Aim: The therapeutic targeting of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors in cancer, including non-small cell lung cancer (NSCLC), is a widely studied approach for tumor selective apoptotic cell death therapy. However, apoptosis resistance is often encountered. The main aim of this study was to investigate the apoptotic mechanism underlying TRAIL sensitivity in three bortezomib (BTZ)-resistant NSCLC variants, combining induction of both the intrinsic and extrinsic pathways. Methods: Sensitivity to TRAIL in BTZ-resistant variants was determined using a tetrazolium (MTT) and a clonogenic assay. A RT-qPCR profiling mRNA array was used to determine apoptosis pathway-specific gene expression. The expression of these proteins was determined through ELISA assays and western Blotting, while apoptosis (sub-G1) and cytokine expression were determined using flow cytometry. Apoptotic genes were silenced by specific siRNAs. Lipid rafts were isolated with fractional ultracentrifugation. Results: A549BTZR (BTZ-resistant) cells were sensitive to TRAIL in contrast to parental A549 cells, which are resistant to TRAIL. TRAIL-sensitive H460 cells remained equally sensitive for TRAIL as H460BTZR. In A549BTZR cells, we identified an increased mRNA expression of TNFRSF11B [osteoprotegerin (OPG)] and caspase-1, -4 and -5 mRNAs involved in cytokine activation and immunogenic cell death. Although the OPG, interleukin-6 (IL-6), and interleukin-8 (IL-8) protein levels were markedly enhanced (122-, 103-, and 11-fold, respectively) in the A549BTZR cells, this was not sufficient to trigger TRAIL-induced apoptosis in the parental A549 cells. Regarding the extrinsic apoptotic pathway, the A549BTZR cells showed TRAIL-R1-dependent TRAIL sensitivity. The shift of TRAIL-R1 from non-lipid into lipid rafts enhanced TRAIL-induced apoptosis. In the intrinsic apoptotic pathway, a strong increase in the mRNA and protein levels of the anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) and B-cell leukemia/lymphoma 2 (Bcl-2) was found, whereas the B-cell lymphoma-extra large (Bcl-xL) expression was reduced. However, the stable overexpression of Bcl-xL in the A549BTZR cells did not reverse the TRAIL sensitivity in the A549BTZR cells, but silencing of the BH3 Interacting Domain Death Agonist (BID) protein demonstrated the importance of the intrinsic apoptotic pathway, regardless of Bcl-xL. Conclusion: In summary, increased sensitivity to TRAIL-R1 seems predominantly related to the relocalization into lipid rafts and increased extrinsic and intrinsic apoptotic pathways.

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) can induce apoptosis in a wide variety of cancer cells, both in vitro and in vivo, importantly without killing any essential normal cells. These findings formed the basis for the development of TRAIL-receptor agonists (TRAs) for cancer therapy. However, clinical trials conducted with different types of TRAs have, thus far, afforded only limited therapeutic benefit, as either the respectively chosen agonist showed insufficient anticancer activity or signs of toxicity, or the right TRAIL-comprising combination therapy was not employed. Therefore, in this review we will discuss molecular determinants of TRAIL resistance, the most promising TRAIL-sensitizing agents discovered to date and, importantly, whether any of these could also prove therapeutically efficacious upon cancer relapse following conventional first-line therapies. We will also discuss the more recent progress made with regards to the clinical development of highly active non-immunogenic next generation TRAs. Based thereupon, we next propose how TRAIL resistance might be successfully overcome, leading to the possible future development of highly potent, cancer-selective combination therapies that are based on our current understanding of biology TRAIL-induced cell death. It is possible that such therapies may offer the opportunity to tackle one of the major current obstacles to effective cancer therapy, namely overcoming chemo- and/or targeted-therapy resistance. Even if this were achievable only for certain types of therapy resistance and only for particular types of cancer, this would be a significant and meaningful achievement.

Pyroptosis is an active and ordered form of programmed cell death. The signaling pathways of pyroptosis are mainly divided into canonical pathways mediated by caspase-1 and noncanonical pathways mediated by caspase-11. Cell pyroptosis is characterized by the activation of inflammatory caspases (mainly caspase-1, 4, 5, 11) and cleavage of various members of the Gasdermin family to form membrane perforation components, leading to cell membrane rupture, inflammatory mediators release, and cell death. Moderate pyroptosis is an innate immune response that fights against infection and plays an important role in the occurrence and development of the normal function of the immune system. However, excessive pyroptosis occurs and leads to immune disorders in many pathological conditions. Based on canonical pathways, research on pyroptosis regulation has demonstrated several pyroptotic inhibitors, including small-molecule drugs, natural products, and formulations of traditional Chinese medicines. In this paper, we review the characteristics and molecular mechanisms of pyroptosis, summarize inhibitors of pyroptosis, and propound that herbal medicines should be a focus on the research and development for pyroptosis blockers.

Breast cancer prognosis is frequently good but a substantial number of patients suffer from relapse. The death receptor ligand TRAIL can in combination with Smac mimetics induce apoptosis in some luminal-like ER-positive breast cancer cell lines, such as CAMA-1, but not in MCF-7 cells. Here we show that TRAIL and the Smac mimetic LCL161 induce non-canonical NF-κB and IFN signaling in ER-positive MCF-7 cells and in CAMA-1 breast cancer cells when apoptosis is blocked by caspase inhibition. Levels of p52 are increased and STAT1 gets phosphorylated. STAT1 phosphorylation is induced by TRAIL alone in MCF-7 cells and is independent of non-canonical NF-κB since downregulation of NIK has no effect. The phosphorylation of STAT1 is a rather late event, appearing after 24 hours of TRAIL stimulation. It is preceded by an increase in IFNB1 mRNA levels and can be blocked by siRNA targeting the type I IFN receptor IFNAR1 and by inhibition of Janus kinases by Ruxolitinib. Moreover, downregulation of caspase-8, but not inhibition of caspase activity, blocks TRAIL-mediated STAT1 phosphorylation and induction of IFN-related genes. The data suggest that TRAIL-induced IFNB1 expression in MCF-7 cells is dependent on a non-apoptotic role of caspase-8 and leads to autocrine interferon-β signaling.

To investigate the role and mechanism of insulin-like growth factor 1(IGF-1)-mediated EMT on multiple myeloma (MM) growth and metastasis.

The expression data from GEO datasets were utilized to explore the expression levels of IGF-1 and epithelial-mesenchymal transition (EMT) markers in MM. Western blotting and flow cytometry analysis were performed to detect the protein levels of EMT markers as well as key components of the PI3K/Akt pathway. Cell proliferation ability was assessed using colony formation assay and EdU incorporation assays. Transwell migration and invasion assays were performed to assess cell metastasis properties. Vimentin was knocked down by using electro-transfection with small interfering RNA (siRNA) to detect the effect of IGF-1-mediated EMT on MM cell growth and metastasis.

First of all, the analysis of GEO database revealed that IGF-1 was excessively expressed and closely correlated with the expression of the EMT markers in MM patients. Furthermore, we demonstrated that IGF-1 enhanced the acquisition of mesenchymal features in a time-dependent manner. Additionally, in vitro studies revealed that IGF-1-mediated mesenchymal phenotype promoted MM migration, invasion and colony formation. Finally, the mechanism study showed PI3K/Akt signaling pathway was involved in the IGF-1-induced EMT in MM cells.

IGF-1-induced mesenchymal phenotype contributed to MM progression via the PI3K/Akt pathway regulation.

TRAIL, a promising antitumor immuno-agent, exerted limited efficacy in clinical trials. The third disulfide loop of TGF-α (TGF3L peptide) with a very low affinity for EGFR has been reported to enhance the activity of fused antigens or cytokines. We wondered whether fusion of this peptide could enhance TRAIL activity and what the underlying mechanism for this enhancement would be. The TGF3L-TRAIL showed greatly enhanced cytotoxicity in a variety of cancer cell lines while spared normal cells unharmed. Typical apoptosis and cellular caspase activation were potently induced by TGF3L-TRAIL at the concentration levels corresponding to its cytotoxicity. TGF3L-TRAIL was able to activate both DR4 and DR5 the same as TRAIL did. It induced complete cell death in Colo205 through only one receptor when the other one was blocked, different from TRAIL-induced cell death (through DR4 dominantly). TGF3L-TRAIL cytotoxicity was not reduced in some cell lines even if both receptors are blocked simultaneously. Surprisingly, TGF3L-TRAIL self-assembled into stable polymers, which was responsible for its enhanced cytotoxicity. In human tumor xenograft mouse models, TGF3L-TRAIL showed anti-tumor activity similar to or better than TRAIL in different cancer cell types, consistent with its differing enhancement of cytotoxicity in vitro. Taken together, TGF3L fusion of TRAIL obviously enhances the anticancer activity of TRAIL by promoting assembly into polymers, which presents a novel fusion strategy for improving TRAIL function.

Despite recent advances in oncology, diagnosis, and therapy, treatment of pancreatic ductal adenocarcinoma (PDAC) is still exceedingly challenging. PDAC remains the fourth leading cause of cancer-related deaths worldwide. Poor prognosis is due to the aggressive growth behavior with early invasion and distant metastasis, chemoresistance, and a current lack of adequate screening methods for early detection. Consequently, novel therapeutic approaches are urgently needed. Many hopes for cancer treatment have been placed in the death ligand tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) since it was reported to induce apoptosis selectively in tumor cells in vitro and in vivo. TRAIL triggers apoptosis through binding of the trans-membrane death receptors TRAIL receptor 1 (TRAIL-R1) also death receptor 4 (DR4) and TRAIL receptor 2 (TRAIL-R2) also death receptor 5 (DR5) thereby inducing the formation of the death-inducing signaling complex (DISC) and activation of the apoptotic cascade. Unlike chemotherapeutics, TRAIL was shown to be able to induce apoptosis in a p53-independent manner, making TRAIL a promising anticancer approach for p53-mutated tumors. These cancer-selective traits of TRAIL led to the development of TRAIL-R agonists, categorized into either recombinant variants of TRAIL or agonistic antibodies against TRAIL-R1 or TRAIL-R2. However, clinical trials making use of these agonists in various tumor entities including pancreatic cancer were disappointing so far. This is thought to be caused by TRAIL resistance of numerous primary tumor cells, an insufficient agonistic activity of the drug candidates tested, and a lack of suitable biomarkers for patient stratification. Nevertheless, recently gained knowledge on the biology of the TRAIL-TRAIL-R system might now provide the chance to overcome intrinsic or acquired resistance against TRAIL and TRAIL-R agonists. In this review, we summarize the status quo of clinical studies involving TRAIL-R agonists for the treatment of pancreatic cancer and critically discuss the suitability of utilizing the TRAIL-TRAIL-R system for successful treatment.

Macrophage elastase (matrix metalloproteinase [MMP]-12) is a potent protease that contributes to the lung destruction that accompanies cigarette smoking; it simultaneously inhibits lung tumor angiogenesis and metastasis by catalyzing the formation of antiangiogenic peptides. Recent studies have revealed novel nonproteolytic functions of MMP12, including antimicrobial activity through a peptide within its C-terminal domain (CTD).

To determine whether the MMP12 CTD contributes to its antitumor activity in lung cancer.

We used recombinant MMP12 peptide fragments, including its catalytic domain, CTD, and a 20 amino acid peptide within the CTD (SR20), in an in vitro system to delineate their effects on non-small cell lung cancer cell proliferation and apoptosis. We translated our findings to two murine models of lung cancer, including orthotopic human xenograft and Kras^LSL/G12D mouse models of lung cancer.

We show that SR20 triggers tumor apoptosis by up-regulation of gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4, sensitizing cells to an autocrine loop of TRAIL-mediated cell death. We then demonstrate the therapeutic efficacy of SR20 against two murine models of lung cancer.

The MMP12 CTD initiates TRAIL-mediated tumor cell death through its conserved SR20 peptide.

TNF ligand superfamily member 10 (TRAIL) is a member of the tumor necrosis factor superfamily. The present study was performed in an effort to increase the expression of soluble (s)TRAIL by rebuilding the gene sequence of TRAIL. Three principles based on the codon bias of Escherichia coli were put forward to design the rebuild strategy. Relying on these three principles, a P7R mutation near the N‑terminal region of sTRAIL, named TRAIL‑Mu, was designed. TRAIL‑Mu was subsequently cloned into the PTWIN1 plasmid and expressed in E. coli BL21 (DE3). Using a high‑level expression system and a three‑step purification method, soluble TRAIL‑Mu protein reached ~90% of total cellular protein and purity was >95%, demonstrating success in overcoming inclusion body formation. The cytotoxic effect of TRAIL‑Mu was evaluated by sulforhodamine B assay in the MD‑MB‑231, A549, NCI‑H460 and L02 cell lines. The results demonstrated that TRAIL‑Mu exerted stronger antitumor effects on TRAIL‑sensitive tumor cell lines, and was able to partially reverse the resistance of a TRAIL‑resistant tumor cell line. In addition, TRAIL‑Mu exhibited no notable biological effects in a normal liver cell line. The novel TRAIL variant generated in the present study may be useful for the mass production of this important protein for therapeutic purposes.

Stem-cell based gene therapy is a promising novel therapeutic approach for inoperable invasive tumors, including brainstem glioma. Previously, we demonstrated the therapeutic potential of human adipose tissue-derived mesenchymal stem cells (hAT-MSC) genetically engineered to express a secreted form of tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) against brainstem glioma. However, safety concerns should be comprehensively investigated before clinical applications of hAT-MSC.sTRAIL. At first, we injected stereotactically low (1.2 × 10(5) cells/18 μL), medium (2.4 × 10(5)/18 μL), or high dose (3.6 × 10(5)/18 μL) of hAT-MSC.sTRAIL into the brainstems of immunodeficient mice reflecting the plan of the future clinical trial. Local toxicity, systemic toxicity, secondary tumor formation, and biodistribution of hAT-MSC.sTRAIL were investigated. Next, presence of hAT-MSC.sTRAIL was confirmed in the brain and major organs at 4, 9, and 14 weeks in brainstem glioma-bearing mice. In the 15-week subchronic toxicity test, no serious adverse events in terms of body weight, food consumption, clinical symptom, urinalysis, hematology, clinical chemistry, organ weight, and histopathology were observed. In the 26-week tumorigenicity test, hAT-MSC.sTRAIL made no detectable tumors, whereas positive control U-87 MG cells made huge tumors in the brainstem. No remaining hAT-MSC.sTRAIL was observed in any organs examined, including the brainstem at 15 or 26 weeks. In brainstem glioma-bearing mice, injected hAT-MSC.sTRAIL was observed, but gradually decreased over time in the brain. The mRNA of human specific GAPDH and TRAIL was not detected in all major organs. These results indicate that the hAT-MSC.sTRAIL could be applicable to the future clinical trials in terms of biosafety.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of TNF family, is an interesting ligand which selectively induces apoptosis in tumor cells and, therefore, it has been developed for cancer therapy. This ligand has been produced by various hosts such as E.coli. However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream. The aim of this study is to develop an expression system for the production of recombinant TRAIL secreted to the E.coli periplasm instead of cytoplasm. By using Overlapping Extension PCR, an OmpA signal sequence was fused to TRAIL cDNA and OmpA-TRAIL fragment was then cloned in pET-22b plasmid. This construct was confirmed by PCR and DNA sequencing. Promoter was induced in E.coli BL21 (DE3) and periplasmic expressed proteins were released using osmotic shock procedure. SDS-PAGE analysis showed that about 37% of recombinant TRAIL was transferred into the periplasm and its identity was confirmed by western blot analysis. Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay. The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space.

One of the major challenges in the treatment of lung cancer is the development of drug resistance. This represents a major obstacle in the treatment of patients, limiting the efficacy of both conventional chemotherapy and biological therapies. Deciphering the mechanisms of resistance is critical to further understanding the multifactorial pathways involved, and in developing more specific targeted treatments. To date, numerous studies have reported the potential role of microRNAs (miRNAs) in resistance to various cancer treatments. MicroRNAs are a family of small non-coding RNAs that regulate gene expression by sequence-specific targeting of mRNAs causing translational repression or mRNA degradation. More than 1200 validated human miRNAs have been identified to date. While as little as one miRNA can regulate hundreds of targets, a single target can also be affected by multiple miRNAs. Evidence suggests that dysregulation of specific miRNAs may be involved in the acquisition of resistance to a number of cancer treatments, thereby modulating the sensitivity of cancer cells to such therapies. Therefore, targeting miRNAs may be an attractive strategy for developing novel and more effective individualized therapies, improving drug efficiency, and for predicting patient response to different treatments. In this review, we provide an overview on the role of miRNAs in resistance to current lung cancer therapies and novel biological agents.

Basophilia is associated with allergic and parasitic diseases and advanced chronic myeloid leukemia. In the present study, we characterized the expression and function of the death receptors Fas/CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors in basophils from healthy donors compared to neoplastic basophils. Peripheral blood basophils obtained from healthy donors (HD-PBB) and from patients with chronic myeloid leukemia (CML-PBB) were found to express high levels of Fas/CD95 and low levels of TRAIL-R2, whereas the basophil-like chronic myeloid leukemia cell line KU-812 expressed significant levels of TRAIL-R1 and TRAIL-R2. HD-PBB underwent apoptosis in response to anti-Fas/CD95, but showed resistance to TRAIL, unless they were co-treated with actinomycin D. Interestingly, CML-PBB and KU-812 cells exhibited the opposite response pattern with resistance to anti-Fas/CD95, but significant susceptibility to TRAIL-induced apoptosis. Our data show that anti-Fas/CD95 and TRAIL differentially regulate apoptosis of normal and neoplastic human basophils, which may direct the development of novel therapeutic strategies.

Non-small cell lung cancer (NSCLC) accounts for ∼80% of all lung cancers. Although some advances in lung cancer therapy have been made, patient survival is still quite poor. Two microRNAs, miR-221 and miR-222, upregulated by the MET proto-oncogene, have been already described to enhance cell survival and to induce TNF-related apoptosis-inducing ligand (TRAIL) resistance in NSCLC cell lines, through the downregulation of p27(kip1), PTEN and TIMP3. Here, we further investigated this pathway and showed that miR-130a, expressed at low level in lung cancer cell lines, by targeting MET was able to reduce TRAIL resistance in NSCLC cells through the c-Jun-mediated downregulation of miR-221 and miR-222. Moreover, we found that miR-130a reduced migratory capacity of NSCLC. A better understanding of MET-miR-221 and 222 axis regulation in drug resistance is the key in developing new strategies in NSCLC therapy.

Human tumor necrosis factor related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in a variety of transformed cells and is currently being developed as a cancer therapeutic drug. Here we expressed the TRAIL protein including extracellular (114-281aa) without any tag protein named TRAIL-NT, and prepared anti-TRAIL polyclonal antibodies (Poly-Ab). The human TRAIL extracellular gene was amplified from PBMC and cloned into pGEM-T-Easy vector for sequence analysis. The expression vector pET-28a/TRAIL was constructed using the DNA recombinant method, and the recombinant protein without any tag protein was expressed in Escherichia coli BL21(DE3). The TRAIL-NT protein was purified by cation ion-exchange column and identified by SDS-PAGE and Western blot analysis. The proliferation inhibition activity of TRAIL-NT was detected by the MTT method, Wright-Giemsa staining assay, and FACS. The polyclonal antibody of TRAIL-NT was obtained after the BALB/C mice were immunized with purificated TRAIL-NT protein. Results showed that the target protein expressed in E. coli BL21(DE3) has the same molecular weight as that expected and could be recognized by anti-TRIAL Poly-Ab. The TRAIL-NT protein could also inhibit proliferation and induced apoptosis of Jurkat cells but no cytotoxicity to human liver cells and PBMC was observed. This preliminary research laid a solid foundation for further research on its biological activity and application in anti-tumor therapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is one of the tumor necrosis factor (TNF) superfamily members, participating in many biological processes including apoptosis and immune responses. In present study, a novel human soluble TRAIL (sTRAIL) homolog, CasTRAIL was identified from the oyster, Crassostrea ariakensis. CasTRAIL has a 99% and 98% similarity to human sTRAIL over the cDNA sequence and the amino acid sequence, respectively. It mostly distributes in tissues of the oyster defense system and was mainly localized at cell membrane, and has no cytotoxicity to normal hemocytes of oyster. The phosphorylation state of MAP kinases revealed that CasTRAIL induced a rapid increase in the phospho-ERK and phospho-p38 levels, which indicated that the MAPK pathway was involved in CasTRAIL-mediated signaling. In addition, CasTRAIL also showed an ability of anti-RLO infection which might be through the p38-MAPK activation pathway. Present studies provide an understanding and insight of the biological functions of CasTRAIL.

We earlier reported that PU.1 was downregulated in myeloma cell lines and myeloma cells in a subset of myeloma patients, and that conditional PU.1 expression in PU.1-negative myeloma cell lines, U266 and KMS12PE, induced growth arrest and apoptosis. To elucidate the molecular mechanisms of the growth arrest and apoptosis, we performed DNA microarray analyses to compare the difference in gene expression before and after PU.1 induction in U266 cells. Among cell cycle-related genes, cyclin A2, cyclin B1, CDK2 and CDK4 were downregulated and p21 was upregulated, although among apoptosis-related genes, tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) was found highly upregulated. When TRAIL was knocked down by small interference RNAs, apoptosis of PU-1-expressing cells was inhibited, suggesting that TRAIL has a critical role in PU.1-induced apoptosis in both U266 and KMS12PE myeloma cells. In both U266 and KMS12PE cells expressing PU.1, PU.1 directly bound to a region 30 bp downstream of the transcription start site of the TRAIL gene. Upregulation of PU.1-induced transactivation of the TRAIL promoter in reporter assays, and disruption of the PU.1-binding site in the TRAIL promoter eliminated this transactivation. Therefore, we conclude that PU.1 is capable of inducing apoptosis in certain myeloma cells by direct transactivation of TRAIL.

The Death Receptor 5 (DR5), a member of tumor necrosis factor receptor (TNFR) superfamily of receptors, triggers apoptosis (programmed cell death) when stimulated by its tridentate ligand TRAIL. Until recently it was generally assumed that the activation of DR5 resulted from the recruitment of three independent receptor units, leading to the trimerization of intracellular domains. However, there is mounting evidence to suggest that, in the absence of ligand, such cytokine receptors primarily reside as preformed complexes. In this work, molecular dynamics simulations of the TRAIL-DR5 complex, the unbound receptor trimer and individual receptor monomers are compared to gain insight in the mechanism of activation. The results suggest that, in the absence of TRAIL, DR5 has a strong propensity to self-associate and that this is primarily mediated through interactions of the membrane proximal domains. The association of the free receptors leads to a loss of the threefold symmetry found within the receptor-ligand complex. The simulations suggest that the primary role of TRAIL is to induce threefold-symmetry within the DR5 complex and to constrain the receptor to a specific conformation. The implications of this in terms of the mechanism by which the receptor switches from an inactive to an active state are discussed.

Juvenile dermatomyositis (JDM) is the most common myopathy in children with characteristic skin rash and muscle weakness, in which longer duration of untreated disease was associated with less muscle weakness. The duration of untreated inflammation may alter the apoptotic pathways involved in skeletal muscle damage. Diagnostic muscle biopsies from 14 untreated patients were stained for apoptosis markers. TUNEL-positive nuclei and caspase 3 were detected within the laminin layer, indicating apoptosis of skeletal muscle nuclei. Untreated JDM disease duration greater than 2 months ("long"), was associated with higher Fas-positive cell counts in the perivascular region compared with the "short" disease duration group, 2 months or less. Within the "long" duration group, higher Fas-positive cell counts were positively associated with increased TUNEL-positive nuclei and caspase 3. We conclude that the duration of untreated disease (chronic inflammation) influences the mode of continuing cell damage and death in children with JDM.

Based on preclinical studies demonstrating that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts a potent and cancer cell-specific proapoptotic activity, recombinant TRAIL as well as agonistic anti-TRAIL-R1 and anti-TRAIL-R2 antibodies recently entered clinical trials. Additionally, gene therapy approaches using TRAIL-encoding adenovirus (Ad-TRAIL) are currently being developed to overcome the limitations inherent to TRAIL receptor targeting, i.e., pharmacokinetic of soluble TRAIL, pattern of receptor expression, and tumor cell resistance. To optimize gene therapy approaches, CD34+ cells transduced with Ad-TRAIL (CD34-TRAIL+) have been investigated as cellular vehicles for TRAIL delivery. Transduced cells exhibit a potent tumor killing activity on a variety of tumor cell types both in vitro and in vivo and are also cytotoxic against tumor cells resistant to soluble TRAIL. Studies in tumor-bearing nonobese diabetic/severe combined immunodeficient mice suggest that the antitumor effect of CD34-TRAIL+ cells is mediated by both direct tumor cell killing due to apoptosis and indirect tumor cell killing due to vascular-disrupting mechanisms. The clinical translation of cell and gene therapy approaches represent a challenging strategy that might achieve systemic tumor targeting and increased intratumor delivery of the therapeutic agent.

Previous studies indicated that signalling via CD95 and DR5 is greatly enhanced by the formation of ceramide-enriched membrane platforms. Here, we employed this concept to convert doses of subtherapeutic TRAIL that were unable to release ceramide and kill leukemic B-cells or ex vivo T lymphocytes, into a very effective apoptotic stimulus. Ceramide production was induced by application of sub-toxic doses of doxorubicin that resulted in an activation of the acid sphingomyelinase (ASM), release of ceramide and formation of ceramide-enriched membrane platforms. The latter served DR5 to cluster after application of very low doses of TRAIL in combination with doxorubicin. Genetic deficiency of the ASM abrogated doxorubicin-induced ceramide release, as well as clustering of DR5 and apoptosis induced by the combined treatment of doxorubicin and TRAIL. These data show that local release of ceramide potentiates very low, otherwise inactive doses of TRAIL that may represent a novel therapeutic concept to treat tumors.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of transformed cells while sparing normal cells. To enhance the therapeutic index of soluble (s)TRAIL, we used CD34+ cells transduced with a replication-deficient adenovirus encoding the human TRAIL gene (CD34-TRAIL+) for the systemic delivery of membrane-bound (m)TRAIL to lymphoid tumors. CD34-TRAIL+ cells were evaluated for their activity in vitro and in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with sTRAIL-sensitive and -resistant tumors. In vitro, coculturing CD34-TRAIL+ cells with sTRAIL-sensitive or -resistant lymphoma cell lines induced significant levels of caspase-dependent tumor cell death. In vivo, CD34-TRAIL+ cells significantly increased the survival of NOD/SCID mice bearing sTRAIL-sensitive or -resistant lymphoid tumors at an early or advanced stage of disease. No obvious toxicity was observed on administration of CD34-TRAIL+ cells. Histological analysis revealed high-level expression of the agonistic receptor TRAIL-R2 by tumor endothelial cells, and efficient tumor homing of transduced cells. Injection of CD34-TRAIL+ cells resulted in extensive damage of tumor vasculature followed by hemorrhagic necrosis exhibiting a perivascular distribution. These results show that CD34-TRAIL+ cells might be an efficient vehicle for mTRAIL delivery to tumors, where they exert a potent antitumor effect possibly mediated by both direct tumor cell killing and indirect vascular-disrupting mechanisms.

We recently reported that, in cultured leukemic T lymphocytes and promyelocytic cells, a mild heat shock treatment (1 h at 42 degrees C) induced a long lasting stimulation of the apoptosis induced by TNF-related apoptosis inducing ligand (TRAIL). On the opposite, no effects were recorded toward normal human T lymphocytes. The apoptogenic efficiency of TRAIL in leukemic lymphocytes is linked to the long lasting increased ability of TRAIL to recognize and bind DR4 and DR5 receptors during hyperthermia. Here, we have analyzed whether this new apoptotic co-treatment could be relevant toward primary cells from patients suffering of chronic lymphocytic leukemia. Analysis of samples from 24 patients with different ages, sex and disease stages revealed that half of them had lymphocytes that, once isolated and analyzed in vitro, positively responded (increase of cell death) to the heat shock plus TRAIL co-treatment. Analysis of the level of expression of various anti-apoptotic proteins in the cell samples revealed a great heterogeneity between patients and no clear relationships could be drawn. Nevertheless, most cell samples that were sensitive to TRAIL plus heat shock induced apoptosis displayed a higher level of cell surface DR4 and DR5 receptors than the non-sensitive counterparts. Hence, analysis of the level of TRAIL surface receptors is a prerequisite for future clinical applications based on this protocol.

Metastatic liver tumors are highly malignant and refractory to conventional therapies. TRAIL-resistant CT-26 cells underwent apoptosis in vitro in the presence of both recombinant TRAIL (rTRAIL) and a suboptimal dose of actinomycin D (ACD). Co-administration of soluble TRAIL (sTRAIL) gene and ACD suppressed the metastatic liver tumors of CT-26, significantly inducing apoptosis in the tumors, while such effects were not demonstrated in mice that received either the sTRAIL gene or ACD alone. The gene therapy of sTRAIL with a suboptimal dose of an anticancer drug is a new strategy for treatment of multiple liver metastasis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of malignant cells, but not in normal cells. This preferential toxicity to the abnormal cells renders TRAIL potentially a very powerful therapeutic weapon against cancer. However, a requirement for large quantities of TRAIL to suppress tumor growth in vivo is one of the major factors that has hindered it from being widely applied clinically. To overcome this, we constructed a replication-deficient adenovirus that carries a human full-length TRAIL gene (Ad-TRAIL) and tested its efficacy against a lung cancer model system in comparison to that of the recombinant soluble TRAIL protein.

To investigate the antitumor activity and therapeutic value of the Ad-TRAIL on the non-small cell lung cancer (NSCLC), four NSCLC cell lines, namely, YTMLC, GLC, A549, and H460 cells, were used. TRAIL protein expression was determined by Western blotting and flow cytometry. Cell viability was analyzed by proliferation assay, and DNA ladder and cell-cycle analysis were used to identify apoptosis. To further evaluate the effect of Ad-TRAIL in vivo, YTMLC cells were inoculated to the subcutis of nude mice. The Ad-TRAIL was subsequently administered into the established tumors. Tumor growth and the TRAIL toxicity were evaluated after treatment.

YTMLC cells infected with Ad-TRAIL showed decreased cell viability and a higher percentage of apoptosis. Similar, Ad-TRAIL treatment also significantly suppressed tumor growth in vivo.

TRAIL gene therapy provides a promising therapy for the treatment of NSCLC.

Cancer is a widespread disease, with half of all men and one-third of all women in the United States developing cancer during their lifetime. The efficacy of many cancer treatments including radiotherapy, chemotherapy and immunotherapy is due to their ability to induce tumor cell apoptosis. Recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is currently being developed as a cancer therapeutic since it selectively induces apoptosis in a variety of transformed cells, but not in most normal cells. Agonistic monoclonal antibodies (mAbs) specific for human death-inducing TRAIL receptors (DR4 or DR5) are also being actively pursued. Importantly, in experimental mice, synergistic anti-tumor effects have been observed with a combination treatment of agonistic mAb against DR5 together with either IL-21 or agonistic mAbs against CD40 and CD137. Together, these findings suggest that antibody-based therapies that cause tumor cell apoptosis and promote T cell memory or function may be effective in fighting cancer.

The combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cisplatin resulted in a greater cytotoxicity than could be accounted for by the addition of the cytotoxic effects of the agents alone. In this study, we hypothesized that the synergistic interaction between the two modalities can be changed when both the sequence and the time interval between the two treatments are varied. To test the hypothesis, human head-and-neck squamous-cell carcinoma (HNSCC)-6 cells were either pretreated with 0.01-0.5 microg/ml TRAIL for various times (0-24 h) followed by treatment with 5 microg/ml cisplatin or pretreated with 5 microg/ml cisplatin for various times (0-24 h) followed by treatment with 0.5 microg/ml TRAIL. In latter case, the synergistic effect was gradually increased when the time interval between the two treatments was increased. In former case, a maximal synergy occurred within 0-4 h of pretreatment with TRAIL. However, the synergistic effect was gradually decreased when the time interval between the two treatments was increased. Data from immunoblotting analysis reveal that a similar pattern emerged for the PARP cleavage and caspase activation. The synergistic effect is not associated with DR4, DR5, FADD, and FLIP(L). Interestingly, a complex pattern of synergistic interaction between TRAIL and cisplatin is related to the cleavage of FLIP(S). Although overexpression of FLIP(S) protected cells from FLIP(S) cleavage and apoptotic death, blockage of FLIP(S) cleavage by replacing Asp(39) and Asp(42) residues with alanine did not further enhance FLIP(S)-mediated protection. Taken together, FLIP(S) cleavage reflects apoptotic damage, but it does not cause apoptosis.

The brain is particularly vulnerable to oxygen free radicals, and these radicals have been implicated in the pathology of several neurological disorders. In this study, the modulation of TNF-related apoptosis-inducing ligand (TRAIL) expression by oxidative stress was shown in LN215 cells, an astroglioma cell line. Hydrogen peroxide (H2O2) treatment increased TRAIL expression in LN215 cells and H2O2-induced TRAIL augmented apoptosis in Peer cells, a cell line sensitive to TRAIL- mediated cell death. Our findings suggest that the upregulation of TRAIL in astroglial cells may abrogate immune cell effector functions.

The cell-extrinsic apoptotic pathway triggers programmed cell death in response to certain ligands that bind to cell-surface death receptors. Apoptosis is essential for normal development and homeostasis in metazoans, and furthermore, selective activation of the cell-extrinsic pathway in tumor cells holds considerable promise for cancer therapy. We used phage display to identify peptides and synthetic antibodies that specifically bind to the human proapoptotic death receptor DR5. Despite great differences in overall size and structure, the DR5-binding peptides and antibodies shared a tripeptide motif, which was conserved within a disulfide-constrained loop of the peptides and the third complementarity determining region of the antibody heavy chains. The X-ray crystal structure of an antibody in complex with DR5 revealed that the tripeptide motif is buried at the core of the interface, confirming its central role in antigen recognition. We found that certain peptides and antibodies exhibited potent proapoptotic activity against DR5-expressing SK-MES-1 lung carcinoma cells. These phage-derived ligands may be useful for elucidating DR5 activation at the molecular level and for creating synthetic agonists of proapoptotic death receptors.

TRAIL/Apo2L is a CD95 ligand-related member of the TNF family that initiates apoptosis in immune and neoplastic cells after binding to specific surface receptors. The authors previously reported a specific topographic pattern of TRAIL expression in the normal colonic mucosa and the loss of TRAIL expression in tubular adenomas as well as in most colon carcinomas. Therefore, they hypothesized that similar changes may occur during the malignant transformation of Barrett's esophagus. The aim of this study was to compare TRAIL/Apo2L expression in normal gastroesophageal (GE) junction, Barrett's esophagus with and without dysplasia, and associated adenocarcinoma. Immunohistochemical evaluation of TRAIL expression was performed on formalin-fixed paraffin-embedded sections from 29 GE junction/esophageal biopsies, 20 gastric biopsies, 6 esophagectomies, 2 small bowel resection specimens, and 5 colon biopsies. The expression was graded semiquantitatively on a 4-point scale (0-3). TRAIL was expressed in the foveolar epithelium of the histologically normal GE junctional mucosa and stomach as well as in the normal intestinal epithelium, with maximal expression in the surface epithelium. TRAIL was always detected in Barrett's metaplasia (21/21, 100%), and the overall expression was similar to that of the columnar portion of the normal GE junction (8/8, 100%). TRAIL was rarely and weakly (1+) expressed in Barrett's esophagus with dysplasia (3/18, 16.7%) and adenocarcinoma (1/10, 10.0%) (P<0.001). Similarities in the topographic pattern of TRAIL expression in the normal GE junction, stomach, small intestine, and colon suggest a common function of TRAIL throughout the gastrointestinal tract. These results show that the downregulation of TRAIL is associated with development of dysplasia in Barrett's esophagus. Thus, the immunohistochemically detected downregulation of TRAIL expression appears to be a promising indicator of dysplasia in Barrett's esophagus.

We have previously shown that activation of the acid sphingomyelinase (ASM), the release of ceramide and the formation of ceramide-enriched membrane domains are central for the induction of apoptosis by CD95. Here, we demonstrate that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 activate the ASM via a redox mechanism resulting in release of ceramide and formation of ceramide-enriched membrane platforms. Ceramide-enriched membrane platforms serve to cluster DR5 upon stimulation. Antioxidants prevent TRAIL-mediated stimulation of ASM, the release of ceramide, the formation of ceramide-enriched membrane platforms and the induction of apoptosis by TRAIL. Further, ASM-deficient splenocytes fail to cluster DR5 in ceramide-enriched membrane domains upon TRAIL stimulation and resist TRAIL-induced apoptosis, events that were restored by addition of natural C(16)-ceramide. A dose-response analysis indicates that ceramide-enriched membrane platforms greatly sensitized tumor cells to TRAIL-induced apoptosis. Our data indicate that ceramide-enriched membrane platforms are required for the signaling of TRAIL-DR5 complexes under physiological conditions.

Overexpression of anti-apoptotic Bcl-2 family proteins may play an important role in the aggressive behavior of prostate cancer cells and their resistance to therapy. The Bcl-2 homology 3 domain (BH3) is a uniquely important functional element within the pro-apoptotic class of the Bcl-2-related proteins, mediating their ability to dimerize with other Bcl-2-related proteins and promote apoptosis. The BH3 inhibitors (BH3Is) function by disrupting the interactions mediated by the BH3 domain between pro- and anti-apoptotic members of the Bcl-2 family and liberating more Bax/Bak to induce mitochondrial membrane permeabilization. LNCaP-derived C4-2 human prostate cancer cells are quite resistant to non-tagged, human recombinant soluble Apo2 ligand [Apo2L, also Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL], a tumor specific drug that is now in clinical trials. However, when Apo2L/TRAIL was combined with the Bcl-xL inhibitor, BH3I-2', it induced apoptosis synergistically through activation of Caspase-8 and the proapoptotic Bcl-2 family member Bid, resulting in the activation of effector Caspase-3 and proteolytic cleavage of Poly(ADP-ribose) polymerase, events that were blocked by the pan-caspase inhibitor zVAD-fmk. Our data indicate that, in combination with the BH3 mimetic, BH3I-2', Apo2L/TRAIL synergistically induces apoptosis in C4-2 human prostate cancer cells through both the extrinsic and intrinsic apoptotic pathways.

To investigate the antitumor activity and safety of a novel recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmh TRAIL).

Antitumor activity of rmh TRAIL was evaluated by using several tumor cell lines by MTT assay in vitro, and by using a mouse xenograft model in vivo. rmh TRAIL-induced apoptosis in tumor cells was detected by cell death enzyme-linked immunosorbent assay (ELISA), TdT-mediated dUTP nick-end labeling (TUNEL) assay and flow cytometry. The safety of rmh TRAIL was also evaluated in several normal human cell lines.

At the concentration of 0.32-1 000 ng/mL, rmh TRAIL remarkably inhibited the proliferation of 5 tumor cell lines from lung, colon, and breast cancer compared with wild type (wt TRAIL) in vitro, whereas at the concentration of 1 ng/mL-10 microg/mL, rmh TRAIL showed no or mild cytotoxicity in the normal cell lines. rmh TRAIL (3, 15 mg/kg, ip, once daily for 10 d) exerted a significant inhibition on the growth of xenograft tumor NCI-H460 in nude mice compared with the saline group (P<0.01), and was more potent than wt TRAIL, a positive control. The apoptosis of NCI-H460 cells was markedly induced in a concentration-dependent and time-dependent manner after rmh TRAIL treatment. The percentage of apoptotic cells induced by rmh TRAIL in NCI-H460 cells was significantly higher than that by wt TRAIL.

rmh TRAIL provided potent antitumor activity in vivo and in vitro, whereas most normal human cells were resistant to rmh TRAIL. The results suggested that rmh TRAIL might be a useful anticancer agent in future.

Death receptor 5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L). In this study, we showed that tunicamycin, a naturally occurring antibiotic, is a potent enhancer of TRAIL-induced apoptosis through up-regulation of DR5 expression. Tunicamycin significantly sensitized PC-3, androgen-independent human prostate cancer cells, to TRAIL-induced apoptosis. The tunicamycin-mediated enhancement of TRAIL-induced apoptosis was markedly blocked by a recombinant human DR5/Fc chimeric protein. Tunicamycin and TRAIL cooperatively activated caspase-8, -10, -9, and -3 and Bid cleavage and this activation was also blocked in the presence of the DR5/Fc chimera. Tunicamycin up-regulated DR5 expression at the mRNA and protein levels in a dose-dependent manner. Furthermore, the tunicamycin-mediated sensitization to TRAIL was efficiently reduced by DR5 small interfering RNA, suggesting that the sensitization was mediated through induction of DR5 expression. Tunicamycin increased DR5 promoter activity and this enhanced activity was diminished by mutation of a CHOP-binding site. In addition, suppression of CHOP expression by small interfering RNA reduced the tunicamycin-mediated induction of DR5. Of note, tunicamycin-mediated induction of CHOP and DR5 protein expression was not observed in normal human peripheral blood mononuclear cells. Moreover, tunicamycin did not sensitize the cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may be a promising candidate for prostate cancer therapy.

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis in various tumor cell types and is under investigation as a cancer therapeutic. The development of a recombinant adenovirus encoding the full-length human TRAIL gene (Ad5-TRAIL) replaces the need for large quantities of soluble TRAIL protein in tumor suppressive therapies. However, the full potential of Ad5-TRAIL has not yet been maximized. Recent investigation of a histone deacetylase inhibitor, depsipeptide (FR901228), has demonstrated that it increases cellular susceptibility to adenovirus infection and augments adenoviral transgene expression. Thus, studies were initiated to evaluate the ability of depsipeptide to enhance the cytotoxic activity of Ad5-TRAIL against human prostate tumor cells. In vitro, depsipeptide increased expression of coxsackie-adenovirus receptor, leading to increased adenoviral infection and transgene expression. Additionally, tumor cell killing by Ad5-TRAIL was higher following depsipeptide pretreatment. More surprisingly, depsipeptide also increased prostate tumor cell sensitivity to TRAIL-induced apoptosis. Investigation into the mechanism responsible for increased TRAIL responsiveness revealed increased levels of TRAIL-R1 and -R2 in membrane lipid rafts following depsipeptide treatment. These results indicate that depsipeptide is a potent agent for enhancing the activity of Ad5-TRAIL by multiple mechanisms, allowing for a more efficient use of Ad5-TRAIL as an antitumor therapy.

Death receptor 5 (DR5) is a receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL is a promising candidate for cancer therapeutics due to its ability to induce apoptosis selectively in cancer cells. Here, we report that histone deacetylase inhibitors (HDACIs) such as trichostatin A (TSA), sodium butyrate, and suberoylanilide hydroxamic acid (SAHA) upregulated DR5 expression in various human malignant tumor cells. An RNase protection assay demonstrated that HDACIs induced DR5 mRNA markedly but not that of other death receptor family members in Jurkat cells. HDACIs increased DR5 mRNA and protein in a dose- and time-dependent manner. We also show TSA increased DR5 promoter activity using a luciferase promoter assay. Furthermore, we demonstrated that HDACIs strongly sensitized exogenous soluble recombinant human TRAIL-induced apoptosis synergistically in Jurkat and HL-60 cells that were tolerant to TRAIL alone. The combined use of HDACIs and TRAIL in suboptimal concentrations induced Bid cleavage and activation of caspase-8, -10, -3, and -9. Human recombinant DR5/Fc chimera protein, zVAD-fmk pancaspase inhibitor, and caspase-8 and -10 inhibitors efficiently reduced apoptosis induced by cotreatment with HDACIs and TRAIL. Furthermore, TSA did not significantly induce DR5 protein and HDACIs did not enhance TRAIL-induced apoptosis in normal human peripheral blood mononuclear cells. These results suggest that this combined treatment with HDACIs and TRAIL is a promising strategy for new cancer therapeutics.

TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) and KILLER are a death-inducing ligand and receptor pair that belong to the TNF and TNF-receptor superfamilies, respectively. To date, only one apoptosis-inducing TRAIL receptor (murine KILLER [MK]) has been identified in mice, and it is a homologue of human Death Receptor 5. Whereas the expression of other death receptors, such as Fas and TNF receptor 1 have been documented in mammalian preimplantation embryos, no evidence currently demonstrates either the presence or the function of TRAIL and its corresponding death receptor, MK. Using reverse transcription-polymerase chain reaction and confocal immunofluorescent microscopy, we found that both TRAIL and MK are expressed from the 1-cell through the blastocyst stage of murine preimplantation embryo development. These proteins are localized mainly at the cell surface from the 1-cell through the morula stage. At the blastocyst stage, both TRAIL and MK exhibit an apical staining pattern in the trophectoderm cells. Finally, using the TUNEL assay, we demonstrated that MK induces apoptosis in blastocysts sensitized to TRAIL via actinomycin D. Taken together, these data are the first to demonstrate the presence and function of TRAIL and MK, a death-inducing ligand and its receptor, in mammalian preimplantation embryos.

Renal cell carcinoma (RCC) is one of the most drug-resistant malignancies in humans. We show that adriamycin (ADR) and TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L have a synergistic cytotoxic effect against RCC cells. This synergistic cytotoxicity was obtained in ACHN, A704, Caki-1 and Caki-2 human RCC cell lines and freshly derived RCC cells from 6 patients. This synergistic effect, however, was not achieved in 5 samples of freshly isolated normal kidney cells. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of TRAIL/Apo2L and ADR was realized by inducing apoptosis. Sequential treatment with ADR followed by TRAIL/Apo2L induced significantly more cytotoxicity than the reverse treatment. ADR increased the expression of DR4 and DR5 in RCC cells, but not in the normal kidney cells. Furthermore, the synergistic cytotoxicity was significantly inhibited by DR4:Fc and DR5:Fc fusion proteins, which inhibit TRAIL/Apo2L-mediated apoptosis. In addition, caspase activity assays and treatment of caspase inhibitors demonstrated that the combination treatment with ADR and TRAIL/Apo2L activated caspase cascade, including caspase-9, -8, -6 and -3, which were the downstream molecules of death receptors. These findings indicate that ADR sensitizes RCC cells to TRAIL/Apo2L-mediated apoptosis through induction of DR4 and DR5, suggesting that the combination therapy of TRAIL/Apo2L and ADR might be effective for RCC therapy.

Stroke is one of the leading causes of death and severe disability in most industrialized countries. Despite the extensive research efforts of both academic and industrial laboratories during the last few decades, no changes have been brought about by the design of neuroprotective therapies. The progressive decrease of stroke-induced death and disability is entirely attributable to improvements in the identification and reduction of risk factors. Over the past few years, experimental research has led to the emergence of a wealth of information regarding the complex and interrelated processes of neuronal degeneration and death triggered by ischemia. This unprecedented insight has led to new theories on the mechanisms of ischemic damage, and has suggested new targets and strategies for therapeutic intervention designed to reduce the clinical consequences of stroke. Among current developments, three strategies seem particularly appealing namely, the limitation of initial or secondary neuronal death by inhibition of apoptotic mechanisms, the enhancement of the endogenous capacity of nervous structures to restore lost function, and the replacement of lost cells by transplantation therapy.

Severe bone destruction due to inappropriate osteoclastogenesis is a prominent feature of multiple myeloma (MM). MM increases bone loss by disrupting the checks that normally control signaling by receptor activator of nuclear factor kappaB ligand (RANK-L, also called TRANCE [tumor necrosis factor-related, activation-induced cytokine], osteoprotegerin ligand [OPG-L], osteoclast differentiation factor [ODF], and tumor necrosis factor superfamily member 11 [TNFSF11]), a TNF-family cytokine required for osteoclast differentiation and activation. RANK-L binds to its functional receptor RANK (TNF receptor superfamily member 11a [TNF RSF11a]) to stimulate osteoclastogenesis. Osteotropic cytokines regulate this process by controlling bone marrow stromal expression of RANK-L. Further control over osteoclastogenesis is maintained by regulated expression of osteoprotegerin (OPG, also called osteoclastogenesis inhibitory factor and TNFRSF11b), a soluble decoy receptor for RANK-L. In normal bone marrow, abundant stores of OPG in stroma, megakaryocytes, and myeloid cells provide a natural buffer against increased RANK-L. MM disrupts these controls by increasing expression of RANK-L and decreasing expression of OPG. Concurrent deregulation of RANK-L and OPG expression is found in bone marrow biopsies from patients with MM but not in specimens from patients with non-MM hematologic malignancies.

RANK-Fc is a recombinant RANK-L antagonist that is formed by fusing the extracellular domain of RANK to the Fc portion of human immunoglobulin G(1) (hIgG(1)). In vitro, addition of RANK-Fc virtually eliminates the formation of osteoclasts in cocultures of MM with bone marrow and osteoblast/stromal cells. The severe combined immunodeficiency (SCID)/ARH77 mouse model and the SCID-hu-MM mouse model of human MM were used to assess the ability of RANK-Fc to block the development of MM-induced bone disease in vivo. Mice received either RANK-Fc or hIgG(1) 200 microg intravenously three times per week.

RANK-Fc limited bone destruction in both the SCID/ARH-77 model and the SCID-hu-MM model. Administration of RANK-Fc also caused a marked reduction in tumor burden and serum paraprotein in SCID-hu-MM mice that was associated with the restoration of OPG and a reduction in RANK-L expression in the xenograft.

MM-induced bone destruction requires increased RANK-L expression and is facilitated by a concurrent reduction in OPG, a natural decoy receptor for RANK-L. Administration of the RANK-L antagonist RANK-Fc limits MM-induced osteoclastogenesis, development of bone disease, and MM tumor progression.

Apoptosis has emerged as an important mechanism to reduce numbers of activated stellate cells during the resolution phase of hepatic fibrosis. These observations suggest that activated stellate cells may be more susceptible to apoptotic stimuli than their quiescent counterparts. Because other activated cell types are more sensitive than their quiescent phenotypes to apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we examined the expression of TRAIL death receptors (DRs) and susceptibility to TRAIL cytotoxicity in stellate cells undergoing progressive activation. A spontaneously immortalized human stellate cell line, LX-2, was analyzed during 14 days of progressive activation following plating, during which time alpha-smooth muscle actin (alpha-SMA) and a beta-crystallin (markers of stellate cell activation) messenger RNA (mRNA) increased 7-fold and 5-fold, respectively. During this same interval, TRAIL-R1/DR4 and TRAIL-R2/DR5 mRNA expression increased 18-fold and 17.6-fold, although TRAIL-R2/DR5 expression was quantitatively 103-fold greater than TRAIL-R1/DR4; parallel changes occurred in TRAIL/DR5 protein expression and cellular susceptibility to TRAIL-mediated apoptosis. Similar findings were observed in primary murine stellate cells undergoing activation on a plastic surface. In conclusion, stellate cells show activation-dependent TRAIL-R2/DR5 expression and TRAIL-mediated apoptosis. Because TRAIL-R2/DR5 is not expressed by hepatocytes, TRAIL/DR5 agonists may be useful in reducing fibrosis by inducing stellate cell apoptosis.

This study describes experiments on cytotoxic effects and the production of oxidative radicals and the proinflammatory cytokine tumor growth factor alpha (TNFalpha) in a cell line of rat lung macrophages exposed to aqueous extracts from ambient air particles <10 microm (PM(10)) collected on Teflon filters. The particles were collected during the four seasons at two urban sites, one rural site, and one alpine site in Switzerland. Cytotoxic effects, determined as a reduction in the metabolic activity, were found in particle extracts from all sites and seasons. Taking together the data from all sites and seasons, a dose-response function was observed between the particle mass on the filter and toxicity (r(2)=0.633, linear regression). The release of the pro-inflammatory cytokine TNFalpha as well as of oxidative radicals was most pronounced in particles collected in spring-summer and autumn. While at Montana (alpine), the stimulation of the cells was positively correlated with the particle mass on the filters, this correlation was negative at the urban sites Zürich and Lugano. It is interpreted that at high PM(10) levels, as in these cities, macrophages are inhibited by increasing air pollution due to toxic effects. Cytotoxic effects and the release of oxidative radicals could be inhibited when the extracts were treated with an endotoxin-neutralizing protein. This suggests that endotoxin, a cell-wall constituent of gram-negative bacteria, is one of the factors which modulates macrophage activity. All together, the experiments indicate that in the PM(10) fraction, water-soluble macrophage-toxic and macrophage-stimulating compounds are present. The data offer an explanation for at least some of the known harmful effects of PM(10), and confirm endotoxin as a possible reactant.

The possible expression of FasL in gilthead seabream leucocytes was studied by flow cytometry, immunoblotting and immunocytochemistry, using an anti-mouse FasL monoclonal antibody. The results pointed to a cytosolic FasL-like, but not a membrane-like form, in resting leucocytes from head-kidney, thymus, spleen, blood and peritoneal exudate. Immunoblotting revealed a 19kDa band in resting leucocytes, while activated leucocytes showed the same band and another of 39kDa. The FasL-like molecule is identified in lymphocytes, monocyte-macrophages and acidophilic granulocytes. Phylogenetical and functional implications are suggested.

Adenoviruses (Ads) are endemic in the human population and the well-studied group C Ads typically cause an acute infection in the respiratory epithelium. A growing body of evidence suggests that these viruses also establish a persistent infection. The Ad genome encodes several proteins that counteract the host anti-viral mechanisms, which function to limit viral infections. This review describes the adenovirus immuno-regulatory proteins and how they function to block apoptosis of infected cells. In addition to facilitating the successful completion of the viral replication cycle and spread of progeny virus, these functions may help maintain the virus in a persistent state.