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.     

Interleukin-2 activated NK cells do not use the CD95L- and TRAIL-pathways in the rapid induction of apoptosis of rat colon carcinoma CC531s cells

Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-α pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-α pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.     

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.     

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.     

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 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.     

Tumor necrosis factor-related apoptosis-inducing ligand and chemotherapy cooperate to induce apoptosis in mesothelioma cell lines

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in certain tumor cells. In addition, TRAIL and chemotherapy can act cooperatively, possibly as a result of chemotherapy-induced increases in expression of a TRAIL receptor, DR5. We used cell lines derived from a highly chemoresistant tumor, malignant mesothelioma, to learn whether TRAIL was effective alone or together with chemotherapy and whether cooperativity depended on increases in DR5 expression. TRAIL (codons 95-285) was expressed in a bacterial expression vector and purified by nickel affinity chromatography. TRAIL alone (25 to 500 ng/ml) had little effect on mesothelioma cells. TRAIL plus chemotherapy (doxorubicin, cis-platinum, etoposide, or gemcitabine) acted cooperatively to induce apoptosis in mesothelioma cells (M28, REN, VAMT, and MS-1). For example, in M28 cells treated for 18 h, apoptosis from TRAIL (100 ng/ml) plus doxorubicin (0.6 microg/ml; 71 +/- 11%) greatly exceeded that from TRAIL alone (21 +/- 8%) or from doxorubicin alone (6 +/- 2%) (means +/- standard deviation; P < 0.03). Mesothelioma cells treated with chemotherapy showed no change in DR5 protein by Western analysis or by immunocytochemistry. TRAIL plus chemotherapy was associated with an increase in mitochondrial cytochrome c release and mitochondrial depolarization. We conclude that TRAIL and chemotherapy act cooperatively to kill mesothelioma cell lines, not by increases in DR5 receptor but in association with mitochondrial amplification of apoptotic signals.     

Over-expression of Smac promotes TRAIL-induced cell death in human hepatocellular carcinoma

The second mitochondria-derived activator of caspase, Smac, is an apoptosis-related protein. Smac releases inhibition of the IAP family from caspase-3 to induce apoptosis. Smac is expressed in some malignant tumor cells and is released from mitochondria into the cytosol after death receptor stimulation to promote apoptosis of tumor cells. In this study, we found down-regulated Smac protein expression in hepatocellular carcinoma (HCC) tissues, compared to that in non-tumor hepatic tissues. Simultaneously, caspase-3 expression also decreased in HCC tissues. HCC cell lines did not undergo apoptosis after TRAIL stimulation, although Smac was expressed in these HCC cells. Ectopic Smac alone did not induce cell death, but could sensitize HCC cells to TRAIL stimulation. With over-expression of Smac in HCC cells, TRAIL induced by 10% HCC cell death. The role of Smac in apoptosis signaling pathway in HCC cells warrants further study.     

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.     

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.     

CD95-mediated Apoptosis of Human Glioma Cells: Modulation by Epidermal Growth Factor Receptor Activity

The death ligands CD95L and Apo2L/TRAIL are promising investigational agents for the treatment of malignant glioma. EGFR is overexpressed in a significant proportion of malignant gliomas in vivo. Here, we report that CD95L-induced cell death is enhanced by EGFR inhibition using tyrphostine AG1478 in 7 of 12 human malignant glioma cell lines. Conversely, CD95-mediated and Apo2L-induced cell death are both inhibited by overexpression of EGFR in LN-229 cells. CD95L-induced cell death augmented by AG1478 is accompanied by enhanced processing of caspase 8. LN-229 cells overexpressing the viral caspase inhibitor, crm-A, are not sensitized to CD95L-induced cell death by AG1478, indicating that EGFR exerts its antiapoptotic properties through a caspase 8-dependent pathway. These data define a modulatory effect of EGFR-activity on death ligand-induced apoptosis and indicate that EGFR inhibition is likely to improve the efficacy of death ligand-based cancer therapies. Furthermore, it is tempting to speculate that EGFR amplification protects tumor cells from death ligand-mediated host immune responses in vivo and that EGFR's effects on death receptor-mediated apoptosis may explain the anti-tumor effects of non-cytotoxic, unarmed anti-EGFR family antibodies.     

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.     

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.     

膜结合配体依赖的TRAIL抗肿瘤细胞旁观者效应

目的：研究TRAIL基因抗肿瘤作用旁观者效应的发生机制。方法：腺病毒Ad/Lac-Z转染肿瘤细胞后作为标记的旁观者细胞，腺病毒Ad/g-TRAIL 转染肿瘤细胞作为效应细胞，在6孔Transwell培养板上同一孔内应用隔离或混合培养旁观者细胞和效应细胞的方法，通过检测旁观者细胞Lac-Z酶的活性，以明确TRAIL基因旁观者效应的发生机制。结果：检测了不同来源的肿瘤细胞株，包括大肠癌细胞株DLD1和Lovo、肺癌细胞株A549、肝癌细胞株HepG2、乳腺癌细胞株MDA-MB231、卵巢癌细胞株DOV13。各株肿瘤细胞转染Ad/g-TRAIL后ELISA方法均未检测出培养液中出现可溶性TRAIL（s-TRAIL）；隔离培养效应细胞与旁观者细胞，旁观者细胞的Lac-Z活性与PBS及腺病毒Ad/CMV-GFP对照组无明显差异，而混合培养组其活性明显低于对照组（P<0.05）；混合培养效应细胞与旁观者细胞（1∶1），发现高密度细胞生长组其旁观者效应明显大于低密度生长组（P<0.05 ）；FACS检测Sub G0/G1 发现DOV13细胞在高密度生长组显著高于低密度生长组（P<0.05）；荧光及相差显微镜下观察发现TRAIL转染细胞可诱导周围旁观者细胞发生凋亡而自己并不首先发生凋亡。结论：腺病毒介导的TRAIL基因的旁观者效应是通过TRIAL蛋白表达并定位于细胞膜上，通过细胞接触结合于邻近肿瘤细胞膜上的TRAIL受体而诱导旁观者肿瘤细胞的凋亡，而不是通过水解膜配体形成s-TRAIL而发生旁观者效应，这种作用是细胞接触依赖的。     

COX-2 inhibitors sensitize human hepatocellular carcinoma cells to TRAIL-induced apoptosis

Cyclooxygenase (COX)-2 is upregulated in a variety of human cancers, including in hepatocellular carcinoma (HCC), whereas it is undetectable in most normal tissue. Evidence suggests that COX-2 is likely to be involved in hepatocarcinogenesis and, thus, COX-2 may be involved in an early process in carcinogenesis, dedifferentiation. To address this possibility, we investigated the effect of COX-2 inhibitors on TNF-related apoptosis, inducing ligand (TRAIL) sensitivity and its molecular mechanisms, with special attention to anti-apoptotic proteins. We used the highly selective COX-2 inhibitors, NS398 and CAY10404. We also used the MTT assay and cytological analysis of DAPI-stained DNA to assess viability and apoptosis in two HCC cells (SK-Hep1 and HLE). In order to ask what led to increased sensitivity to TRAIL in HCC cells, cell surface expression of TRAIL and TRAIL-receptors was investigated using flow cytometry analysis. Expression of survivin, X-chromosome-linked IAP (XIAP), Bcl-xL, AKT and phospho-AKT was also investigated using immunoblotting. COX-2 inhibitors resulted in a concentration-dependent decrease in cell viability in the two HCC cell lines tested. Subtoxic levels of COX-2 inhibitors did not significantly augment TNFalpha-induced apoptosis but did dramatically enhance TRAIL-induced apoptosis in both cell lines. TRAIL receptor 2/death receptor 5 (TRAIL-R2/DR5) expression was significantly up-regulated in SH-Hep1 and HLE cells. TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) expression was up-regulated only in SK-Hep1. Expression of survivin and Bcl-xL was down-regulated in SK-Hep1 and HLE cells in the presence of CAY10404 but XIAP was not affected. Expression of survivin, Bcl-xL and XIAP was down-regulated in SK-Hep1 cells in the presence of NS398. Survivin expression was also down-regulated in the presence of NS398 in HLE cells. Finally, NS398 also decreased phospho-AKT in SK-Hep1 cells. These results demonstrate that COX-2 inhibitors can induce apoptosis and augment TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of both survivin and AKT signaling.     

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.     

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.