Rapid and significant induction of TRAIL-mediated type II cells in apoptosis of primary salivary epithelial cells in primary Sjögren’s syndrome

Expressions of the effector molecules of Fas-mediated apoptosis in primary cultured salivary gland epithelial cells (SGEC) of primary Sjögren’s syndrome (pSS) remain to be clarified. We focused on Fas-mediated caspase cleavage compared to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Induction of apoptosis was performed by anti-Fas antibody coupled with PI3K inhibitor, or TRAIL. Activation of caspases, cytochrome C, and apoptotic protease activating factor-1 (Apaf-1) was determined by western blotting or immunofluorescence observed by confocal microscopy. Fas-mediated apoptosis and activation of caspase 3/8 were induced in the presence of LY294002. TRAIL-induced apoptosis in SGEC, which was stronger than that induced by anti-Fas antibody. TRAIL-induced caspase 9 cleavage accompanied by activation of cytochrome C and Apaf-1 were not mediated by anti-Fas antibody. Our results suggest that death receptor-dependent apoptosis in primary cultured SGEC is regulated by the engagement of type II cells in pSS.     

Inhibition of phosphatidylinositol 3-kinase sensitizes vascular endothelial cells to cytokine-initiated cathepsin-dependent apoptosis

In the presence of cycloheximide, tumor necrosis factor or interleukin-1 initiates caspase activation, loss of mitochondrial membrane potential (DeltaPsi), DNA degradation, and nuclear condensation and fragmentation characteristic of apoptotic cell death in human vascular endothelial cells (EC). Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002, but not inhibition of Akt by dominant-negative mutation, also sensitizes EC to cytokine-initiated apoptosis. Cytokine-initiated caspase activation is slower and comparatively less with LY294002 than with cycloheximide. Cycloheximide but not LY294002 decreases expression of c-FLIP (cellular FLICE inhibitory protein), an inhibitor of caspase-8 activation. The caspase inhibitor zVADfmk completely blocks caspase activation, DNA degradation, and nuclear fragmentation in both cases but only prevents loss of DeltaPsi and cell death for cytokine plus cycloheximide treatment. In contrast, overexpression of Bcl-2 protects EC treated with cytokine plus LY294002 but not EC treated with cytokine plus cycloheximide. The cathepsin B inhibitor CA-074-Me prevents loss of DeltaPsi, caspase activation, and cell death for EC treated with cytokine plus LY294002 but has no effect on EC treated with cytokine plus cycloheximide. Cathepsin B translocates from lysosomes to cytosol following treatment with LY294002 prior to the activation of caspases. These results suggest that inhibition of PI3K allows cytokines to activate a cathepsin-dependent, mitochondrial death pathway in which caspase activation is secondary, is not inhibited by c-FLIP, and is not essential for cell death.     

TRAIL-induced apoptosis in gliomas is enhanced by Akt-inhibition and is independent of JNK activation

Patients with malignant gliomas have a poor prognosis and new treatment paradigms are needed against this disease. TRAIL/Apo2L selectively induces apoptosis in malignant cells sparing normal cells and is hence of interest as a potential therapeutic agent against gliomas. To determine the factors that modulate sensitivity to TRAIL, we examined the differences in TRAIL-activated signaling pathways in glioma cells with variable sensitivities to the agent. Apoptosis in response to TRAIL was unrelated to DR5 expression or endogenous p53 status in a panel of 8 glioma cell lines. TRAIL activated the extrinsic (cleavage of caspase-8, caspase-3 and PARP) and mitochondrial apoptotic pathways and reduced FLIP levels. It also induced caspase-dependent JNK activation, which did not influence TRAIL-induced apoptosis. Because the pro-survival PI3K/Akt pathway is highly relevant to gliomas, we assessed whether Akt could protect against TRAIL-induced apoptosis. Pretreatment with SH-6, a novel Akt inhibitor, enhanced TRAIL-induced apoptosis, suggesting a protective role for Akt. Conversely, TRAIL induced caspase-dependent cleavage of Akt neutralizing its anti-apoptotic effects. These results demonstrate that TRAIL-induced apoptosis in gliomas involves both activation of death pathways and downregulation of survival pathways. Additional studies are warranted to determine the therapeutic potential of TRAIL against gliomas.Supported in part by the NIH grant PO1 CA55261     

Glucose—a sweet way to die

TRAIL, a putative anticancer cytokine, induces extrinsic cell death by activating the caspase cascade directly (Type I cells) via the death-inducing signaling complex (DISC) or indirectly (Type II cells) by caspase-8 cleavage of Bid and activation of the mitochondrial cell death pathway. Cancer cells are characterized by their dependence on aerobic glycolysis, which, although inefficient in terms of ATP production, facilitates tumor metabolism. Our studies show that TRAIL-induced cell death is significantly affected by the metabolic status of the cell. Inhibiting glycolysis with 2-deoxyglucose potentiates TRAIL-induced cell death, whereas glucose deprivation can paradoxically inhibit apoptosis. These conflicting responses to glycolysis inhibition are modulated by the balance between the Akt and AMPK pathways and their subsequent downstream regulation of mTORC1. This results in marked changes in protein translation, in which the equilibrium between anti- and pro-apoptotic Bcl-2 family member proteins is decided by their individual degradation rates. This regulates the mitochondrial cell death pathway and alters its sensitivity not only to TRAIL, but to ABT-737, a Bcl-2 inhibitor. Taken together, our studies show that the sensitivity of cancer cells to apoptosis can be modulated by targeting their unique metabolism in order to enhance sensitivity to apoptotic agents.     

Glucose—a sweet way to die: Metabolic switching modulates tumor cell death

TRAIL, a putative anticancer cytokine, induces extrinsic cell death by activating the caspase cascade directly (Type I cells) via the death-inducing signaling complex (DISC) or indirectly (Type II cells) by caspase-8 cleavage of Bid and activation of the mitochondrial cell death pathway. Cancer cells are characterized by their dependence on aerobic glycolysis, which, although inefficient in terms of ATP production, facilitates tumor metabolism. Our studies show that TRAIL-induced cell death is significantly affected by the metabolic status of the cell. Inhibiting glycolysis with 2-deoxyglucose potentiates TRAIL-induced cell death, whereas glucose deprivation can paradoxically inhibit apoptosis. These conflicting responses to glycolysis inhibition are modulated by the balance between the Akt and AMPK pathways and their subsequent downstream regulation of mTORC1. This results in marked changes in protein translation, in which the equilibrium between anti- and pro-apoptotic Bcl-2 family member proteins is decided by their individual degradation rates. This regulates the mitochondrial cell death pathway and alters its sensitivity not only to TRAIL, but to ABT-737, a Bcl-2 inhibitor. Taken together, our studies show that the sensitivity of cancer cells to apoptosis can be modulated by targeting their unique metabolism in order to enhance sensitivity to apoptotic agents.     

Tumor-cell resistance to death receptor--induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax

The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor--mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive. Bax was dispensable for apical death-receptor signaling events including caspase-8 activation, but crucial for mitochondrial changes and downstream caspase activation. Treatment of colon tumor cells deficient in DNA mismatch repair with the death-receptor ligand apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selected in vitro or in vivo for refractory subclones with Bax frameshift mutations including deletions at a novel site. Chemotherapeutic agents upregulated expression of the Apo2L/TRAIL receptor DR5 and the Bax homolog Bak in Baxminus sign/minus sign cells, and restored Apo2L/TRAIL sensitivity in vitro and in vivo. Thus, Bax mutation in mismatch repair--deficient tumors can cause resistance to death receptor--targeted therapy, but pre-exposure to chemotherapy rescues tumor sensitivity.     

The TRAIL apoptotic pathway mediates proteasome inhibitor induced apoptosis in primary chronic lymphocytic leukemia cells

The proteasome inhibitors are a new class of antitumor agents. These inhibitors cause the accumulation of many proteins in the cell with the induction of apoptosis including TRAIL death receptors DR4 and DR5, but the role of the TRAIL apoptotic pathway in proteasome inhibitor cytotoxicity is unknown. Herein, we have demonstrated that the induction of apoptosis by the proteasome inhibitors, MG-132 and PS-341 (bortezomib, Velcade), in primary CLL cells and the Burkitt lymphoma cell line, BJAB, is associated with up-regulation of TRAIL and its death receptors, DR4 and DR5. In addition, FLICE-like inhibitory protein (c-FLIP) protein is decreased. MG-132 treatment increases binding of DR5 to the adaptor protein FADD, and causes caspase-8 activation and cleavage of pro-apoptotic BID. Moreover, DR4:Fc or blockage of DR4 and DR5 expression using RNA interference, which prevents TRAIL apoptotic signaling, blocks proteasome inhibitor induced apoptosis. MG-132 also increases apoptosis and DR5 expression in normal B-cells. However, when the proteasome inhibitors are combined with TRAIL or TRAIL receptor activating antibodies the amount of apoptosis is increased in CLL cells but not in normal B cells. Thus, activation of the TRAIL apoptotic pathway contributes to proteasome inhibitor induced apoptosis in CLL cells.     

Bile acids stimulate cFLIP phosphorylation enhancing TRAIL-mediated apoptosis

Bile acids induce hepatocyte injury by enhancing death receptor-mediated apoptosis. In this study, bile acid effects on TRAIL-mediated apoptosis were examined to gain insight into bile acid potentiation of death receptor signaling. TRAIL-induced apoptosis of HuH-7 cells, stably transfected with a bile acid transporter, was enhanced by bile acids. Caspase 8 and 10 activation, bid cleavage, cytosolic cytochrome c, and caspase 3 activation by TRAIL were all increased by the bile acid glycochenodeoxycholate (GCDCA). GCDCA (100 microm) did not alter expression of TRAIL-R1/DR4, TRAIL-R2/DR5, procaspase 8, cFLIP-L, cFLIP-s, Bax, Bcl-xL, or Bax. However, both caspase 8 and caspase 10 recruitment and processing within the TRAIL death-inducing signaling complex (DISC) were greater in GCDCA-treated cells whereas recruitment of cFLIP long and short was reduced. GCDCA stimulated phosphorylation of both cFLIP isoforms, which was associated with decreased binding to GST-FADD. The protein kinase C antagonist chelerythrine prevented bile acid-stimulated cFLIP-L and -s phosphorylation, restored cFLIP binding to GST-FADD, and attenuated bile acid potentiation of TRAIL-induced apoptosis. These results provide new insights into the mechanisms of bile acid cytotoxicity and the proapoptotic effects of cFLIP phosphorylation in TRAIL signaling.     

Radicicol, an inhibitor of Hsp90, enhances TRAIL-induced apoptosis in human epithelial ovarian carcinoma cells by promoting activation of apoptosis-related proteins

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various cancer cells. Hsp90 is known to be involved in cell survival and growth in tumor cells. Nevertheless, Hsp90 inhibitors exhibit a variable effect on the cytotoxicity of anticancer drugs. Furthermore, the combined effect of Hsp90 inhibitors on TRAIL-induced apoptosis in epithelial ovarian cancer cells has not been determined. To assess the ability of an inhibitor of Hsp90 inhibitor radicicol to promote apoptosis, we investigated the effect of radicicol on TRAIL-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. TRAIL induced a decrease in Bid, Bcl-2, Bcl-xL, and survivin protein levels, increase in Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-8, -9, and -3), cleavage of PARP-1 and an increase in the tumor suppressor p53 levels. Radicicol enhanced TRAIL-induced apoptosis-related protein activation, nuclear damage and cell death. These results suggest that radicicol may potentiate the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathway and the mitochondria-mediated apoptotic pathway, leading to caspase activation. Radicicol may confer a benefit in the TRAIL treatment of epithelial ovarian adenocarcinoma.     

Hepatitis C virus core protein modulates TRAIL-mediated apoptosis by enhancing Bid cleavage and activation of mitochondria apoptosis signaling pathway

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease, which leads to cirrhosis of liver and hepatocellular carcinoma. The HCV core protein, a viral nucleocapsid, has been shown to affect various intracellular events, including cell proliferation and apoptosis. However, the precise mechanisms of the effects are not fully understood. In this study, we show that HCV core protein sensitizes human hepatocellular carcinoma cell line, Huh7, conferred sensitivity to TRAIL-, but not Fas ligand-mediated apoptosis. Huh7 cells are resistant to TRAIL, despite the induction of caspase-8 after TRAIL engagement. However, HCV core protein induces TRAIL apoptosis signaling via sequential induction of caspase-8, Bid cleavage, activation of mitochondrial pathway, and effector caspase-3. HCV core protein also induces activation of caspase-9 after TRAIL engagement, and the induction of TRAIL sensitivity by HCV core protein could be reversed by caspase-9 inhibitor. Therefore, the HCV core protein-induced TRAIL-mediated apoptosis is dependent upon activation of caspase-8 downstream pathway to convey the death signal to mitochondria, leading to activation of mitochondrial signaling pathway and breaking the apoptosis resistance. These results combined indicate that the HCV core protein enhances TRAIL-, but not Fas ligand-mediated apoptotic cell death in Huh7 cells via a mechanism dependent on the activation of mitochondria apoptosis signaling pathway. These results suggest that HCV core protein may have a role in immune-mediated liver cell injury by modulation of TRAIL-induced apoptosis.     

Overexpression of BCL2 blocks TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells

The tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL or Apo2L) and its receptors are members of the tumor necrosis factor superfamily. TRAIL triggers apoptosis by binding to its two proapoptotic receptors DR4 and DR5, a process which is negatively regulated by binding of TRAIL to its two decoy receptors TRID and TRUNDD. Here, we show that TRAIL effectively induces apoptosis in H460 human non-small-cell lung carcinoma cells via cleavage of caspases 8, 9, 7, 3, and BID, release of cytochrome c from the mitochondria, and cleavage of poly (ADP-ribose) polymerase (PARP). However, overexpression of Bcl2 blocked TRAIL-induced apoptosis in H460 cells, which correlated with the Bcl2 protein levels. Importantly, the release of cytochrome c and cleavage of caspase 7 triggered by TRAIL were considerably blocked in Bcl2 overexpressing cells as compared to vector control cells. Moreover, inhibition of TRAIL-mediated cytochrome c release and caspase 7 activation by Bcl2 correlated with the inability of PARP to be cleaved and the inability of the Bcl2 transfectants to undergo apoptosis. Thus, these results suggest that Bcl2 can serve an anti-apoptotic function during TRAIL-dependent apoptosis by inhibiting the release of cytochrome c and activation of caspase 7, thereby blocking caspase 7-dependent cleavage of cellular substrates.     

Overexpression of cFLIPs inhibits oxaliplatin-mediated apoptosis through enhanced XIAP stability and Akt activation in human renal cancer cells

cFLIP inhibits caspase 8 recruitment and processing at the death-inducing signaling complex (DISC), which is known to inhibits apoptosis mediated by death receptors such as Fas and death receptor 5 (DR5) as well as apoptosis mediated by anticancer therapeutic drugs. We observed that oxaliplatin induced apoptosis, the activation of DEVDase activity, DNA fragmentation, and cleavage of PLC-gamma1 and degradation of XIAP protein in dose-dependent manners, which was prevented by pretreatment with z-VAD or NAC, suggesting that oxaliplatin-induced apoptosis was mediated by caspase- or reactive oxygen species (ROS)-dependent pathways. Furthermore, ectopic expression of cFLIPs potently attenuated oxaliplatin-induced apoptosis, whereas cFLIP(L) had less effect. Interestingly, we found that the protein level of XIAP was sustained in oxaliplatin-treated cFLIPs overexpressing cell, which was caused by the increased XIAP protein stability and that the phospho-Akt level was high compared to vector-transfected cell. The increased XIAP protein stability was lessened by PI3K inhibitor LY294002 treatment in cFLIPs overexpressing cells. Thus, our findings imply that the anti-apoptotic functions of cFLIPs may be attributed to inhibit oxaliplatin-induced apoptosis through the sustained XIAP protein level and Akt activation.     

Codium fragile F2 sensitize colorectal cancer cells to TRAIL-induced apoptosis via c-FLIP ubiquitination

This study demonstrates that combined treatment with subtoxic doses of Codium extracts (CE), a flavonoid found in many fruits and vegetables, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induces apoptosis in TRAIL-resistant colorectal cancer (CRC) cells. Effective induction of apoptosis by combined treatment with CE and TRAIL was not blocked by Bcl-xL overexpression, which is known to confer resistance to various chemotherapeutic agents. While TRAIL-mediated proteolytic processing of procaspase-3 was partially blocked in various CRC cells treated with TRAIL alone, co-treatment with CE efficiently recovered TRAIL-induced caspase activation. We observed that CE treatment of CRC cells did not change the expression of anti-apoptotic proteins and pro-apoptotic proteins, including death receptors (DR4 and DR5). However, CE treatment markedly reduced the protein level of the short form of the cellular FLICE-inhibitory protein (c-FLIPS), an inhibitor of caspase-8, via proteasome-mediated degradation. Collectively, these observations show that CE recovers TRAIL sensitivity in various CRC cells via down-regulation of c-FLIPS.     

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Induces Caspase-Dependent Interleukin-8 Expression and Apoptosis in Human Astroglioma Cells

Among the tumor necrosis factor (TNF) family of cytokines, FasL and TNF-related apoptosis-inducing ligand (TRAIL) are known to induce cell death via caspase activation. Recently, other biological functions of these death ligands have been postulated in vitro and in vivo. It was previously shown that Fas ligation induces chemokine expression in human glioma cells. In this study, we investigated whether the TRAIL-DR5 system transduces signals similar to those induced by other TNF family ligands and receptors. To address this issue, two human glioma cell lines, CRT-MG and U87-MG, were used, and an agonistic antibody against DR5 (TRA-8) and human recombinant TRAIL were used to ligate DR5. We demonstrate that DR5 ligation by either TRAIL or TRA-8 induces two functional outcomes, apoptosis and expression of the chemokine interleukin-8 (IL-8); the nonspecific caspase inhibitor Boc-D-Fmk blocks both TRAIL-mediated cell death and IL-8 production; the caspase 3-specific inhibitor z-DEVD-Fmk suppresses TRAIL-mediated apoptosis but not IL-8 induction; caspase 1- and 8-specific inhibitors block both TRAIL-mediated cell death and IL-8 production; and DR5 ligation by TRAIL mediates AP-1 and NF-kappaB activation, which can be inhibited by caspase 1- and 8-specific inhibitors. These findings collectively indicate that DR5 ligation on human glioma cells leads to apoptosis and that the activation of AP-1 and NF-kappaB leads to the induction of IL-8 expression; these responses are dependent on caspase activation. Therefore, the TRAIL-DR5 system has a role not only as an inducer of apoptotic cell death but also as a transducer for proinflammatory and angiogenic signals in human brain tumors.     

p21(WAF1/CIP1) inhibits initiator caspase cleavage by TRAIL death receptor DR4

Death receptors of the Tumor Necrosis Factor (TNF) family form membrane-bound self-activating signaling complexes that initiate apoptosis through cleavage of proximal caspases including CASP8 and 10. Here we show that overexpression of the cytoplasmic domain (CD) of the DR4 TRAIL receptor (TNFRSF10A, TRAIL R1) in human breast, lung, and colon cancer cell lines, using an adenovirus vector (Ad-DR4-CD), leads to p53-independent apoptotic cell death involving cleavage of CASP8 and 10 proximally and CASP3, 6, and 7 distally. DR4-CD overexpression also leads to cleavage of poly(ADP-ribose) polymerase (PARP) and the DNA fragmentation factor (DFF45; ICAD). Importantly, normal lung fibroblasts are resistant to DR4-CD overexpression and show no evidence of PARP-, CASP8- or CASP3-cleavage despite similar levels of adenovirus-delivered DR4-CD protein as the cancer cells. These results suggest that DR4 may signal death through known caspases and that further studies are required to evaluate Ad-DR4-CD as a novel anti-cancer agent. Finally, we show that overexpression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (CDKN1A), or its N-terminal 91 amino acids containing cell cycle-inhibitory activity, inhibits DR4-CD-dependent proximal caspase cleavage. The blockage of initiator caspase activation provides a novel insight into how p21 may suppress apoptosis and enhance cell survival.     

The combination of TRAIL and luteolin enhances apoptosis in human cervical cancer HeLa cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for cancer therapeutics. However, some tumor cells are resistant to TRAIL-induced apoptosis. Our previous studies have shown that luteolin, a naturally occurring flavonoid, induces the up-regulation of death receptor 5 (DR5), which is a receptor for TRAIL. Here, we show for the first time that luteolin synergistically acts with exogenous soluble recombinant human TRAIL to induce apoptosis in HeLa cells, but not in normal human peripheral blood mononuclear cells. The combined use of luteolin and TRAIL induced Bid cleavage and the activation of caspase-8. Also, human recombinant DR5/Fc chimera protein, caspase inhibitors, and DR5 siRNA efficiently reduced apoptosis induced by co-treatment with luteolin and TRAIL. These results raise the possibility that this combined treatment with luteolin and TRAIL might be promising as a new therapy against cancer.     

Synergistic induction of cell death in liver tumor cells by TRAIL and chemotherapeutic drugs via the BH3-only proteins Bim and Bid

Although death receptors and chemotherapeutic drugs activate distinct apoptosis signaling cascades, crosstalk between the extrinsic and intrinsic apoptosis pathway has been recognized as an important amplification mechanism. Best known in this regard is the amplification of the Fas (CD95) signal in hepatocytes via caspase 8-mediated cleavage of Bid and activation of the mitochondrial apoptosis pathway. Recent evidence, however, indicates that activation of other BH3-only proteins may also be critical for the crosstalk between death receptors and mitochondrial triggers. In this study, we show that TNF-related apoptosis-inducing ligand (TRAIL) and chemotherapeutic drugs synergistically induce apoptosis in various transformed and untransformed liver-derived cell lines, as well as in primary human hepatocytes. Both, preincubation with TRAIL as well as chemotherapeutic drugs could sensitize cells for apoptosis induction by the other respective trigger. TRAIL induced a strong and long lasting activation of Jun kinase, and activation of the BH3-only protein Bim. Consequently, synergistic induction of apoptosis by TRAIL and chemotherapeutic drugs was dependent on Jun kinase activity, and expression of Bim and Bid. These findings confirm a previously defined role of TRAIL and Bim in the regulation of hepatocyte apoptosis, and demonstrate that the TRAIL–Jun kinase–Bim axis is a major and important apoptosis amplification pathway in primary hepatocytes and liver tumor cells.