ZD1839 (Gefitinib, 'Iressa'), an epidermal growth factor receptor-tyrosine kinase inhibitor, enhances the anti-cancer effects of TRAIL in human esophageal squamous cell carcinoma

The EGF (epidermal growth factor) receptor-tyrosine kinase inhibitor ZD1839 (Gefitinib, 'Iressa') blocks the cell signaling pathways involved in cell proliferation, survival, and angiogenesis in various cancer cells. TNF-related death apoptosis inducing ligand (TRAIL) acts as an anticancer agent. We investigated the antitumor effects of ZD1839 alone or in combination with TRAIL against human esophageal squamous cell cancer (ESCC) lines. Although all ESCC cells expressed EGF receptor at a protein level, the effect of ZD1839 on cell growth did not correlate with the level of EGFR expression and phosphorylation of EGF receptor protein in ESCC lines. ZD1839 caused a dose-dependent growth arrest at G0-G1 phase associated with increased p27 expression. As TE8 cells are resistant to TRAIL, we tested whether ZD1839 combined with TRAIL induced apoptosis of TE8 cells via the inhibition of EGF receptor signaling by ZD1839. ZD1839 inhibited the phosphorylation of Akt, and enhanced TRAIL-induced apoptosis via activation of caspase-3 and caspase-9, and inactivation of Bcl-xL. Our results indicated that ZD1839 has anti-cancer properties against human esophageal cancer cells. ZD1839 also augmented the anti-cancer activity of TRAIL, even in TRAIL-resistant tumors. These results suggest that treatment with ZD1839 and TRAIL may have potential in the treatment of ESCC patients.     

Pro-survival function of Akt/protein kinase B in prostate cancer cells. Relationship with TRAIL resistance.

Tumor necrosis factor superfamily member TRAIL/Apo-2L has recently been shown to induce apoptosis in transformed and cancer cells. Some prostate cancer cells express constitutively active Akt/protein kinase B due to a complete loss of lipid phosphatase PTEN gene, a negative regulator of phosphatidylinositol 3-kinase pathway. Constitutively active Akt promotes cellular survival and resistance to chemotherapy and radiation. We have recently noticed that some human prostate cancer cells are resistant to TRAIL. We therefore examined the intracellular mechanisms of cellular resistance to TRAIL. The cell lines expressing the highest level of constitutively active Akt were more resistant to undergo apoptosis by TRAIL than those expressing the lowest level. Down-regulation of constitutively active Akt by phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, reversed cellular resistance to TRAIL. Treatment of resistant cells with cycloheximide (a protein synthesis inhibitor) rendered cells sensitive to TRAIL. Transfecting dominant negative Akt decreased Akt activity and increased TRAIL-induced apoptosis in cells with high Akt activity. Conversely, transfecting constitutively active Akt into cells with low Akt activity increased Akt activity and attenuated TRAIL-induced apoptosis. Inhibition of TRAIL sensitivity occurs at the level of BID cleavage, as caspase-8 activity was not affected. Enforced expression of anti-apoptotic protein Bcl-2 or Bcl-X(L) inhibited TRAIL-induced mitochondrial dysfunction and apoptosis. We therefore identify Akt as a constitutively active kinase that promotes survival of prostate cancer cells and demonstrate that modulation of Akt activity, by pharmacological or genetic approaches, alters the cellular responsiveness to TRAIL. Thus, TRAIL in combination with agents that down-regulate Akt activity can be used to treat prostate cancer.     

Lithium enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

Non-small cell lung cancer (NSCLC) A549 cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Therefore, combination therapy using sensitizing agents to overcome TRAIL resistance may provide new strategies for treatment of NSCLC. Here, we investigated whether lithium chloride (LiCl), a drug for mental illness, could sensitize A549 cells to TRAIL-induced apoptosis. We observed that LiCl significantly enhanced A549 cells apoptosis through up-regulation of death receptors DR4 and DR5 and activation of caspase cascades. In addition, G2/M arrest induced by LiCl also contributed to TRAIL-induced apoptosis. Concomitantly, LiCl strongly inhibited the activity of c-Jun N-terminal kinases (JNKs), and the inhibition of JNKs by SP600125 also induced G2/M arrest and augmented cell death caused by TRAIL or TRAIL plus LiCl. However, glycogen synthase kinase-3β (GSK3β) inhibition was not involved in TRAIL sensitization induced by LiCl. Collectively, these findings indicated that LiCl sensitized A549 cells to TRAIL-induced apoptosis through caspases-dependent apoptotic pathway via death receptors signaling and G2/M arrest induced by inhibition of JNK activation, but independent of GSK3β.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rheumatoid arthritis synovial fibroblast proliferation through mitogen-activated protein kinases and phosphatidylinositol 3-kinase/Akt

A hallmark of rheumatoid arthritis (RA) is the pseudo-tumoral expansion of fibroblast-like synoviocytes (FLSs), and the RA FLS has therefore been proposed as a therapeutic target. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been described as a pro-apoptotic factor on RA FLSs and, therefore, suggested as a potential drug. Here we report that exposure to TRAIL-induced apoptosis in a portion (up to 30%) of RA FLSs within the first 24 h. In the cells that survived, TRAIL induced RA FLS proliferation in a dose-dependent manner, with maximal proliferation observed at 0.25 nm. This was blocked by a neutralizing anti-TRAIL antibody. RA FLSs were found to express constitutively TRAIL receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) on the cell surface. TRAIL-R2 appears to be the main mediator of TRAIL-induced stimulation, as RA FLS proliferation induced by an agonistic anti-TRAIL-R2 antibody was comparable with that induced by TRAIL. TRAIL activated the mitogen-activated protein kinases ERK and p38, as well as the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway with kinetics similar to those of TNF-alpha. Moreover, TRAIL-induced RA FLS proliferation was inhibited by the protein kinase inhibitors PD98059, SB203580, and LY294002, confirming the involvement of the ERK, p38, and PI3 kinase/Akt signaling pathways. This dual functionality of TRAIL in stimulating apoptosis and proliferation has important implications for its use in the treatment of RA.     

Hypoxia inhibition of apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Hypoxia is a common environmental stress. Particularly, the center of rapidly growing solid tumors is easily exposed to hypoxic conditions. Thus, tumor cell response to hypoxia plays an important role in tumor progression as well as tumor therapy. However, little is known about hypoxic effect on apoptotic cell death. To examine the effects of hypoxia on TRAIL-induced apoptosis, human lung carcinoma A549 cells were exposed to hypoxia and treated with TRAIL protein. Hypoxia significantly protected A549 cells from apoptosis induced by TRAIL. Western blotting analysis demonstrated that hypoxia increased expression of antiapoptotic proteins such as Bcl-2, Bcl-XL, and IAP family members. The increase of these antiapoptotic molecules is believed to play an hypoxia-mediated protective role in TRAIL-induced apoptosis. Our findings suggest that an increase of antiapoptotic proteins induced by hypoxia may regulate the therapeutic activity of TRAIL protein in cancer therapy.     

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     

Amiloride potentiates TRAIL-induced tumor cell apoptosis by intracellular acidification-dependent Akt inactivation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor gene family, is considered as one of the most promising cancer therapeutic agents due to its ability to selectively induce tumor cell apoptosis. In this study, we investigated whether the Na(+)/H(+) exchanger inhibitor, amiloride, promotes TRAIL-induced apoptotic death both in sensitive and resistant tumor cells, HeLa and LNCaP cells, respectively, and its underlying molecular mechanism. Amiloride enhanced TRAIL-induced apoptosis and activation of caspase-3 and -8 in both cells. This compound increased TRAIL-induced mitochondrial cytochrome c release and poly(ADP-ribose) polymerase cleavage. Moreover, amiloride-induced intracellular acidification, and inhibited the phosphorylated activation of the serine/threonine kinase Akt, which is known to promote cell survival, in both tumor cells. These data suggest that amiloride sensitizes both tumor cells to TRAIL-induced apoptosis by promoting Akt dephosphorylation and caspase-8 activation via the intracellular acidification and that Na(+)/H(+) exchanger inhibitors may play an important role in the anti-cancer activity of TRAIL, especially, in TRAIL-resistant tumors with highly active and expressed Akt.     

Reconstitution of galectin-3 alters glutathione content and potentiates TRAIL-induced cytotoxicity by dephosphorylation of Akt

We investigated the role of galectin-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic death in human breast carcinoma BT549 cells. We observed that parental galectin-3 null BT549 cells (BT549(par)) as well as control vector transfected (BT549(neo)) cells were resistant to TRAIL, while galectin-3 cDNA-transfected BT549 cells (BT549(gal-3)) were sensitive to TRAIL. Data from flow cytometry and immunoblotting analyses reveal that reconstitution of galectin-3 promoted cell death and PARP cleavage as well as caspase (-8, -9, and -3) activation during TRAIL treatment. However, unlike TRAIL treatment, galectin-3 transfectants were resistant to UV-B-induced PARP cleavage. Data from cDNA array analysis show that galectin-3 did not significantly enhance or reduce any apoptosis-related gene expression. Moreover, although galectin-3 restored pre-mRNA splicing activity and resulted in elevation of FLIPs protein, experiments with FLIPs cDNA-transfected cells show that overexpression of FLIPs did not sensitize cells to TRAIL. Interestingly, BT549(gal-3) cells demonstrated a approximately 2-fold increase in total glutathione content as well as a approximately 5-fold increase in GSSG content in comparison to BT549(par) and BT549(neo) cells, suggesting that galectin-3 overexpression may alter intraceullular oxidation/reduction reactions affecting the metabolism of glutathione and other thiols. In addition, galectin-3 overexpression inactivated Akt by dephosphorylation. Finally, overexpression of constitutively activated Akt protected BT549(gal-3) cells from TRAIL-induced cytotoxicity. Taken together, our data suggest that galectin-3-enhanced TRAIL-induced cytotoxicity is mediated through dephosphorylation of Akt, possibly through a redox-dependent process.     

Genistein enhances TRAIL-induced apoptosis through inhibition of p38 MAPK signaling in human hepatocellular carcinoma Hep3B cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some carcinoma cancer cells. However, it was found that treatment with TRAIL in combination with nontoxic concentrations of genistein sensitized TRAIL-resistant human hepatocellular carcinoma Hep3B cells to TRAIL-mediated apoptosis. Combined treatment with genistein and TRAIL-induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis were correlated with the induction of caspase activity that resulted in the cleavage of poly(ADP-ribose) polymerase (PARP). Both cell viability and the cleavage of PARP induced by combined treatment were significantly inhibited by caspase-3, -8 and -9 inhibitors, which demonstrates the important roles of caspases in the observed cytotoxic effects. Genistein treatment also triggered the inhibition of p38-β mitogen-activated protein kinase (MAPK) activation. Pretreatment with SB203580 resulted in significantly increased sub-G1 population and loss of mitochondrial membrane potential (MMP) in TRAIL-induced apoptosis. By contrast, overexpression of p38 MAPK protected apoptosis by co-treatment with genistein and TRAIL, suggesting that the p38 MAPK act as key regulators of apoptosis in response to treatment with a combination of genistein and TRAIL in human hepatocellular carcinoma Hep3B cells.     

IFN-gamma inhibition of TRAIL-induced IAP-2 upregulation, a possible mechanism of IFN-gamma-enhanced TRAIL-induced apoptosis

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

IFN-gamma enhances TRAIL-induced apoptosis through IRF-1.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family and a potent inducer of apoptosis. TRAIL has been shown to effectively limit tumor growth in vivo without detectable cytotoxic side-effects. Interferon (IFN)-gamma often modulates the anticancer activities of TNF family members including TRAIL. However, little is known about the mechanism. To explore the mechanism, A549, HeLa, LNCaP, Hep3B and HepG2 cells were pretreated with IFN-gamma, and then exposed to TRAIL. IFN-gamma pretreatment augmented TRAIL-induced apoptosis in all these cell lines. A549 cells were selected and further characterized for IFN-gamma action in TRAIL-induced apoptosis. Western blotting analyses revealed that IFN-gamma dramatically increased the protein levels of interferon regulatory factor (IRF)-1, but not TRAIL receptors (DR4 and DR5) and pro-apoptotic (FADD and Bax) and anti-apoptotic factors (Bcl-2, Bcl-XL, cIAP-1, cIAP-2 and XIAP). To elucidate the functional role of IRF-1 in IFN-gamma-enhanced TRAIL-induced apoptosis, IRF-1 was first overexpressed by using an adenoviral vector AdIRF-1. IRF-1 overexpression minimally increased apoptotic cell death, but significantly enhanced apoptotic cell death induced by TRAIL when infected cells were treated with TRAIL. In further experiments using an antisense oligonucleotide, a specific repression of IRF-1 expression abolished enhancer activity of IFN-gamma for TRAIL-induced apoptosis. Therefore, our data indicate that IFN-gamma enhances TRAIL-induced apoptosis through IRF-1.     

Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression.     

Elevated AKT activity protects the prostate cancer cell line LNCaP from TRAIL-induced apoptosis

We find that the prostate cancer cell lines ALVA-31, PC-3, and DU 145 are highly sensitive to apoptosis induced by TRAIL (tumor-necrosis factor-related apoptosis-inducing ligand), while the cell lines TSU-Pr1 and JCA-1 are moderately sensitive, and the LNCaP cell line is resistant. LNCaP cells lack active lipid phosphatase PTEN, a negative regulator of the phosphatidylinositol (PI) 3-kinase/Akt pathway, and demonstrate a high constitutive Akt activity. Inhibition of PI 3-kinase using wortmannin and LY-294002 suppressed constitutive Akt activity and sensitized LNCaP cells to TRAIL. Treatment of LNCaP cells with TRAIL alone induced cleavage of the caspase 8 and XIAP proteins. However, processing of BID, mitochondrial release of cytochrome c, activation of caspases 7 and 9, and apoptosis did not occur unless TRAIL was combined with either wortmannin, LY-294002, or cycloheximide. Blocking cytochrome c release by Bcl-2 overexpression rendered LNCaP cells resistant to TRAIL plus wortmannin treatment but did not affect caspase 8 or BID processing. This indicates that in these cells mitochondria are required for the propagation rather than the initiation of the apoptotic cascade. Infection of LNCaP cells with an adenovirus expressing a constitutively active Akt reversed the ability of wortmannin to potentiate TRAIL-induced BID cleavage. Thus, the PI 3-kinase-dependent blockage of TRAIL-induced apoptosis in LNCaP cells appears to be mediated by Akt through the inhibition of BID cleavage.     

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.     

TRAIL apoptosis is enhanced by quercetin through Akt dephosphorylation

TNF-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy that preferentially induces apoptosis in cancer cells. However, many neoplasms are resistant to TRAIL by mechanisms that are poorly understood. Here we demonstrated that human prostate cancer cells, but not normal prostate cells, are dramatically sensitized to TRAIL-induced apoptosis and caspase activation by quercetin. Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells. We have shown that quercetin can potentiate TRAIL-induced apoptotic death. Human prostate adenocarcinoma DU-145 and LNCaP cells were treated with various concentrations of TRAIL (10-200 ng/ml) and/or quercetin (10-200 microM) for 4 h. Quercetin, which caused no cytotoxicity by itself, promoted TRAIL-induced apoptosis. The TRAIL-mediated activation of caspase, and PARP (poly(ADP-ribose) polymerase) cleavage were both enhanced by quercetin. Western blot analysis showed that combined treatment with TRAIL and quercetin did not change the levels of TRAIL receptors (death receptors DR4 and DR5, and DcR2 (decoy receptor 2)) or anti-apoptotic proteins (FLICE-inhibitory protein (FLIP), inhibitor of apoptosis (IAP), and Bcl-2). However, quercetin promoted the dephosphorylation of Akt. Quercetin-induced potent inhibition of Akt phosphorylation. Taken together, the present studies suggest that quercetin enhances TRAIL-induced cytotoxicity by activating caspases and inhibiting phosphorylation of Akt.     

Acrolein sensitizes human renal cancer Caki cells to TRAIL-induced apoptosis via ROS-mediated up-regulation of death receptor-5 (DR5) and down-regulation of Bcl-2

TRAIL resistance in many cancer cells is one of the major problems in TRAIL-based cancer therapy. Thus, the agents that can sensitize the tumor cells to TRAIL-mediated apoptosis are strictly needed for the improvement of anti-cancer effect of TRAIL. Acrolein is a byproduct of lipid peroxidation, which has been involved in pulmonary, cardiac and neurodegenerative diseases. We investigated whether acrolein, an α,β-unsaturated aldehyde, can potentiate TRAIL-induced apoptosis in human renal cancer cells. The combined treatment with acrolein and TRAIL significantly induced apoptosis, and stimulated of caspase-3 activity, DNA fragmentation, and cleavage of PARP. We found that acrolein down-regulated the protein level of Bcl-2 and Bcl-2 overexpression inhibited the cell death induced by the combined treatment with acrolein and TRAIL. In addition, acrolein up-regulated C/EBP homologous protein (CHOP) and TRAIL death receptor 5 (DR5) and down-regulation of CHOP or DR5 expression using the respective small interfering RNA significantly attenuated the apoptosis induced by acrolein plus TRAIL. Interestingly, pretreatment with an antioxidant, N-acetylcysteine (NAC), inhibited not only CHOP and DR5 up-regulation but also the cell death induced by acrolein plus TRAIL. Taken together, our results demonstrated that acrolein enhances TRAIL-induced apoptosis in Caki cells through down-regulation of Bcl-2 and ROS dependent up-regulation of DR5.     

Aspirin enhances TRAIL-induced apoptosis via regulation of ERK1/2 activation in human cervical cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers tumor-specific apoptosis. However, some tumors and cancer cell lines are resistant to TRAIL. Here, the effect of the non-steroidal anti-inflammatory drug aspirin on sensitization of human cervical cancer cells to TRAIL and the underlying mechanism(s) of the effect were explored. Combination treatment with aspirin and TRAIL markedly enhanced apoptotic cell death, as assessed by lactate dehydrogenase (LDH) assay and analysis of cell cycle sub-G1 phase. The two agents together activated the several caspases and mitochondrial signaling pathway. Whereas Mcl-1 protein level was increased and extracellular signal-related kinase (ERK)1/2 was activated in cells treated with TRAIL alone, combination treatment dramatically inhibited ERK1/2 activation and down-regulated Mcl-1 protein level. An inhibitor of ERK1/2 activation, PD98059, also augmented TRAIL-induced apoptosis. Combination treatment with PD98059 and TRAIL showed the activation of caspases and mitochondrial pathway, and the down-regulation of Mcl-1 level. These results suggest that cancer cells can be sensitized to TRAIL-induced apoptosis by pre-treatment with aspirin via suppression of ERK1/2 activation. These findings provide a basis for further exploring the potential applications of this combination approach for the treatment of cancer, including cervical cancer.     

Curcumin enhances TRAIL-induced apoptosis of breast cancer cells by regulating apoptosis-related proteins

The TNF-related apoptosis inducing ligand (TRAIL) has promising anti-cancer therapeutic activity, although significant percentage of primary tumors resistant to TRAIL-induced apoptosis remains an obstacle to the extensive use of TRAIL-based mono-therapies. Natural compound curcumin could potentially sensitize resistant cancer cells to TRAIL. We found that the combination of TRAIL with curcumin can synergistically induces apoptosis in three TRAIL-resistant breast cancer cell lines. The mechanism behind this synergistic cell death was investigated by examining an effect of curcumin on the expression and activation of TRAIL-associated cell death proteins. Immunoblotting, RNA interference, and use of chemical inhibitors of TRAIL-activate signaling revealed differential effects of curcumin on the expression of Mcl-1 and activities of ERK and Akt. Curcumin-induced production of reactive oxygen species did not affect total expression of DR5 but it enhanced mobilization of DR5 to the plasma membrane. In these breast cancer cells curcumin also induced downregulation of IAP proteins. Taken together, our data suggest that a combination of TRAIL and curcumin is a potentially promising treatment for breast cancer, although the specific mechanisms involved in this sensitization could differ even among breast cancer cells of different origins.     

cFLIPL prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis

Sensitivity to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and the lysosomal pathway of cell death are features of cancer cells. However, it is unknown if TRAIL cytotoxic signaling engages the lysosomal pathway of cell death. Our aim, therefore, was to ascertain if TRAIL killing involves lysosomal permeabilization. TRAIL-induced apoptosis of hepatocellular carcinoma cells (HuH-7, Hep3B) was associated with lysosomal permeabilization, as demonstrated by redistribution of the lysosomal protease cathepsin B into the cytosol. Pharmacological and short hairpin RNA-targeted inhibition of cathepsin B reduced apoptosis. Because cellular FLICE-inhibitory protein (cFLIP) inhibits TRAIL-induced cell death and is frequently overexpressed by human cancers, the ability of cFLIP to prevent lysosomal permeabilization during TRAIL treatment was examined. Enforced long-form cFLIP (cFLIP(L)) expression reduced release of cathepsin B from lysosomes and attenuated apoptosis. cFLIP(L) overexpression was also associated with robust p42/44 MAPK activation following exposure to TRAIL. In contrast, cFLIP(L) overexpression attenuated p38 MAPK activation and had no significant effect on JNK and NF-kappaB activation. Inhibition of p42/44 MAPK by PD98059 restored TRAIL-mediated lysosomal permeabilization and apoptosis in cFLIP-overexpressing cells. In conclusion, these results demonstrate that lysosomal permeabilization contributes to TRAIL-induced apoptosis of hepatocellular carcinoma cells and suggest that cFLIP(L) cytoprotection is, in part, due to p42/44 MAPK-dependent inhibition of lysosomal breakdown.