Functional expression of tumor necrosis factor-related apoptosis-inducing ligand in human colonic adenocarcinoma cells

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in various transformed cell lines. Therefore, we investigated TRAIL sensitivity, TRAIL-induced nuclear factor-kappaB (NF-kappaB) activation, and expression of TRAIL in human colonic adenocarcinoma cell lines (HT-29, LS180, SK-CO-1). All four TRAIL receptors (TRAIL-R1 through TRAIL-R4) are expressed in these cell lines. TRAIL sensitivity was assessed by assay of cell viability. Cancer cell viabilities were 83 +/- 3.1% (HT-29), 90 +/- 4.3% (LS180), and 88 +/- 6.3% (SK-CO-1) at 24 hours after the addition of 100 ng/ml TRAIL, indicating that these cell lines were relatively resistant to TRAIL. Activation of NF-kappaB was variably influenced by TRAIL administration, with no consistent tendency among the cell lines, indicating that TRAIL-induced NF-kappaB activation might be cell-type dependent. In contrast, TRAIL was expressed in the human colonic adenocarcinoma cell lines by Western blotting and RT-PCR. Increased expression of TRAIL on tumor cells was observed by flow cytometry after cytokine stimulation (IFN-gamma, TNF-alpha) or the addition of chemotherapeutic agents (camptothecin, doxolubicin hydrochloride). TRAIL on HT-29 cells was functional and able to induce apoptosis in Jurkat cells. Jurkat cell viability was increased by the addition of TRAILR1-R4-Fc. In the presence of various cytokines or chemotherapeutic agents, functional TRAIL is expressed on the surface of tumor cells, and this expressed TRAIL might contribute to tumor immune privilege by inducing apoptosis of activated human lymphocytes.     

FLIP as an anti-cancer therapeutic target

Suppression of apoptosis is one of the hallmarks of carcinogenesis. Tumor cells endure apoptotic pressure by overexpressing several antiapoptotic proteins, and FLICE inhibitory protein (FLIP) is one of the important antiapoptotic proteins that have been shown to be overexpressed in various primary tumor cells. FLIP has two death-effector domains in tandem, mimicking the prodomain of procaspase-8. It is recruited to Fadd in death-inducing signaling complex, thereby preventing the activation of procaspase-8. To date, three isoforms of human cytosolic FLIP (c-FLIP) and six viral homologs (v-FLIP) have been identified. Recently, the crystal structure of v-FLIP MC159 was determined for the first time as an atomic-detail FLIP structure, which revealed that two death effector domains are packed tightly against each other mainly through conserved hydrophobic interactions. The overexpression of c-FLIP in tumor cells has been shown to be the determinant of the tumor's resistance to death ligands such as FasL and TRAIL. It has also been shown that the down-regulation of c-FLIP results in sensitizing resistant tumor cells. Therefore, the agents directly targeting c-FLIP at mRNA and protein levels are expected to be developed in near future and tested for the potential as a new class of anti-cancer drugs.     

Sensitizing TRAIL response via differential modulation of anti- and pro-apoptotic factors by AZD5582 combined with ER nanosomal TRAIL in neuroblastoma

Neuroblastoma is a metastatic brain tumor particularly common in children. The cure rate is below 50% for patients of high-risk condition. Novel therapeutic agents and approaches are needed to improve the cure rate. Tumor necrosis factor-related and apoptosis-inducing ligand (TRAIL) is a promising proapoptotic factor that rapidly induces apoptosis preferentially in transformed and cancerous cells. Unfortunately, the common TRAIL resistance in cancers has hampered the clinical application of the ligand. Previously we prepared a novel TRAIL-armed ER derived nanosomal agent (ERN-T) that overcomes TRAIL resistance in some cancer lines when combined with a synthetic antagonist of inhibitors of apoptosis proteins (IAPs), AZD5582. However, how AZD5582 sensitizes cancer cells to ERN-T remains not well understood. In this study we continued to test the therapeutic efficacy of the combinatory therapy of ERN-T and AZD5582 on neuroblastoma, aiming to reveal the molecular mechanism underlying the synergism between AZD5582 and ERN-T. The obtained data revealed that ERN-Ts overcame TRAIL resistance and showed significant cytotoxicity on the resistant neuroblastoma line SH-SH5Y when combined with AZD5582 whilst sparing normal cells. The combination of low doses of ERN-Ts and AZD5582 induced intensive apoptosis in SH-SY5Y but not in normal skin fibroblasts (NSFs). Importantly we discovered that TRAIL sensitization in SH-SY5Y was associated with the concomitant downregulation of antiapoptotic factors cFLIP, MCL-1 and IAPs and upregulation of proapoptotic protein BAX and the death receptor 5 (DR5) by the cotreatment of ERN-T and AZD5582. In vivo study demonstrated that the combination of ERN-T and AZD5582 constituted a highly effective and safe therapy for subcutaneous SH-SY5Y xenograft neuroblastoma in nude mice. In conclusion, we identified that the concomitant regulation of both antiapoptotic and proapoptotic factors and DR5 is an essential molecular mechanism for overcoming TRAIL resistance in SH-SY5Y and the combination of ERN-T and AZD5582 potentially constitutes a novel therapeutic strategy, which is highly effective and safe for neuroblastoma.     

Adenoviral-mediated transfer of p53 gene enhances TRAIL-induced apoptosis in human hepatocellular carcinoma cells

p53 is a tumor suppressor protein with numerous biological functions including transformation, regulation of cell growth, differentiation and apoptosis. The TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in various transformed cell lines. We investigated the effects of combining wild-type p53 gene transduction by adenoviral infection (Ad-p53) with addition of TRAIL on cell death, expression levels of TRAIL receptors (TRAIL-R1, TRAIL-R2), FLICE inhibitory protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) on human hepatocellular carcinoma (HCC) cell lines. HCC cell death was increased by combination of Ad-p53 infection and addition of TRAIL compared to either alone. Western blotting demonstrated decreased TRAIL-R1 and TRAIL-R2 levels after infection with Ad-p53. FLIP levels decreased in Huh7 cells and Hep3B cells, and XIAP levels decreased in all three HCC cell lines after infection with Ad-p53. Thus, death of HCC cells due to combined p53 gene transduction and exogenous TRAIL may be due to down regulation of FLIP or XIAP.     

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.     

Genistein enhances proteasomal degradation of the short isoform of FLIP in malignant glioma cells and thereby augments TRAIL-mediated apoptosis

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer drug. One obstacle in TRAIL-based therapies is that many cancer cells, including gliomas, are resistant towards TRAIL. In this study one glioblastoma cell line, one human short-term glioblastoma culture and human astrocytes were treated with genistein, tumour necrosis factor-related apoptosis-inducing ligand or the combination of both. Single treatment with genistein or TRAIL does not induce cytotoxicity in malignant glioma cells. However, treatment with genistein in combination with TRAIL induces rapid apoptosis in TRAIL-resistant glioma cells. Notably, normal human astrocytes were not affected by the combination treatment consisting of genistein and TRAIL. Genistein enhanced proteasomal degradation of the short isoform of c-FLIP. Importantly, over-expression of only the short isoform of c-FLIP attenuated genistein TRAIL-mediated cytotoxicity. Taken together, we gave evidence that genistein facilitated TRAIL-mediated apoptosis at the level of the extrinsic apoptotic pathways in malignant glioma cells.     

FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2

Apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO-2L) has been shown to exert important functions during various immunological processes. The involvement of the death adaptor proteins FADD/MORT1, TRADD, and RIP and the apoptosis-initiating caspases-8 and -10 in death signaling by the two death-inducing TRAIL receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are controversial. Analysis of the native TRAIL death-inducing signaling complex (DISC) revealed ligand-dependent recruitment of FADD/MORT1 and caspase-8. Differential precipitation of ligand-stimulated TRAIL receptors demonstrated that FADD/MORT1 and caspase-8 were recruited to TRAIL-R1 and TRAIL-R2 independently of each other. FADD/MORT1- and caspase-8-deficient Jurkat cells expressing only TRAIL-R2 were resistant to TRAIL-induced apoptosis. Thus, FADD/MORT1 and caspase-8 are essential for apoptosis induction via TRAIL-R2.     

To die or not to die--the quest of the TRAIL receptors.

The last 18 months have witnessed the characterization of several new members of the tumor necrosis factor (TNF) receptor family. Among these are five receptors for the cytotoxic ligand TRAIL (TNF-related apoptosis-inducing ligand). Two of these receptors, TRAIL-R1 and TRAIL-R2, contain classical cytoplasmic death domains and are able to transduce an apoptotic signal. The others lack functional death domains and are not able to promote cell death. Indeed, one of the receptors for TRAIL, osteoprotegerin (OPG) is a soluble protein whose activities so far have been shown to be inhibition of osteoclastogenesis and increased bone density in vivo. The existence of multiple receptors for TRAIL suggests an unexpected complexity to TRAIL-mediated biological functions.     

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF-alpha promote the NF-kappaB-dependent maturation of normal and leukemic myeloid cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF-alpha induced monocytic maturation of primary normal CD34-derived myeloid precursors and of the M2/M3-type acute myeloid leukemia HL-60 cell line, associated to increased nuclear factor (NF)-kappaB activity and nuclear translocation of p75, p65, and p50 NF-kappaB family members. Consistently, both cytokines also induced the degradation of the NF-kappaB inhibitors, IkappaBalpha and IkappaB epsilon, and up-regulated the surface expression of TRAIL-R3, a known NF-kappaB target. However, NF-kappaB activation and IkappaB degradation occurred with different time-courses, since TNF-alpha was more potent, rapid, and transient than TRAIL. Of the two TRAIL receptors constitutively expressed by HL-60 (TRAIL-R1 and TRAIL-R2), only the former was involved in IkappaB degradation, as demonstrated by using agonistic anti-TRAIL receptor antibodies. Moreover, NF-kappaB nuclear translocation induced by TRAIL but not by TNF-alpha was abrogated by z-IETD-fmk, a caspase-8-specific inhibitor. The key role of NF-kappaB in mediating the biological effects of TNF-alpha and TRAIL was demonstrated by the ability of unrelated pharmacological inhibitors of the NF-kappaB pathway (parthenolide and MG-132) to abrogate TNF-alpha- and TRAIL-induced monocytic maturation. These findings demonstrate that NF-kappaB is essential for monocytic maturation and is activated via distinct pathways, involving or not involving caspases, by the related cytokines TRAIL and TNF-alpha.     

Protective effect of RIP and c-FLIP in preventing liver cancer cell apoptosis induced by TRAIL

TRAIL (TNF-related apoptosis-inducing ligand) is a member of the tumor necrosis factor superfamily that can induce tumor selective death by up-regulating death receptor 4 (DR4) and DR5 expression. The study aimed to explore the role of RIP and c-FLIP genes in TRAIL induced liver cancer cell HepG2 and Hep3B apoptosis and related mechanism. RIP and c-FLIP silenced HepG2 and Hep3B cell model were established through siRNA. Western blot was applied to test c-FLIP, RIP, DR4, DR5, FADD, Caspase-3/8/9, ERK1/2, and DFF45 protein expression. Caspase-8 kit was used to detect Caspase-8 expression. Flow cytometry was performed to measure cell apoptosis rate. Acid phosphatase method was applied to determine cell cycle. TRAIL had no significant effect on Caspase-3/8/9, DR4, DR5, ERK1/2, and DFF45 protein expression, but up-regulated c-FLIP and RIP protein expression and reduced FADD expression level. After treated by the chemotherapy drug mitomycin and adriamycin, c-FLIP and RIP expression decreased significantly, while FADD increased. After knockout c-FLIP and RIP gene, HepG2 and Hep3B cell apoptosis rate induced by TRAIL increased obviously. Meanwhile, cell subG1 percentage increased markedly and exhibited G1 phase growth retardation. In addition, after two kinds of gene knockout, Caspase-8 was activated and produce Caspase-3 P20 and P24, leading DFF45 appeared DNA fragment P17 and P25. c-FLIP and RIP can inhibit Caspase-8 activation and prompting HepG2 and Hep3B resistant to cell apoptosis induced by TRAIL.     

Involvement of tumor necrosis factor-related apoptosis-inducing ligand and tumor necrosis factor-related apoptosis-inducing ligand receptors in viral hepatic diseases

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor cells, but not in most normal cells. The role of TRAIL in hepatic cell death and hepatic diseases is not well understood. The present study investigated the expression of TRAIL and TRAIL receptors (TRAIL-Rs) in patients with hepatitis C virus infection using immunohistochemistry and examined physiological roles under viral infection in the HepG2 cell line. Staining of TRAIL or TRAIL-Rs was prominent in the cytoplasm and membrane of hepatocytes in the periportal area. Some liver-infiltrating lymphocytes also displayed positive staining for TRAIL. Staining intensity was significantly increased with disease progression, particularly in the periportal area. AdCMVLacZ (Q-BIOgene, Carisbad, Calif) infection was also found to induce apoptosis in HepG2 cells and significantly augment TRAIL-induced apoptosis. Anti-TRAIL antibody significantly inhibited apoptosis induced by AdCMVLacZ infection. Flow cytometry analysis revealed that both TRAIL-R2 and TRAIL were up-regulated on the cell surface of HepG2 cells with AdCMVLacZ infection. Transforming growth factor-beta1 also enhanced TRAIL expression in HepG2 cells. These results indicate that TRAIL/TRAIL-R apoptotic pathways play important roles in the hepatic cell death during viral infection.     

Characterization of tumour necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in the adult human testis

Tumour necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of the tumour necrosis factor-alpha (TNF-alpha) family of cytokines which is known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, the presence of TRAIL and its receptors was investigated by immunohistochemistry in adult human testes. In addition, TRAIL and its receptors were studied in terms of protein and mRNA using western blot analysis and RT-PCR respectively. TRAIL and its receptors were immunodetected according to the different testicular cell types: TRAIL, DR5/TRAIL-R2 and DcR2/TRAIL-R4 were localized in Leydig cells, DR4/TRAIL-R1 was seen in peritubular and Sertoli cells whereas ligand and all receptors were detected in germ cells. Proteins and mRNA corresponding to TRAIL and its receptors were also identified in adult human testes. In conclusion, TRAIL and its receptors DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1/TRAIL-R3 and DcR2/TRAIL-R4 are expressed in the human testis, and are predominantly localized in different germ cell types.     

Daidzein overcomes TRAIL-resistance in malignant glioma cells by modulating the expression of the intrinsic apoptotic inhibitor,bcl-2

The soy isoflavone Daidzein has been reported to exhibit therapeutic activity in cancer. In this study glioblastoma cells and human astrocytes were treated with Daidzein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of Daidzein in combination with TRAIL induces rapid apoptosis in glioma cells. Notably, human astrocytes were not affected by the combined treatment consisting of Daidzein and TRAIL. Combined treatment with Daidzein and TRAIL augmented the activation of caspase-9, suggesting that Daidzein modulated the intrinsic apoptotic pathway. Daidzein did not modulate the expression of death receptors, c-FLIP, XIAP and survivin. However, Daidzein down-regulated bcl-2 and over-expression of bcl-2 attenuated apoptosis induced by the combination of Daidzein and TRAIL. In summary, bcl-2 is a key regulator in TRAIL–Daidzein mediated cell death in malignant glioma.     

In chronic pancreatitis,widespread emergence of TRAIL receptors in epithelia coincides with neoexpression of TRAIL by pancreatic stellate cells of early fibrotic areas

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis by cross-linking of the two TRAIL receptors that contain a death domain, TRAIL-R1 and TRAIL-R2. TRAIL-R3 and TRAIL-R4 are receptors that do not transmit an apoptotic signal. Our aim was to determine the expression of TRAIL and its receptors in normal pancreas and chronic pancreatitis. We applied real-time PCR, immunohisto(cyto)chemistry, and nick-end labeling of apoptosis. In normal pancreas, a minor subset of acinar cells coexpressed TRAIL-R2 and TRAIL-R4, whereas ductular epithelium and interstitial fibroblast-like cells (FLC) expressed TRAIL-R4. TRAIL-R1 and TRAIL-R3 were not detected in normal pancreas. In chronic pancreatitis, the exocrine epithelium strongly expressed TRAIL-R1, -R2, -R4, and, to a lesser extent, TRAIL-R3. Islets focally neoexpressed TRAIL-R1 and -R2 and intensely expressed TRAIL-R4. Changes in TRAIL receptor expression were most pronounced in areas of inflammatory infiltration and active fibrosis. In normal pancreas, expression of TRAIL was low on the mRNA level and undetectable on the protein level. In chronic pancreatitis, FLC in areas of active fibrosis expressed TRAIL. In addition, apoptosis were most numerous in these areas. We show that these FLC are pancreatic stellate cells. Pancreatic stellate cells express TRAIL in vivo and in vitro, and TRAIL expression is enhanced by IFN-gamma. Our findings indicate that the TRAIL/TRAIL receptor system is likely to be involved in chronic pancreatitis and suggest that pancreatic stellate cells may directly contribute to acinar regression by inducing apoptosis of parenchymal cells in a TRAIL-dependent manner.     

Cellular FLICE/caspase-8-inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma

Human hepatocellular carcinomas (HCCs) show resistance to apoptosis mediated by several death receptors. Because cellular FLICE/caspase-8-inhibitory protein (cFLIP) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs), we investigated the expression and function of cFLIP in human HCCs. We found that cFLIP is constitutively expressed in all human HCC cell lines and is expressed more in human HCC tissues than in nontumor liver tissues. Metabolic inhibitors, actinomycin D (ActD) or cycloheximide (CHX), dramatically rendered HCC cells sensitive to Fas-mediated apoptosis. Neither caspase-8 nor caspase-3 was activated by agonistic anti-Fas antibody alone, but both caspases were activated by Fas stimulation in the presence of ActD or CHX, indicating the importance of caspase-8 inhibitors that are sensitive to metabolic inhibitors. Actually, cFLIP expression was decreased in ActD or CHX treatment. cFLIP down-regulation induced by cFLIP antisense oligodeoxynucleotides sensitized HLE cells to Fas, TNF-R, and TRAIL-R-mediated apoptosis. Furthermore, cFLIP over-expression activated nuclear factor (NF)-kappaB and cFLIP down-regulation attenuated NF-kappaB activation induced by TNF-alpha or TRAIL. Pretreatment with pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), restored NF-kappaB activity attenuated by cFLIP down-regulation. cFLIP expression was increased by TNF-alpha, TRAIL, or vascular endothelial growth factor but decreased by wortmannin, indicating that cFLIP expression is regulated by both the NF-kappaB and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. These results suggest that cFLIP plays an important role in cell survival not simply by inhibiting death-receptor-mediated apoptosis but also by regulating NF-kappaB activation in human HCCs.     

Differential effects of phosphatidylinositol-3/Akt-kinase inhibition on apoptotic sensitization to cytokines in LNCaP and PCc-3 prostate cancer cells.

Alterations in phosphatidylinositol 3'-kinase (PI3'-kinase) and Akt activation frequently occur in prostate cancer and may disrupt apoptotic induction by such cytokines as tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). To examine the role of PI3' phosphorylation in the cellular response to cytokines, two prostate cancer cell lines with constitutively activated PI3'-kinase cascades (LNCaP and PC-3) were examined for direct sensitivity to cytokines. TNF or TRAIL alone failed to activate apoptosis in either LNCaP or PC-3 cells, and drug-mediated inhibition of the PI3k/Akt cascade caused only minimal activation of apoptosis in either cell line. Suppression of PI3'-kinase/Akt signaling markedly enhanced the apoptotic activity of both TNF and TRAIL in LNCaP cells but not in PC-3 cells. Adenovirus-mediated PTEN/MMAC1 expression in LNCaP cells reduced Akt activation, activated apoptosis, and sensitized cells to TNF but not to TRAIL. Together, these results suggest that PI3'-kinase signaling inhibits both TNF-mediated and TRAIL-mediated apoptosis but may represent one of several apoptotic resistance mechanisms that inhibit cytokine-mediated killing of prostate cancer cells.