Tumor Necrosis Factor-related Apoptosis-inducing Ligand Gene Research Articles

Inhibition ofTrailGene Expression by Cyclopentenonic Prostaglandin 15-Deoxy-Δ12,14-Prostaglandin J2in T Lymphocytes

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a cyclopentenonic prostaglandin endowed with powerful anti-inflammatory activities, as shown in animal models of inflammatory/autoimmune diseases, where pharmacological administration of this prostanoid can ameliorate inflammation and local tissue damage via activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) and/or covalent modifications of cellular proteins. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily expressed in most of the cells, including those of immune system such as T lymphocytes, in which it is up-regulated upon antigen-specific stimulation. This cytokine plays an important role in regulating various physiological and immunopathological processes, such as immunosurveillance of tumors and tissue destruction associated with different inflammatory and autoimmune diseases. Here, we demonstrate that 15d-PGJ(2) inhibits trail mRNA and protein expression by down-regulating the activity of its promoter in human T lymphocytes. Our data indicate that both the chemically reactive cyclopentenone moiety of 15d-PGJ(2) and the activation of PPARgamma may be involved in this repressive mechanism. We identified nuclear factor kappaB (NF-kappaB) as a direct target of the prostanoid. 15d-PGJ(2) significantly decreases the expression and/or DNA binding of c-rel, RelA, and p50 transcription factors to the NF-kappaB1 site of trail promoter. Moreover, 15d-PGJ(2)-mediated activation of the transcription factor heat shock factor-1 may contribute to inhibit trail promoter activity in transfected Jurkat T cells. These results suggest that modulation of TRAIL gene expression by 15d-PGJ(2) in T cells may provide a novel pharmacological tool to modify the onset and the progression of specific autoimmune and inflammatory disorders.

The histone deacetylase inhibitors depsipeptide and MS-275, enhance TRAIL gene therapy of LNCaP prostate cancer cells without adverse effects in normal prostate epithelial cells

Gene therapy of cancer using adenovirus as a single treatment modality has met limited success and efforts to enhance therapeutic outcomes have included combination of gene therapy with chemotherapy. The goal of this study was to investigate which chemotherapeutic agents may be suitable for combination with gene therapy of prostate cancer. Using an adenovirus expressing green fluorescent protein (GFP), we determined the effect of cisplatin, gemcitabine, doxorubicin, depsipeptide and MS-275 on adenoviral infectivity and transgene expression in LNCaP cells. We found that the two histone deacetylase inhibitors (HDACi), depsipeptide and MS-275, and to a lesser extent doxorubicin, increased infectivity and transgene expression. However, only the HDACi selectively increased infectivity in LNCaP cells while doxorubicin increased infectivity to a greater extent in normal prostate epithelial cells (PrEC). The increase in infectivity but not transgene expression correlated to increased surface expression of coxsackie and adenovirus receptor (CAR). Increased transgene expression following infection with an adenovirus expressing tumor necrosis factor-related apoptosis inducing ligand (TRAIL) was observed only in LNCaP cells treated with depsipeptide or MS-275. Combination of TRAIL gene therapy with HDACi but not doxorubicin resulted in increased induction of apoptosis in LNCaP cells. In contrast, apoptosis was not enhanced by HDACi in normal PrEC. These results suggest that combination of HDACi with adenoviral TRAIL gene therapy may be a new therapeutic approach for the treatment of prostate cancer that warrants further investigation.

Antitumor Activity of Human CD34+ Cells Expressing Membrane-Bound Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (mTRAIL).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expected to play a key role in anti-cancer therapy due to its high cancer cell-specificity and potent antitumor activity. The clinical development of soluble (s)TRAIL is however hampered by several limitations, includingshort plasma half-life;liver toxicity;tumor cell resistance.To overcome these limitations, we used CD34+ cells transduced with an adenovirus encoding the full-length human TRAIL gene (CD34−TRAIL+) as vehicles for intra-tumor delivery of membrane-bound (m)TRAIL. The mean (±SD) transduction efficiency of CD34+ cells exposed to a multiplicity of infection (MOI) of 500 was 83 ± 8% (range 70 – 95%) with a cell viability ≥85%. In vitro, exposure of the sTRAIL-sensitive KMS-11 cell line to CD34−TRAIL+, but not mock-transduced CD34+ cells consistently resulted in caspase-3, -8, and -9 activation and in PARP cleavage, as well as in potent induction of apoptosis (up to 80% after a 48-hour co-culture). Exposure of the sTRAIL-resistant JVM-2 cell line to CD34−TRAIL+ cells resulted in significant levels of tumor cell death (up to 50% after a 48-hour co-culture). Studies in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with KMS-11 cell line showed that CD34−TRAIL+ cells significantly increased the median survival of mice bearing early-stage (92 vs 55 days, P ≤ 0.0001) and advanced-stage (83 vs 55 days, P ≤ 0.0001) disease, as compared with controls. Additionally, CD34−TRAIL+ cells significantly prolonged the median survival of mice xenografted with the sTRAIL-resistant JVM-2 (40 vs 31 days, P ≤ 0.0001) and SU-DHL-4V (38 vs 30 days, P ≤ 0.0001) cell lines. No obvious toxicity was observed upon administration of CD34−TRAIL+ cells. Histological analysis of subcutaneous lymphoma revealed an efficient tumor homing of transduced cells and high level expression of the agonistic TRAIL-R2 receptor by tumor endothelial cells. Following injection of CD34−TRAIL+ cells, but not mock-transduced CD34+ cells, TUNEL staining revealed increasing amounts of apoptotic cells with a 21-fold increase of the apoptotic index at 120 hour post-injection. Additionally, CD34−TRAIL+ cells induced signs of vascular damage leading to a progressive disintegration of the vascular bed, suggesting that tumor endothelial cells represent an early target of CD34−TRAIL+ cells. Our experiments show that:in vitro, the co-culture of tumor cells and CD34−TRAIL+ cells resulted in a marked apoptosis of both sTRAIL-sensitive and sTRAIL-resistant tumor cells;in vivo, injection of CD34−TRAIL+ cells in mice bearing advanced-stage tumors as well as sTRAIL-resistant tumors was associated with a significant prolongation of survival.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.

Antitumor Activity of Human CD34+Cells Expressing Membrane-Bound Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

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.

Systemic Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Delivery Shows Antiatherosclerotic Activity in Apolipoprotein E–Null Diabetic Mice

Although in vitro studies have suggested that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) might be involved in vascular biology, its potential role in the pathogenesis and/or treatment of atherosclerosis has not been investigated. Both recombinant human TRAIL and an adeno-associated virus vector expressing human TRAIL were used to deliver TRAIL in apolipoprotein E (apoE)-null mice in which diabetes mellitus was induced by destruction of islet cells with streptozotocin. Diabetes in apoE-null mice was associated with a significant increase in atherosclerotic plaque area and complexity in the aorta as assessed by a marked increase in interstitial collagen, cellular proliferation, and macrophage infiltration and a focal loss of endothelial coverage. Repeated intraperitoneal injections of recombinant human TRAIL and a single intravenous injection of adeno-associated virus-human TRAIL significantly attenuated the development of atherosclerotic plaques in apoE-null animals. TRAIL also markedly affected the cellular composition of plaque lesions by inducing apoptosis of infiltrating macrophages and increasing the vascular smooth muscle cell content. Moreover, TRAIL promoted the in vitro migration of cultured human aortic vascular smooth muscle cells but not of monocytes or macrophages. Conversely, TRAIL selectively induced apoptosis of human cultured macrophages but not of vascular smooth muscle cells. Overall, data from the present study indicate that atherosclerosis in diabetic apoE-null mice is ameliorated by systemic TRAIL administration and that adeno-associated virus-mediated TRAIL gene delivery might represent an innovative method for the therapy of diabetic vascular diseases.

Adenoviral vector expressing CYLD augments antitumor activity of TRAIL by suppression of NF-κB survival signaling in hepatocellular carcinoma

CYLD is a tumor suppressor gene related to cylindroma and is negative regulator of NF-κB. However, antitumor effect of CYLD has not been reported. The activation of NF-κB induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) renders hepatocellular carcinoma (HCC) resistant to TRAIL-mediated cell apoptosis. Here we described that the adenoviral vector expressing CYLD (Ad/hTERT-CYLD) augmented the cytotoxicity of TRAIL in HCC cells by negatively regulating NF-κB activity since CYLD could reverse the ubiquitination of TNF receptor-associated factor 2 (TRAF2) and interact with the I B kinase? (IKKγ). The combined treatment of Ad/hTERT-CYLD and a conditionally replicating adenovirus carrying TRAIL gene (ZD55-TRAIL) induced rapid and potent apoptosis in HCC cells, characterized by activation of caspase-3, caspase-8, PARP and the reduction of X-linked inhibitor of apoptosis protein (XIAP). In animal study, the combined treatment could eradicate the BEL7404 xenograft tumors. In contrast, treatment with Ad/hTERT-CYLD or ZD55-TRAIL alone achieved less antitumor effect. In conclusion: CYLD inhibits TRAIL-mediated NF-κB activation and enhances the sensitivity of HCC cells to TRAIL-triggered apoptosis. The combined delivery of Ad/hTERT-CYLD and ZD55-TRAIL may be a new useful strategy for HCC or other tumor cells with enhanced NF-κB activity.

Tumor-specific apoptotic gene targeting overcomes radiation resistance in esophageal adenocarcinoma

To overcome radiation resistance in esophageal adenocarcinoma by tumor-specific apoptotic gene targeting using tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Adenoviral vector Ad/TRAIL-F/RGD with a tumor-specific human telomerase reverse transcription promoter was used to transfer TRAIL gene to human esophageal adenocarcinoma and normal human lung fibroblastic cells (NHLF). Activation of apoptosis was analyzed by Western blot, fluorescent activated cell sorting, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate labeling (TUNEL) assay. A human esophageal adenocarcinoma mouse model was treated with intratumoral injections of Ad/TRAIL-F/RGD plus local radiotherapy. The combination of Ad/TRAIL-F/RGD and radiotherapy increased the cell-killing effect in all esophageal adenocarcinoma cell lines but not in NHLF cells. This combination also significantly reduced clonogenic formation (p < 0.05) and increased sub-G1 deoxyribonucleic acid accumulation in cancer cells (p < 0.05). Activation of apoptosis by Ad/TRAIL-F/RGD plus radiotherapy was demonstrated by activation of caspase-9, caspase-8, and caspase-3 and cleaved poly (adenosine diphosphate-ribose) polymerase in vitro and TUNEL assay in vivo. Combined Ad/TRAIL-F/RGD and radiotherapy dramatically inhibited tumor growth and prolonged mean survival in the esophageal adenocarcinoma model to 31.6 days from 16.7 days for radiotherapy alone and 21.5 days for Ad/TRAIL-F/RGD alone (p < 0.05). The combination of tumor-specific TRAIL gene targeting and radiotherapy enhances the effect of suppressing esophageal adenocarcinoma growth and prolonging survival.

Recombinant Adenoviruses Expressing TRAIL Demonstrate Antitumor Effects on Non-Small Cell Lung Cancer (NSCLC)

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.

Intra-articular injection of recombinant TRAIL induces synovial apoptosis and reduces inflammation in a rabbit knee model of arthritis

We demonstrated previously that local, intra-articular injection of an adenoviral vector expressing human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a rabbit knee model of inflammatory arthritis stimulated synovial apoptosis and reduced inflammation. To examine whether intra-articular injection of recombinant chimeric human TRAIL protein (rTRAIL) also induces apoptosis of proliferating rabbit synovium and reduces inflammation, we used an experimental rabbit arthritis model of rheumatoid arthritis, induced by intra-articular introduction of allogeneic fibroblasts genetically engineered to secrete human IL-1β. Analysis of synovium isolated from the rabbits treated with intra-articular injection of rTRAIL, relative to saline control, showed areas of extensive acellular debris and large fibrous regions devoid of intact cells, similar to adenoviral mediated TRAIL gene transfer. Extensive apoptosis of the synovial lining was demonstrated using TUNEL analysis of the sections, corresponding to the microscopic findings in hematoxylin and eosin staining. In addition, leukocyte infiltration into the synovial fluid of the inflamed knee joints following rTRAIL treatment was reduced more than 50% compared with the saline control. Analysis of the glycosaminoglycan synthetic rate by cultured cartilage using radiolabeled sulfur and cartilage histology demonstrated that rTRAIL did not adversely affect cartilage metabolism and structure. Analysis of serum alanine aminotransferase showed that intra-articular injection of rTRAIL did not have adverse effects on hepatic function. These results demonstrate that intra-articular injection of rTRAIL could be therapeutic for treating pathologies associated with rheumatoid arthritis.

Conditionally replicative adenovirus vector carrying TRAIL gene for enhanced oncolysis of human hepatocellular carcinoma

Conditionally replicative adenovirus (CRAd) mediated tumor specific gene therapy based on transcriptional control is considered a new direction for the treatment of cancer. Our previous studies showed that an HS4 insulator increased the alpha-fetoprotein (AFP) promoter-driven expression in the context of an adenovirus (Ad) vector, while retaining the highly specific gene expression in hepatoma cells in vitro and in vivo. In this study, we constructed two HS4-AFP promoter based CRAd vectors (Ad.HS4.AFP.E1a/TRAIL and Ad.HS4.AFP.E1a) with and without the expression cassette of TNF-related apoptosis-inducing ligand (TRAIL). The TRAIL-expressing virus vector, Ad.HS4.AFP.E1a/TRAIL, exhibited more obvious oncolytic effect than Ad.HS4.AFP.E1a in both high-AFP-producing HCC cell lines (Hep3B and HUH7) and a low-AFP-producing HCC cell line (PLC/PRF/5) examined, indicating endogenous TRAIL over-expression increased CRAd potency. The enhanced hepatoma cell death was mainly mediated through apoptotic mechanism, as evidenced by the activation of caspase-3, binding of annexin V and inhibition by caspase inhibitor z-vad-fmk. In s.c. xenograft of low-AFP-producing PLC/PRF/5 hepatoma model, the administration of Ad.HS4.AFP.E1a/TRAIL resulted in a more potent oncolytic effect compared with the same dose of Ad.HS4.AFP.E1a 28 days after virus exposure. This study demonstrated that the TRAIL in the context of a CRAd vector was able to increase the oncolytic activity in low-AFP-producing HCC cells in vitro and in vivo. Considering that oncolytic viruses destroy tumor cells expressing low levels of the tumor marker is a clinical concern, TRAIL might be a useful tool to improve the efficacy of these vectors.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrane protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAIL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRF1 sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish ( Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen.

Cancer gene therapy using a novel secretable trimeric TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, is a type II transmembrane cytokine molecule. Soluble TRAIL has been shown to induce apoptosis in a wide variety of cancer cells in vitro and to suppress tumor growth specifically without damaging normal cells and tissues in vivo. In our previous report, we have demonstrated that an artificial gene encoding the polypeptide composed of the three functional elements (a secretion signal, a trimerization domain and an apoptosis-inducing moiety of TRAIL gene sequence) expresses and secretes highly apoptotic trimeric TRAIL into the culture supernatant. Here, as an approach to TRAIL-based cancer gene therapy, we developed an adenoviral vector delivering the gene that encodes our secretable trimeric TRAIL (stTRAIL). This adenovirus (Ad-stTRAIL) potently induced apoptosis in vitro in cancer cell lines such as HeLa, MDA-MB-231, A549, HCT116 and U-87MG. In an animal xenograft tumor model bearing a human glioma cell line U-87MG, intratumoral delivery of Ad-stTRAIL dramatically suppressed tumor growth without showing detectable adverse side effects. Histological analysis revealed that Ad-stTRAIL suppresses tumor growth by inducing apoptotic cell death. Contrary to the known rapid clearance of systemically delivered TRAIL protein from the blood circulation, stTRAIL expressed by Ad-stTRAIL in tumor tissues persisted for more than 4 days. In a comparison of tumor suppressor activity between Ad-stTRAIL and Ad-flTRAIL (delivering the full-length TRAIL gene) after mixing infected cells with uninfected cells and implanting these mixed cells in nude mice, Ad-stTRAIL showed higher tumor suppressor activity than that of Ad-flTRAIL. Our data reveal that a gene therapy using Ad-stTRAIL has a promising potential to treat human cancers including gliomas.

Combination of Radiotherapy and Tumor Specific TRAIL Gene Targeting Improved Survival of Mice Bearing Human Esophageal Cancer

Purpose/Objective: The clinical outcome for locally advanced esophageal cancer is dismal. Radiotherapy plays an important role in locoregional control. However, many patients experience resistance to radiotherapy. We developed a novel tumor-specific apoptotic gene targeting using tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to overcome radiation resistance and thus improve the therapeutic ratio.

Requirement of c-Jun NH2-terminal kinase activation in interferon-α-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.

Combination effect of oncolytic adenovirotherapy and TRAIL gene therapy in syngeneic murine breast cancer models.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and oncolytic adenovirotherapy have been investigated extensively in xenografic human tumor models established in immunocompromised nude mice. However, the effects of these therapies on syngeneic murine tumors in immunocompetent settings were not well documented. We hypothesized that TRAIL gene therapy used with an oncolytic adenovirus would overcome the weaknesses of the two therapies used individually. In this study, we evaluated the antitumor effects of an oncolytic adenovirus, Delta24, in both human and murine breast cancer cell lines. We also analyzed the effects of TRAIL gene therapy combined with oncolytic virotherapy in these cancer cells. Our results showed that Delta24 can replicate and help the E1-deleted adenovector replicate in murine cancer cells. We also found that these two therapies combined had greater antitumor activity than either one alone in both human and murine breast cancer cells lines and in the syngeneic breast cancer models established in immunocompetent mice. Moreover, Delta24 virotherapy alone and combined with TRAIL gene therapy dramatically reduced the spontaneous liver metastasis that originated in the subcutaneous 4T1 tumor established in Balb/c mice. These findings provide important considerations in the development and preclinical assessments of oncolytic virotherapy.

Depsipeptide (FR901228) Enhances the Cytotoxic Activity of TRAIL by Redistributing TRAIL Receptor to Membrane Lipid Rafts

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.

Absence of Gene Mutation in TRAIL Receptor 1 (TRAIL-R1) and TRAIL Receptor 2 (TRAIL-R2) in Chronic Myelogenous Leukemia and Myelodysplastic Syndrome, and Analysis of mRNA Expressions of TRAIL and TRAIL-Related Genes in Chronic Myelogenous Leukemia

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an interferon (IFN)-induced molecule with apoptotic activity. We examined gene mutations in the death domains of TRAIL receptor 1 (TRAIL-R1) and TRAIL receptor 2 (TRAIL-R2), and in the TRAIL gene promoter in 46 chronic myelogenous leukemia (CML) patients. In 23 of the 46 patients, all the coding regions of TRAIL-R2 were also examined. However, no mutation or loss of heterozygosity was found. Furthermore, no mutation in the death domains of TRAIL-R1 and TRAIL-R2 genes, which causes amino acid change, was found in 18 myelodysplastic syndrome (MDS) patients. Ribonuclease protection assay (RPA) and real-time quantitative polymerase chain reaction using polymorphonuclear neutrophils of five new CML patients showed that the TRAIL mRNA expression was very low before in vitro IFN-α stimulation and markedly upregulated after IFN-α stimulation. FAS mRNA was also upregulated with IFN-α stimulation but the fold induction was far lower than that of TRAIL mRNA. In addition, RPA revealed that the ratio of (TRAIL-R1 plus TRAIL-R2) to TRAIL-R3 was also increased after IFN-α stimulation. Taken together, gene mutations of TRAIL-R1, TRAIL-R2 are infrequent in patients with CML and MDS. Andso is the TRAIL promoter for CML. These mutations seem unrelated to tumorigenesis, disease progression, and response to IFN-α therapy in CML. A markedly high induction of TRAIL mRNA by IFN-α may have some relevance to IFN-α action in CML patients.

Antitumor Activity of TRAIL Recombinant Adenovirus in Human Malignant Glioma Cells

Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. One of disadvantages to previous in vivo protocols was the need for large quantities of TRAIL recombinant protein to suppress tumor growth. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene (Ad.hTRAIL) and transferred them into malignant glioma cells in vitro and tumors in vivo, as an alternative to recombinant soluble TRAIL protein. The results show that TRAIL-sensitive glioma cells infected Ad.hTRAIL undergo apoptosis through the production and expression of TRAIL protein. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of cleavage of poly (ADP-ribose) polymerase. Furthermore, in vivo administration of Ad.hTRAIL at the site of tumor implantation suppressed the outgrowth of human glioma xenografts in SCID mice. These results further define Ad.hTRAIL as an anti-tumor therapeutic and demonstrate its potential use as an alternative approach to treatment for malignant glioma.

Fiber-modified adenoviral vector expressing the tumor necrosis factor-related apoptosis-inducing ligand gene from the human telomerase reverse transcriptase promoter induces apoptosis in human hepatocellular carcinoma cells

Because of a major resistance to chemotherapy, prognosis of hepatocellular carcinoma (HCC) is still poor. New treatments are required and gene therapy may be an option. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in multiple malignant tumors, and using adenoviral vectors has shown a targeted tumor-specific therapy. However, repeated administration of adenoviral vectors can lead to cell resistance, which may be caused by the initial coxsackie-adenovirus receptor (CAR). One technique to overcome resistance is the use of modified adenoviral vectors containing an Arg-Gly-Asp (RGD) sequence. In this study we constructed an adenoviral vector (designated Ad/TRAIL-F/RGD) with RGD-modified fibers, expressing the TRAIL gene from the human telomerase reverse transcriptase (hTERT) promoter, and evaluated its antitumor activity in HCC cell lines. To investigate the effects of Ad/TRAIL-F/RGD in human HCC cell lines Hep G2 and Hep 3b, cells were infected with Ad/CMV-GFP (vector control), Ad/gTRAIL (positive control), and Ad/TRAIL-F/RGD. Phosphate-buffered saline (PBS) was used as control. Cell viability was determined by proliferation assay (XTT), and apoptosis induction by fluorescence activated cell sorting (FACS). Cells treated with Ad/TRAIL-F/RGD and Ad/gTRAIL showed a significantly reduced cell viability in comparison to PBS and Ad/CMV-GFP treatment in both cell lines. Whereas, treatment with PBS and Ad/CMV-GFP had no cell-killing effect. The reduced cell viability was caused by induction of apoptosis as shown by FACS analysis. The amount of apoptotic cells was similar after incubation with Ad/gTRAIL and Ad/TRAIL-F/RGD. The new RGD modified vector Ad/TRAIL-F/RGD could become a potent therapeutic agent for the treatment of HCC, adenovirus resistant tumors, and CAR low or negative cancer cells.

Suppression of pancreatic tumor growth in the liver by systemic administration of the TRAIL gene driven by the hTERT promoter.

Local and locoregional administration of adenovectors expressing the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene has been demonstrated to be useful in treating established tumors in animals. Moreover, expression of the TRAIL gene from the human telomerase reverse transcriptase (hTERT) promoter can be used to prevent possible liver toxicity of the TRAIL gene. However, it remains unknown whether systemic administration of the TRAIL-expressing adenovector can be used for cancer therapy. Here, we showed that a combination of TRAIL gene therapy and gemcitabine, the first-line chemotheraphy agent for pancreatic cancer, had a synergistic effect on the induction of apoptosis in human pancreatic cancer cell lines in vitro. Systemic administration of an adenovector that contains an insertion of integrin-binding motif argine-glycine-aspartate (RGD) in the HI loop of the adenoviral fiber protein and expresses the human TRAIL gene from the hTERT promoter (designated Ad/TRAIL-F/RGD) suppressed the growth of human pancreatic tumor cells inoculated in the liver of nu/nu nude mice. Furthermore, Ad/TRAIL-F/RGD in combination with gemcitabine suppressed the tumor growth of pancreatic cancer in the liver more than did treatments consisting of each agent alone. No obvious liver toxicity was detected in any of the treatment groups. Our results suggest that TRAIL gene therapy in combination with gemcitabine might be a useful therapeutic approach for treating metastatic pancreatic cancers.