticulum (ER) stress plays an important role in physiology and disease, but the death-inducing signaling mechanisms are incompletely understood. To gain more insight into these mechanisms, the ER stressor thapsigargin (Tg) is an instrumental experimental tool. Additionally, Tg forms the basis for analog prodrugs designed for cell killing in targeted cancer therapy. Tg induces apoptosis via the unfolded protein response (UPR), but how apoptosis is initiated, and how individual effects of the various UPR components are integrated, is unclear. Furthermore, the role of autophagy and autophagy-related (ATG) proteins remains elusive.MethodsTo systematically address these key questions, we analyzed the effects of Tg and therapeutically relevant Tg analogs in two human cancer cell lines of different origin (LNCaP prostate- and HCT116 colon cancer cells), using RNAi and inhibitory drugs to target death receptors, UPR components and ATG proteins, in combination with measurements of cell death by fluorescence imaging and propidium iodide staining, as well as real-time RT-PCR and western blotting to monitor caspase activity, expression of ATG proteins, UPR components, and downstream ER stress signaling.ResultsIn both cell lines, Tg-induced cell death depended on death receptor 5 and caspase-8. Optimal cytotoxicity involved a non-autophagic function of MAP1LC3B upstream of procaspase-8 cleavage. PERK, ATF4 and CHOP were required for Tg-induced cell death, but surprisingly acted in parallel rather than as a linear pathway; ATF4 and CHOP were independently required for Tg-mediated upregulation of death receptor 5 and MAP1LC3B proteins, whereas PERK acted via other pathways. Interestingly, IRE1 contributed to Tg-induced cell death in a cell type-specific manner. This was linked to an XBP1-dependent activation of c-Jun N-terminal kinase, which was pro-apoptotic in LNCaP but not HCT116 cells. Molecular requirements for cell death induction by therapy-relevant Tg analogs were identical to those observed with Tg.ConclusionsTogether, our results provide a new, integrated understanding of UPR signaling mechanisms and downstream mediators that induce cell death upon Tg-triggered, unmitigated ER stress.1yG1LZYrokkFLLYyz3UJEsVideo Graphical abstract

Self-assembled peptide nanostructures targeting death receptor 5 and encapsulating paclitaxel as a multifunctional cancer therapy.

The development of tumor-targeted nanoscale carriers for the delivery of cancer therapeutics offers the ability to increase efficacy while limiting off-target toxicity. In this work we focused on targeting death receptor 5 (DR5), which is highly expressed by cancer cells, and upon binding, triggers programmed cell death. Hence, a nanostructure targeting DR5 would act as a dual targeting and therapeutic agent. We report here on a peptide amphiphile (PA) containing a dimeric, cyclic peptide that self-assembles into cylindrical supramolecular nanofibers and targets DR5. Coassembly of the DR5-targeting PA and a pegylated PA creates a supramolecular nanoscale construct with enhanced binding affinity to DR5 relative to a monomeric targeting PA, and was found to be cytotoxic in vitro. When combined with the chemotherapy paclitaxel, DR5-targeting carriers showed potent antitumor activity in vivo, demonstrating the multifunctional capabilities of peptide-based supramolecular nanostructures.

TRAIL-Inspired Multivalent Dextran Conjugates Efficiently Induce Apoptosis upon DR5 Receptor Clustering.

Triggering apoptosis of tumor cells has been in focus of cancer-inspired research since decades. As clustering of death receptor 5 (DR5), which is overexpressed on various cancer cells, leads to formation of the death-inducing signaling cascade (DISC), DR5 has recently become a promising target for tumor treatment. Herein, we demonstrate that covalent multimerization of a death receptor targeting peptide (DR5TP) on a dextran scaffold generates potent apoptosis-inducing conjugates (EC50 =2-20 nm). A higher conformational flexibility compared to reported DR5TP multimerization approaches, introduced by the polysaccharide framework compensates the reported need for the defined ligand orientation that was considered as essential prerequisite for effective receptor clustering and apoptosis induction. Enzyme-catalyzed ligation of a hydrophilic dextran conjugate bearing multiple DR5-targeting sites to a human fragment crystallizable (Fc) receptor did not affect the potency (EC50 =2-7 nm), providing an option for improved in vivo half-life and prospective conjugation to an antibody of interest in view of bispecific tumor targeting.

Molecular Mode of Action of TRAIL Receptor Agonists-Common Principles and Their Translational Exploitation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAILR1/death receptor 4 (DR4) and TRAILR2/DR5 trigger cell death in many cancer cells but rarely exert cytotoxic activity on non-transformed cells. Against this background, a variety of recombinant TRAIL variants and anti-TRAIL death receptor antibodies have been developed and tested in preclinical and clinical studies. Despite promising results from mice tumor models, TRAIL death receptor targeting has failed so far in clinical studies to show satisfying anti-tumor efficacy. These disappointing results can largely be explained by two issues: First, tumor cells can acquire TRAIL resistance by several mechanisms defining a need for combination therapies with appropriate sensitizing drugs. Second, there is now growing preclinical evidence that soluble TRAIL variants but also bivalent anti-TRAIL death receptor antibodies typically require oligomerization or plasma membrane anchoring to achieve maximum activity. This review discusses the need for oligomerization and plasma membrane attachment for the activity of TRAIL death receptor agonists in view of what is known about the molecular mechanisms of how TRAIL death receptors trigger intracellular cell death signaling. In particular, it will be highlighted which consequences this has for the development of next generation TRAIL death receptor agonists and their potential clinical application.

Silica nanoparticles induce spermatocyte cell apoptosis through microRNA-2861 targeting death receptor pathway

Silica nanoparticles (SiNPs) are found in the environmental particulate matter and have been proved to pose an adverse effect on fertility. However, the relationship between miRNA and apoptosis induced by SiNPs in spermatogenesis and its underlying mechanism remains confusing. Therefore, the present study was designed to investigate the toxic effects of SiNPs on spermatogenic cells mediated through miRNAs. Spermatocyte cells were divided into 0 μg/mL and 5 μg/mL SiNPs groups, and the cells were collected and analyzed after passaging for 1, 10, 20, and 30 generations. miRNA profile and mRNA profile of spermatocyte cells were measured after exposure to SiNPs for 30 generations. Further, mimics and inhibitors of miRNA were used to verify the relationship between miRNA and their predicted target genes in the 30th-generation cells. The results showed that the degree of cell apoptosis in the SiNPs group significantly increased in the 30th generation. After exposure to SiNPs for 30 generations, the expression of 15 miRNAs was altered, including 5 upregulated miRNAs and 10 downregulated miRNAs. Of the 15 miRNAs, miR-138 and miR-2861 were related to the death receptor pathway. The miR-2861 mimic could target to regulate the mRNA expression of fas/fasl/ripk1 and increase the protein expression of Fas/FasL/RIPK1/FADD/caspase-8/caspase-3 of spermatogenic cells in the 30th generation, while the miR-138 inhibitor could not. In conclusion, SiNPs could cause apoptosis of spermatocyte cells by inhibiting the expression of miRNA-2861, thereby resulting in the upregulation of mRNA expression of fas/fasl/ripk1 and activating the death receptor pathway of spermatocyte cells. miRNA-2861 could be considered a biomarker of the toxic effect of SiNPs on spermatocyte cells.The main finding: Silica nanoparticles induce apoptosis in spermatocyte cells through microRNA-2861 inhibition, thereby upregulating mRNA expression of fas/fasl/ripk1 and activating the death receptor pathway of spermatocyte cells.

SMAC mimetics as potential cancer therapeutics in myeloid malignancies.

Evasion of apoptosis has been identified as one of the essential hallmarks of cancer. Inhibitor of apoptosis proteins (IAPs) are implicated in a host of myeloid malignancies, providing the rationale for strategies aimed at neutralizing IAPs to lower the cancer cell apoptosis threshold. Modes of IAP antagonism may include down-regulating IAP expression, up-regulating endogenous pro-apoptotic proteins, such as tumour necrosis factor-α or Fas ligand, or directly antagonizing IAP activity against caspases. Direct targeting of IAPs using mimetics of the second mitochondria-derived activator of caspase (SMAC) protein has shown therapeutic promise by sensitizing the effect of chemotherapy on malignant cells. In pre-clinical studies, SMAC mimetics have demonstrated broad synergistic activity with a wide range of therapeutics, including cytotoxic chemotherapy, receptor tyrosine kinase inhibitors, agents targeting death receptors and alternative mechanisms of cell death, such as necroptosis or autophagy and immune check point blockade. SMAC mimetics represent a novel approach for further investigation in patients with high-risk, chemo-refractory blood cancers, as single agents or in thoughtfully selected combinations. In this review, we discuss the development and therapeutic rationale of small molecule SMAC mimetics, with an emphasis on agents in clinical development for myeloid malignancies.

The anticonvulsive Phenhydan\xae suppresses extrinsic cell death

Different forms of regulated cell death-like apoptosis and necroptosis contribute to the pathophysiology of clinical conditions including ischemia-reperfusion injury, myocardial infarction, sepsis, and multiple sclerosis. In particular, the kinase activity of the receptor-interacting serine/threonine protein kinase 1 (RIPK1) is crucial for cell fate in inflammation and cell death. However, despite its involvement in pathological conditions, no pharmacologic inhibitor of RIPK1-mediated cell death is currently in clinical use. Herein, we screened a collection of clinical compounds to assess their ability to modulate RIPK1-mediated cell death. Our small-scale screen identified the anti-epilepsy drug Phenhydan® as a potent inhibitor of death receptor-induced necroptosis and apoptosis. Accordingly, Phenhydan® blocked activation of necrosome formation/activation as well as death receptor-induced NF-κB signaling by influencing the membrane function of cells, such as lipid raft formation, thus exerting an inhibitory effect on pathophysiologic cell death processes. By targeting death receptor signaling, the already FDA-approved Phenhydan® may provide new therapeutic strategies for inflammation-driven diseases caused by aberrant cell death.

A Single-Agent Dual-Specificity Targeting of FOLR1 and DR5 as an Effective Strategy for Ovarian Cancer

Therapeutic antibodies targeting ovarian cancer (OvCa)-enriched receptors have largely been disappointing due to limited tumor-specific antibody-dependent cellular cytotoxicity. Here we report a symbiotic approach that is highly selective and superior compared with investigational clinical antibodies. This bispecific-anchored cytotoxicity activator antibody is rationally designed to instigate "cis" and "trans" cytotoxicity by combining specificities against folate receptor alpha-1 (FOLR1) and death receptor 5 (DR5). Whereas the invivo agonist DR5 signaling requires FcγRIIB interaction, the FOLR1 anchor functions as a primary clustering point to retain and maintain a high level of tumor-specific apoptosis. The presented proof of concept study strategically makes use of a tumor cell-enriched anchor receptor for agonist death receptor targeting to potentially generate a clinically viable strategy for OvCa.

Combination of novel DR5 targeting agonistic scFv antibody TR2-3 with cisplatin shows enhanced synergistic antitumor activity in vitro and in vivo

ObjectivesTo investigate the antitumor activity of a novel agonistic single chain fragment variable (scFv) antibody TR2-3 targeting death receptor 5 (DR5) combined with cisplatin in vitro and in vivo. MethodsThe in vitro cytotoxic effects of TR2-3 and cisplatin, alone or in combination on human cancer cell lines COLO205 and MDA-MB-231 were evaluated using the MTT assay. The apoptosis in cancer cells was evaluated by an Annexin V-PE apoptosis detection kit and flow cytometry. The mRNA and protein levels of DR5 were analyzed by real-time PCR and Western blot, respectively. Additionally, the in vivo antitumor activity of TR2-3 combined with cisplatin was evaluated in a xenograft model. ResultsThe combination treatment with TR2-3 and cisplatin for 24 h on COLO205 and MDA-MB-231 cells showed significant cytotoxicity effects by MTT assay, compared with the alone treatment. Consistent with cell viability results, the cisplatin enhanced the apoptosis-inducing effects of TR2-3 in the COLO205 cells and MDA-MB-231 cells by flow cytometry. In addition, treatment with cisplatin alone for 24 h resulted in significantly up-regulating the mRNA and protein levels of DR5 in both COLO205 and MDA-MB-231 cell lines by q-PCR and Western blot assay. Moreover, the cytotoxic effects of TR2-3 can be blocked by adding the soluble DR5, and the blocking rate can be greatly reduced by co-treatment with cisplatin. These results indicated that cisplatin sensitized COLO205 and MDA-MB-231 cancer cells to TR2-3-mediated apoptosis by up-regulation of DR5 expression. Furthermore, combination therapy with TR2-3 and cisplatin enhanced tumor growth inhibition compared to treatment with TR2-3 or cisplatin alone in mice bearing COLO205 xenograft tumors. ConclusionsOur findings suggest that cisplatin enhanced the antitumor activity of TR2-3 in COLO205 and MDA-MB-231 cancer cells through up-regulation of DR5 expression. The TR2-3 combined with cisplatin may be a promising treatment for cancer therapy.

Targeting death receptors for drug-resistant cancer therapy: Codelivery of pTRAIL and monensin using dual-targeting and stimuli-responsive self-assembling nanocomposites

Chemoresistance remains a formidable hurdle against cancer therapy. Seeking for novel therapy strategies is an urgent need for those who no longer benefit from chemotherapy. Chemoresistance is usually associated with the dysfunction of intrinsic apoptosis. Targeting extrinsic apoptosis via TRAIL signaling and the death receptors could be a potential solution to treat chemoresistant cancer. A highly biocompatible nano system for codelivery of the TRAIL DNA and the death receptor sensitizer monensin was developed, in which low-molecular-weight PEI (LMW-PEI) was crosslinked by the sulfhydryl cyclodextrin via disulfide bonds, and then bound with DNA, thus forming the bioreducible polyplex cores. In addition, the cyclodextrin also functioned as a carrier for the hydrophobic monensin via host-guest inclusion. Poly-γ-glutamic acid (γ-PGA) was used to modify the polyplex core via charge interaction. The γ-PGA corona can specifically bind with the tumor-associated gamma-glutamyl transpeptidase (GGT) overexpressed on the tumor cells, and achieve tumor-targeting delivery. Moreover, the tumor-homing peptide RGD-modified γ-PGA was also prepared as the surface coating materials for further improving gene delivery efficiency. This gene delivery system was characterized by the dual ligand-targeting, dual stimuli-responsive features. The ligands of RGD and γ-PGA can target the tumor-associated receptors (i.e., integrin and GGT). The conformation of γ-PGA is pH-sensitive, and the tumor acidic micro environments could trigger the detachment of surface-coating γ-PGA. The disulfide crosslinking LMW-PEI is redox-sensitive, and its fast disassembling in the tumor cells could favor the efficient gene delivery. The anti-tumor efficacy was demonstrated both in vitro and in vivo. Moreover, MYC-mediated synthetic lethality could be an important mechanism for overcoming the drug resistance. An important finding of our studies is the demonstration of the in vivo treatment efficacy of TRAIL/monensin, thus providing a potential novel therapeutic strategy for overcoming drug-resistant cancer.

Antibodies and Derivatives Targeting DR4 and DR5 for Cancer Therapy.

Developing therapeutics that induce apoptosis in cancer cells has become an increasingly attractive approach for the past 30 years. The discovery of tumor necrosis factor (TNF) superfamily members and more specifically TNF-related apoptosis-inducing ligand (TRAIL), the only cytokine of the family capable of eradicating selectively cancer cells, led to the development of numerous TRAIL derivatives targeting death receptor 4 (DR4) and death receptor 5 (DR5) for cancer therapy. With a few exceptions, preliminary attempts to use recombinant TRAIL, agonistic antibodies, or derivatives to target TRAIL agonist receptors in the clinic have been fairly disappointing. Nonetheless, a tremendous effort, worldwide, is being put into the development of novel strategic options to target TRAIL receptors. Antibodies and derivatives allow for the design of novel and efficient agonists. We summarize and discuss here the advantages and drawbacks of the soar of TRAIL therapeutics, from the first developments to the next generation of agonistic products, with a particular insight on new concepts.

A Novel Fully Human Agonistic Single Chain Fragment Variable Antibody Targeting Death Receptor 5 with Potent Antitumor Activity In Vitro and In Vivo.

Agonistic antibodies, which bind specifically to death receptor 5 (DR5), can trigger apoptosis in tumor cells through the extrinsic pathway. In this present study, we describe the use of a phage display to isolate a novel fully human agonistic single chain fragment variable (scFv) antibody, which targets DR5. After five rounds of panning a large (1.2 × 108 clones) phage display library on DR5, a total of over 4000 scFv clones were screened by the phage ELISA. After screening for agonism in a cell-viability assay in vitro, a novel DR5-specific scFv antibody TR2-3 was isolated, which inhibited COLO205 and MDA-MB-231 tumor cell growth without any cross-linking agents. The activity of TR2-3 in inducing apoptosis in cancer cells was evaluated by using an Annexin V-PE apoptosis detection kit in combination with flow cytometry and the Hoechst 33342 and propidium iodide double staining analysis. In addition, the activation of caspase-dependent apoptosis was evaluated by Western blot assays. The results indicated that TR2-3 induced robust apoptosis of the COLO205 and MDA-MB-231 cells in a dose-dependent and time-dependent manner, while it remarkably upregulated the cleavage of caspase-3 and caspase-8. Furthermore, TR2-3 suppressed the tumor growth significantly in the xenograft model. Taken together, these data suggest that TR2-3 exhibited potent antitumor activity both in vitro and in vivo. This work provides a novel human antibody, which might be a promising candidate for cancer therapy by targeting DR5.

N-myc downstream-regulated gene 1 promotes apoptosis in colorectal cancer via up-regulating death receptor 4.

The aim of this study was to evaluate the clinical significance of N-myc downstream-regulated gene 1 (NDRG1) in colorectal cancer (CRC) patients and to explore the mechanisms governing the role of NDRG1 in apoptosis of CRC cells. In the current study, we found that NDRG1 was a prognostic marker of CRC patients. Moreover, NDRG1 expression negatively correlated to tumor size and clinical TNM stage, suggesting that NDRG1 might act as a tumor suppressor by inhibiting proliferation or inducing apoptosis in CRC. Consistently, substantial apoptosis was observed in vitro and in vivo in the presence of NDRG1. From a mechanistic standpoint, we discovered that NDRG1 was able to prevent death receptor 4 from degradation induced by MARCH-8, a member of the membrane-associated RING-CH (MARCH) ubiquitin ligase family. As a consequence, CRC cells expressing NDRG1 were more sensitive to reagents targeting death receptors such as tumor necrosis factor-related apoptosis-inducing ligands (TRAIL). Additionally, the pro-apoptotic effect of NDRG1 was also validated in mouse xenograft model. In conclusion, our results provided further insights of the pivotal role of NDRG1 in apoptosis initiated by death receptors and demonstrated a novel marker to predict the sensitivity of CRC to TRAIL treatment in future clinical study.