Presence Of TNF-related Apoptosis-inducing Ligand Research Articles

TRAIL-Dependent Resolution of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung disease characterized by the persistence of activated myofibroblasts resulting in excessive deposition of extracellular matrix proteins and profound tissue remodeling. In the present study, the expression of tumor necrosis factor- (TNF-) related apoptosis-inducing ligand (TRAIL) was key to the resolution of bleomycin-induced pulmonary fibrosis. Both in vivo and in vitro studies demonstrated that Gr-1+TRAIL+ bone marrow-derived myeloid cells blocked the activation of lung myofibroblasts. Although soluble TRAIL was increased in plasma from IPF patients, the presence of TRAIL+ myeloid cells was markedly reduced in IPF lung biopsies, and primary lung fibroblasts from this patient group expressed little of the TRAIL receptor-2 (DR5) when compared with appropriate normal samples. IL-13 was a potent inhibitor of DR5 expression in normal fibroblasts. Together, these results identified TRAIL+ myeloid cells as a critical mechanism in the resolution of pulmonary fibrosis, and strategies directed at promoting its function might have therapeutic potential in IPF.

Abstract 3901: Polymeric mechanical amplifiers of tumor cell receptor-mediated apoptosis

Abstract Introduction: It has become evident that tumor cells are responsive to mechanical forces in vivo, and prove critical to tumor cell proliferation and death. Recent work has shown that tumor cells exposed to fluid shear forces increase receptor-mediated signaling. We hypothesized that biocompatible, polymeric particles conjugated to the tumor cell surface act as mechanical amplifiers in presence of fluid shear forces to increase mechanotransduction, and can be exploited to increase the efficacy of therapeutic ligands. Methods: Polymeric particles (size:100 nm-1 μm) were conjugated to free amines on tumor cells via NHS cross linker chemistry. Particles bound to tumor cells were assessed using flow cytometry, brightfield, and confocal fluorescence microscopy. A cone-and-plate viscometer was used to apply a fluid shear force (2.0 dyn/cm⁁2) to tumor cell suspensions and to amplify the force exerted by polymeric particles on tumor cells. Tumor cells (COLO 205, PC-3) were treated with 0.1 μg/mL of a TNF-related apoptosis-inducing ligand (TRAIL) to assess amplified mechanotransduction and receptor-mediated apoptosis in the presence of polymeric particles. An annexin-V apoptosis assay was used to characterize the mode of cell death. Caspase colorimetric assays and inhibitors (Z-VAD-FMK) were utilized to assess caspase-dependence in the mechanotransduction response. Tumor cells were injected into C57/BL6 via tail vein to assess amplification of TRAIL apoptosis in vivo. Results and Discussion: NHS crosslinker chemistry was successfully used to conjugate polymeric particles to the tumor cell surface. In the presence of fluid shear forces, it was found that polymeric particles act to mechanical amplify tumor cell mechanotransduction, as evidenced by increased receptor-mediated apoptosis and decreased tumor cell viability in the presence of the therapeutic ligand TRAIL. Additionally, amplification of TRAIL-mediated apoptosis was increased with particles of increasing size, demonstrating that increasing the force exerted on the cell surface with larger particles amplified the therapeutic response. Annexin-V apoptosis assays showed the addition of conjugated polymeric particles to the cell surfaces nearly doubled tumor cell apoptosis in the presence of TRAIL under shear forces, and inhibition assays revealed the response to be caspase-dependent apoptosis. Polymeric particles amplified TRAIL-mediated tumor cell killing in mice, and reduced both circulating tumor cells in blood and overall tumor cell burden in vivo by over 90%. Conclusions: These data demonstrate that polymeric particles, both degradable and non-degradable, act as mechanical amplifiers of tumor mechanotransduction in the presence of shear forces in vitro and in vivo, and are exploited to increase therapeutic efficacy. Clinically, this approach shows that increased mechanical force applied to target tumor cells can increase sensitivity to therapeutic ligands. Citation Format: Michael J. Mitchell, Robert Langer. Polymeric mechanical amplifiers of tumor cell receptor-mediated apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3901.

Polymeric mechanical amplifiers of receptor-mediated apoptosis

Polymeric mechanical amplifiers of receptor-mediated apoptosis

Cytokines from the tumor microenvironment modulate sirtinol cytotoxicity in A549 lung carcinoma cells

Cytokines in tumor microenvironment play an important role in the success or failure of molecular targeted therapies. We have chosen tumor necrosis factor α (TNF-α), TNF related apoptosis inducing ligand (TRAIL), insulin-like growth factor 1 (IGF-1) and transforming growth factor β (TGF-β) as representative pro-inflammatory, pro-apoptotic, anti-apoptotic and anti-inflammatory tumor derived cytokines. Analysis of Oncomine database revealed the differential expression of these cytokines in a subset of cancer patients. The effects of these cytokines on cytotoxicity of FDA approved drugs – cisplatin and taxol and inhibitors of epidermal growth factor receptor – AG658, Janus kinase – AG490 and SIRT1 – sirtinol were assessed in A549 lung cancer cells. TRAIL augmented cytotoxicity of sirtinol and IGF-1 had a sparing effect. Since TRAIL and IGF-1 differentially modulated sirtinol cytotoxicity, further studies were carried out to identify the mechanisms. Sirtinol or knockdown of SIRT1 increased the expression of death receptors DR4 and DR5 and sensitized A549 cells to TRAIL. Increased cell death in presence of TRAIL and sirtinol was caspase independent and demonstrated classical features of necroptosis. Inhibition of iNOS increased caspase activity and switched the mode of cell death to caspase mediated apoptosis. Interestingly, sirtinol or SIRT1 knockdown did not increase IGF-1R expression. Instead, it abrogated ligand induced downregulation of IGF-1R and increased cell survival through PI3K–AKT pathway. In conclusion, these findings reveal that the tumor microenvironment contributes to modulation of cytotoxicity of drugs and that combination therapy, with agents that increase TRAIL signaling and suppress IGF-1 pathway may potentiate anticancer effect.

Human Colostrum and Breast Milk Contain High Levels of TNF-Related Apoptosis-Inducing Ligand (TRAIL)

TNF-related apoptosis inducing ligand (TRAIL) is a pleiotropic cytokine, which plays a key role in the immune system as well as in controlling the balance of apoptosis and proliferation in various organs and tissues. To investigate the presence and levels of soluble TRAIL in human colostrum and milk. The levels of soluble human TRAIL were measured in human colostrum (day 2 after delivery) and breast milk (day 5 after delivery). The presence of TRAIL was also measured in infant formula. Levels of soluble TRAIL in the colostrum and mature human milk were, respectively, at least 400 and 100 fold higher than those detected in human serum. No TRAIL was detected in formula. Human soluble TRAIL is present at extremely high levels in human colostrum and human milk and might have a significant role in mediating the anti-cancer activity of human milk.

Dental Pulp Stem Cells (DPSCs), differentiating into osteoblasts, become a source of the pro-apoptotic factor TRAIL: evaluation of an experimental model for cancer therapy

The DPSCs belong to the family of mesenchymal stem cells (MSCs) and are capable, if properly stimulated to differentiate into different cell types including osteoblasts. It has been shown, in animal models, that MSCs derived from bone marrow, producing TNF-related apoptosis-inducing ligand (TRAIL), inhibit the growth of tumors that metastatize to the bone tissue, including lung cancer; however, the expression of TRAIL by the MSCs to be effective, must be stimulated with genetic engineering techniques with the involvement of bacterial vectors. TRAIL is a pro-apoptotic factor, member of the super-family of tumor necrosis factors, known for its peculiarity to selectively induce apoptosis in cancer cells. TRAIL can activate apoptotic signals by binding two different death receptors, DR4 and DR5. In our work we evaluated the production of TRAIL by DPSCs differentiated toward the osteoblastic phenotype. Microarray experiments, also supported by real-time PCR, showed that in DPSCs after differentiation, the expression of TRAIL increased fifteen times. Moreover, cell viability tests have shown that DPSCs differentiating into osteoblasts become resistant to the pro-apoptotic effect of the molecule. This strong resistance derives from a significant decrease in the expression of TRAIL receptors DR4 and DR5, and is confirmed by the weak activation of intracellular apoptotic signal (caspase 3) following the exposure to the molecule. Conversely, the tumor cell lines expressed high levels of DR4 and DR5, and in the presence of TRAIL, activate the intracellular apoptotic signals (caspase 3-8). Thus, the DPSCs, differentiated into osteoblasts, expressing high levels of TRAIL and developing a resistance to the apoptotic effect of the molecule, could represent a valuable therapeutic approach to cancer therapy.

TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation from monocyte/macrophage lineage precursor cells via TRAF-6 dependent pathway (92.14)

Abstract Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. Recent evidence indicates that the TNF-related apoptosis-inducing ligand (TRAIL) of the TNF ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated T cells. We show in this work that TRAIL can induce osteoclast formation from human monocytes and murine RAW264.7 macrophages. We demonstrated that both cell models differentiate into osteoclast-like cells in the presence of TRAIL in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. The TRAIL-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. However, TRAIL-induced osteoclastogenesis is dependent on activation of NF-£eB, ERK, and p38 MAP kinase. The TRAIL-induced osteoclastogenesis was significantly inhibited by treatment with TRAF-6 siRNA and TRAF-6 decoy peptide, indicating this pathway is TRAF-6 dependent. Thus, our data demonstrate that TRAIL induces osteoclast differentiation via direct engagement with the TRAIL death receptor through a signaling pathway distinct from apoptosis. Our results indicate that in addition to triggering apoptosis, TRAIL induces osteoclast differentiation. It provides a novel role for TRAIL in regulating osteoclast differentiation and in osteoimmunology.

Osteoblast Apoptosis in Periodontal Disease: Role of TNF-Related Apoptosis-Inducing Ligand

Periodontal disease (Pd) is characterized by an increased osteoclast resorption and a decreased osteoblast (OB) bone formation. OBs obtained from alveolar bone of Periodontitis patients (Pp) undergo apoptosis in the presence of TNF-related apoptosis-inducing ligand (TRAIL). We studied the intracellular apoptotic pathway induced by TRAIL; TRAIL death (DR4, DR5) and decoy (DcR1, DcR2) receptors expression in Periodontitis patients OBs (PpOBs), and we measured the concentration of TRAIL in the serum of Pp. We demonstrated that DNA fragmentation and activation of caspase-8 and caspase-3 in PpOBs, following TRAIL stimulation, occurred in shorter time; moreover, a higher amount of both caspases was activated in order to direct OBs. Down-regulation of DcR2 in PpOBs was demonstrated and high TRAIL levels were detected in the serum of Pp. In conclusion, our data suggest that PpOBs are more sensitive to TRAIL-induced apoptosis when compared to the control group. The down-regulation of DcR2 possibly leads to an imbalanced ratio between death and decoy receptors. Our findings highlight a role of TRAIL in the pathogenesis of Pd.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Inhibits Experimental Autoimmune Thyroiditis by the Expansion of CD4+CD25+ Regulatory T Cells

There have been several reports that TNF-related apoptosis-inducing ligand (TRAIL) has the ability to suppress the development of experimental autoimmune diseases, including a mouse model of experimental autoimmune encephalomyelitis, a rabbit model of rheumatoid arthritis, type 1 diabetes mellitus, in mice and experimental autoimmune thyroiditis (EAT) in mice. However, the mechanism underlying TRAIL effect is not well defined. In the present study, we specifically examined TRAIL effects on CD4(+)CD25(+) regulatory T cells. CD4(+)CD25(+) T cells prepared from mouse thyroglobulin (mTg)-immunized CBA/J mice proliferate in the presence of TRAIL and dendritic cells in vitro. These CD4(+)CD25(+) T cells included both CD4(+)CD25(+)CD45RB(Low) (regulatory) and CD4(+)CD25(+)CD45RB(High) (effector) T cells. Our results demonstrated that mTg-immunized mice treated with TRAIL showed significant increases in the number of CD4(+)CD25(+)CD45RB(Low) T cells compared with mice immunized with mTg alone. CD4(+)CD25(+)CD45RB(Low) T cells expressed much higher levels of the forkhead family transcription factor, IL-10, and TGFbeta1 than CD4(+)CD25(+)CD45RB(High) T cells, and these cells can completely suppress the proliferation of the mTg-primed splenocytes in lower concentrations than the unfractionated CD4(+)CD25(+) T cells. Furthermore, transfer of these cells into CBA/J mice prior to mTg-primed splenocyte injection could markedly reduce the frequency and severity of EAT development. CD4(+)CD25(+)CD45RB(Low) T cells were more effective at suppressing histological thyroiditis than unfractionated cells. These results indicated that TRAIL can increase the number of mTg-specific CD4(+)CD25(+)CD45RB(Low) T cells, inhibiting autoimmune responses and preventing the progression of EAT. These findings reveal a novel mechanism by which TRAIL could inhibit autoimmune disease.

TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation from monocyte/macrophage lineage precursor cells

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. Recent evidence indicates that the TNF-related apoptosis-inducing ligand (TRAIL) of the TNF ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated T cells. We show in this work that TRAIL can induce osteoclast formation from human monocytes and murine RAW264.7 macrophages. We demonstrated that both cell models differentiate into osteoclast-like cells in the presence of TRAIL in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. The TRAIL-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. However, TRAIL-induced osteoclastogenesis is dependent on activation of NF-κB, ERK, and p38 MAP kinase. Thus, our data demonstrate that TRAIL induces osteoclast differentiation via direct engagement with the TRAIL death receptor through a signaling pathway distinct from apoptosis. Our results indicate that in addition to triggering apoptosis, TRAIL induces osteoclast differentiation. It provides a novel role for TRAIL in regulating osteoclast differentiation and in osteoimmunology.

Soluble TRAIL does not impair the anti‐osteoclastic activity of osteoprotegerin

Dear Editor: It has been extensively documented that osteoprotegerin (OPG), a soluble member of the TNF receptor superfamily, inhibits osteoclastogenesis by binding to receptor activator of NF-kB ligand (RANKL) and preventing interaction with its cognate transmembrane receptor RANK [reviewed in 1]. However, OPG can also interact with, and neutralize, TNF-related apoptosis inducing ligand (TRAIL), whose extracellular domain shares a 30% homology with the extracellular domain of RANKL [reviewed in 2]. Although some inconsistencies on the differential binding affinity of OPG for RANKL versus TRAIL were present in initial studies [2, 3], it has been recently demonstrated that the affinity of native OPG for native TRAIL is comparable to that for RANKL (45 nM versus 23 nM, respectively) at 37°C, as determined by plasmon surface resonance analysis [4]. Consistently with this biochemical study, OPG has been shown to act in a paracrine and autocrine manner by binding TRAIL and promoting the survival of multiple myeloma [5], prostate cancer [6], ameloblastoma cells [7] and synovial fibroblasts [8]. Interestingly, previous data from different groups have shown that recombinant TRAIL modulates the differentiation of erythroid and myeloid precursors [9–10], while inhibits both human and mouse osteoclastogenesis when added to pre-osteoclast cultures induced to differentiate with recombinant macrophage colony-stimulating factor (M-CSF) +RANKL as well as to mature osteoclasts [11–14]. On the other hand, a couple of studies suggested that recombinant soluble TRAIL might promote osteoclastogenesis [4, 15], and the proposed molecular mechanism to explain such observation was a competition between TRAIL and RANKL for OPG binding. However, it should be noticed that Vitovsky et al. used much higher concentrations of TRAIL (500 ng/ml) than RANKL (30 ng/ml) or OPG (50 ng/ml) and more importantly used mouse bone marrow pre-osteoclasts [4]. In this respect, it has been clearly shown that mouse pre-osteoclasts only express TRAIL-R2 [2], while human peripheral blood-derived pre-osteoclasts express both death receptors TRAIL-R1 and TRAIL-R2 as well as TRAIL-R4 [11–14]. Therefore, to further elucidate the important issue of the interplay between RANKL, TRAIL and OPG in human osteoclastogenesis, we have cultured adherent PBMC with M-CSF+RANKL for 14 days in the absence or presence of recombinant OPG and recombinant TRAIL, prepared as previously described [16]. TRAIL and OPG were added alone or in combination. Importantly, all cytokines were used at the same concentration (50 ng/ml). As expected [11–14], the addition of TRAIL to M-CSF+RANKL significantly (P < 0.05) inhibited osteoclast formation (Fig. 1A and B). Of note, recombinant OPG completely abrograted (P < 0.05) osteoclast formation irrespectively of the presence of recombinant TRAIL in culture (Fig. 1A and B). The anti-osteoclastic activity of OPG could not be ascribed to a low affinity of OPG for TRAIL since OPG (50 ng/ml) efficiently inhibited the apoptosis induced by TRAIL (50 ng/ml) in HL-60 leukemic cells (data not shown). Effect of combined treatment of RANKL, TRAIL and OPG on osteoclastic differentiation. Adherent PBMC were cultured with M-CSF alone for 6 days. Then cells were cultured for additional 14 days with the addition of RANKL in the absence or presence of TRAIL and/or OPG, as indicated. Cultures were analysed for osteoclastic differentiation by scoring the number of TRAP+ multinucleated cells (A), and measuring the levels of TRAP 5b (B), a specific marker of resorption activity, in culture supernatants by ELISA. Data represent the means ±SD of three different experiments performed in duplicate. Our current observations on one hand confirm that TRAIL has anti-osteoclastic activity and on the other hand indicate that it does not affect the potent anti-osteoclastic activity of OPG at least in the simplified model of osteoclastogenesis represented by human PBMC induced to differentiate by M-CSF+RANKL. Taken together with previous studies [4, 11–15], these data also suggest that the relative concentrations of TRAIL, RANKL and OPG in the local microenvironment are likely key determinant for the regulation of osteoclastogenesis.

Long lasting heat shock stimulation of TRAIL-induced apoptosis in transformed T lymphocytes

We report that a mild heat shock, that did not impair cell growth, stimulated TNF-related apoptosis inducing ligand (TRAIL)-mediated apoptosis of leukemic T lymphocytes and promyelocytic cells, but not normal human T lymphocytes. The death stimulation was maximal when the heat shock was performed at the beginning of the exposure to TRAIL. However, enhanced apoptosis was still observed when TRAIL was added one day after heat shock. The phenomenon was transcription and translation independent suggesting that newly made heat shock proteins were not involved. TRAIL-induced apoptosis after heat shock was dependent on caspases and FADD and an enhanced FlipL/S processing was noticed. However, since after the heat shock FlipL/S processing was transient, events upstream of caspase 8 and FADD may be responsible of the long lasting enhanced TRAIL apoptosis observed after heat shock. No heat-mediated alteration in the antibody recognition of cell surface DR4 and DR5 TRAIL receptors was observed. However, in the presence of TRAIL, a long lasting attenuation in the antibody detection of DR4 and DR5 was observed in heat shock-treated cells that correlated with the enhanced apoptogenic efficiency of TRAIL.