Soluble TRAIL Research Articles

Tumor cell-selective apoptosis induction through targeting of KV10.1 via bifunctional TRAIL antibody

BackgroundThe search for strategies to target ion channels for therapeutic applications has become of increasing interest. Especially, the potassium channel KV10.1 (Ether-á-go-go) is attractive as target since this surface protein is virtually not detected in normal tissue outside the central nervous system, but is expressed in approximately 70% of tumors from different origins.MethodsWe designed a single-chain antibody against an extracellular region of KV10.1 (scFv62) and fused it to the human soluble TRAIL. The KV10.1-specific scFv62 antibody -TRAIL fusion protein was expressed in CHO-K1 cells, purified by chromatography and tested for biological activity.ResultsProstate cancer cells, either positive or negative for KV10.1 were treated with the purified construct. After sensitization with cytotoxic drugs, scFv62-TRAIL induced apoptosis only in KV10.1-positive cancer cells, but not in non-tumor cells, nor in tumor cells lacking KV10.1 expression. In co-cultures with KV10.1-positive cancer cells the fusion protein also induced apoptosis in bystander KV10.1-negative cancer cells, while normal prostate epithelial cells were not affected when present as bystander.ConclusionsKV10.1 represents a novel therapeutic target for cancer. We could design a strategy that selectively kills tumor cells based on a KV10.1-specific antibody.

Plumbagin treatment leads to apoptosis in human K562 leukemia cells through increased ROS and elevated TRAIL receptor expression

This study examined the ability of plumbagin to induce apoptosis in chronic myelogenous leukemia (CML). Plumbagin exposure led to a significant reduction in cell viability and the induction of apoptosis. Mechanistically, plumbagin treatment led to elevated levels of ROS. Plumbagin-induced apoptosis was inhibited by N-acetyl l-cysteine (NAC) and PEG-catalase. Furthermore, plumbagin exposure led to elevated expression of DR4 and DR5 and increased killing through soluble TRAIL. The plumbagin-induced increase in DR4 and DR5 was inhibited by treatment with NAC. Together, this study suggests that plumbagin may be an effective treatment of CML through increased sensitivity to TRAIL-mediated killing.

Therapeutic effect of neural stem cells expressing TRAIL and bortezomib in mice with glioma xenografts

Treatment of glioblastoma remains a challenge in neuro-oncology. We investigated if treatment with neural stem cells engineered to express membrane-bound TRAIL (NSCs-mTRAIL) alone or in combination with proteasome inhibitors is a feasible therapeutic approach for experimental glioma. Glioma cells showed resistance to soluble TRAIL and proteasome inhibitors alone, but responded well to their combined treatment. In co-culture with NSCs-mTRAIL, glioma cells appeared to be more prone to apoptosis than to treatment with soluble TRAIL, which was enhanced by proteasome inhibitor bortezomib. In vivo, the survival of animals bearing intracranial glial xenografts was significantly improved by NSCs-mTRAIL. The addition of bortezomib further enhanced the efficacy of NSCs-TRAIL treated group in one of examined tumor models. These data demonstrate that therapy with NSCs-mTRAIL is a potent cell based approach for treatment of glioma. Such an approach warrants further search for therapeutics capable of increasing sensitivity of glioma cells to mTRAIL in vivo.

Molecular identification of an MHC-independent ligand recognized by a human α/β T-cell receptor

AbstractDuring an analysis of T-cell responses against human renal cell carcinoma (RCC), we identified a CD4+ T-cell line that showed TCR-mediated recognition and lysis of nearly all RCC lines regardless of MHC type. We have now elucidated the nature of the ligand for this α/β TCR, and it contains no MHC-related moiety and does not involve classic peptide processing. First, matrix metalloproteinase 14 (MMP14) expressed on RCC cells releases membrane-bound TRAIL expressed by the T cell; then, soluble TRAIL binds to its receptor DR4 (TRAIL-R1), which is expressed on tumor cells, and this TRAIL-DR4 complex is recognized by the TCR through a complementarity-determining region 3α (CDR3α)–mediated interaction. Direct and specific antigen-TCR interaction was demonstrated when the immobilized recombinant TRAIL/DR4 complex stimulated the TCR. In addition, amino acid substitutions in the CDR3α of the TCR either obliterated or enhanced target-specific recognition. This description of the molecular nature of a non-MHC target structure recognized by a naturally occurring α/β TCR not only broadens our concept of what the TCR can recognize, but also raises the question of whether such a T cell could be of clinical utility against RCC.

Abstract 2695: Lactobacillus plantarum induces TRAIL production and facilitates natural killer activity against cancer cells

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent due to its induction of apoptosis in various types of malignant tumor cells with little or no toxicity in normal cells. TRAIL is an endogeneous cytokine, and thus, it may be possible to develop agents that can induce endogeneous TRAIL production as an anti-tumor strategy. Here, we show for the first time that lactobacilli induce TRAIL production in normal human peripheral blood mononuclear cells (PBMC). Treatment with heat-killed Lactobacillus (L.) plantarum, a type of lactobacilli, induced TRAIL expression on the surface of PBMC. L. plantarum also enhanced the release of soluble TRAIL into the culture medium. Moreover, L. plantarum increased the level of TRAIL mRNA as well as TRAIL protein, indicating that L. plantarum up-regulates TRAIL gene expression. L. plantarum also increased the secretion of interferon-γ (IFN-γ) from PBMC. The release of soluble TRAIL from PBMC was partially blocked by IFN-γ neutralization antibody, indicating that the TRAIL production induced by L. plantarum at least partially depends on IFN-γ. To examine whether TRAIL production by L. plantarum leads to cytotoxicity in cancer cells, we co-cultured prostate cancer cells with L. plantarum-treated PBMC and measured natural killer (NK) activity. L. plantarum facilitated NK activity, and the dominant negative form of TRAIL receptor and TRAIL neutralization antibody efficiently prevented NK activity. Our results indicate that L. plantarum facilitates TRAIL-dependent NK activity through TRAIL production, and raise the possibility of a new strategy for TRAIL-based prevention or therapy against malignant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2695. doi:10.1158/1538-7445.AM2011-2695

HIV infection enhances TRAIL-induced cell death in macrophage by down-regulating decoy receptor expression and generation of reactive oxygen species.

BackgroundTumor 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.Methodology/Principal FindingsBy 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.Conclusions/SignificanceHIV 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.

Combination of vorinostat and adenovirus-TRAIL exhibits a synergistic antitumor effect by increasing transduction and transcription of TRAIL in lung cancer cells

Soluble TRAIL and adenovirus (ad)-TRAIL exhibit a strong antitumor effect by inducing apoptosis. Vorinostat is the histone deacetylase (HDAC) inhibitor that induces cell death in cancer cell lines and regulates the expression of epigenetically silenced genes, such as Coxackie adenoviral receptor (CAR), the receptor for adenoviral entry. We propose a new strategy in which vorinostat will induce high expression of ad-TRAIL and a strong antitumor response, and investigated the mechanism involved. The effect of vorinostat on transcription and expression of TRAIL from ad-TRAIL-transduced lung cancer cells were confirmed by reverse transciption-PCR (RT-PCR), quantitative real time-PCR and western blot assay. Anti-tumor effects were measured after cotreatment of vorinostat and ad-TRAIL, and the drug interactions were analyzed. After combined treatment of vorinostat and ad-TRAIL, apoptosis and western blot assays for Akt, Bcl-2 and caspase were performed. Vorinostat increased the expression of CAR in lung cancer cell lines and increased the expression of luciferase (luc) from ad-luc-transduced cells and TRAIL from ad-TRAIL-transduced cells. RT-PCR and quantitative real time-PCR, after sequential vorinostat treatment, revealed that vorinostat may enhance TRAIL expression from ad-TRAIL by increasing transduction through enhanced CAR expression and increasing adenoviral transgene transcription. Combined vorinostat and ad-TRAIL treatment showed the synergistic anti-tumor effect in lung cancer cell lines. Combined vorinostat and ad-TRAIL induced stronger apoptosis induction, suppression of NF-κB activation and breakdown of the anti-apoptotic molecule Bcl-2. In conclusion, the vorinostat synergistically enhanced the anti-tumor effect of ad-TRAIL by (1) increasing adenoviral transduction through the increased expression of CAR and (2) increasing adenoviral transgene (TRAIL) transcription in lung cancer cell lines.

Soluble FasL therapy reduces Herpetic Stromal Keratitis (154.23)

Abstract Fas ligand (FasL) signals apoptosis in Fas+ cells, and plays a vital role in controlling the entrance of inflammatory cells and neovascularization of ocular tissue. We tested the therapeutic value of soluble FasL in both an acute and recurrent model of HSK using BALB/c and BALB-lpr mice. sFasL was administered in a topical ointment and by subconjunctival injection following infection or reactivation. The controls were treatment with ointment alone or with soluble TRAIL (sTRAIL). These mice were evaluated for corneal disease by a masked observer who scored the corneas for opacity and neovascularization. Following acute infection, wild-type BALB/c mice treated with sFasL displayed reduced incidence of opacity and neovascularization when compared to the sTRAIL and ointment groups post infection. When the Fas deficient BALB-lpr mice were used, results showed no difference in HSK between sFasL and sTRAIL treated groups, as expected. When BALB/c mice undergoing recurrent HSK were treated with sFasL, we saw a decrease in both opacity and neovascularization as compared to the sTRAIL treated mice. These data we believe are due to the apoptotic signaling by sFasL of the Fas molecule found on inflammatory cells and vascular endothelium of newly growing vessels which are attempting to invade the cornea following infection or reactivation.

CCL18-mediated pulmonary infiltration of T lymphocytes is independent of TRAIL but requires DR5 (102.20)

Abstract Activities of T lymphocytes are centrally regulated by interactions between multiple members of the TNF/TNF-R family. TRAIL is a member of the TNF family with pro-apoptotic activity. The only signaling TRAIL receptor in mice is death receptor (DR) 5. CCL18, a chemokine with selective chemotactic activity on T lymphocytes, is known for its involvement in pulmonary inflammation and fibrosis associated with autoimmune diseases. We measured soluble TRAIL in bronchoalveolar lavage (BAL) fluids. The levels of TRAIL were significantly elevated (p < 0.01, ANOVA) in BAL from 16 patients with scleroderma lung disease (211±110 pg/ml) compared to 16 patients without lung involvement (115±58 pg/ml), or 8 healthy controls (73±41 pg/ml). To determine whether TRAIL is important for CCL18-induced pulmonary infiltration of lymphocytes, recombinant adenovirus-mediated gene delivery of CCL18 was performed in TRAIL KO, DR5 KO, and WT mice. Overexpression of CCL18 caused profound pulmonary lymphocytic infiltration in WT and TRAIL KO mice, compared to control mice infected with AdV-NULL. In contrast, infiltration of T lymphocytes was completely abrogated in CCL18-expressing DR5 KO mice, based on lung histology. Thus, the levels of TRAIL are elevated in the lungs of patients with autoimmunity-associated inflammation and fibrosis, yet TRAIL does not appear to be involved in the CCL18-mediated T lymphocytic infiltration, whereas its receptor DR5 is required for such T cell response to CCL18.

Functional plasticity of regulatory T cells (168.7)

Abstract Regulatory T cells (Tregs) play a pivotal role in maintaining immune homeostasis, preventing autoimmunity, controlling exaggerated immune responses against infections and suppressing anti-tumor immunity. Tregs utilize multiple mechanisms to mediate their function. However, the number of mechanisms critical to exert maximal suppressive activity is unclear. It is conceivable that suppressive mechanisms could be tightly regulated by cross-regulatory networks. It has been shown that interleukin 10 (IL10) and IL35 have critical role in Treg function. Therefore, we assessed the functional capacity of Tregs lacking the ability to secrete IL10 and IL35. Surprisingly, our study revealed that Tregs lacking both IL10 and IL35 (Ebi3-/-Il10-/- or Il12a-/-Il10-/-) were fully functional and were able to suppress Tconv cells both in vitro and in vivo . Absence of IL10 and IL35 was compensated by an increase in cathepsin E (CTSE), influencing the release of soluble TRAIL and thereby resulting in suppression of target cells in a TRAIL-dependent manner. Inhibiting TRAIL activity disrupted the function of Ebi3-/-Il10-/- Tregs. The loss of suppressive function was maximal in Tregs lacking IL10, IL35 and TRAIL (Ebi3-/-Il10-/-Tnfsf10-/-Tregs). These data suggests the existence of cross-regulatory networks which controls the utilization of suppressive factors, thereby maintaining Treg functional plasticity.

TRAIL shows potential cardioprotective activity

Recent clinical trials carried out in patients with advanced cancer have shown that recombinant TRAIL administration is usually safe and well tolerated when used either alone or in association with chemotherapeutic drugs. Notably, anticancer chemotherapy can be associated to cardiomiopathy. We have here demonstrated that TRAIL (administrated as either recombinant soluble TRAIL or as AAV-TRAIL expression viral vector) reduced the development of cardiomyopathy in the ApoE(-/-) diabetic mouse model. These data suggest, for the first time, that therapeutically administration of TRAIL might have a cardioprotective effect.

Expression of Human Soluble Trail Protein in Transgenic Tobacco Nc89

Plant bioreactors have been considered as an efficient system for biopharmaceutical production. It offers many advantages, such as low cost of production, eukaryotic expression way and absence of human pathogens. In this study, we tried to express the soluble extracellular domain of human tumor necrosis factor related apoptosis-inducing ligand (sTRAIL) in transgenic plants of NC89, a tobacco variety that was planted widely. Using the chloroplast-targeted expression vector pGTPsT, which was constructed previously, we transformed NC89 tobacco leaf explants with the mediation of Agrobacterium tumefaciens LBA4404. We used kanamycin resistance to select transformants and PCR analysis to confirm the presence of sTRAIL gene. Then, we performed RT-PCR analysis to detect the transcription of sTRAIL gene and Western blot to analyze sTRAIL protein accumulation in the leaves of the transformed tobacco plants. The results showed that sTRAIL was successfully inserted into NC89 genome of 8 independent transgenic lines and was expressed in all tested lines. However, no significant protein accumulation was detected at present analysis conditions. This suggests that the accumulation of sTRAIL in transgenic plants of NC89 is much lower than that in transgenic Petit Havana, another variety, which we have investigated previously. NC89, therefore, may not be a suitable plant variety used as plant bioreactor for foreign protein expression and accumulation. A strong protein degradation system may exist in the chloroplast of this variety. However, this point should by experimently tested in the future study.

Effects of IFN-B on TRAIL and Decoy Receptor Expression in Different Immune Cell Populations from MS Patients with Distinct Disease Subtypes

Using quantitative RT-PCR, we compared mRNA levels for TRAIL [tumor necrosis factor (TNF)–related apoptosis-inducing ligand] and its receptors in various immune cell subsets derived from the peripheral blood of untreated normal subjects (NS) and patients with distinct subtypes of multiple sclerosis (MS): active relapsing-remitting MS (RRA), quiescent relapsing-remitting MS (RRQ), secondary-progressive MS (SPMS) or primary-progressive MS (PPMS). Consistent with a role for TRAIL in the mechanism of action of interferon-β (IFN-β), TRAIL mRNA levels were increased in monocytes from patients clinically responsive to IFN-β (RRQ) but not those unresponsive to this therapeutic (RRA). TRAIL-R3 (decoy receptor) expression was elevated in T cells from untreated RRMS patients while IFN-β therapy reversed this increase suggesting that IFN-β may promote the apoptotic elimination of autoreactive T cells by increasing the amount of TRAIL available to activate TRAIL death receptors. Serum concentrations of soluble TRAIL were increased to a similar extent by IFN-β therapy in RRQ, RRA and SPMS patients that had not generated neutralizing antibodies against this cytokine. Although our findings suggest altered TRAIL signaling may play a role in MS pathogenesis and IFN-β therapy, they do not support use of TRAIL as a surrogate marker for clinical responsiveness to this therapeutic.

TRAIL-Induced Apoptosis Is Preferentially Mediated via TRAIL Receptor 1 in Pancreatic Carcinoma Cells and Profoundly Enhanced by XIAP Inhibitors

We previously reported that small molecule X-linked inhibitor of apoptosis (XIAP) inhibitors synergize with soluble TRAIL to trigger apoptosis in pancreatic carcinoma cells. Because cancers may preferentially signal via 1 of the 2 agonistic TRAIL receptors, we investigated these receptors as a therapeutic target in pancreatic cancer in the present study. We examined TRAIL receptor expression and cytotoxicity of specific monoclonal antibodies to TRAIL-R1 (HGS-ETR1, mapatumumab) or TRAIL-R2 (HGS-ETR2, lexatumumab) and of TRAIL receptor selective mutants alone and in combination with small molecule XIAP inhibitors in pancreatic cancer cell lines, in primary specimens, and in a xenotransplant model in vivo. The majority of primary pancreatic carcinoma samples and all cell lines express one or both agonistic TRAIL receptors. Nine of 13 cell lines are more sensitive to mapatumumab-induced apoptosis, whereas lexatumumab requires cross-linking for maximal activity. Similarly, TRAIL-R1 selective mutants display higher cytotoxicity than TRAIL-R2 selective mutants. Small molecule XIAP inhibitors preferentially act in concert with mapatumumab to trigger caspase activation, caspase-dependent apoptosis, and suppress clonogenic survival. Also, primary cultured pancreatic carcinoma cells are more susceptible to mapatumumab than lexatumumab, which is significantly enhanced by a XIAP inhibitor. Importantly, combined treatment with mapatumumab and a XIAP inhibitor cooperates to suppress tumor growth in vivo. Mapatumumab exerts antitumor activity, especially in combination with XIAP inhibitors against most pancreatic carcinoma cell lines, whereas lexatumumab requires cross-linking for optimal cytotoxicity. These findings have important implications for the design of TRAIL-based protocols for pancreatic cancer.

Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP)-targeted delivery of soluble TRAIL potently inhibits melanoma outgrowth in vitro and in vivo

BackgroundAdvanced melanoma is characterized by a pronounced resistance to therapy leading to a limited patient survival of ~6 - 9 months. Here, we report on a novel bifunctional therapeutic fusion protein, designated anti-MCSP:TRAIL, that is comprised of a melanoma-associated chondroitin sulfate proteoglycan (MCSP)-specific antibody fragment (scFv) fused to soluble human TRAIL. MCSP is a well-established target for melanoma immunotherapy and has recently been shown to provide important tumorigenic signals to melanoma cells. TRAIL is a highly promising tumoricidal cytokine with no or minimal toxicity towards normal cells. Anti-MCSP:TRAIL was designed to 1. selectively accrete at the cell surface of MCSP-positive melanoma cells and inhibit MCSP tumorigenic signaling and 2. activate apoptotic TRAIL-signaling.ResultsTreatment of a panel of MCSP-positive melanoma cell lines with anti-MCSP:TRAIL induced TRAIL-mediated apoptotic cell death within 16 h. Of note, treatment with anti-MCSP:sTRAIL was also characterized by a rapid dephosphorylation of key proteins, such as FAK, implicated in MCSP-mediated malignant behavior. Importantly, anti-MCSP:TRAIL treatment already inhibited anchorage-independent growth by 50% at low picomolar concentrations, whereas > 100 fold higher concentrations of non-targeted TRAIL failed to reduce colony formation. Daily i.v. treatment with a low dose of anti-MCSP:TRAIL (0.14 mg/kg) resulted in a significant growth retardation of established A375 M xenografts. Anti-MCSP:TRAIL activity was further synergized by co-treatment with rimcazole, a σ-ligand currently in clinical trials for the treatment of various cancers.ConclusionsAnti-MCSP:TRAIL has promising pre-clinical anti-melanoma activity that appears to result from combined inhibition of tumorigenic MCSP-signaling and concordant activation of TRAIL-apoptotic signaling. Anti-MCSP:TRAIL alone, or in combination with rimcazole, may be of potential value for the treatment of malignant melanoma.

Association of tumor necrosis factor-related apoptosis-inducing ligand with total and cardiovascular mortality in older adults

ObjectiveTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibits biological activity on vascular cells in vitro. Rapid variation of circulating TRAIL levels occurs during acute coronary ischemia, suggesting that biological pathways involving TRAIL may be activated during ischemic heart disease. However, whether differential levels of soluble TRAIL in normal individuals are associated with adverse health outcomes has not been investigated. We tested the hypothesis that TRAIL levels predict mortality in a population based sample of community dwelling men and women. MethodsPlasma TRAIL level was measured by ELISA at baseline in 1282 adults (mean age 68 years) enrolled in the InCHIANTI study. Vital status was ascertained over the six-year follow-up. ResultsIn multivariable Cox regression analysis adjusted for potential confounders including prevalent cardiovascular diseases (CVD), ankle-brachial index, electrocardiogram abnormalities, and inflammatory markers, baseline TRAIL levels were inversely related to all-cause mortality (p=0.008). In stratified analyses, the prognostic effect of TRAIL level was strong and highly significant in participants with prevalent CVD (N=321), (lowest versus highest quartile: HR 3.1; 95% CI 1.5–6.5) while it was negligible in those free of CVD (p value for the interaction term between CVD status and TRAIL levels=0.038). Similar findings were obtained when CVD mortality was considered as the outcome of interest. ConclusionsIn older patients with CVD, low levels of TRAIL were associated with increased risk of death over a period of 6 years. Lower concentration of circulating TRAIL may be related to the clinical evolution of older adults with CVD.

A Better TRAIL Variant for Tumor Cell–Specific Targeting? – Letter

Recently, Spitzer and colleagues ([1][1]) reported on a novel recombinant TRAIL variant in which trimerisation of soluble TRAIL, known to be essential for apoptotic activity, is enforced by genetic fusion of three TRAIL monomers. This covalent single-chained TRAIL trimer, designated TR3, was also

Insights on distinct pathways of thiazolidinediones (PPARγ ligand)‐promoted apoptosis in TRAIL‐sensitive or ‐resistant malignant urothelial cells

Thiazolidinediones, including rosiglitazone and troglitazone, are insulin-sensitizing drugs and high-affinity ligands for the peroxisome proliferator-activated receptor gamma (PPARgamma). Apart from their antidiabetic activity, these molecules possess antitumor properties. We investigated their potential apoptotic effects on RT4 (derived from a well-differentiated Grade I papillary tumor) and T24 (derived from an undifferentiated Grade III carcinoma) bladder cancer cells. Rosiglitazone induced G2/M or G0/G1 phase cell cycle arrest in RT4 and T24 cells, respectively. Only troglitazone triggered apoptosis via extrinsic and intrinsic pathways in both cell lines. Interestingly, rosiglitazone amplified TRAIL-induced apoptosis in TRAIL-sensitive RT4 cells or let TRAIL-resistant T24 cells to respond to TRAIL. Thiazolidinediones acted through PPARgamma activation-independent mechanisms. The underlying mechanisms involved for the first time in cancer cells the upregulation of soluble and/or membrane-bound TRAIL. This was associated with increased cell surface death receptor 5 expression and c-FLIP and survivin downregulation, mediated in part through proteasome-dependent degradation in troglitazone-promoted cell death. Therefore, the combination of rosiglitazone and TRAIL could be clinically relevant as chemopreventive or therapeutic agents for the treatment of TRAIL-resistant high-grade urothelial cancers.

TRAIL-induced apoptosis and expression of death receptor TRAIL-R1 and TRAIL-R2 in bladder cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of TNF superfamily able to induce programmed death in cancer cells with no toxicity against normal tissues. TRAIL mediate apoptosis follows binding to the two death receptors, TRAIL-R1 (DR4) and/or TRAIL-R2 (DR5). In this study we investigated the cytotoxic and apoptotic effect of TRAIL on bladder cancer cells and the expression of death receptor TRAIL-R1 and TRAIL-R2 on the surface of these cancer cells. Three human bladder transitional cancer cell (TCC) lines - SW780, 647V and T24 were tested for TRAIL sensitivity. The bladder cancer cells were incubated with human soluble recombinant TRAIL. Cytotoxicity was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-dimethyltetrazolium bromide) and LDH (lactate dyhydrogenase) assays. Apoptosis was detected by flow cytometry with annexin V-FITC/propidium iodide and by fluorescence microscopy with Hoechst 33342/annexin V-FITC/Ethidium Homodimer. The cell surface expression of TRAIL death receptors on bladder cancer were determined using flow cytometry with phycoerythrin-conjugated monoclonal anti-human TRAIL-R1 and TRAIL-R2. Our investigations confirmed that SW780 cells were sensitive to TRAIL, and two other bladder cancer cell lines, 647V and T24, were resistant to TRAIL induced apoptosis. We therefore examined the expression of TRAIL death receptors on bladder cancer cell surfaces. We showed decreased expression of TRAIL-R2 receptor in TRAIL-resistant bladder cancer cells and increased expression of this death receptor in TRAIL-sensitive SW780 cells. The expression of TRAILR1 receptor was similar in all bladder cancer cell lines. TRAIL is one of the promising candidates for cancer therapeutics. However, some cancer cells are resistant to TRAIL-mediated apoptosis. It is therefore important to overcome this resistance for the clinical use of TRAIL in cancer therapy. TRAIL death receptors are attractive therapeutic targets in cancer treatment. The cytotoxic agents capable of up-regulating the expression of TRAIL-R1 and TRAIL-R2 can sensitize cancer cells to TRAIL induced apoptosis.

Abstract 1272: Targeting glioma stem cells through death receptor activation by AdsTRAIL

Abstract Emerging data suggests that human malignant gliomas may be sustained by the activity of a subpopulation of cells which have stem like characteristics; these glioma stem cells (GSC) exhibit resistance to conventional treatments and constitute novel targets for therapeutic strategies against these malignancies. The goal of this study was to examine whether GSC could be targeted using AdsTRAIL, an adenoviral vector expressing soluble TRAIL and to determine if AdsTRAIL effects could be potentiated by suppressing inhibitors of TRAIL-induced apoptosis. Analysis of expression of TRAIL activated death receptor pathway components showed that DR4, DR5, FADD and Caspase 8 were expressed in the majority of GSC although some cell lines expressed only one of the death receptors (DR4 or DR5). Several cell lines also showed high level of expression of survivin, an anti-apoptosis protein. Treatment of GSC, cultured as tumor spheres in stem cell medium, with AdsTRAIL resulted in dissociation of spheres into a single cell suspension and induction of apoptosis dose and time dependent manner, changes which were not seen in the AdEGFP control; no morphologic changes or induction of apoptosis was seen in GSC spheres treated with purified soluble TRAIL. To determine if induction of apoptosis by AdsTRAIL could be enhanced by suppressing inhibitors of TRAIL induced apoptosis, GSC were treated with AdXAF1, an adenoviral construct expressing XAF1, an endogenous inhibitor of XIAP, prior to exposure to AdsTRAIL. Viability was monitored by expression of the fluorescent marker EGFP in the treated cells. The combination of AdsTRAIL and AdXAF1 potently induced apoptosis to a greater degree than either agent alone. These results suggest that despite their resistance to conventional therapeutic agents such as chemotherapy and radiation, glioma stem cells could potentially be targeted by apoptosis inducing biological agents such as AdsTRAIL. Further characterization of the effects of AdsTRAIL against GSC is required to fully assess the potential of this agent against treatment resistant malignant gliomas. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1272.