[Show abstract][Hide abstract]ABSTRACT: Background MicroRNAs have been shown to be important regulators of the immune response and the development of the immune system. It was reported that microRNA-125b (miR-125b) was down-regulated in macrophages challenged with endotoxin. However, little is known about the function and mechanism of action of miR-125b in macrophage activation. Macrophages use L-arginine to synthesize nitric oxide (NO) through inducible NO synthase (iNOS), and the released NO contributes to the tumoricidal activity of macrophages. Methods Luciferase reporter assays were employed to validate regulation of a putative target of miR-125b. The effect of miR-125b on endogenous levels of this target were subsequently confirmed via Western blot. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine the expression level of miR-125b in macrophage. MTS assays were conducted to explore the impact of miR-125b overexpression on the cell viability of 4T1 cells. Results Here, we demonstrate that mmu-miR-125b overexpression suppresses NO production in activated macrophages and that LPS-activated macrophages with overexpressed mmu-miR-125b promote 4T1 tumor cell proliferation in vitro and 4T1 tumor growth in vivo. CCNA2 and eEF2K are the direct and functional targets of mmu-miR-125b in macrophages; CCNA2 and eEF2K expression was knocked down, which mimicked the mmu-miR-125b overexpression phenotype. Conclusions These data suggest that mmu-miR-125b decreases NO production in activated macrophages at least partially by suppressing eEF2K and CCNA2 expression.
[Show abstract][Hide abstract]ABSTRACT: Like anti-Fas monoclonal antibodies, some monoclonal antibod-ies against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors have tumoricidal activity too. In this article we report a novel mouse anti-human DR5 monoclonal antibody, AD5– 10, that induces apoptosis of various tumor cell lines in the absence of second cross-linking in vitro and showed strong tumoricidal activity in vivo. AD5–10 does not compete with TRAIL for binding to DR5 and synergizes with TRAIL to induce apoptosis of tumor cells. AD5–10 induces both caspase-dependent and caspase-inde-pendent cell death in Jurkat cells, whereas TRAIL induces only caspase-dependent cell death. We show for the first time that DR5 can mediate caspase-independent cell death, and DR5 can mediate distinct cell signals when interacting with different extracellular proteins. Studies on AD5–10 help us to understand more on the functions of DR5 and may provide new ideas for cancer immunotherapy. Tumor necrosis factor (TNF) 2 -related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily with the ability to induce apoptosis in a wide variety of transformed cell lines of diverse origin (1). At least five receptors for TRAIL have been identified so far. Two of them, DR4 (TRAIL-R1) and DR5 (TRAIL-R2/TRICK2) (2– 4), are capa-ble of transducing an apoptosis signal, whereas the other three, decoy receptor (DcR) 1 (TRAIL-R3) (5), DcR2 (TRAIL-R4) (6), and osteopro-tegerin (7), serve as decoy receptors to block TRAIL-mediated apopto-sis. DR4 and DR5 share a common intracellular domain, called the death domain (DD), which is indispensable for initiation of the intracellular signaling cascade leading to cell death. The death domain motif is also found in the cytoplasmic adaptor proteins, such as Fas-associated pro-tein with death domain (FADD) (8), TNF-R1-associated death domain protein (TRADD) (9), and receptor-interacting protein (RIP) (10), etc. These adaptor proteins are essential for the intracellular signals medi-ated by DR4 and DR5 (11). TRAIL triggers multiple cell signals, includ-ing the activation of apoptotic caspase cascade, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and NF-B (11–13). In contrast to TNFs and Fas ligand, TRAIL has been known to induce apoptosis in a variety of tumor cells and some virally infected cells but not in most normal cells. The potential and safety of soluble TRAIL (sTRAIL) as an anticancer therapeutic agent have been demonstrated in mice and non-human primates (14, 15). However, increasing experi-mental evidence on TRAIL-inducing apoptosis of normal cells (espe-cially hepatocytes) were reported in recent studies (16, 17), arguing against the potential usefulness and safety of soluble TRAIL in cancer therapy. Meanwhile, there were also reports demonstrating that recom-binant TRAIL without exogenous sequence tags was nontoxic to human hepatocytes both in vitro (18, 19) and in chimeric mice (20). In addition to sTRAIL, monoclonal antibodies (mAbs) against TRAIL receptors with tumoricidal activity are also potential candidates for cancer ther-apy. There are a number of agonistic mAbs against human DR4 or DR5 reported in previous studies (21–23), most of which need cross-linkers to ensure effective killing of tumor cells (21, 22). In 2001, Ichikawa et al. (23) reported a mouse anti-DR5 mAb, TRA-8, that showed strong tumoricidal activity in the absence of cross-linking and had no hepato-cyte toxicity. TRA-8 competes with TRAIL for binding to DR5 and almost entirely mimics the apoptosis-inducing mechanism of TRAIL. The authors believed that DR5 was not sufficient to trigger apoptosis in normal hepatocytes (23). However, a recent study showed that at least some anti-DR5 and anti-DR4 mAbs did induce human hepatocytes apo-ptosis (24). Therefore, we cannot draw a definite conclusion on the hepatocyte toxicity of soluble TRAIL or mAbs against TRAIL receptors now. Studies on the mechanism of anti-DR5 mAbs will help us to under-stand the complicated signal pathways mediated by DR5. In this article, we report studies on AD5–10, a novel monoclonal antibody against human DR5. AD5–10 induces apoptosis in various tumor cell lines in the absence of second cross-linking in vitro and exhibits a strong tumoricidal activity in vivo. AD5–10 does not induce cell death of human normal hepatocytes and primary peripheral blood lymphocytes, and the injection with high doses of AD5–10 in mice causes no toxic reaction in liver, spleen, and kidney. Unlike TRA-8, AD5–10 does not compete with TRAIL for binding to DR5, and there is a synergistic effect between TRAIL and AD5–10 on their tumoricidal activity. Downstream cell signals induced by AD5–10 and TRAIL were also compared. Both TRAIL and AD5–10 activate caspase cascade and induce a classical apoptosis in Jurkat cells. Interestingly, AD5–10 kills
[Show abstract][Hide abstract]ABSTRACT: Unlabelled:
The human T-cell leukemia virus type 1 (HTLV-1) Tax protein is considered to play a central role in the process that leads to adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 Tax-expressing cells show resistance to apoptosis induced by Fas ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). The regulation of Tax on the autophagy pathway in HeLa cells and peripheral T cells was recently reported, but the function and underlying molecular mechanism of the Tax-regulated autophagy are not yet well defined. Here, we report that HTLV-1 Tax deregulates the autophagy pathway, which plays a protective role during the death receptor (DR)-mediated apoptosis of human U251 astroglioma cells. The cellular FLICE-inhibitory protein (c-FLIP), which is upregulated by Tax, also contributes to the resistance against DR-mediated apoptosis. Both Tax-induced autophagy and Tax-induced c-FLIP expression require Tax-induced activation of IκB kinases (IKK). Furthermore, Tax-induced c-FLIP expression is regulated through the Tax-IKK-NF-κB signaling pathway, whereas Tax-triggered autophagy depends on the activation of IKK but not the activation of NF-κB. In addition, DR-mediated apoptosis is correlated with the degradation of Tax, which can be facilitated by the inhibitors of autophagy.
Our study reveals that Tax-deregulated autophagy is a protective mechanism for DR-mediated apoptosis. The molecular mechanism of Tax-induced autophagy is also illuminated, which is different from Tax-increased c-FLIP. Tax can be degraded via manipulation of autophagy and TRAIL-induced apoptosis. These results outline a complex regulatory network between and among apoptosis, autophagy, and Tax and also present evidence that autophagy represents a new possible target for therapeutic intervention for the HTVL-1 related diseases.
Full-text · Article · Dec 2013 · Journal of Virology
[Show abstract][Hide abstract]ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor (TRAIL-R) play important roles in immune regulation and cancer cell death. Although TRAIL has been shown to induce chemokine release in various tumor cells, the function of TRAIL-R in the development of colitis and colitis-associated carcinogenesis has not been explored.In this study, we found that TRAIL-R-deficient mice exhibited a higher incidence of colitis and colitis-associated cancer than that of wild-type (WT) mice, and TRAIL-R expression was down-regulated in WT mice that were fed dextran sulfate sodium. Chemokines, including CCL2 and CXCL1, were highly expressed in the serum and inflammatory colon tissues of TRAIL-R(-/-) mice compared to WT mice, and TRAIL-R(-/-) mice showed a marked infiltration of immune cells during colitis. Hyperactivation of JNK and NF-κB in colon epithelial cells was also observed, which correlated with the severity of colonic inflammation in TRAIL-R(-/-) mice.These data suggest that TRAIL-R plays a protective role in chemical-induced colon injury and negatively regulates mucosal immune responses. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract]ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered a promising agent for cancer therapy, as this molecule induces apoptosis specifically in various cancer cells. Apart from apoptosis, TRAIL also induces non-apoptotic signals, such as those for autophagy, proliferation, and metastasis in cancer cells. In the present study, we report that TRAIL suppressed CXCR4-mediated human breast cancer MDA-MB-231 cell migration by up-regulating miR-146a expression through NF-κB. TRAIL receptor 1 (TRAIL-R1, DR4) was highly expressed in TRAIL-treated MDA-MB-231 cells. A neutralization antibody against DR4 specifically blocked TRAIL-induced NF-κB activation and miR-146a expression. These results were confirmed in a human breast cancer xenograft mouse model, suggesting that TRAIL significantly enhanced miR-146a expression and suppressed CXCR4 expression, indicating that TRAIL-induced miR-146a up-regulation is negatively associated with CXCR4 expression. These findings suggest that TRAIL-induced miR146a expression suppresses CXCR4-mediated human breast cancer migration and provide further insight into the non-apoptotic function of TRAIL in the prevention of metastasis as a therapy for breast cancer. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract]ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a promising and novel anticancer cytokine, specifically kills numerous tumor cells by apoptosis. However, some malignancies are resistant to TRAIL treatment in clinical trials, thus limiting its therapeutic potential. In the present study, the TRAIL-resistant murine hepatocellular carcinoma cell line Hepa1-6 was used to elucidate the physiological significance of TRAIL resistance, especially with respect to the immune regulatory function of TRAIL. Hepa1-6 cells were resistant to TRAIL-induced apoptosis in vitro; however, intratumoral injection of recombinant soluble TRAIL inhibited tumor growth and prolonged survival time in tumor-bearing mice. Local TRAIL treatment decreased the number of intratumoral CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) but did not affect CD4(+)CD25(+)Foxp3(+) Tregs in the draining lymph nodes and spleen. Further investigation showed that TRAIL induced apoptosis of tumor-activated CD4(+)CD25(+)Foxp3(+) Tregs, but not of CD4(+)CD25(-) T cells. Moreover, mouse TRAIL receptor DR5 expression was detected on the surface of the tumor-infiltrating CD4(+)CD25(+)Foxp3(+) Tregs, but not on naïve CD4(+)CD25(+)Foxp3(+) Tregs. Interestingly, intratumoral injection of TRAIL not only decreased the number of CD4(+)CD25(+)Foxp3(+) Tregs but also increased the number of tumor-specific CD8(+) CTL and augmented their cytotoxicity to the tumor cells. These data provide the novel evidence for an immune regulatory function of TRAIL and may shed light on the clinical application of TRAIL.
No preview · Article · Nov 2012 · Cancer Immunology and Immunotherapy
[Show abstract][Hide abstract]ABSTRACT: Construct expression vectors of pCMV-DR4-HA and pCMV-PRMT5-Flag, and transfect them into HEK293 cells to identify the interaction between TRAIL-R1 and PRMT5 and the molecular mechanism underlying DR4-mediated inhibition of chemokine CCL20 release via TRAIL receptor 1 (DR4). Inflammatory cytokine was detected by RT-PCR and ELISA after TRAIL-R1 and/or PRMT5 transfection, respectively. NF-κB activity was detected by Dual Luciferase Reporter Gene Assay. ERK1/2 phosphorylation was analyzed by Western blot. PRMT5 could inhibit DR4-activated NF-κB activity and ERK1/2 phosphorylation. PRMT5 could inhibit NF-κB activition, ERK1/2 phosphorylation as well as CCL20 secretion via binding with DR4 in HEK293 cell, suggesting that PRMT5 may involve in DR4 dependent immune regulation.
Preview · Article · Nov 2012 · Chinese Science Bulletin
[Show abstract][Hide abstract]ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumor cells. TRAIL receptor 2 (DR5) expression is high in tumor cells, transformed cells, and clinical tumor specimens and is low in most normal cells and tissues; therefore, DR5 is considered an attractive target for cancer therapy. In this study, HMCAZ5, a novel mouse-human chimeric antibody based on AD5-10, was generated and stably expressed in CHO-dhfr(-) cells. Highly purified HMCAZ5 exhibits a high affinity for the receptor that is equal to the parental mouse antibody, induces apoptosis in various cancer cells but not in normal hepatocytes, and elicits both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in various human cancer cells. The anthracycline anticancer drug epirubicin (EPB) synergizes the cytotoxicity of HMCAZ5 in cancer cells by upregulating DR5 expression on the cell surfaces, enhancing p53 expression, Bid cleavage, and JNK phosphorylation and downregulating c-FLIP expression and Akt phosphorylation. Moreover, HMCAZ5 alone suppresses tumor growth, and EPB augments the tumoricidal activity in human colorectal and hepatocellular tumor xenografts in athymic nude mice. These data suggest that the anti-DR5 chimeric antibody HMCAZ5 may have a clinical use and represents a useful immunological strategy, in combination with chemotherapy, for the treatment of cancer.
Preview · Article · Sep 2012 · International Union of Biochemistry and Molecular Biology Life
[Show abstract][Hide abstract]ABSTRACT: Age-associated immune dysfunction, characterized by increased systemic levels of cytokines, manifests as an increased susceptibility to infections. Thus, understanding these negative regulators of the immune response has paved the way to delineating signaling pathways that impact immune senescence. In the present study, we found that miR-146a, which negatively regulated the expression of IL-1β and IL-6, was highly expressed in aged mice. However, there was a lack of response to the stimulation of lipopolysaccharide (LPS) and proinflammatory cytokines in macrophages of aged mice. As a result, the negative feedback regulation loop with miR-146a involving down-regulation of inflammation factors was interrupted in aged mice. Aberrant NF-κB binding to the miR-146a promoter was demonstrated to be associated with the abnormal expression of miR-146a in aged mice. The DNA methyltransferase inhibitor (5-aza-2-deoxycytidine) and the histone deacetylase inhibitor [trichostatin A (TSA)] both significantly up-regulated miR-146a transcriptional activation by altering the DNA-binding activity of NF-κB in macrophages isolated from aged mice, which suggests that DNA methylation and histone acetylation are involved in the suppression of age-dependent miR-146a expression. Additionally, high levels of histone deacetylase (HDACs) expressions contributed to the inhibition of miR-146a expression in LPS-stimulated macrophages from aged mice in vitro. While the suppression of HDACs activities by TSA could improve LPS-induced inflammatory responses owing to up-regulation of miR-146a expression in macrophages from aged mice. These data indicate that the dysregulated expression of miR-146a results in the age-associated dysfunction of macrophages, and miR-146a may be a good target for the treatment of age-related inflammatory diseases.
[Show abstract][Hide abstract]ABSTRACT: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces tumor cell death via death receptors on target cells, without adverse effects on most normal cells. Its receptors are therefore an attractive target for antibody-mediated tumor therapy. Here, we report the creation of a lentivirus vector constructed by linking the heavy chain and the light chain of the antibody with a 2A/furin self-processing peptide in a single open reading frame that expresses a novel chimeric antibody (named as zaptuximab) with tumoricidal activity, which is consisted of the variable region of a mouse anti-human DR5 monoclonal antibody, AD5-10, and the constant region of human immunoglobulin G1. Lentivirus-expressed zaptuximab bound specifically to its antigen, DR5, and exhibited significant apoptosis-inducing activity in various tumor cell lines. The packaged recombinant virus lenti-HF2AL showed strong apoptosis-inducing activity in vitro. Meanwhile, inoculated subcutaneous human colon HCT116 tumor formation in nude mice were inhibited significantly. Moreover, there was a synergistic effect of mitomycin C (MMC) on the observed tumoricidal efficacy, prolonging the life span of nude mice with orthotopic human lung tumor cancers. These data suggest that lentivirus-mediated, 2A peptide-based anti-DR5 chimeric antibody expression may have clinical utility as an anticancer treatment and may represent a rational adjuvant therapy in combination with chemotherapy.
[Show abstract][Hide abstract]ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) could induce apoptosis of HIV-1-infected monocyte-derived macrophage (MDM), but the molecular mechanisms are not well understood.
By using an HIV-1 Env-pseudotyped virus (HIV-1 PV)-infected MDM cell model we demonstrate that HIV-1 PV infection down-regulates the expression of TRAIL decoy receptor 1 (DcR1) and 2 (DcR2), and cellular FLICE-inhibitory protein (c-FLIP), but dose not affect the expression of death receptor 4 and 5 (DR4, DR5), and Bcl-2 family members in MDM cells. Furthermore, recombinant soluble TRAIL and an agonistic anti-DR5 antibody, AD5-10, treatment stimulates reactive oxygen species (ROS) generation and JNK phosphorylation.
HIV infection facilitates TRIAL-induced cell death in MDM by down-regulating the expression of TRAIL decoy receptors and intracellular c-FLIP. Meanwhile, the agonistic anti-DR5 antibody, AD5-10, induces apoptosis synergistically with TRAIL in HIV-1-infected cells. ROS generation and JNK phosphorylation are involved in this process. These findings potentiate clinical usage of the combination of TRAIL and AD5-10 in eradication of HIV-infected macrophage and AIDS.
[Show abstract][Hide abstract]ABSTRACT: In the present study we demonstrate that adeno-associated virus (AAV)-mediated anti-DR5 (death receptor 5) mouse-human chimeric antibody (shorten as Adximab) expression significantly suppressed tumor cell growth by inducing apoptosis both in vitro and in vivo. The viral-expressed and cell-secreted Adximab efficiently bound DR5 with an affinity of 0.7nM and induced apoptosis of various tumor cells, but not normal cells. A single intramuscular injection of recombinant AAV particles resulted in a stable expression of Adximab in mouse serum for at least 70days. AAV-mediated Adximab expression led to a significant suppression of tumor growth in nude mice receiving xenografts of human liver and colon cancer. These data suggest that chimeric antibody gene transfer may provide an alternative strategy for the therapy of varieties of cancers.
[Show abstract][Hide abstract]ABSTRACT: Effect of combining rsTRAIL with cisplatin in vitro. KB cells were treated with either rsTRAIL (400 ng/mL) or cisplatin (100-1000 ng/mL) or combination treatment for 24 h. Apoptosis was determined by MTT assay. Values are mean of three independent experiments with error bar representing standard deviation of the mean.
[Show abstract][Hide abstract]ABSTRACT: Adeno-associated virus-2 (AAV-2)-mediated gene therapy is quite suitable for local or regional application in head and neck cancer squamous cell carcinoma (HNSCC). However, its low transduction efficiency has limited its further development as a therapeutic agent. DNA damaging agents have been shown to enhance AAV-mediated transgene expression. Cisplatin, one of the most effective chemotherapeutic agents, has been recognized to cause cancer cell death by apoptosis with a severe toxicity. This study aims to evaluate the role of cisplatin in AAV-mediated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and the effect on HNSCC both in vitro and in vivo.
Five human HNSCC cell lines were treated with recombinant soluble TRAIL (rsTRAIL) and infected with AAV/TRAIL to estimate the sensitivity of the cancer cells to TRAIL-induced cytotoxicity. KB cells were infected with AAV/EGFP with or without cisplatin pretreatment to evaluate the effect of cisplatin on AAV-mediated gene expression. TRAIL expression was detected by ELISA and Western blot. Cytotoxicity was measured by MTT assay and Western blot analysis for caspase-3 and -8 activations. Following the in vitro experiments, TRAIL expression and its tumoricidal activity were analyzed in nude mice with subcutaneous xenografts of HNSCC.
HNSCC cell lines showed different sensitivities to rsTRAIL, and KB cells possessed both highest transduction efficacy of AAV and sensitivity to TRAIL among five cell lines. Preincubation of KB cells with subtherapeutic dosage of cisplatin significantly augmented AAV-mediated transgene expression in a heparin sulfate proteoglycan (HSPG)-dependent manner. Furthermore, cisplatin enhanced the killing efficacy of AAV/TRAIL by 3-fold on KB cell line. The AAV mediated TRAIL expression was observed in the xenografted tumors and significantly enhanced by cisplatin. AAV/TRAIL suppressed the tumors growth and cisplatin augmented the tumoricidal activity by two-fold. Furthermore, Combination treatment reduced cisplatin-caused body weight loss in nude mice.
The combination of AAV-mediated TRAIL gene expression and cisplatin had synergistic therapeutic effects on head and neck cancers and reduced the potential toxicity of cisplatin. These findings suggest that the combination of AAV/TRAIL and cisplatin may be a promising strategy for HNSCC therapy.
[Show abstract][Hide abstract]ABSTRACT: This study tested the hypothesis that TRAIL could play a role in regulating monocyte migration. TRAIL has been widely studied for its anti-tumor function and signaling mechanisms. Using chemotaxis and mouse air-pouch model analyses, we determined that TRAIL-induced chemotactic migration of THP-1 human leukemia and LPS-primed primary human monocytes as well as LPS-stimulated BALB/c mouse monocytes in vivo. To expand the understanding of the TRAIL signaling pathway in this process, we found that the TRAIL receptor DR4 was highly expressed in THP-1 and LPS-primed primary monocytes but not in the non-primed primary monocytes. DR4 neutralization antibody specifically suppressed TRAIL-induced migration of the monocytes. Furthermore, PI3K, Rho GTPase and its downstream effectors, MLC and Pak1, were activated during cell migration. PI3K inhibitors and dominant negative mutants of RhoGTPase blocked monocyte migration toward TRAIL, indicating that PI3K and RhoGTPases were involved in the migration signaling. The DR4 neutralization antibody blocked the activation of PI3K and Rho GTPase effectors in the cells. Thus, these data support the hypothesis that TRAIL induces monocyte migration mediated by TRAIL receptor DR4 via the RhoGTPase signaling pathway. This study is expected to provide novel evidence of the non-apoptotic function of TRAIL in immune defense.
No preview · Article · Sep 2010 · Molecular Immunology
[Show abstract][Hide abstract]ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand receptors death receptor (DR) 4 and DR5 are potential targets for antibody-based cancer therapy. Activation of the proapoptotic DR5 in various cancer cells triggers the extrinsic and/or intrinsic pathway of apoptosis. It has been shown that there are several functional domains in the DR5 extracellular domain. The cysteine-rich domains of DR5 have a conservative role in tumor necrosis factor-related apoptosis-inducing ligand-DR5-mediated apoptosis, and the pre-ligand assembly domain within the N1-cap contributes to the ligand-independent formation of receptor complexes. However, the role of the N-terminal region (NTR) preceding the N1-cap of DR5 remains unclear. In this study, we demonstrate that NTR could mediate DR5 activation that transmits an apoptotic signal when bound to a specific agonistic monoclonal antibody. A novel epitope in the NTR of DR5 was identified by peptide array. Antibodies against the antigenic determinant showed high affinities for DR5 and triggered caspase activation in a time-dependent manner, suggesting the NTR of DR5 might function as a potential death-inducing region. Moreover, permutation analysis showed that Leu(6) was pivotal for the interaction of DR5 and the agonistic antibody. Synthetic wild-type epitopes eliminated the cytotoxicity of all three agonistic monoclonal antibodies, AD5-10, Adie-1, and Adie-2. These results indicate that the NTR of DR5 could be a potential target site for the development of new strategies for cancer immunotherapy. Also, our findings expand the current knowledge about DR5 extracellular functional domains and provide insights into the mechanism of DR5-mediated cell death.
Preview · Article · Mar 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: In the present article, we report that DR4 or DR5 overexpression dramatically activates the release of the inflammatory cytokines IL-8, TNF-alpha, CCL20, MIP-2 and MIP-1beta in an NF-kappaB-dependent manner in 293T, MDA-MB-231 and HCT-116 cells. We showed that death receptor-mediated signals were extracellular domain-independent, whereas the effect of overexpression of the DR4 intracellular domain was much less potent. The TRADD-TRAF2-NIK-IKKalpha/beta signaling cascade, which plays an essential role in TNF-induced NF-kappaB activation, was found to be involved in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated signal transduction. The FADD-caspase signaling pathway, which has been reported to be mostly related to apoptosis, was identified as being essential for DR4 or DR5 overexpression-mediated NF-kappaB activation and cytokine secretion and crosstalks with the TRADD-TRAF2-NIK-IKKalpha/beta signaling cascade. Furthermore, a DR5 agonistic antibody (AD5-10) triggered the inflammatory cytokine release. These data, together with previous reports, provide strong evidence that TRAIL and TRAIL receptors play an important role in inflammation.
[Show abstract][Hide abstract]ABSTRACT: It is reported that the agonistic antibodies against death receptors 4 and 5 (DR4, DR5) are cytotoxic to various cancer cells. In the present study, the sensitivity of five human lung cancer cell lines to previously reported AD5-10 agonistic antibody against DR5 were investigated. Of these cell lines, A549 and small cell lung cancer showed a moderate sensitivity to AD5-10 and three other cell lines were resistant. Cell line H460 is resistant to AD5-10 despite a high level of cell-surface DR5 expression. We demonstrated that the resistance of H460 cells to AD5-10 was not related to the expression level of DR5, but the expression and cleavage of c-FLIP(L) in the cells. Inhibition of endogenous c-FLIP(L) expression by siRNA significantly enhanced AD5-10-induced cell death in these lung cancer cells. We further showed that this sensitizing effect was associated with decreased expression of Bcl-2 family proteins Bid and Bcl-X(L), change of mitochondrial membrane potential, release of cytochrome c from mitochondria, and caspase activation. Therefore, these data provide evidence that c-FLIP(L) is involved in the resistance of lung cancer cells to AD5-10-induced apoptosis. Moreover, immunohistochemistry on paraffin-embedded tissue revealed that c-FLIP(L) was expressed in 87.9% (29 of 33) of lung carcinoma tissues from the patients, but little in tissues from normal controls. This suggests that inhibition of c-FLIP(L) expression might be a potential strategy for lung cancer therapy, especially for those lung cancers resistant to the agonistic antibody against death receptors.
[Show abstract][Hide abstract]ABSTRACT: In the present study we demonstrated Jurkat leukemia cell lines of TIB152 and TIB153 with different sensitivities to recombinant soluble TRAIL cytotoxicity. TRAIL receptor death receptor 5 (DR5) was constitutively localized in the rafts in both cell lines. FADD, caspase-8, and PI3K-p85 subunit were recruited into DR5 lipid rafts of TIB152 but not in TIB153 cells. The expression and enzyme activity of acid sphingomyelinase, which digests sphingomyeline to produce ceramide and plays an essential role in lipid raft assembling, were higher in the rafts of TIB152 than in TIB153. These data provide evidences that DR5-recruited raft components contribute to the different sensitivity of Jurkat leukemia cell lines to TRAIL-induced cell death and may throw some light on the development of better therapeutic strategies for the cancer cells resistant to TRAIL treatment.
Preview · Article · Mar 2009 · International Union of Biochemistry and Molecular Biology Life