Recombinant Tumor Necrosis Factor-related Apoptosis-inducing Ligand Protein Research Articles

Abstract 5607: Combining PD-1 inhibition with the TRAIL receptor agonist TLY012 promotes pancreatic ductal adenocarcinoma tumor regression in an immunocompetent mouse model

Abstract Introduction: An immunosuppressed, apoptosis-resistant phenotype characterizes pancreatic ductal adenocarcinoma (PDAC). Both inflammation and tumorigenesis are influenced by the Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) pathway. TLY012 is pegylated recombinant TRAIL protein and is an orphan drug for systemic sclerosis and chronic pancreatitis. The potential synergy between TLY012 and anti-mouse PD-1 antibody was investigated in a syngeneic immunocompetent heterotopic mouse model. Methods: KPC-luc PDAC cells were subcutaneously implanted into C57Bl/6 mice. Treatment combined 10 mg/kg TLY012 three times per week with 10 mg/kg anti-mouse PD-1 antibody twice weekly and were compared to individual treatment and control groups. Cytokine profiling via 32-plex Luminex immunoassay was completed after 7 days and after three months of treatment. Flow cytometry at three months evaluated NK and T cell profiles in the spleen and tumors. Results: PDAC tumor-bearing mice treated with the combination of anti-mouse PD-1 and TLY012 had slower tumor growth and reduced tumor volume at 70 days compared to either drug alone (all p<0.005). B-cell activating factor, which can be a marker of PDAC tumor progression, was decreased in single and dual treatment groups at three months of treatment. This factor showed the earliest decrease in the dual treatment group, with a cytokine level 44% of that of control mice at 7 days. Long-term dual treatment mice also had the highest levels of proinflammatory cytokines interferon gamma (average 5.6 times control level, p=0.046), CCL5 (average 14.1 times control level, p=0.048), and interleukin-3 (IL-3, average 71.1 times control level, p=0.0053). Flow cytometry showed trends toward decreased circulating regulatory T cells, increased NK cells, and a higher proportion of CD8+ T cells within tumors in the dual treatment group. Conclusions: The combination of anti-mouse PD-1 and TLY012 prevents growth of PDAC in an immunocompetent mouse model. The combination also increases tumor-infiltrating CD8+ T cells, decreases circulating Treg cells and alters cytokine expression. CCL5 is a T-cell chemokine, and both interferon gamma and IL-3 are produced by activated T cells and macrophages, and have proinflammatory, antitumor effects. Combining TLY012 and anti-mouse PD-1 creates these changes in immune cell and cytokine levels, suggesting induction of a more proinflammatory immune environment. The proinflammatory state may contribute to decreased tumor growth. Citation Format: Anna D. Louie, Kelsey E. Huntington, Young Lee, Jared Mompoint, Jinxuan Wu, Seulki Lee, Wafik S. El-Deiry. Combining PD-1 inhibition with the TRAIL receptor agonist TLY012 promotes pancreatic ductal adenocarcinoma tumor regression in an immunocompetent mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5607.

Tumor necrosis factor‑related apoptosis‑inducing ligand is a novel transcriptional target of runt‑related transcription factor1.

Runt-related transcription factor 1 (RUNX1), which is also known as acute myeloid leukemia 1 (AML1), has been frequently found with genomic aberrations in human leukemia. RUNX1 encodes a transcription factor that can regulate the expression of hematopoietic genes. In addition, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) performs an important function for malignant tumors in immune surveillance. However, the regulatory mechanism of TRAIL expression remain to be fully elucidated. In the present study, tetradecanoylphorbol 13-acetate-treated megakaryocytic differentiated K562 cells was used to examine the effect of RUNX1 on TRAIL expression. Luciferase assay series of TRAIL promoters for the cells co-transfected with RUNX1 and core-binding factor β (CBFβ) expression vectors were performed to evaluate the nature of TRAIL transcriptional regulation. Electrophoresis mobility shift assay of the RUNX1 consensus sequence of the TRAIL promoter with recombinant RUNX1 and CBFβ proteins was also performed. BloodSpot database analysis for TRAIL expression in patients with acute myeloid leukemia were performed. The expression of TRAIL, its receptor Death receptor 4 and 5 and RUNX1 in K562 cells transfected with the RUNX1 expression vector and RUNX1 siRNA were evaluated by reverse transcription-quantitative PCR (RT-qPCR). TRAIL and RUNX1-ETO expression was also measured in Kasumi-1 cells transfected with RUNX1-ETO siRNA and in KG-1 cells transfected with RUNX1-ETO expression plasmid, both by RT-qPCR. Cell counting, lactate dehydrogenase assay and cell cycle analysis by flow cytometry were performed on Kasumi-1, KG-1, SKNO-1 and K562 cells treated with TRAIL and HDAC inhibitors sodium butyrate or valproic acid. The present study demonstrated that RUNX1 is a transcriptional regulator of TRAIL. It was initially found that the induction of TRAIL expression following the megakaryocytic differentiation of human leukemia cells was RUNX1-dependent. Subsequently, overexpression of RUNX1 was found to increase TRAIL mRNA expression by activating its promoter activity. Additional analyses revealed that RUNX1 regulated the expression of TRAIL in an indirect manner, because RUNX1 retained its ability to activate this promoter following the mutation of all possible RUNX1 consensus sites. Furthermore, TRAIL expression was reduced in leukemia cells carrying the t(8;21) translocation, where the RUNX1-ETO chimeric protein interfere with normal RUNX1 function. Exogenous treatment of recombinant TRAIL proteins was found to induce leukemia cell death. To conclude, the present study provided a novel mechanism, whereby TRAIL is a target gene of RUNX1 and TRAIL expression was inhibited by RUNX1-ETO. These results suggest that TRAIL is a promising agent for the clinical treatment of t(8;21) AML.

TRAIL therapy and prospective developments for cancer treatment

Tumor Necrosis Factor (TNF) Related Apoptosis-Inducing Ligand (TRAIL), an immune cytokine of TNF-family, has received much attention in late 1990s as a potential cancer therapeutics due to its selective ability to induce apoptosis in cancer cells. TRAIL binds to cell surface death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) and facilitates formation of death-inducing signaling complex (DISC), eventually activating the p53-independent apoptotic cascade. This unique mechanism makes the TRAIL a potential anticancer therapeutic especially for p53-mutated tumors. However, recombinant human TRAIL protein (rhTRAIL) and TRAIL-R agonist monoclonal antibodies (mAb) failed to exert robust anticancer activities due to inherent and/or acquired resistance, poor pharmacokinetics and weak potencies for apoptosis induction. To get TRAIL back on track as a cancer therapeutic, multiple strategies including protein modification, combinatorial approach and TRAIL gene therapy are being extensively explored. These strategies aim to enhance the half-life and bioavailability of TRAIL and synergize with TRAIL action ultimately sensitizing the resistant and non-responsive cells. We summarize emerging strategies for enhanced TRAIL therapy in this review and cover a wide range of recent technologies that will provide impetus to rejuvenate the TRAIL therapeutics in the clinical realm.

Receptor-specific TRAIL as a means to achieve targeted elimination of activated hepatic stellate cells

Activated hepatic stellate cells (HSCs) are known to play a central role in liver fibrosis and their elimination is a crucial step toward the resolution and reversion of liver fibrosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a molecule that may contribute to the apoptotic removal of activated HSC through binding to its dedicated receptors. In the present study, we investigated the potential application of recombinant receptor-specific TRAIL proteins in the efficient elimination of activated HSCs. Our finding revealed differential contribution of TRAIL receptors among HSCs populations with activated hepatic stellate cells expresses more TRAIL receptors DR5. In vitro treatment of activated HSCs with DR5-specific or wild-type TRAIL variants induced a significant reduction in viability and extracellular matrix production, whereas no significant decrease in viability was associated with the treatment of cells by DR4-specific TRAIL. Our analysis indicate the successful application of the DR5 receptor-specific TRAIL variant in the targeted elimination of activated HSCs via interference with collagen production and simultaneous induction of apoptosis via activation of the caspase pathway. DR5 receptor-specific TRAIL may thus represent a new therapeutic compound for the treatment of liver fibrosis.

Salmonella‐mediated tumor‐targeting TRAIL gene therapy significantly suppresses melanoma growth in mouse model

Attenuated Salmonella typhimurium (S. typhimurium) strains can selectively grow and express exogenous genes in tumors for targeted therapy. We engineered S. typhimurium strain VNP20009 to secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) under the control of a hypoxia-induced nirB promoter and examined the efficacy of Salmonella-mediated targeted expression of TRAIL in mice bearing melanoma tumor and in TRAIL-resistant RM-1 tumor. We found that VNP preferentially accumulated in tumor tissues and the nirB promoter effectively drove targeted expression of TRAIL. Compared with recombinant TRAIL protein and VNP20009 combination therapy, VNP20009 expressing TRAIL significantly suppressed melanoma growth but failed to suppress RM-1 tumor growth. Furthermore, we confirmed that VNP20009 expressing TRAIL yielded its antitumor effect by inducing melanoma apoptosis. Our findings indicate that Salmonella-mediated tumor-targeted therapy with TRAIL could reduce tumor growth and extend host survival.

TRAIL contributes to the apoptotic effect of 13-cis retinoic acid in human sebaceous gland cells

The full mechanism of action of isotretinoin [13-cis retinoic acid (13-cis RA)] in treating acne is unknown. 13-cis RA induces key genes in sebocytes that are involved in apoptosis, including Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL). In this study, we investigated the role of 13-cis RA-induced TRAIL within SEB-1 sebocytes. Using 13-cis RA and recombinant human TRAIL (rhTRAIL) protein, we assessed induction of TRAIL and apoptosis in SEB-1 sebocytes, normal keratinocytes and patient skin biopsies. Treatment with rhTRAIL protein increased TUNEL-positive staining in SEB-1 sebocytes. TRAIL siRNA significantly decreased the percentage of TUNEL-positive SEB-1 sebocytes in response to 13-cis RA treatment. Furthermore, TRAIL expression increased in the skin of patients with acne after 1 week of isotretinoin therapy compared with baseline. TRAIL expression localized within sebaceous glands. Unlike sebocytes, TRAIL protein expression was not increased in normal human epidermal keratinocytes in response to 13-cis RA, nor did rhTRAIL induce apoptosis in keratinocytes, suggesting that TRAIL is key in the sebocyte-specific apoptotic effects of 13-cis RA. Taken together, our data suggest that TRAIL, like the neutrophil gelatinase-associated lipocalin, is involved in mediating 13-cis RA apoptosis of sebocytes.

Inhibition of the ubiquitin-proteasome system sensitizes TRAIL-resistant prostate cancer cells by up-regulation of death receptor 5

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine and a potent inducer of apoptosis in cancer cells. However, some cancer cells, particularly prostate cancer cells, such as LNCaP cells, were found to be resistant to TRAIL. In the present study, we demonstrate that the proteasome inhibitor ALLN significantly enhanced TRAIL-induced apoptosis by up-regulating TRAIL/Apo2L death receptor 5 expression in LNCaP cells. LNCaP cells were exposed to ALLN for 3 h and treated with recombinant TRAIL protein. ALLN alone induced a 20% cell death after a 3‑h treatment; however, pretreatment with ALLN induced death to more than 80% of cells after 3 h of TRAIL treatment. ALLN also enhanced the cell death of TRAIL-sensitive/resistant prostate cancer and other cancer cell lines. Western blotting results showed that the combination of ALLN and TRAIL increased the levels of activated caspase-8, -3 and DR-5 in LNCaP cells. Furthermore, we observed an increase in DR-5 expression following 3 h of treatment of ALLN alone. Taken together, our findings indicate that ALLN enhances TRAIL-induced apoptosis in LNCaP cells by up-regulating DR-5 expression. Thus, our results suggest that the combination of ALLN and TRAIL is a novel therapeutic strategy in TRAIL-resistant tumors.

Targeted knockdown of death-associated protein kinase expression induces TRAIL-mediated apoptosis in human endometrial adenocarcinoma cells

Death-associated protein kinase (DAPK) is a serine/threonine kinase that participates in the modulation of apoptosis and tumor suppression. Our previous study revealed high levels of DAPK protein expression in differentiated endometrial adenocarcinoma cells. To clarify the role of DAPK in human endometrial adenocarcinomas, we down-regulated endogenous DAPK expression in HHUA cells, a well-differentiated endometrial adenocarcinoma cell line, using specific small-interfering RNAs (siRNAs). The suppression of endogenous DAPK expression triggered apoptosis in HHUA cells, as evidenced by an increase in the sub-G1 DNA content in flow cytometric analyses. The apoptosis induced by the DAPK siRNA transfections was caspase-dependent, as characterized by the activations of caspase-3, -8 and -9. RNase protection assays detected higher levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), DR4 and DR5 transcripts in the DAPK siRNA-transfected HHUA cells than in the control siRNA-transfected cells. Consistent with these findings, enzyme-linked immunosorbent assays revealed that the DAPK siRNA transfections significantly increased the secretion of TRAIL protein from the cells. Treatment with recombinant human TRAIL protein dose-dependently suppressed the cell viability of HHUA cells. The present findings reveal that down-regulation of endogenous DAPK expression in HHUA cells induces caspase-dependent apoptosis, possibly through increased TRAIL, DR4 and DR5 signaling, thereby suggesting that DAPK expression is essential for HHUA cell survival. Consequently, endogenous DAPK mRNA may represent a potential candidate for molecularly targeted anticancer therapies.

TRAIL and cancer therapy

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors are promising targets for the selective eradication of tumor cells while sparing normal cells. Currently, both recombinant TRAIL proteins and TRAIL receptor agonistic antibodies are being tested in the clinic, showing encouraging antitumor activities and mild side effects. Unfortunately, resistance to TRAIL therapy is frequently encountered requiring combined treatments with sensitizing agents. Standard chemotherapeutics can enhance TRAIL sensitivity; however, more specific and less toxic agents are needed to exploit the full antitumor potential of TRAIL. Here, a brief overview of the TRAIL signaling pathway is given together with a short description of early results obtained with TRAIL therapy in the clinic. Mechanisms of TRAIL resistance and ways to overcome these by targeted agents that either neutralize apoptotic blockades or suppress prosurvival signals also triggered by TRAIL are highlighted, such as inhibitors of IAPs, Bcl-2 family members, HDACi, and modulators of NF-κB, Raf and EGFR signaling.

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.

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.

Differential susceptibility of metastatic and primary oral cancer cells to TRAIL-induced apoptosis

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) preferentially induces apoptosis of cancer cells without toxicity in normal cells. TRAIL plays an important role in host immune surveillance against tumor metastasis. Cathepsin B (CB) is a mediator of apoptosis whose activity is regulated by its inhibitors, known as cystatins. We examined the TRAIL-mediated cytotoxicity rates of clonally-related primary and metastatic oral cancer (OC) cells and correlated them with the expression levels of TRAIL receptors, cathepsin B and cystatins A, B, C and M. Two pairs of primary (686Tu and 101A) and metastatic (686Ln and 101B) OC cell lines were treated with various concentrations (5 to 1000 ng/ml) of recombinant human TRAIL protein for 14 h, and cell viability and apoptotic rate were determined. In both pairs of cell lines, primary OC cells revealed greater susceptibility to TRAIL than their metastatic counterparts. The protein synthesis inhibitor cycloheximide markedly increased the TRAIL sensitivity of these cell lines, whereas the CB-specific chemical inhibitor CA-074 markedly reduced the sensitivity of primary OC cells to TRAIL. DNA laddering and M30 CytoDEATH immunodetection assays confirmed that TRAIL-induced OC cell death is an apoptotic process. Expression levels of TRAIL death (DR4 and DR5) and decoy (DcR1 and DcR2) receptors were not different between primary and metastatic OC cells. However, expression levels of cystatins were higher in metastatic OC cells than in their respective primary cells, whereas CB levels remain unchanged. Cathepsin B is a mediator of TRAIL-induced apoptosis in OC cells. Elevated levels of cystatins in metastatic OC cells may cause their greater resistance to TRAIL-induced apoptosis. Our data suggest that high expression of cystatins in OC cells may confer a metastatic phenotype by enhancing their resistance to TRAIL.

Death Induction by Recombinant Native TRAIL and Its Prevention by a Caspase 9 Inhibitor in Primary Human Esophageal Epithelial Cells

The cytotoxic death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a tumor-specific agent under development as a novel anticancer therapeutic agent. However, some reports have demonstrated toxicity of certain TRAIL preparations toward human hepatocytes and keratinocytes through a caspase-dependent mechanism that involves activation of the extrinsic death pathway and Type II signaling through the mitochondria. We have isolated and purified both His-tagged protein and three versions of native recombinant human TRAIL protein from Escherichia coli. We found that 5 mm dithiothreitol in the purification process enhanced oligomerization of TRAIL and resulted in the formation of hyper-oligomerized TRAILs, including hexamers and nonomers with an extremely high potency in apoptosis induction. Although death-inducing signaling complex formation was much more efficient in cells treated with hyper-oligomerized TRAILs, this did not convert TRAIL-sensitive Type II HCT116 colon tumor cells to a Type I death pattern as judged by their continued sensitivity to a caspase 9 inhibitor. Moreover, TRAIL-resistant Type II Bax-null colon carcinoma cells were not converted to a TRAIL-sensitive Type I state by hyper-oligomerized TRAIL. Primary human esophageal epithelial 2 cells were found to be sensitive to all TRAIL preparations used, including trimer TRAIL. TRAIL-induced death in esophageal epithelial 2 cells was prevented by caspase 9 inhibition for up to 4 h after TRAIL exposure. This result suggests a possible therapeutic application of caspase 9 inhibition as a strategy to reverse TRAIL toxicity. Hyper-oligomerized TRAIL may be considered as an alternative agent for testing in clinical trials.

Elevated Cystatin expression in metastatic oral cancer cells confers greater resistance to TRAIL-induced apoptosis

Background TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) preferentially induces apoptosis of cancer cells without toxicity in normal cells. TRAIL plays an important role in host immunosurveillance against tumor metastasis. Cathepsin B (CB) is a mediator of apoptosis whose activity is regulated by inhibitors known as cystatins (CY). Objective To relate the TRAIL sensitivity of clonally related primary and metastatic oral cancer (OC) cells with their CB and CY levels. Study design Two pairs of primary (686Tu and 101Tu) and metastatic (686Ln and 101Ln) OC cell lines were treated with various concentrations (10-1000 ng/mL) recombinant human TRAIL protein for 14 hours. Cell viability was quantified by MTT assay. Apoptosis rate among TRAIL-treated cells was measured using the TUNEL and M30 CytoDEATH immunodetection assays. CB and CY (A, B, C, and M) levels in these cells lines were analyzed by RT-PCR and Western blots. Results Primary cells revealed greater susceptibility to TRAIL-induced killing (ED 50 of 50 and 250 ng/mL for 686Tu and 101Tu, respectively) than their metastatic clones (ED 50 of 250 and 1000 ng/mL for 686Ln and 101Ln, respectively). Primary cells in the presence of CB-specific chemical inhibitor CA074 revealed markedly increased resistance to TRAIL (ED 50 > 1000 ng/mL). Expression levels of CY were markedly higher in metastatic OC cells than in their respective primary cells whereas CB levels remain unchanged. Conclusion CB is a mediator of TRAIL-induced apoptosis in OC cells. Elevated levels of cystatins in metastatic OC cells correlate with their greater resistance to TRAIL-induced apoptosis. Our data suggest that overexpression of CY in OC cells may confer a metastatic phenotype by enhancing their resistance to TRAIL.

Overcoming Acquired Resistance to TRAIL by Chemotherapeutic Agents and Calpain Inhibitor I through Distinct Mechanisms

We recently found that repeated application of adenovectors expressing the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or recombinant TRAIL proteins to TRAIL-susceptible cancer cells resulted in selection and expansion of TRAIL-resistant cells. Overcoming this acquired resistance to TRAIL is desirable for TRAIL-mediated cancer therapy. Here we demonstrate that several chemotherapeutic agents, including 5-fluorouracil (5-FU) and mitomycin, and calpain inhibitor I, an NFκB inhibitor, can overcome acquired resistance to TRAIL in DLD1 colon cancer cells. The combination of TRAIL (approved gene symbol TNFSF10) gene therapy and 5-FU enhanced tumor suppression in vivo in nude mice bearing subcutaneous tumors established from TRAIL-resistant colon cancer cells. Whereas treatment with the combination of TRAIL and 5-FU or mitomycin led to enhanced activation of caspase-3, the combination of TRAIL and calpain inhibitor I resulted in enhanced activation of both caspase-8 and caspase-3. Moreover, mitomycin, but not 5-FU or calpain inhibitor I, induced overexpression of the BAX gene, which was correlated with enhanced TRAIL-induced cell killing in TRAIL-resistant DLD1 cells. Together, these results suggest that acquired resistance to TRAIL can be overcome by different mechanisms and that combinations of TRAIL gene therapy and chemotherapy may be a useful approach for cancer treatment.

Helicobacter pylori enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human gastric epithelial cells.

To investigate the relations between tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Helicobacter pylori (H pylori) infection in apoptosis of gastric epithelial cells and to assess the expression of TRAIL on the surface of infiltrating T-cells in H pylori-infected gastric mucosa. Human gastric epithelial cell lines and primary gastric epithelial cells were co-cultured with H pylori in vitro, then recombinant TRAIL proteins were added to the culture. Apoptosis of gastric epithelial cells was determined by a specific ELISA for cell death. Infiltrating lymphocytes were isolated from H pylori-infected gastric mucosa, and expression of TRAIL in T cells was analyzed by flow cytometry. The apoptosis of gastric epithelial cell lines and primary human gastric epithelial cells was mildly increased by interaction with either TRAIL or H pylori alone. Interestingly, the apoptotic indices were markedly elevated when gastric epithelial cells were incubated with both TRAIL and H pylori (Control vs TRAIL and H pylori: 0.51+/-0.06 vs 2.29+/-0.27, P = 0.018). A soluble TRAIL receptor (DR4-Fc) could specifically block the TRAIL-mediated apoptosis. Further studies demonstrated that infiltrating T-cells in gastric mucosa expressed TRAIL on their surfaces, and the induction of TRAIL sensitivity by H pylori was dependent upon direct cell contact of viable bacteria, but not CagA and VacA of H pylori. H pylori can sensitize human gastric epithelial cells and enhance susceptibility to TRAIL-mediated apoptosis. Modulation of host cell sensitivity to apoptosis by bacterial interaction adds a new dimension to the immunopathogenesis of H pylori infection.

Identification of X-linked Inhibitor of Apoptosis-associated Factor-1 as an Interferon-stimulated Gene That Augments TRAIL Apo2L-induced Apoptosis

In the course of gene array studies aimed at identifying IFN-stimulated genes associated with interferon beta (IFN-beta)-induced apoptosis, we identified X-linked inhibitor of apoptosis-associated factor-1 (XAF1) as a novel IFN-stimulated gene. XAF1 mRNA was up-regulated by IFN-alpha and IFN-beta in all cells examined. However, IFNs induced high levels of XAF1 protein predominantly in cell lines sensitive to the proapoptotic effects of IFN-beta. In apoptosis-resistant cells including WM164 melanoma, WM35 melanoma, U937 pro-monocytic leukemia, and HT1080 fibrosarcoma cells, XAF1 mRNA was strongly up-regulated but XAF1 protein was up-regulated only weakly or not at all. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a critical mediator of IFN-beta-induced apoptosis, but most melanoma cell lines were resistant to recombinant TRAIL protein. For example, A375 melanoma cells were defective in TRAIL induction by IFN-beta and were resistant to TRAIL-induced apoptosis. However, IFN-beta pretreatment sensitized them to subsequent recombinant TRAIL-induced apoptosis. A375 cells expressing XAF1 constitutively were more sensitive to TRAIL-induced apoptosis compared with empty vector-transfected cells. The degree of sensitization by XAF1 was similar to that provided by IFN pretreatment and was correlated with the level of XAF1 expressed. Furthermore, the overexpression of the zinc-finger portion of XAF1 blocked IFN-dependent sensitization of A375 melanoma cells to the proapoptotic effects of TRAIL. These results suggested that IFN-dependent induction of XAF1 strongly influenced cellular sensitivity to the proapoptotic actions of TRAIL.

Regulation of Akt by EGF-R inhibitors, a possible mechanism of EGF-R inhibitor-enhanced TRAIL-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine and a potent inducer of apoptosis. Epidermal growth factor (EGF) signaling is well known to involve in tumor survival and overexpression of EGF receptor (EGF-R) attributes to decreased responsiveness to many available therapies in cancer treatment. We investigated whether EGF-R inhibitors enhance TRAIL-induced apoptosis. We exposed A549 cells to Genistein, PD153035, and PD158780 for 12 h and then treated with recombinant TRAIL protein. TRAIL alone induced 25% cell death after a 3-h treatment, but in cells pretreated with EGF-R inhibitors, TRAIL induced cell death to more than 70% after 3 h treatment. Genistein enhanced TRAIL-induced apoptosis in a time- and dose-dependent manner. Western blot analyses showed that pretreatment with Genistein down-regulated the protein levels of total Akt and phosphorylated active Akt. Genistein also decreased the protein level of Bcl-XL that is regulated by Akt. These molecules are well characterized to act against induction of apoptotic cell death. Therefore, our data suggest that EGF-R inhibitor may sensitize A549 cells to TRAIL-induced apoptosis by regulating expression of these proteins. EGF-R inhibitors may play an important role in the anti-cancer activity of TRAIL protein, especially in TRAIL-resistant tumors that arise by expressing constitutively active Akt.

IFN-γ Inhibition of TRAIL-Induced IAP-2 Upregulation, a Possible Mechanism of IFN-γ-Enhanced TRAIL-Induced Apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine molecule of TNF family and a potent inducer of apoptosis. The anticancer activities of TNF family members are often modulated by interferon (IFN)-γ. Thus, we investigated whether IFN-γ enhances TRAIL-induced apoptosis. We exposed HeLa cells to IFN-γ for 12 h and then treated with recombinant TRAIL protein. No apoptosis was induced in cells pretreated with IFN-γ, and TRAIL induced 25% cell death after 3 h treatment. In HeLa cells pretreated with IFN-γ, TRAIL induced cell death to more than 70% at 3 h, indicating that IFN-γ pretreatment sensitized HeLa cells to TRAIL-induced apoptosis. We investigated molecules that might be regulated by IFN-γ pretreatment that would affect TRAIL-induced apoptosis. Western blotting analyses demonstrated that TRAIL treatment increased the level of IAP-2 protein and IFN-γ pretreatment inhibited the upregulation of IAP-2 protein by TRAIL protein. Our data indicate that TRAIL can signal to activate both apoptosis induction and antiapoptotic mechanism, at least, through IAP-2 simultaneously. IFN-γ or TRAIL treatment alone did not change expression of other pro- or antiapoptotic proteins such as DR4, DR5, FADD, Bax, IAP-1, XIAP, Bcl-2, and Bcl-XL. Our findings suggest that IFN-γ may sensitize HeLa cells to TRAIL-induced apoptosis by preventing TRAIL-induced IAP-2 upregulation, and IFN-γ may play a role in anticancer therapy of TRAIL protein through such mechanism.

CD95 Ligand (Fas-L/APO-1L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Mediate Ischemia-Induced Apoptosis in Neurons

Programmed cell death plays an important role in the neuronal degeneration after cerebral ischemia, but the underlying mechanisms are not fully understood. Here we examined, in vivo and in vitro, whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain. Further recombinant CD95 ligand and TRAIL proteins induced apoptosis in primary neurons and neuron-like cells in vitro. The immunosuppressant FK506, which most effectively protects against ischemic neurodegeneration, prevented postischemic expression of these death-inducing ligands both in vivo and in vitro. FK506 also abolished phosphorylation, but not expression, of the c-Jun transcription factor involved in the transcriptional control of CD95 ligand. Most importantly, in lpr mice expressing dysfunctional CD95, reversible middle cerebral artery occlusion resulted in infarct volumes significantly smaller than those found in wild-type animals. These results suggest an involvement of CD95 ligand and TRAIL in the pathophysiology of postischemic neurodegeneration and offer alternative strategies for the treatment of cardiovascular brain disease.