Cetuximab enhances TRAIL-induced gastric cancer cell apoptosis by promoting DISC formation in lipid rafts

TRAIL is a member of the tumor necrosis factor family that selectively induces cancer cell apoptosis. However, gastric cancer cells are insensitive to TRAIL. Our and others studies showed that the inhibition of EGFR pathway activation could increase the sensitivity of TRAIL in cancer cells. But the detailed mechanism is not fully understood. In the present study, compared with TRAIL or cetuximab (an anti-EGFR monoclonal antibody) alone, treatment with the TRAIL/cetuximab combination significantly promoted death receptor 4 (DR4) clustering as well as the translocation of both DR4 and Fas-associated death domain-containing protein (FADD) into lipid rafts. This in turn resulted in caspase-8 cleavage and the formation of the death-inducing signaling complex (DISC) in these lipid rafts. Cholesterol-depletion with methyl-β-cyclodextrin partially prevented DR4 clustering and DISC formation, and thus partially reversed apoptosis induced by the TRAIL/cetuximab dual treatment. These results indicate that cetuximab increases TRAIL-induced gastric cancer cell apoptosis at least partially through the promotion of DISC formation in lipid rafts.     

Targeting AML through DR4 with a novel variant of rhTRAIL

Despite progress in the treatment of acute myelogenous leukaemia (AML) the outcome often remains poor. Tumour necrosis factor related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent in many different types of tumours, but AML cells are relatively insensitive to TRAIL-induced apoptosis. Here we show that TRAIL-induced apoptosis in AML cells is predominantly mediated by death receptor 4 (DR4) and not DR5. Therefore, we constructed a variant of TRAIL (rhTRAIL-C3) that is a strong inducer of DR4-mediated apoptosis. TRAIL-C3 demonstrated much stronger pro-apoptotic activity than wild-type (WT) TRAIL in a panel of AML cell lines as well as in primary AML blasts. The higher pro-apoptotic potential was further enhanced when the TRAIL mutant was used in combination with BMS-345541, a selective inhibitor of inhibitor-κB kinases. It illustrates that combination of this TRAIL variant with chemotherapeutics or other targeted agents can kill AML with high efficacy. This may represent a major advantage over the currently used therapies that have serious toxic side effects. The high efficacy of rhTRAIL-C3 containing therapies may enable the use of lower drug doses to reduce the toxic side effects and improve patient outcome. Our findings suggest that the rational design of TRAIL variants that target DR4 potentiate the death-inducing activity of TRAIL and offer a novel therapeutic strategy for the treatment of AML.     

Differential inhibition of TRAIL-mediated DR5-DISC formation by decoy receptors 1 and 2

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces cancer cell death by apoptosis with some selectivity. TRAIL-induced apoptosis is mediated by the transmembrane receptors death receptor 4 (DR4) (also known as TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind decoy receptor 1 (DcR1) (TRAIL-R3) and DcR2 (TRAIL-R4) that fail to induce apoptosis since they lack and have a truncated cytoplasmic death domain, respectively. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and we demonstrate here that this occurs through distinct mechanisms. While DcR1 prevents the assembly of the death-inducing signaling complex (DISC) by titrating TRAIL within lipid rafts, DcR2 is corecruited with DR5 within the DISC, where it inhibits initiator caspase activation. In addition, DcR2 prevents DR4 recruitment within the DR5 DISC. The specificity of DcR1- and DcR2-mediated TRAIL inhibition reveals an additional level of complexity for the regulation of TRAIL signaling.     

Apple procyanidins activate apoptotic signaling pathway in human colon adenocarcinoma cells by a lipid-raft independent mechanism

Flavonoids are polyphenolic compounds able to favour cholesterol-lipid-raft formation and control cell signaling pathways by targeting receptors at the cell surface. Procyanidins (Pcy) are oligomeric and polymeric flavonoids formed by catechins and epicatechins monomers trigger apoptosis by activating TRAIL-death receptors in human colon adenocarcinoma SW480 cells. Here, we investigated whether the apoptotic process triggered by apple procyanidins involving the up-regulation of TRAIL-death receptors DR4/DR5 at the cell surface was dependent on cell membrane lipid-raft formation. We report that Pcy-induced apoptosis was enhanced in presence of nystatin, a cholesterol-sequestering compound inhibiting lipid-raft formation, without changing DR4/DR5 receptor expression. Treatment of SW480 cells with TRAIL caused a 3.5-fold increased level of caveolin together with a 2- to 2.5-fold increased amount of DR4/DR5 proteins in lipid rafts. Pcy-treatment did not induce any alteration in the expression of DR4/DR5 proteins as well as of caveolin present in lipid-raft fractions. Pcy induced an activation of TRAIL-death receptor-mediated apoptosis by a mechanism independent of lipid-raft formation. These results highlight the potential of Pcy as a direct activator of TRAIL-death receptors in cell membrane even in the absence of lipid rafts.     

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.     

Caveolin-1 negatively regulates TRAIL-induced apoptosis in human hepatocarcinoma cells

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) offers promising therapeutic potential based on its ability to induce apoptosis in various cancer cell lines without obvious adverse effect to normal cells. However, the mechanism of the differential sensitivity towards TRAIL-induced apoptosis remains unclear. Here, we demonstrate that caveolin-1 directly regulated TRAIL-induced apoptosis in HepG2 cells. ShRNA-mediated caveolin knockdown sensitized TRAIL-induced apoptosis and disruption of caveolae structure by the cholesterol-extracting reagent, methyl-β-cyclodextrin (MCD), enhanced TRAIL-induced apoptosis. Over-expression of caveolin-1 partially blocked TRAIL-induced apoptosis. The engagement of TRAIL with its receptor DR4 reduced the localization of DR4 in caveolae and resulted in its internalization. Blockade of caveolae-mediated internalization of DR4 by filipin III effectively enhanced TRAIL-induced apoptosis. Collectively, our results reveal a new mechanism by which caveolin-1 negatively regulates TRAIL-induced apoptosis in human hepatocarcinoma cells.     

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.     

Sub-toxic dose of apigenin sensitizes HepG2 cells to TRAIL through ERK-dependent up-regulation of TRAIL receptor DR5

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a promising candidate for anticancer therapy due to its selective toxicity to cancer cells. Nevertheless, because of TRAIL resistance in some cancer cells, combined treatment with sensitizing agents is required to enhance the anticancer potential of TRAIL. In this study, we investigated the underlying mechanism of apigenin-induced sensitization of HepG2 cells to TRAIL-induced cell death. Synergistic induction of apoptosis by combination was confirmed by examining the typical morphology changes of apoptosis, PARP-cleavage, and activation of effector caspases. Z-VAD-fmk, a pan-caspase inhibitor, inhibited the enhanced cell death by combined treatment of apigenin and TRAIL, demonstrating that a caspase-dependent pathway is involved in apigenin/TRAIL-mediated apoptosis. In addition, we found that apigenin/ TRAIL co-treatment up-regulates DR5 cell surface expression. The synergistic induction of cell death by the apigenin/ TRAIL combination was significantly attenuated by DR5 blocking chimera antibody. Next, using pharmacological inhibitors, we found that ERK activation is involved in the induction of DR5 expression. Inhibition of ERK1/2 by U0126 significantly decreased the apigenin/TRAIL-induced DR5 expression and apoptosis. Taken together, our results indicate that apigenin can enhance the apoptotic effect of TRAIL via ERK-induced up-regulation of DR5.     

Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53?/?) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.     

Phenethyl isothiocyanate sensitizes glioma cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a promising antitumor therapy. However, many cancer cells, including malignant glioma cells, tend to be resistant to TRAIL, highlighting the need for strategies to overcome TRAIL resistance. Here we show that in combination with phenethyl isothiocyanate (PEITC), exposure to TRAIL induced apoptosis in TRAIL-resistant glioma cells. Subtoxic concentrations of PEITC significantly potentiated TRAIL-induced cytotoxicity and apoptosis in glioma cells. PEITC dramatically upregulated DR5 receptor expression but had no effects on DR4 receptor. PEITC enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-induced-p53.     

Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors: Evidence for the role of c-Jun N-terminal kinase activation

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53−/−) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.     

Lipid raft-regulated IGF-1R activation antagonizes TRAIL-induced apoptosis in gastric cancer cells

Gastric cancer cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the resistance mechanism is not fully understood. In human gastric cancer MGC803 and BGC823 cells, TRAIL induces insulin-like growth factor-1 receptor (IGF-1R) pathway activation. Treatment with IGF-1R inhibitor OSI-906 or small interfering RNAs against IGF-1R, prevents IGF-1R pathway activation and increases TRAIL-induced apoptosis. The TRAIL-induced IGF-1R pathway activation is promoted by IGF-1R translocation into lipid rafts. Moreover, the translocation of IGF-1R into lipid rafts is regulated by Casitas B-lineage lymphoma b (Cbl-b). Taken together, TRAIL-induced IGF-1R activation antagonizes TRAIL-induced apoptosis by Cbl-b-regulated distribution of IGF-1R in lipid rafts.     

Constitutive localization of DR4 in lipid rafts is mandatory for TRAIL-induced apoptosis in B-cell hematologic malignancies

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) acts as an apoptosis inducer for cancer cells sparing non-tumor cell targets. However, several phase I/II clinical trials have shown limited benefits of this molecule. In the present work, we investigated whether cell susceptibility to TRAIL ligation could be due to the presence of TRAIL death receptors (DRs) 4 and 5 in membrane microdomains called lipid rafts. We performed a series of analyses, either by biochemical methods or fluorescence resonance energy transfer (FRET) technique, on normal cells (i.e. lymphocytes, fibroblasts, endothelial cells), on a panel of human cancer B-cell lines as well as on CD19⁺ lymphocytes from patients with B-chronic lymphocytic leukemia, treated with different TRAIL ligands, that is, recombinant soluble TRAIL, specific agonistic antibodies to DR4 and DR5, or CD34⁺ TRAIL-armed cells. Irrespective to the expression levels of DRs, a molecular interaction between ganglioside GM3, abundant in lymphoid cells, and DR4 was detected. This association was negligible in all non-transformed cells and was strictly related to TRAIL susceptibility of cancer cells. Interestingly, lipid raft disruptor methyl-beta-cyclodextrin abrogated this susceptibility, whereas the chemotherapic drug perifosine, which induced the recruitment of TRAIL into lipid microdomains, improved TRAIL-induced apoptosis. Accordingly, in ex vivo samples from patients with B-chronic lymphocytic leukemia, the constitutive embedding of DR4 in lipid microdomains was associated per se with cell death susceptibility, whereas its exclusion was associated with TRAIL resistance. These results provide a key mechanism for TRAIL sensitivity in B-cell malignances: the association, within lipid microdomains, of DR4 but not DR5, with a specific ganglioside, that is the monosialoganglioside GM3. On these bases we suggest that lipid microdomains could exert a catalytic role for DR4-mediated cell death and that an ex vivo quantitative FRET analysis could be predictive of cancer cell sensitivity to TRAIL.     

Lipid raft connection between extrinsic and intrinsic apoptotic pathways

Apoptosis in mammalian cells is modulated by extrinsic and intrinsic signaling pathways through the formation of death receptor-mediated death-inducing signaling complex (DISC) and mitochondrial-derived apoptosome, respectively. We found by ultrastructural approaches that the antitumor drug edelfosine induced aggregates of lipid rafts containing Fas/CD95 receptor and Fas-associated death domain-containing protein in leukemic cells. Death receptors together with DISC and apoptosome constituents were recruited in rafts during edelfosine treatment in multiple myeloma cells. This apoptotic response involved caspases-8/-9/-10 that were translocated to rafts. Lipid raft disruption by cholesterol depletion inhibited loss of mitochondrial transmembrane potential, caspase activation and apoptosis, whereas cholesterol replenishment restored these responses. Our data indicate that rafts act as scaffolds where extrinsic and intrinsic apoptotic signaling pathways concentrate, forming clusters of apoptotic signaling molecule-enriched rafts (CASMER), which function as novel supramolecular entities in the triggering of apoptosis, and play an important role in edelfosine-induced apoptosis in blood cancer cells.     

TRAIL induces apoptosis but not necroptosis in colorectal and pancreatic cancer cells preferentially via the TRAIL-R2/DR5 receptor

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine that can trigger apoptosis in many types of human cancer cells via engagement of its two pro-apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). TRAIL can also activate several other signaling pathways such as activation of stress kinases, canonical NF-κB signaling and necroptosis. Though both receptors are ubiquitously expressed, their relative participation in TRAIL-induced signaling is still largely unknown. To analyze TRAIL receptor-specific signaling, we prepared Strep-tagged, trimerized variants of recombinant human TRAIL with high affinity for either DR4 or DR5 receptor. Using these receptor-specific ligands, we examined the contribution of individual pro-apoptotic receptors to TRAIL-induced signaling pathways. We found that in TRAIL-resistant colorectal HT-29 cells but not in pancreatic PANC-1 cancer cells, DISC formation and initial caspase-8 processing proceeds comparably via both DR4- and DR5-activated signaling. TRAIL-induced apoptosis, enhanced by the inhibitor of the Bcl-2 family ABT-737, or by the translation inhibitor homoharringtonine, proceeded in both cell lines predominantly via the DR5 receptor. ShRNA-mediated downregulation of DR4 or DR5 receptors in HT-29 cells also pointed to a stronger contribution of DR5 in TRAIL-induced apoptosis. In contrast to apoptosis, necroptotic signaling was activated similarly by both DR4- or DR5-specific ligands. Activation of auxiliary signaling pathways involving NF-κB or stress kinases proceeded under apoptotic conditions mainly in a DR5-dependent manner, while these signaling pathways were during necroptosis similarly activated by either of these ligands. Our study provides the first systematic insight into DR4 ?/DR5-specific signaling in colorectal and pancreatic cancer cells.