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.     

Involvement of lipid rafts in the localization and dysfunction effect of the antitumor ether phospholipid edelfosine in mitochondria

Lipid rafts and mitochondria are promising targets in cancer therapy. The synthetic antitumor alkyl-lysophospholipid analog edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) has been reported to target lipid rafts. Here, we have found that edelfosine induced loss of mitochondrial membrane potential and apoptosis in human cervical carcinoma HeLa cells, both responses being abrogated by Bcl-x_L overexpression. We synthesized a number of new fluorescent edelfosine analogs, which preserved the proapoptotic activity of the parent drug, and colocalized with mitochondria in HeLa cells. Edelfosine induced swelling in isolated mitochondria, indicating an increase in mitochondrial membrane permeability. This mitochondrial swelling was independent of reactive oxygen species generation. A structurally related inactive analog was unable to promote mitochondrial swelling, highlighting the importance of edelfosine molecular structure in its effect on mitochondria. Raft disruption inhibited mitochondrial localization of the drug in cells and edelfosine-induced swelling in isolated mitochondria. Edelfosine promoted a redistribution of lipid rafts from the plasma membrane to mitochondria, suggesting a raft-mediated link between plasma membrane and mitochondria. Our data suggest that direct interaction of edelfosine with mitochondria eventually leads to mitochondrial dysfunction and apoptosis. These observations unveil a new framework in cancer chemotherapy that involves a link between lipid rafts and mitochondria in the mechanism of action of an antitumor drug, thus opening new avenues for cancer treatment.     

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     

Oxaliplatin enhances TRAIL-induced apoptosis in gastric cancer cells by CBL-regulated death receptor redistribution in lipid rafts

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family that selectively induces apoptosis in cancer cells. However, gastric cancer cells are insensitive to TRAIL. In the present study, we show that oxaliplatin enhanced TRAIL-induced apoptosis of MGC803, BGC823, and SGC7901 cells. Oxaliplatin promoted death receptor 4 (DR4) and death receptor 5 (DR5) clustering into aggregated lipid rafts, while the cholesterol-sequestering agent nystatin partially prevented lipid raft aggregation, DR4 and DR5 clustering, and reduced apoptosis. Furthermore, the expression of the casitas B-lineage lymphoma (Cbl) family was downregulated by oxaliplatin. Transfection of c-Cbl or Cbl-b partially reversed oxaliplatin-induced lipid raft aggregation. These results indicated that oxaliplatin enhanced TRAIL-induced gastric cancer cell apoptosis at least partially through Cbl-regulated death receptor redistribution in lipid rafts.     

Compartmentalized DCC signalling is distinct from DCC localized to lipid rafts

Background information. Netrin‐1 is a bi‐functional cue that attracts or repels different classes of neurons during development. The netrin‐1 receptor DCC (deleted in colorectal cancer) acts as a tyrosine kinase‐associated receptor to mediate the attractive response towards netrin‐1. The lipid raft‐localized Src family kinase Fyn is required for DCC‐mediated axon guidance. DCC functions are also dependent on lipid rafts, membrane microdomains corresponding to a low‐density, detergent‐resistant membrane fraction. However, it remains unclear how the association of DCC with lipid rafts controls netrin‐1 signalling. Results. DCC targeted to lipid rafts represented a minor proportion of total DCC inside the cell, but predominated on the cell surface of both IMR‐32 human neuroblastoma cells and embryonic cortical neurons. Netrin‐1 accumulated in lipid rafts, but had no effect on the targeting of DCC to that compartment, with DCC remaining on the cell surface in lipid rafts through 60 min post‐treatment. However, DCC was able to interact with Fyn, both in the lipid rafts and soluble compartments isolated from embryonic E19 rat brains, whereas early downstream signalling components such as Nck‐1, and total and active focal adhesion kinase were mainly localized to the non‐lipid raft compartment. Conclusions. Together, these results suggest that DCC can be found in raft and non‐raft portions of the plasma membrane, with early signalling events propagated by non‐raft associated DCC.     

Increased expression of Mcl-1 is responsible for the blockage of TRAIL-induced apoptosis mediated by EGF/ErbB1 signaling pathway

Epidermal growth factor (EGF) protects against death receptor induced apoptosis in epithelial cells. Herein, we demonstrate that EGF protection against tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced apoptosis is mediated by increased expression of the Bcl-2 family member myeloid cell leukemia 1 (Mcl-1). EGF increased the mRNA and protein levels of Mcl-1. Furthermore, expression of ErbB1 alone or in combination with ErbB2 in NIH3T3 cells up-regulates Mcl-1 following EGF treatment. In addition, up-regulation of Mcl-1 by EGF is mediated through AKT and NFkappaB activation since kinase inactive AKT and DeltaIkappaB effectively blocks this up-regulation. NFkappaB was also critical for the ability of EGF to prevent TRAIL induced apoptosis as a dominant negative IkappaB (DeltaIkappaB) blocked NFkappaB activation, and relieved EGF protection against TRAIL mediated mitochondrial cytochrome-c release and apoptosis. Finally, anti-sense oligonucleotides directed against Mcl-1 effectively reduced the protein levels of Mcl-1 and blocked EGF protection against TRAIL induced mitochondrial cytochrome-c release and apoptosis. Taken together, EGF signaling leads to increased Mcl-1 expression that is required for blockage of TRAIL induced apoptosis.     

The death receptor DR5 is involved in TRAIL-mediated human osteoclast apoptosis

The number and activity of osteoclasts (OCs) are critical for maintaining normal bone turnover. The number is determined by the rates of cell differentiation and death. TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis by interacting with its death receptors, (DR4, DR5). However, its activity can be modulated by two decoy receptors, (DcR1 and DcR2). In this paper we show that TRAIL treatment causes reduced OC viability as well as an increased apoptotic OC number. Loss of nuclei integrity and derangement of the actin microfilament were also induced by TRAIL in OCs. Moreover, we demonstrated the expression of all TRAIL receptors in both precursors and differentiated OCs, and the upregulation of DR5 during OC differentiation. Interestingly, DcR2 was upregulated in the early stage of osteoclastogenesis and downregulated at the end of the differentiation process. We showed that DR5, upregulated by TRAIL, could be the mediator of TRAIL-induced OC apoptosis, since the addition of anti-DR5 neutralizing antibodies restores the OC viability previously reduced by TRAIL. Furthermore, the intracellular pathway induced by TRAIL in OCs involves caspase-8 and Bid activation. In conclusion, our data highlight an important role for the TRAIL/TRAIL receptor system in the regulation of OC apoptosis.     

Cosignaling of NCAM via lipid rafts and the FGF receptor is required for neuritogenesis

The neural cell adhesion molecule (NCAM) has been reported to stimulate neuritogenesis either via nonreceptor tyrosine kinases or fibroblast growth factor (FGF) receptor. Here we show that lipid raft association of NCAM is crucial for activation of the nonreceptor tyrosine kinase pathway and induction of neurite outgrowth. Transfection of hippocampal neurons of NCAM-deficient mice revealed that of the three major NCAM isoforms only NCAM140 can act as a homophilic receptor that induces neurite outgrowth. Disruption of NCAM140 raft association either by mutation of NCAM140 palmitoylation sites or by lipid raft destruction attenuates activation of the tyrosine focal adhesion kinase and extracellular signal-regulated kinase 1/2, completely blocking neurite outgrowth. Likewise, NCAM-triggered neurite outgrowth is also completely blocked by a specific FGF receptor inhibitor, indicating that cosignaling via raft-associated kinases and FGF receptor is essential for neuritogenesis.     

Suppression of TRPM7 enhances TRAIL-induced apoptosis in triple-negative breast cancer cells

Transient receptor potential cation channel subfamily M member 7 (TRPM7) composed of an ion channel and a kinase domain regulates triple-negative breast cancer (TNBC) cell migration, invasion, and metastasis, but it does not modulate TNBC proliferation. However, previous studies have shown that the combination treatment of nonselective TRPM7 channel inhibitors (2-aminoethoxydiphenyl borate and Gd³⁺) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases antiproliferative effects and apoptosis in prostate cancer cells and hepatic stellate cells. We, therefore, investigated the potential role of TRPM7 in proliferation and apoptosis of TNBC cells (MDA-MB-231 and MDA-MB-468 cells) with TRAIL. We demonstrated that suppression of TRPM7 via TRPM7 knockdown or pharmacological inhibition synergistically increases TRAIL-induced antiproliferative effects and apoptosis in TNBC cells. Furthermore, we showed that the synergistic interaction might be associated with TRPM7 channel activities using combination treatments of TRAIL and TRPM7 inhibitors (NS8593 as a TRPM7 channel inhibitor and TG100-115 as a TRPM7 kinase inhibitor). We reveal that downregulation of cellular FLICE-inhibitory protein via inhibition of Ca²⁺ influx might be involved in the synergistic interaction. Our study would provide both a new role of TRPM7 in TNBC cell apoptosis and a potential combinatorial therapeutic strategy using TRPM7 inhibitors with TRAIL in the treatment of TNBC.     

Inhibition of TRAIL-induced apoptosis by IL-8 is mediated by the p38-MAPK pathway in OVCAR3 cells

Introduction: TRAIL (TNF-Related Apoptosis Inducing Ligand) is a member of the TNF superfamily of cell death inducing ligands. Interestingly, while malignant cells are responsive to TRAIL-induced cell death when used alone or in combination with other agents, normal cells do not appear to be sensitive to this ligand, making it a desirable therapeutic compound against many cancers, including many ovarian carcinomas. Interleukin-8 (IL-8), a member of the C-X-C chemokine family, has been found to be at significantly higher level in the ascites from patients with ovarian cancer. We have previously demonstrated a role for IL-8 in blocking TRAIL's ability to induce apoptosis in the ovarian cancer cell line, OVCAR3, possibly by repressing the DR4 TRAIL receptor expression and blocking caspase-8 cleavage. In addition, we showed a member of the mitogen-activated protein kinase (MAPK) superfamily, p38γ, is among the genes regulated in OVCAR3 cells by TRAIL and IL-8. The present study further investigates involvement of the p38 MAPK pathway in IL-8's ability to block TRAIL-induced apoptosis in the ovarian surface epithelial cancer cell line, OVCAR3. Results: In this study we demonstrate that p38γ as well as p38α play a significant role in IL-8's ability to block TRAIL-induced apoptosis. Through array analysis, as well as confirmation with other methods, we detected regulation of p38γ and p38α following treatment of the cancer cell line with IL-8 or TRAIL. We also tested two other isoforms of p38 MAPK, p38β and p38δ, but did not find significant regulation by IL-8 or TRAIL. We also examined activation of the p38 MAPK pathway, up-stream as well as down-stream, and noticed activation of the pathway following treatment with TRAIL and decreased activity when IL-8 was introduced. With the use of specific inhibitors, we were able to further confirm the role of this pathway in TRAIL-induced apoptosis, and IL-8's ability to block this apoptosis, in ovarian cancer cell lines. Conclusion: Taken together, these results further solidify the role of IL-8 in blocking the TRAIL-induced apoptosis in these ovarian carcinoma cells and provide new molecular insight into this potentially important therapeutic target.     

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and α2β1 integrin) for efficient platelet activation. Pascal Maurice and Ludovic Waeckel have contributed equally to this work.     

Glut-4 is translocated to both caveolae and non-caveolar lipid rafts, but is partially internalized through caveolae in insulin-stimulated adipocytes

Caveolae and non-caveolar lipid rafts are two types of membrane lipid microdomains that play important roles in insulin-stimulated glucose uptake in adipocytes. In order to ascertain their specific functions in this process, caveolae were ablated by caveolin-1 RNA interference. In Cav-1 RNAi adipocytes, neither insulin-stimulated glucose uptake nor Glut-4 （glucose transporter 4） translocation to membrane lipid microdomains was affected by the ablation of caveolae. With a modified sucrose density gradient, caveolae and non-caveolar lipid rafts could be separated. In the wild-type 3T3- L l adipocytes, Glut-4 was found to be translocated into both caveolae and non-caveolar lipid rafts. However, in Cav1 RNAi adipocytes, Glut-4 was localized predominantly in non-caveolar lipid rafts. After the removal of insulin, caveolaelocalized Glut-4 was internalized faster than non-caveolar lipid raft-associated Glut-4. The internalization of Glut-4 from plasma membrane was significantly decreased in Cav-1 RNAi adipocytes. These results suggest that insulin-stimulated Glut-4 translocation and glucose uptake are caveolae-independent events. Caveolae play a role in the internalization of Glut-4 from plasma membrane after the removal of insulin.     

Ethanol mimics ligand-mediated activation and endocytosis of IL-1RI/TLR4 receptors via lipid rafts caveolae in astroglial cells

We have recently reported that ethanol-induced inflammatory processes in the brain and glial cells are mediated via the activation of interleukin-1 beta receptor type I (IL-1RI)/toll-like receptor type 4 (TLR4) signalling. The mechanism(s) by which ethanol activates these receptors in astroglial cells remains unknown. Recently, plasma membrane microdomains, lipid rafts, have been identified as platforms for receptor signalling and, in astrocytes, rafts /caveolae constitute an important integrators of signal events and trafficking. Here we show that stimulation of astrocytes with IL-1β, lipopolysaccharide or ethanol (10 and 50 mM), triggers the translocation of IL-1RI and/or TLR4 into lipid rafts caveolae-enriched fractions, promoting the recruitment of signalling molecules (phospho-IL-1R-associated kinase and phospho-extracellular regulated-kinase) into these microdomains. With confocal microscopy, we further demonstrate that IL-1RI is internalized by caveolar endocytosis via enlarged caveosomes organelles upon IL-1β or ethanol treatment, which sorted their IL-1RI cargo into the endoplasmic reticulum–Golgi compartment and into the nucleus of astrocytes. In short, our findings demonstrate that rafts /caveolae are critical for IL-1RI and TLR4 signalling in astrocytes, and reveal a novel mechanism by which ethanol, by interacting with lipid rafts caveolae, promotes IL-1RI and TLR4 receptors recruitment, triggering their endocytosis via caveosomes and downstream signalling stimulation. These results suggest that TLRs receptors are important targets of ethanol-induced inflammatory damage in the brain.     

The γ-aminobutyric acid receptor B, but not the metabotropic glutamate receptor type-1, associates with lipid rafts in the rat cerebellum

Recent evidence suggests that specialized microdomains, called lipid rafts, exist within plasma membranes. These domains are enriched in cholesterol and sphingolipids and are resistant to non-ionic detergent-extraction at 4 degrees C. They contain specific populations of membrane proteins, and can change their size and composition in response to cellular signals, resulting in activation of signalling cascades. Here, we demonstrate that both the metabotropic gamma-aminobutyric acid receptor B (GABA(B) receptor) and the metabotropic glutamate receptor-1 from rat cerebellum are insoluble in the non-ionic detergent Triton X-100. However, only the GABA(B) receptor associates with raft fractions isolated from rat brain by sucrose gradient centrifugation. Moreover, increasing the stringency of isolation by decreasing the protein : detergent ratio caused an enrichment of the GABA(B) receptor in raft fractions. In contrast, depletion of cholesterol from cerebellar membranes by either saponin or methyl-beta-cyclodextrin treatment, which solubilize known raft markers, also increased the solubility of the GABA(B) receptor. These properties are all consistent with an association of the GABA(B) receptor with lipid raft microdomains.     

Comparative binding to DR4 and DR5 receptors of TRAIL and BNNTs/PAHE/mPEG‐DSPE/TRAIL nanoparticles

TRAIL is a member of the tumor necrosis factor family of cytokines, which induces apoptosis of cancer cells, thanks to its binding to its cognate receptors DR5 and DR4. We have recently demonstrated that nanovectorization of TRAIL with single‐walled carbon nanotubes enhanced TRAIL affinity to DR5. In this paper, 1‐pyrenebutyric acid N‐hydroxysuccinimide ester functionalized boron nitride nanotubes (BNNTs) were used to anchor the TRAIL protein. The resulting BNNT/1‐pyrenebutyric acid N‐hydroxysuccinimide ester nanotubes were mixed with methoxy‐poly(ethylene glycol)‐1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐conjugates so as to allow a good dispersion of these nanoparticle TRAIL (NPT) in aqueous solution. The difference of binding between NPT and soluble TRAIL to DR4 and DR5 receptors was then studied by the use of affinity chromatography. DR4 and DR5 receptors were thus immobilized on a chromatographic support, and the binding of the 2 ligands TRAIL and NPT to DR4 and DR5 was studied in the temperature range 30°C to 50°C. Negative enthalpy (ΔH ) values indicated that van der Waals interactions and hydrogen bonding are engaged favorably at the ligand‐receptor interface. It was shown that their rank‐ordered affinities were strongly different in the sequence TRAIL_DR4 < NPT_DR4 < TRAIL_DR5 < NPT_DR5, and the highest affinity for NPT to DR4 and DR5 receptors observed at low pHs was due to the less accessibility of the His molecular switch to be protonated when TRAIL was immobilized on BNNTs. Taken together, our results demonstrated that nanovectorization of TRAIL with BNNTs enhanced its binding to both DR4 and DR5 receptors at 37°C. Our novel nanovector could potentially be used for delivering TRAIL to cells for cancer treatment.     

Functional roles of glycosphingolipids in signal transduction via lipid rafts

The formation of glycosphingolipid (GSL)-cholesterol microdomains in cell membranes has been proposed to function as platforms for the attachment of lipid-modified proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins and src-family tyrosine kinases. The microdomains are postulated to be involved in GPI-anchored protein signaling via src-family kinase. Here, the functional roles of GSLs in signal transduction mediated by the microdomains are discussed. Antibodies against GSLs co-precipitate GPI-anchored proteins, src-family kinases and several components of the microdomains. Antibody-mediated crosslinking of GSLs, as well as that of GPI-anchored proteins, induces a rapid activation of src-family kinases and a transient increase in the tyrosine phosphorylation of several substrates. Enzymatic degradation of GSLs reduces the activation of src-family kinase and tyrosine phosphorylation by antibody-mediated crosslinking of GPI-anchored protein. Furthermore, GSLs can also modulate signal transduction of immunoreceptors and growth factor receptors in the microdomains. Thus, GSLs have important roles in signal transduction mediated by the microdomains.     

Localization of receptors in lipid rafts can inhibit signal transduction

Processes of cell survival, division, differentiation, and death are guided by the binding of signal molecules to receptors, which activates intracellular signaling networks and ultimately elicits genetic, biochemical, or biomechanical responses within the cell. While intracellular mechanisms for these processes have been well studied, little attention has been given to the role extracellular ligand transport and binding may play in signal initiation. Recent studies have found that the localization of receptors in lipid rafts is critical for the functions of many signaling pathways. By concentrating membrane components, rafts may promote essential interactions for signaling. Lipid rafts can also have negative effects on signaling, but mechanisms remain elusive. We propose that raft-mediated receptor clustering can reduce signaling by prolonging the diffusion of ligands to their receptors. We quantify this effect using a simple diffusion-limited binding model that accounts for the spatial distribution of lipid rafts and receptors on the cell surface. We find that receptor clustering can reduce the apparent rate of receptor binding by up to 80%, consistent with observed increases in epidermal growth factor (EGF) binding by up to 100% following disruption of lipid rafts (Pike and Casey 2002 Biochemistry 41:10315-10322; Roepstorff et al. 2002 J Biol Chem 277:18954-18960). Failure to account for the effects of receptor clustering on rates of ligand binding can skew the interpretation of current methods of cancer diagnosis and treatment. Finally, we discuss how the activation of particular signaling pathways can change over time, depending, in part, on the overall level and spatial distribution of the receptors.     

Long chain‐polyunsaturated fatty acids modulate membrane phospholipid composition and protein localization in lipid rafts of neural stem cell cultures

Rat neural stem cells/neural progenitors (NSC/NP) are generally grown in serum‐free medium. In this study, NSC/NP were supplemented with the main long‐chain polyunsaturated fatty acids (PUFAs) present in the brain, arachidonic acid (AA), or docosahexaenoic acid (DHA), and were monitored for their growth. Lipid and fatty acid contents of the cells were also determined. Under standard conditions, the cells were characterized by phospholipids displaying a highly saturated profile, and very low levels of PUFAs. When cultured in the presence of PUFAs, the cells easily incorporated them into the phospholipid fraction. We also compared the presence of three membrane proteins in the lipid raft fractions: GFR and connexin 43 contents in the rafts were increased by DHA supplementation, whereas Gβ subunit content was not significantly modified. The restoration of DHA levels in the phospholipids could profoundly affect protein localization and, consequently, their functionalities. J. Cell. Biochem. 110: 1356–1364, 2010. © 2010 Wiley‐Liss, Inc.