Recombinant Human TRAIL Research Articles

Induction of RKIP and Inhibition of the Transcription Repressor YY1 in B-NHL by the Novel Proteasome Inhibitor NPI-0052 and Sensitization to TRAIL-Induced Apoptosis.

Proteasome inhibitors have been shown to exert multiple effects including direct cytotoxic activity in sensitive cancer cells. In addition, proteasome inhibitors have recently been used therapeutically for certain cancers such as multiple myeloma and mantle cell lymphoma. We have recently reported that the novel proteasome inhibitor, NPI-0052 (Nereus Pharmaceuticals), can sensitize drug-resistant B-NHL tumor cells to apoptosis by various chemotherapeutic drugs. Also, NPI-0052 inhibits the NF-κB survival pathway and inhibition of NF-κB sensitizes tumor cells to TRAIL-induced apoptosis. We hypothesized that inhibition of NF-κB by NPI-0052 may also lead to sensitization of TRAIL-resistant B-NHL cells to TRAIL-mediated apoptosis. Human Burkitt's lymphoma,Ramos cells, were treated with various concentrations of NPI-0052 (0–10ng/ml for 24h) and were then treated with recombinant human TRAIL (0–100ng/ml for 24h). The cells were examined for apoptosis by Annexin V/PI. The combination treatment resulted in significant potentiation of cytotoxicity and synergy in apoptosis was achieved. We have then examined a potential underlying mechanism of NPI-0052-mediated sensitization. The transcription repressor YY1 was recently shown to negatively regulate the transcription of the TRAIL death receptor DR5 and thus overexpression of YY1 is a resistant factor for TRAIL-induced apoptosis. Hence, treatment of Ramos cells with NPI-0052 resulted in significant inhibition of YY1 expression and upregulation of surface DR5 expression. We have also examined the effect of NPI-0052 on RKIP (Raf-kinase inhibitor protein) (Yeung et al., Molecular Cellular Biology; 21:7207, 2001) expression. Since both NPI-0052 and RKIP inhibit the NF-κB pathway, we hypothesized that treatment of Ramos cells with NPI-0052 may result in significant upregulation of RKIP expression. The findings, indeed, demonstrate that NPI-0052 significantly induced RKIP expression in Ramos cells. Overall, the findings show, for the first time, that NPI-0052 sensitizes B-NHL tumor cells to TRAIL-mediated apoptosis. Sensitization by NPI-0052 was correlated with both the induction of RKIP expression and inhibition of YY1 and upregulation of DR5 expression. Currently, TRAIL or agonist monoclonal antibodies against DR4 or DR5 are being examined clinically for anti-tumor activity. Therefore, our findings suggest the potential application of the combination of NPI-0052 and TRAIL or agonistic monoclonal antibodies against DR4 or DR5 in the treatment of tumor cells that are resistant to drugs and TRAIL.

Loss of Caspase-9 Provides Genetic Evidence for the Type I/II Concept of CD95-mediated Apoptosis

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.

TRAIL-Mediated Apoptosis in HIV-1-Infected Macrophages Is Dependent on the Inhibition of Akt-1 Phosphorylation

HIV-1 uses mononuclear phagocytes (monocytes, tissue macrophages, and dendritic cells) as a vehicle for its own dissemination and as a reservoir for continuous viral replication. The mechanism by which the host immune system clears HIV-1-infected macrophages is not understood. TRAIL may play a role in this process. TRAIL is expressed on the cell membrane of peripheral immune cells and can be cleaved into a soluble, secreted form. The plasma level of TRAIL is increased in HIV-1-infected patients, particularly those with high viral loads. To study the effect of elevated TRAIL on mononuclear phagocytes, we used recombinant human (rh) TRAIL and human monocyte-derived macrophages (MDM) as an in vitro model. Our results demonstrated rhTRAIL-induced apoptosis in HIV-1-infected MDM and inhibited viral replication, while having a reduced effect on uninfected MDM. HIV-1 infection significantly decreased Akt-1 phosphorylation; rhTRAIL exposure further decreased Akt-1 phosphorylation. Infection with a dominant-negative Akt-1 adenovirus potentiated rhTRAIL-induced apoptosis, while constitutively active Akt-1 blocked rhTRAIL-induced apoptosis in HIV-1-infected MDM. From this data we conclude the death ligand TRAIL preferentially provokes apoptosis of HIV-1-infected MDM, and the mechanism is reliant upon the inhibition of Akt-1 phosphorylation. Understanding this mechanism may facilitate the elimination of HIV-1-infected macrophages and lead to new therapeutic avenues for treatment of HIV-1 infection.

Expression and function of tumor necrosis factor receptor superfamily in human intrahepatic biliary epithelial cells

Although apoptosis of biliary epithelial cells may play an important role in the pathogenesis of vanishing bile duct diseases such as primary biliary cirrhosis, its mechanism is still unknown. TNF receptor 1 (TNFR1), CD95, and TRAIL-R1 and -R2 belonging to TNF receptor superfamily contain a death domain and induce apoptosis by cross-linking of their ligands. The aim of this study was to investigate the expression and functions of apoptosis-inducing receptors in intrahepatic biliary cells. The expression of receptors was studied by RT-PCR and the susceptibility to apoptosis was evaluated using 3 cultured human intrahepatic biliary epithelial cells (HIBECs). All HIBEC cells express CD95 and TRAIL-R1 and -R2, but were completely resistant to an agonist anti-CD95 antibody and recombinant human TRAIL under basal conditions. All HIBEC cells lack TNFR1 expression and TNF-induced apoptosis. However, HIBEC cells could be sensitized to undergo apoptosis in response to CD95 and TRAIL by the proteasome inhibitor MG132. Moreover, this apoptosis induction was significantly enhanced by bcl-2 inhibitors and eliminated by caspase inhibitors. In conclusion, HIBEC cells are resistant to CD95-, TRAIL-, and TNF-induced apoptosis, but proteasome inhibitors sensitize HIBEC cells to CD95- and TRAIL-induced apoptosis through the mitochondrial pathway. This suggests that the proteasome may be an important regulator of apoptosis in biliary epithelial cells and additional factor(s) are necessary for the induction of biliary cell apoptosis in vivo. This is important for the understanding of intrahepatic biliary cell kinetics in patients with biliary diseases.

Bortezomib Partially Overcomes TNF-Related Apoptosis Inducing ligand/Apo-2L (TRAIL/Apo-2L) Environment Mediated-Drug Resistance (EM-DR).

Multiple Myeloma (MM) is an incurable disease that warrants novel treatments. MM survival is dependent on resistance conferred by interactions with the bone marrow (BM) microenvironment (stroma and/or extracellular matrix proteins): Environment Mediated-Drug Resistance (EM-DR). We have shown that adhesion to fibronectin inhibits CD95-induced caspase-8 activation resulting in apoptosis by regulating the cellular localization and availability of c- FLIPL (Shain K. et al. J Immunol . 2002; 168(5):2544–53). TNF-related apoptosis inducing ligand/Apo-2L (TRAIL/Apo-2L) is a member of the TNF superfamily of death ligands that induce apoptosis of resistant MM cell lines and patient's cells while cultured in suspension. TRAIL/Apo-2L also inhibits the growth of human plasmocytomas in xenografted NOD/SCID mice (Mitsiades C. et al. Blood 2001; 98(3):795–804). To explore if the tumor microenvironment confers resistance to TRAIL/Apo-2L mediated apoptosis, we treated RPMI 8226 cells with recombinant human killer TRAIL (Alexis Biochemicals). In a series of 3 experiments, we observed a 50.8 ± 10 % specific apoptosis, measured by annexin/PI staining, when cells were treated with TRAIL (10ng/mL) in suspension (Su) media. In contrast, a dramatic resistance to TRAIL mediated apoptosis (6.9 ± 1.2 %) was observed when RPMI 8226 cells were adhered (Ad) to stroma cells (HS-5 and HS-5 transfected with green fluorescent protein-GFP) suggesting the EM-DR phenotype. We hypothesized that EM-DR can potentially be reversed by Bortezomib, a proteosome inhibitor, via NF-κB inhibition resulting in reduced transcription of cellular adhesion proteins genes (Mitsiades et al. Blood 2002; 99(11): 4079–4086). Resistance to drug-induced apoptosis was also observed when RPMI 8226 cells were adhered to HS-5 and HS5-GFP and treated with Bortezomib alone at 5, 10 and 15nM (55.6 ± 11.6 % Su and 12.1 ± 1.6 % Ad). Microarray analysis of RPMI 8226 cells treated with Bortezomib showed that death receptor 4 (DR4) and DR5 expression was up-regulated suggesting a potential interaction between Bortezomib and TRAIL pathway. When RPMI 8226 cells were treated with Bortezomib (10nM) for 20 Hrs and subsequently treated with TRAIL for additional 4 Hrs, we observed an enhancement to 77.7 ± 3.64 % (Su) and 47.22 ± 13.6 % (Ad) specific apoptosis. In addition, in a series of 3 experiments using transwell inserts, RPMI 8226 cells (upper well) separated from HS-5 (lower well) were treated with Bortezomib (10nM), TRAIL (10ng/ML) or Bortezomib and TRAIL combined as described above. Results were comparable with direct contact experiments described, suggesting that cytokines and/or chemokines released by stroma may be responsible for EM-DR phenotype (Figure 1). Further mechanistic studies elucidating TRAIL and Bortezomib combination are currently under investigation. These preclinical studies provide the basis for clinical trial testing the combination of these agents. [Display omitted]

463 Translation targeting TRAIL receptors to the clinic

Background and purpose: Previously it was shown that combination of death ligand TRAIL and irradiation strongly increases cell kill in several human tumour cell lines. Since Bcl-2 overexpression did not strongly interfere with the efficacy, components of the mitochondrial death pathway are not required for an effective combined treatment. In the present study the minimal molecular prerequisites for the efficacy of a combined treatment were determined.Materials and methods: Apoptosis induction in control, caspase-8 and FADD negative Jurkat cells, BJAB control and FADD-DN cells was analysed by FACS. Activation of caspase-8, -10 and -3 and cleavage of PARP was determined by immunoblotting. TRAIL receptors were activated using recombinant human TRAIL. Surface expression of TRAIL receptors DR4 and DR5 was analysed by FACS.Results: Jurkat T-cells express the agonistic DR5 receptor but not DR4. Presence of FADD was found to be essential for TRAIL induced apoptosis. Caspase-8 negative cells show very low rates of apoptosis after prolonged stimulation with TRAIL. No combined effects of TRAIL with irradiation could be found in FADD-DN overexpressing and FADD deficient cells. However, the combination of TRAIL and irradiation clearly lead to a combined effect in caspase-8 negative Jurkat cells, albeit with reduced death rates. In these cells activation of the alternative initiator caspase-10 could be detected after combined treatment.Conclusion: Our data show that a combined therapy with TRAIL and irradiation will only be effective in cells expressing at least one agonistic TRAIL receptor, FADD and caspase-8 or caspase-10.

Expression of TRAIL receptors in human hepatocellular carcinoma and apoptosis induced by TRAIL

Objective: To investigate the expression of TRAIL receptors in human hepatocellular carcinoma and neighboring non-tumor liver tissues and to study its roles in apoptosis of SK-Hep1 cell line.Methods: The expression of TRAIL receptors in hepatocellular carcinoma and in SK-Hep1 cell line was detected by immunohistochemical staining and RT-PCR, and the apoptosis of SK-Hep1 cell line induced by recombinant human TRAIL together with or without INF-γ was measured by FCM.Results: The expression of four TRAIL receptors was detectable in human hepatocellular carcinoma and neighboring non-tumor liver tissues. High expression of DR4 and DR5 was detected in tumor and neighboring non-tumor liver tissues whereas low expression of DcR1 and DcR2 was found in tumor, significantly lower than that in normal liver tissues (67.86% vs 100%, 78.57% vs 100%, respectively,P<0.01. TRAIL together with INF-γ strongly increased the apoptosis of SK Hep1 cell line.Conclusion: The four TRAIL receptors can be detected in hepatocellular carcinoma and in SK-Hep1 cell line. Combinations of TRAIL and INF-γ may be a useful alternative to HCC.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/APO2L) and its receptors expression in human squamous cell carcinoma of the oral cavity

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/APO2L) is a novel member of the tumor necrosis factor cytokine family and a potent inducer of apoptosis in tumor cells. TRAIL is expressed in most normal human cells and tissues, including peripheral blood leukocytes, spleen, lung and prostate. However, TRAIL expression in human neoplasms is largely unknown. In this study, we investigated whether TRAIL and its receptors are expressed in human oral squamous cell carcinomas (HOSCCs) or HOSCC cell lines (HSC-2, HSC-3, HSC-4, Ca9-22 and KB) and whether these cells are sensitive to TRAIL-induced apoptosis. TRAIL and its receptor transcripts and proteins were detected in all HOSCC cell lines. A cell viability (MTT) assay performed after exposure to recombinant human (rh) TRAIL for 16 h showed that KB cells were the most sensitive. Immunohistochemical examinations for TRAIL expression were also carried out in a total of 50 HOSCC cases with various types of differentiation. In 17 of 27 WHO Grade 1 SCCs, the tumor invasive areas with keratinization showed diffuse and moderate TRAIL expression. In 6 of 20 WHO Grade 2 SCCs, TRAIL expression was slightly to moderately detected. In 1 of 3 WHO Grade 3 SCCs, a slight TRAIL expression was observed. Thus, there is considerable heterogeneity of TRAIL expression and susceptibility to TRAIL-induced apoptosis among human oral tumors.

Molecular requirements for the combined effects of TRAIL and ionising radiation

Background and purpose: Previously it was shown that combination of death ligand TRAIL and irradiation strongly increases cell kill in several human tumour cell lines. Since Bcl-2 overexpression did not strongly interfere with the efficacy, components of the mitochondrial death pathway are not required for an effective combined treatment. In the present study the minimal molecular prerequisites for the efficacy of a combined treatment were determined. Materials and methods: Apoptosis induction in control, caspase-8 and FADD negative Jurkat cells, BJAB control and FADD-DN cells was analysed by FACS. Activation of caspase-8, -10 and -3 and cleavage of PARP was determined by immunoblotting. TRAIL receptors were activated using recombinant human TRAIL. Surface expression of TRAIL receptors DR4 and DR5 was analysed by FACS. Results: Jurkat T-cells express the agonistic DR5 receptor but not DR4. Presence of FADD was found to be essential for TRAIL induced apoptosis. Caspase-8 negative cells show very low rates of apoptosis after prolonged stimulation with TRAIL. No combined effects of TRAIL with irradiation could be found in FADD-DN overexpressing and FADD deficient cells. However, the combination of TRAIL and irradiation clearly lead to a combined effect in caspase-8 negative Jurkat cells, albeit with reduced death rates. In these cells activation of the alternative initiator caspase-10 could be detected after combined treatment. Conclusion: Our data show that a combined therapy with TRAIL and irradiation will only be effective in cells expressing at least one agonistic TRAIL receptor, FADD and caspase-8 or caspase-10.

TRAIL (APO-2L) induces apoptosis in human prostate cancer cells that is inhibitable by Bcl-2.

To determine if TRAIL-induced apoptosis in human prostate tumor cells was suppressed by bcl-2, we compared the levels of apoptosis induced by recombinant human TRAIL in pairs of isogenic cell lines that do or do not express bcl-2. Three human prostate tumor cell lines (PC3, DU145 and LNCaP) and their bcl-2-expressing counterparts were tested for their susceptibility to TRAIL. Cells were exposed to TRAIL in the presence of cycloheximide which acted as a sensitizer. Apoptosis was induced rapidly in PC3 and DU145 neo-control transfected cells, whereas induction in LNCaP required 24 h. All three cell line variants expressing bcl-2 were resistant to the apoptotic effects of TRAIL. Caspase 3 and 8 activation was also detected in the neo control cells after treatment with TRAIL, demonstrating the rapid activation of the caspase cascade similar to that seen with other death receptors. Bcl-2 overexpression in these cells blocked activation of these caspases, suggesting that bcl-2 expression of human cancer cells may be a critical factor in the therapeutic efficacy of TRAIL.

Green fluorescent protein as a novel tool to measure apoptosis and necrosis.

Several apoptosis-detecting methods are currently available. Many of them are work intensive and require the additional use of antibodies, dyes, specific substrates, or enzymatic reactions. A simple, fast, and reliable method was developed to test for apoptosis or necrosis using mouse and human cell lines (e.g., Jurkat, A20.2J, and PB3c cells) stably transfected with a vector coding for green fluorescent protein (GFP) as indicator cells. Apoptosis in GFP-transfected cell lines was induced either by soluble Fas-Ligand (sFasL), recombinant human TRAIL (rhTRAIL), or interleukin-3 (IL-3) deprivation. Necrosis was induced by polyclonal anti-A20 and complement treatment of GFP-transfected A20. Cells were analyzed by flow cytometry for GFP fluorescence. Propidium iodide and Annexin V staining were used to confirm the results obtained with the GFP-method. Live GFP-transfected cells show a strong fluorescence intensity, which is significantly diminished upon induction of apoptosis, whereas necrotic GFP-transfected cells almost completely lose their GFP-associated fluorescence. Apoptosis but not necrosis of GFP-transfected cells was blocked by the use of a caspase inhibitor. The results are highly comparable to conventional apoptosis-detecting methods. The advantage of our GFP-based assay compared with other methods is the analysis of apoptosis or necrosis without the necessity for additional staining or washing steps, making it an ideal tool for screening apoptotic or necrotic stimuli.

Expression, purification and crystallization of recombinant human TRAIL.

TRAIL (also known as Apo-2L) belongs to the tumour necrosis factor (TNF) cytokine family and induces rapid apoptosis in a wide variety of tumour cell lines upon binding to the death-signalling receptors on the cell membrane. Normal cells are resistant to TRAIL, owing to the expression of decoy receptors which lack functional death domains and antagonize TRAIL-induced apoptosis. Soluble and functional human TRAIL, expressed in Escherichia coli and refolded into a functional form, has been crystallized. The crystals belong to space group P63 with unit-cell dimensions a = b = 65.61, c = 131. 70 A. The asymmetric unit contains two molecules of TRAIL, with a crystal volume per protein mass (Vm) of 2.41 A3 Da-1 and a solvent content of about 42% by volume. A native and a platinum-derivative data set to 2.8 and 3.5 A resolution, respectively, were obtained from frozen crystals. Structure determination by a combined molecular replacement and isomorphous replacement method is in progress.