The Potential of the Tumor Microenvironment to Influence Apo2L/TRAIL Induced Apoptosis

Apo2L/TRAIL ligation of specific cell surface receptors (DR4 and DR5) induces apoptosis of many malignant cells with little effect on normal cells. This anti-tumor capability has been demonstrated using cell lines of many tumor types, both in vitro and in vivo when the cells are grown as xenografts. We have extended these studies to investigate the efficacy of Apo2L/TRAIL against patient tumor xenografts in SCID mice and found that the growth of many tumors, both of primary and metastatic origin, can be inhibited by Apo2L/TRAIL. The basis of resistance to Apo2L/TRAIL induced apoptosis in malignant cells and normal cells is not completely understood, but it is known that a variety of factors including hypoxia, MMPs and cytokines present in the tumor microenvironment can influence the response of malignant cells to Apo2L/TRAIL. Currently, the clinical potential of several molecules targeting the Apo2L/TRAIL receptors DR4 and DR5 is being investigated. Our goal in this review is to provide a brief overview of a number of factors that have potential to influence the response of patient tumors to Apo2L/TRAIL.     

The Potential of the Tumor Microenvironment to Influence Apo2L/TRAIL Induced Apoptosis

Apo2L/TRAIL ligation of specific cell surface receptors (DR4 and DR5) induces apoptosis of many malignant cells with little effect on normal cells. This anti-tumor capability has been demonstrated using cell lines of many tumor types, both in vitro and in vivo when the cells are grown as xenografts. We have extended these studies to investigate the efficacy of Apo2L/TRAIL against patient tumor xenografts in SCID mice and found that the growth of many tumors, both of primary and metastatic origin, can be inhibited by Apo2L/TRAIL. The basis of resistance to Apo2L/TRAIL induced apoptosis in malignant cells and normal cells is not completely understood, but it is known that a variety of factors including hypoxia, MMPs and cytokines present in the tumor microenvironment can influence the response of malignant cells to Apo2L/TRAIL. Currently, the clinical potential of several molecules targeting the Apo2L/TRAIL receptors DR4 and DR5 is being investigated. Our goal in this review is to provide a brief overview of a number of factors that have potential to influence the response of patient tumors to Apo2L/TRAIL.     

The anti-tumor effect of Apo2L/TRAIL on patient pancreatic adenocarcinomas grown as xenografts in SCID mice

Background  

Apo2L/TRAIL has considerable promise for cancer therapy based on the fact that this member of the tumor necrosis factor family induces apoptosis in the majority of malignant cells, while normal cells are more resistant. Furthermore, in many cells, when Apo2L/TRAIL is combined with chemotherapy, the effect is synergistic. The majority of this work has been carried out using cell lines. Therefore, investigation of how patient tumors respond to Apo2L/TRAIL can validate and/or complement information obtained from cell lines and prove valuable in the design of future clinical trials.     

Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5

Fas (APO-1/CD95) and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis by recruiting the apoptosis initiator caspase-8 through the adaptor FADD. Fas binds FADD directly, whereas TNFR1 binds FADD indirectly, through TRADD. TRADD alternatively recruits the NF-kappaB-inducing adaptor RIP. The TNF homolog Apo2L/TRAIL triggers apoptosis through two distinct death receptors, DR4 and DR5; however, receptor over-expression studies have yielded conflicting results on the ligand's signaling mechanism. Apo2L/TRAIL induced homomeric and heteromeric complexes of DR4 and DR5 and stimulated recruitment of FADD and caspase-8 and caspase-8 activation in nontransfected cells. TRADD and RIP, which bound TNFR1, did not bind DR4 and DR5. Thus, Apo2L/TRAIL and FasL initiate apoptosis through similar mechanisms, and FADD may be a universal adaptor for death receptors.     

Characterization of the in vivo function of TNF-alpha-related apoptosis-inducing ligand,TRAIL/Apo2L,using TRAIL/Apo2L gene-deficient mice

To define the normal physiological role for the TRAIL/Apo2L in vivo, we generated TRAIL/Apo2L gene-targeted mice. These mice develop normally and show no defects in lymphoid or myeloid cell homeostasis or function. Although TRAIL/Apo2L kills transformed cells in vitro, TRAIL/Apo2L(-/-) mice do not spontaneously develop overt tumors at an early age. However, in the A20 B cell lymphoma-transferred tumor model, TRAIL/Apo2L(-/-) mice are clearly more susceptible to death from overwhelming tumor burden, due to increased lymphoma load in the liver. A20 tumors are susceptible to TRAIL/Apo2L killing in vitro, indicating that TRAIL/Apo2L may act directly to control A20 cells in vivo. Despite the fact that TRAIL binds osteoprotegerin and osteoprotegerin-transgenic mice are osteopetrotic, TRAIL/Apo2L(-/-) mice show no evidence of altered gross bone density, and no alterations in frequency or in vitro differentiation of bone marrow precursor osteoclasts. Moreover, leucine zipper TRAIL has no toxicity when repeatedly administered to osteoprotegerin(-/-) mice. Thus, TRAIL/Apo2L is important in controlling tumors in vivo, but is not an essential regulator of osteoprotegerin-mediated biology, under normal physiological conditions.     

TNF相关的凋亡诱导配体与肿瘤

TNF相关的凋亡诱导配体（TNF-related apoptosis-inducing ligand, TRAIL），属于TNF 超家族成员，又称为Apo-2L，TRAIL能诱导多种肿瘤细胞的凋亡，而正常的细胞却对其不敏感，TRAIL主要通过与其受体结合激活caspase-8，启动非线粒体和线粒体依赖途径导致细胞凋亡。部分肿瘤细胞对TRAIL的敏感性较差，其原因与TRAIL的受体、信号转导途径激酶以及相关蛋白存在密切联系。     

Expression of TRAIL (TNF-related apoptosis-inducing ligand) and its receptors in normal colonic mucosa,adenomas, and carcinomas

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumour cell lines. Four membrane-bound receptors for TRAIL have been identified, two apoptosis-mediating receptors, DR4 and DR5, and two apoptosis-inhibiting receptors, DcR1 and DcR2. The aim of this study was to examine the role of TRAIL and its receptors in colorectal cancer development. The immunohistochemical expression and localization of TRAIL and its receptors were investigated in normal mucosa (n=10), adenomas (n=19), and carcinomas (n=21). Correlations between the expression of TRAIL and its receptors and the degree of apoptosis (assessed by M30 expression) and histopathological characteristics were explored. TRAIL and its receptors were expressed in normal mucosal epithelium. Expression of the receptors was seen in adenomas and carcinomas. TRAIL expression was lost in a subset of colorectal tumours, more frequently in carcinomas than in adenomas (p<0.05). DR4 and DR5 staining was stronger in neoplastic cells than in normal cells and was accompanied by a higher degree of apoptosis. No differences were found between tumour and normal cells regarding DcR1 and DcR2 expression. No correlations were found between TRAIL or TRAIL receptor expression and histopathological characteristics. In conclusion, marked changes were seen in the course of the adenoma-carcinoma sequence with respect to the expression of TRAIL and TRAIL receptors DR4 and DR5. The stronger expression of DR4 and DR5 in neoplastic cells than in normal cells, together with a higher degree of apoptosis, suggests a possible functional role for these receptors in apoptosis induction in neoplastic colorectal cells.     

Regulation of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in thyroid carcinoma cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand selectively kills neoplastic cells, including thyroid carcinoma cells (Mitsiades et al: Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res 2000, 60:4122-41299). We investigated the mechanisms regulating Apo2L/TRAIL-induced apoptosis in thyroid carcinoma cells, as well as the impact of insulin-like growth factor (IGF)-1, interferon-gamma, and TNF-alpha. We found that the emergence of resistance to Apo2L/TRAIL, after prolonged incubation with this cytokine, was associated with increased levels of FLICE inhibitory protein (FLIP), and was overcome by cycloheximide and bisindolylmaleimide, that specifically down-regulated FLIP expression, as well as by transfection of a FLIP anti-sense oligonucleotide. IGF-1 activated Akt; up-regulated the caspase inhibitors FLIP, cIAP-2, XIAP, and survivin; and attenuated Apo2L/TRAIL-induced apoptosis. This effect was inhibited by the IGF-1 receptor neutralizing antibody aIR3, the PI-3K inhibitor wortmannin, and the heat shock protein-90 chaperone inhibitor geldanamycin. Transfection of constitutively active Akt protected from TRAIL. Conversely, interferon-gamma and TNF-alpha had a sensitizing effect. We conclude that FLIP may negatively regulate Apo2L/TRAIL-induced apoptosis in thyroid carcinomas. Microenvironmental paracrine survival factors, such as IGF-1, up-regulate caspase inhibitors, including FLIP, and protect from Apo2L/TRAIL in a PI-3K/Akt-dependent manner. T helper-1 cytokines and compounds that selectively abrogate the IGF-1 signaling pathway may be helpful adjunct agents in Apo2L/TRAIL-based anti-cancer therapeutic regimens.     

TRAIL triggers apoptosis in human malignant glioma cells through extrinsic and intrinsic pathways

Many malignant glioma cells express death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), yet some of these cells are resistant to TRAIL. Here, we examined signaling events in TRAIL-induced apoptosis and searched for therapeutic agents that could overcome TRAIL resistance in glioma cells. TRAIL induced apoptosis through death receptor 5 (DR5) and was mediated by caspase-8-initiated extrinsic and intrinsic mitochondrial pathways in sensitive glioma cell lines. TRAIL also triggered apoptosis in resistant glioma cell lines through the same pathways, but only if the cells were pretreated with chemotherapeutic agents, cisplatin, camptothecin and etoposide. Previous studies suggested that this was due to an increase in DR5 expression in wild-type TP53 cells, but this mechanism did not account for cells with mutant TP53. Here, we show that a more general effect of these agents is to downregulate caspase-8 inhibitor c-FLIP(S) (the short form of cellular Fas-associated death domain-fike interleukin-1-converting enzyme-inhibitory protein) and up-regulate Bak, a pro-apoptotic Bcl-2 family member, independently of cell's TP53 status. Furthermore, we showed that TRAIL alone or in combination with chemotherapeutic agents, induced apoptosis in primary tumor cultures from patients with malignant gliomas, reinforcing the potential of TRAIL as an effective therapeutic agent for malignant gliomas.     

Application of flow cytometry to molecular medicine: detection of tumor necrosis factor-related apoptosis-inducing ligand receptors in acute myeloid leukaemia blasts

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), a cytokine belonging to the TNF (tumor necrosis factor) family, is currently regarded as a potential anti-cancer agent. Nevertheless, several types of cancer cells display a low sensitivity to TRAIL or are completely resistant to this pro-apoptotic cytokine. TRAIL signalling is dependent on four receptors. Two of them, death receptors 4 and 5 (DR4 and DR5), induce apoptosis, whereas decoy receptors 1 and 2 (DcR1 and DcR2) are unable to evoke cell death upon TRAIL binding. TRAIL resistance may be related to the expression of TRAIL decoy receptors. TRAIL has been proposed as a novel therapeutic agent for the treatment of haematological disorders, including acute myeloid leukaemia (AML). Surprisingly, however, very limited information is available concerning the expression of TRAIL receptors in AML blasts. Here, we have evaluated, using flow cytometry, TRAIL receptor surface expression and sensitivity to TRAIL-dependent apoptosis of AML blasts from 30 patients. We observed frequent expression of TRAIL DcR1 and DcR2, while expression of DR4 and DR5 was less frequent. Nevertheless, the expression of DR4 or DR5 in leukaemic cells was always matched by a similar expression of one of the decoy receptors. Leukaemic blasts were invariably resistant, even to a high concentration (1000 ng/ml) of TRAIL. We suggest that AML blasts are resistant to TRAIL apoptosis in vitro. Therefore, it is unlikely that TRAIL alone might be used in the future as an innovative pharmacological agent for the treatment of AML.     

The characteristic of an anti-human DR5 antibody A6

The efficacy of many cancer treatments is due to their ability to induce apoptosis. DR5 can activate apoptosis pathway after binding with its natural ligand, tumour necrosis factor-related apoptosis-inducing ligand （TRAIL/ Apo2L）. Both TRAIL and agonistic anti-DR5 monoclonal antibody are currently being explored for cancer therapy. The mechanisms of cytotoxicity of our previously prepared monoclonal antibody A6 against DR5 were investigated here. A6 could cause viability loss of Jurkat cells in both time- and dose-dependent manner which could be attributed to the activation of apoptosis pathway. Caspases 3, 8 and 9 were activated in Jurkat cells and the caspase specific inhibitors, such as broad caspases inhibitor Z-VAD-FMK, caspase 8 specific inhibitor Z-IETDFMK and caspase 9 specific inhibitor Z-LEHD-FMK could recover the viability loss caused by A6. The function and molecular mechanism of TRAIL-mediated apoptosis were also investigated and compared with those of A6. Although A6 and TRAIL recognize a different epitope, they could induce a similar reaction in Jurkat cells.     

肿瘤坏死因子相关凋亡诱导配体在多种疾病中的生物学作用

肿瘤坏死因子相关凋亡诱导配体（Apo2L/TRAIL）是肿瘤坏死因子家族的一员,可以诱导表达特异性死亡受体TRAIL-R1 （DR4）或TRAIL-R2 （DR5）的细胞凋亡.TRAIL可诱导多种肿瘤细胞凋亡,对正常细胞无杀伤性,因此将其作为抗肿瘤靶向治疗候选药物之一,备受关注.近期研究发现,TRAIL既通过其受体介导细胞凋亡信号通路,又可传导非凋亡的信号通路,在自身免疫病和感染性疾病中发挥重要生物学作用.因而简单阐明TRAIL在人类多种疾病中生物学作用的研究进展,对今后TRAIL研究方向和治疗策略的选择具有指导意义.     

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.     

Following TRAIL's path in the immune system

The members of the tumour necrosis factor (TNF) superfamily of cytokines play important roles in the regulation of various immune-cell functions. Likewise, induction of cell death by apoptosis is indispensable for the normal functioning of the immune system. There are two major pathways of apoptosis induction. The intrinsic, or mitochondrial, pathway is regulated by the activation and interaction of members of the Bcl-2 family. The extrinsic, or death receptor, pathway is triggered by certain TNF family members when they engage their respective cognate receptors on the surface of the target cell. Hence, cell-to-cell-mediated death signals are induced by activation of these death receptor–ligand systems. Besides TNF itself and the CD95 (Fas/APO-1) ligand (FasL/Apo1L), the TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) belongs to the subfamily of ligands that is responsible for extrinsic induction of cell death. Depending on their status of stimulation, TRAIL can be expressed by various cells of the immune system, amongst them natural killer (NK) cells, T cells, natural killer T cells (NKT cells), dendritic cells and macrophages. TRAIL has been implicated in immunosuppressive, immunoregulatory and immune-effector functions. With respect to pathological challenges, TRAIL and its receptors have been shown to play important roles in the immune response to viral infections and in immune surveillance of tumours and metastases. In this review we summarize the current knowledge on the role of TRAIL and its receptors in the immune system and, based on this, we discuss future directions of research into the diverse functions of this fascinating receptor–ligand system.     

Downregulation of TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L in Barrett's esophagus with dysplasia and adenocarcinoma.

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.     

The promise of TRAIL—potential and risks of a novel anticancer therapy

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising new anticancer biotherapeutic. As shown by many preclinical studies, TRAIL efficiently induces apoptosis in numerous tumor cell lines but not in the majority of normal cells. However, an increasing number of publications report on a predominance of TRAIL resistance in primary human tumor cells, which require sensitization for TRAIL-induced apoptosis. Sensitization of cancer cells by treatment with chemotherapeutic drugs and irradiation has been shown to restore TRAIL sensitivity in many TRAIL-resistant tumor cells. Accordingly TRAIL treatment has been successfully used in different in vivo models for the treatment of tumors also in combination with chemotherapeutics without significant toxicity. However, some reports demonstrated toxicity of TRAIL alone or in combination with chemotherapeutic drugs in normal cells. This review summarizes data concerning the apoptosis-inducing pathways and efficacy of TRAIL, alone or in combination with chemotherapeutic drugs, in primary cancer cells compared to the unwanted effects of TRAIL treatment on normal tissue. We discuss the different in vitro tumor cell models and the potential of different recombinant forms of TRAIL or agonistic antibodies to TRAIL death receptors. Most preclinical studies show a high efficiency of a combinatorial TRAIL-based therapy in animal models and in primary human ex vivo tumor cells with a low toxicity in normal cells. Accordingly clinical phase I/II studies have begun and will be developed further with caution.     

CD95 Ligand: Lethal Weapon Against Malignant Glioma?

CD95 (Fas/APO-1) and its ligand (CD95L) belong to a growing cytokine and cytokine receptor family that includes nerve growth factor (NGF) and tumor necrosis factor (TNF) and their corresponding receptors. CD95 expression increases during malignant progression from low-grade to anaplastic astrocytoma and is most prominent in perinecrotic areas of glioblastoma. There is, however, no evidence that CD95 expression in malignant gliomas is triggered by hypoxia or ischemia. Agonistic antibodies to CD95, or the natural ligand, CD95L, induce apoptosis in human malignant glioma cells in vitro. Glioma cell sensitivity to CD95-mediated apoptosis is regulated by CD95 expression at the cell surface and by the levels of intracellular apoptosis-regulatory proteins, including bcl-2 family members. Several cytotoxic drugs synergize with CD95L to kill glioma cells. For as yet unknown reasons, glioma cells may co-express CD95 and CD95L in vitro without undergoing suicide or fratricide. Yet, they kill T cells via CD95/CD95L interactions and are sensitive to exogenously added CD95L. Since CD95L is expressed in gliomas in vivo , too, forced induction of CD95 expression might promote therapeutic apoptosis in these tumors. That glioma cells differ from non-transformed T cells in their sensitivity to CD95 antibodies or recombinant ligand, may allow the development of selective CD95 agonists with high antitumor activity that spare normal brain tissue. A family of death ligand/receptor pairs related to CD95L/CD95, including APO2L (TRAIL) and its multiple receptors is beginning to emerge. Although several issues regarding glioma cell sensitivity to CD95L/CD95-mediated apoptosis await elucidation, CD95 is a promising target for the treatment of malignant glioma.