Surface Expression Of Death Receptors Research Articles

Chemotherapeutic Agents Sensitize Resistant Cancer Cells to the DR5-Specific Variant DR5-B more Efficiently than to TRAIL by Modulating the Surface Expression of Death and Decoy Receptors.

TRAIL is considered a promising antitumor agent because it causes apoptosis of transformed cells without affecting normal cells. However, many types of tumors are cytokine resistant, and combination therapy with various chemotherapeutic drugs is being developed to overcome the resistance. We have demonstrated that the combination of TRAIL with doxorubicin, bortezomib, and panobinostat dramatically reduced the viability of TRAIL-resistant A549 and HT-29 cells. Chemotherapy even more efficiently sensitized cells to the DR5-specific mutant variant of TRAIL DR5-B, which does not have an affinity for decoy receptors. Bortezomib and doxorubicin greatly enhanced the surface expression of the death receptors DR5 and DR4, while panobinostat increased expression of DR5 and suppressed expression of DR4 in both cell lines. All drugs increased surface expression of the decoy receptors DcR1 and DcR2. Unlike the combined treatment, if the cells were pretreated with chemotherapy for 24 h, the cytotoxic activity of TRAIL was less pronounced, while sequential treatment of cells enhanced the effectiveness of DR5-B. The same results were obtained with agonistic anti-DR5 antibodies. Thus, the effectiveness of TRAIL was rather limited due to changes in the ratio of death and decoy receptors and DR5-specific agonists may be preferred in combination antitumor therapy regimens.

Tangeretin sensitises human lung cancer cells to TRAILinduced apoptosis via ROS-JNK/ERK-CHOP pathwaymediated up-regulation of death receptor 5

Purpose: To investigate the efficacy of tangeretin, a bioactive flavonoid, as an enhancer of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells (H1299 and H1975). Methods: H1299 and H1975 cells were exposed to tangeretin (25, 50, or 100 μM), and its effect on cell viability was assessed by MTT assay. The cytotoxic effects of tangeretin in combination with TRAIL (25 ng/mL) were determined by live/dead assay and annexin V staining. Intracellular levels of reactive oxygen species (ROS) were determined by DHFDA-flow cytometry analysis. Western blotting was performed to assess the expression of death receptors, apoptosis pathway proteins, JNK and ERK1/2. The cell surface expression of death receptors was determined. RT-PCR was performed to assess the expression of death receptor 5 and CEBP homologous protein (CHOP) mRNA. The cytotoxic effects of tangeretin in the presence of DR5, CHOP siRNA and TRAIL were also detected. Results: MTT assay revealed that tangeretin exerted potent cytotoxic effects and, in combination with TRAIL, exhibited striking apoptosis. Down-regulation of cell survival proteins (Bcl-2, Bcl-xL, survivin, xIAP, c-IAP-1, and c-IAP-2) and elevated expression of Bax and caspases 3, 8, and 9 were observed upon tangeretin exposure. The expression of death receptors DR4 and DR5 was highly up-regulated by tangeretin, while that of decoy receptors DcR1 and DcR2 was down-regulated. DR5 and CHOP siRNA transfection suggested the involvement of CHOP in tangeretin-mediated up-regulation of DR5, leading to more pronounced apoptotic effects. Tangeretin increased ROS levels and effectively caused activation of the mitogen-activated protein kinases JNK, ERK, and p38MAPKs. Expression analysis in the presence of specific MAPK inhibitors suggested the involvement of JNK and ERK in tangeretinmediated TRAIL sensitisation. Conclusions: Tangeretin induces death receptors and enhances TRAIL-induced apoptosis through upregulation of the ROS-JNK/ERK-CHOP pathways. Keywords: Apoptosis, Death receptors, Lung cancer, Tangeretin, Reactive oxygen species, Tumour necrosis factor-related apoptosis-inducing ligand

Targeting miRNAs associated with surface expression of death receptors to modulate TRAIL resistance in breast cancer

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is capable of inducing apoptosis upon engagement of its death receptors (DRs) 4 and 5. TRAIL therapy has garnered intense interest as one of the most promising agents for cancer therapy, for its selective induction of tumor-cell apoptosis while low toxicity to most normal cells. However, a variety of breast cancer cell lines could be resistant to TRAIL-induced apoptosis. Absence of DR4 and DR5 on the breast cancer cell surface has been proposed to be critically involved in resistance to TRAIL and its agonistic antibodies. Moreover, endocytosis and autophagy in breast cancer cells could induce TRAIL resistance through downregulation of surface DR4/5. MicroRNAs (miRNAs), as endogenously expressed small non-coding RNAs, function as regulators of gene expression and involve tremendous biological processes including drug resistance. In this review, we highlight recent advances in the functional role of miRNAs in endocytosis and autophagy pathways. This review aims to present that, through regulation of critical molecules involved in autophagy and endocytosis, miRNAs could lead to mislocalization of DR4/5 in breast cancer cells and therefore play an important role in TRAIL-mediated apoptosis and TRAIL resistance.

Effector function of CTLs is increased by irradiated colorectal tumor cells that modulate OX-40L and 4-1BBL and is reversed following dual blockade.

BackgroundSub-lethal doses of ionizing radiation (IR) can alter the phenotype of target tissue by modulating genes that influence effector T cell activity. Previous studies indicate that cancer cells respond to radiation by up-regulating surface expression of death receptors, cell adhesion molecules and tumor-associated antigens (TAA). However, there is limited information available regarding how T cells themselves are altered following these interactions with irradiated tumor cells.MethodsHere, several human colorectal tumor cell lines were exposed to radiation (0–10 Gy) in vitro and changes in the expression of molecules costimulatory to effector T cells (4-1BBL, OX-40L, CD70, ICOSL) were examined by flow cytometry. T cell effector function was assessed to determine if changes in these proteins were directly related to the changes in T cell function.ResultsWe found OX-40L and 4-1BBL to be the most consistently upregulated proteins on the surface of colorectal tumor cells post-IR while ICOSL and CD70 remained largely unaltered. Expression of these gene products correlated with enhanced killing of irradiated human colorectal tumor cells by TAA-specific T-cells. Importantly, blocking of both OX-40L and 4-1BBL reversed radiation-enhanced T-cell killing of human tumor targets as well as T-cell survival and activation.ConclusionsOverall, results of this study suggest that, beyond simply rendering tumor cells more sensitive to immune attack, radiation can be used to specifically modulate expression of genes that directly stimulate effector T cell activity.

Combination Treatment with Sublethal Ionizing Radiation and the Proteasome Inhibitor, Bortezomib, Enhances Death-Receptor Mediated Apoptosis and Anti-Tumor Immune Attack.

Sub-lethal doses of radiation can modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. Proteasome inhibitors demonstrate broad anti-tumor activity in clinical and pre-clinical cancer models. Here, we use a combination treatment of proteasome inhibition and irradiation to further induce immunomodulation of tumor cells that could enhance tumor-specific immune responses. We investigate the effects of the 26S proteasome inhibitor, bortezomib, alone or in combination with radiotherapy, on the expression of immunogenic genes in normal colon and colorectal cancer cell lines. We examined cells for changes in the expression of several death receptors (DR4, DR5 and Fas) commonly used by T cells for killing of target cells. Our results indicate that the combination treatment resulted in increased cell surface expression of death receptors by increasing their transcript levels. The combination treatment further increases the sensitivity of carcinoma cells to apoptosis through FAS and TRAIL receptors but does not change the sensitivity of normal non-malignant epithelial cells. Furthermore, the combination treatment significantly enhances tumor cell killing by tumor specific CD8+ T cells. This study suggests that combining radiotherapy and proteasome inhibition may simultaneously enhance tumor immunogenicity and the induction of antitumor immunity by enhancing tumor-specific T-cell activity.

Abstract 2555: The mechanism of action of bendamustine alone or in combination with nucleoside analogs in chronic lymphocytic leukemia

Abstract Bendamustine (BEN) is a promising new treatment for chronic lymphocytic leukemia (CLL) and is believed to function as an alkylating agent (eg, chlorambucil, CLB) while sharing structural similarities to a nucleoside analogue (eg, fludarabine, FLU). Enhanced cell death has been observed with the combination of BEN and FLU in primary CLL cells in vitro, likely due to the inhibition of repair of BEN DNA cross-linking by FLU. However, both BEN and FLU are marrow-toxic, limiting the use of this combination in the clinic. The nucleoside analog, pentostatin (PEN), is an adenosine deaminase inhibitor that causes the accumulation of deoxyadenosine (dADO) and is less marrow-suppressive than FLU. However, the cytotoxic effectiveness of dADO/PEN in combination with BEN is not known. Flow cytometry analysis using annexin V/7-ADD and the MTT assay were performed to determine the effectiveness of BEN treatment in primary CLL cells as compared to or in combination with CLB, FLU, or dADO/PEN at varying drug concentrations and time points. Using the MTT assay, there was a linear relationship between cytotoxicity and drug concentration, similar to that seen with CLB. In vitro treatment of CLL cells with BEN, CLB, FLU, or dADO/PEN induced apoptosis, the degree being time- and concentration-dependent. However, the sensitivity of CLL cells to BEN or CLB varied, suggesting different mechanisms of action. Enhanced cell kill was seen with the combination of BEN or CLB with FLU or dADO/PEN, with the extent of increased cytotoxicity being similar for FLU or dADO/PEN. Cell death mechanisms were analyzed via staining for surface expression of death receptors (DR4 and DR5) and mitochondrial stress (DICO6 and DHE). γ-H2AX staining was used to measure DNA double-strand breaks (DSBs) and the alkaline comet assay was used to measure both DNA DSBs and single-strand breaks (SSBs). An increase in DR4 and DR5 surface expression, loss of mitochondrial membrane potential, and increased reactive oxygen species production was observed with all agents. There was an increase in DNA SSB and DSB following FLU, as compared to BEN and CLB, while a combination of BEN and FLU further increased the number of breaks (especially SSBs) compared to FLU alone. Thus, BEN has the properties of an alkylating agent, rather than a nucleoside analog, but has a different spectrum of antitumor activity to CLB. BEN induces apoptosis through both the death receptor and mitochondrial pathways. Increased cell kill is seen on combining BEN with dADO/PEN suggesting that this might be a useful combination in the clinic. Citation Format: Sara E.F. Kost, Eric D.J. Bouchard, William S. Liang, Versha Banerji, Spencer B. Gibson, Sachin Katyal, James B. Johnston. The mechanism of action of bendamustine alone or in combination with nucleoside analogs in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2555. doi:10.1158/1538-7445.AM2015-2555

C-FLIP(L) contributes to TRAIL resistance in HER2-positive breast cancer

Breast cancers with HER2 amplification have a poorer prognosis than the luminal phenotypes. TRAIL activates apoptosis upon binding its receptors in some but not all breast cancer cell lines. Herein, we investigated the expression pattern of c-FLIP(L) in a cohort of 251 invasive breast cancer tissues and explored its potential role in TRAIL resistance. C-FLIP(L) was relatively high-expressed in HER2-positive breast cancer in comparison with other molecular subtypes, co-expressed with TRAIL death receptors, and inversely correlated with the apoptosis index. Downregulation of c-FLIP(L) sensitized SKBR3 cells to TRAIL-induced apoptosis in a concentration- and time-dependent manner and enhanced the activities and cleavages of caspase-8 and caspase-3, without altering the surface expression of death receptors. Together, our results indicate that c-FLIP(L) promotes TRAIL resistance and inhibits caspase-3 and caspase-8 activation in HER2-positive breast cancer.

Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. We have previously shown that deficiency of DR4 and DR5 on the surface membrane is a critical mechanism of cancer cell resistance to the recombinant human TRAIL and its receptor agonistic antibodies, which are being evaluated clinically for treating cancers. In certain cancer cells, DR4 and DR5 were found to be mislocalized in intracellular compartments yet to be characterized. Here, we report a novel role of autophagy in the regulation of dynamics of TRAIL death receptors. We first assessed basal levels of autophagosomes in a panel of 11 breast cancer cell lines using complementary approaches (LC3 immunoblotting, RFP-LC3 fluorescence microscopy, and electron microscopy). We found high levels of basal autophagosomes in TRAIL resistant breast cancer cell lines (e.g. BT474 and AU565) and relevant mouse xenograft models under nutrition-rich conditions. Notably, DR4 and DR5 co-localized with LC3-II in the autophagosomes of TRAIL-resistant cells. Disruption of basal autophagosomes successfully restored the surface expression of the death receptors which was accompanied by sensitization of TRAIL-resistant cells to TRAIL induced apoptosis. By contrast, TRAIL-sensitive cell lines (MDA-MB-231) are characterized by high levels of surface DR4/DR5 and an absence of basal autophagosomes. Inhibition of lysosomal activity induced an accumulation of autophagosomes and a decrease in surface DR4 and DR5, and the cells became less sensitive to TRAIL-induced apoptosis. These findings demonstrate a novel role for the basal autophagosomes in the regulation of TRAIL death receptors. Further studies are warranted to explore the possibility of using autophagosome markers such as LC3-II/LC3-I ratios for prediction of tumor resistance to TRAIL related therapies. The results also provide a rationale for future non-clinical and clinical studies testing TRAIL agonists in combination with agents that directly inhibit autophagosome assembly.

Depolarization potentiates TRAIL-induced apoptosis in human melanoma cells: role for ATP-sensitive K+ channels and endoplasmic reticulum stress.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is promising for cancer treatment owing to its selective cytotoxicity against malignant cells. However, some cancer cell types, including malignant melanoma cells, are resistant to TRAIL-induced apoptosis. Therefore, drugs that can amplify TRAIL cytotoxicity are urgently required. Depolarization of the plasma membrane potential is associated with apoptosis induced by a variety of death-inducing agents but its role in apoptosis remains a matter of debate. We found that TRAIL treatment resulted in robust depolarization in human melanoma cells with a considerable lag (2–4 h). Moreover, membrane-depolarizing agents, including K+ and ATP-sensitive K+ (KATP) channel inhibitors glibenclamide and U37883A enhanced TRAIL-induced apoptosis. On the contrary, inhibitors of calcium- and voltage-dependent K+ channels and mitochondrial KATP channels had no such effects. Melanocytes were insensitive to TRAIL-induced depolarization and apoptosis as well as to the sensitization by membrane-depolarizing agents despite their substantial surface expression of death receptors. TRAIL induced robust activation of X-box-binding protein-1 and caspase-12, both of which were enhanced by the K+ and KATP channel inhibitors, but not by other K+ channel inhibitors. Finally, caspase-12-selective inhibitor completely abolished the amplification of apoptosis. These findings suggest that depolarization promotes endoplasmic reticulum stress-mediated death pathway, thereby amplifying TRAIL cytotoxicity. Thus, membrane-depolarizing agents such as KATP channel inhibitors may have therapeutic potential in the treatment of TRAIL-resistant cancer cells without impairing tumor-selectivity.

WEE1 Inhibition Sensitizes Basal Breast Cancer Cells to TRAIL-Induced Apoptosis

TRAIL is a member of the TNF super family and has been shown to induce apoptosis in many cancer cell lines but not in normal cells. Breast cancers can be divided into different subgroups on the basis of the expression of estrogen and progesterone receptors, HER-2 amplification, or the lack of these three markers (known as triple-negative or basal-type breast cancer). Our group and others have shown previously that triple-negative breast cancer cell lines are sensitive to TRAIL whereas others are relatively resistant. In an earlier study, we reported that inhibition of WEE1, a cell-cycle checkpoint regulator, causes increased cell death in breast cancer cell lines. In this study, we tested the effects of WEE1 inhibition on TRAIL-mediated apoptosis in breast cancer cell lines. Pretreatment with WEE1 inhibitor or knockdown of WEE1 increased the toxicity of TRAIL in the basal/triple-negative breast cancer cell lines compared with WEE1 inhibitor or TRAIL treatment alone. The enhanced cell death is attributed to increased surface expression of death receptors, increased caspase activation which could be blocked by the pan-caspase inhibitor, Z-VAD-FMK, thereby rescuing cells from caspase-mediated apoptosis. The cell death was initiated primarily by caspase-8 because knockdown of caspase-8 and not of any other initiator caspases (i.e., caspase-2, -9, or -10) rescued cells from WEE1 inhibitor-sensitized TRAIL-induced cell death. Taken together, the data suggest that the combination of WEE1 inhibitor and TRAIL could provide a novel combination for the treatment of basal/triple-negative breast cancer.

Highlights of This Issue

The role of G-protein coupled–receptors (GPCR) in cancer and how to best target their function remains incompletely understood. Using The Cancer Genome Atlas, primary tumor specimens, and in vitro studies, Woerner and coworkers found that expression of G-protein coupled–receptor kinases (GRK), critical regulators of GPCR function, is altered in glioblastoma in a molecular subtype–specific fashion. Decreased GRK3 expression in classical glioblastoma was associated with a growth advantage, which was blocked by reconstitution of GRK3 expression. These data indicate that GPCRs function to regulate glioblastoma growth and suggest that modulation of GPCR phosphorylation may be an important therapeutic strategy.The antioxidant enzyme extracellular superoxide dismutase EcSOD is downregulated in a large proportion of human lung cancers. To explore its role in cancer, Teoh-Fitzgerald and colleagues examined EcSOD gene expression, as well as the biological effects of its reexpression in EcSOD-negative tumor cells. Epigenetic silencing by aberrant cytosine methylation of the promoter was the most frequent cause of expression loss in human lung adenocarcinoma compared with normal lung. Reexpression of EcSOD inhibited the in vitro invasiveness of human lung adenocarcinoma cells. This study identifies EcSOD as an epigenetically regulated lung cancer target and a modifier of the malignant phenotype.TRAIL induces apoptosis in basal/triplenegative breast cancer (TNBC) cell lines, whereas other subtypes are relatively resistant. Inhibition of WEE1, a cell-cycle checkpoint regulator, causes increased cell death in cancer cell lines. Here, Garimella and colleagues show that pretreatment with WEE1 inhibitor increased the toxicity of TRAIL in TNBC cell lines. This effect is attributed to increased surface expression of death receptors resulting in increased caspase activation initiated by caspase-8. These data suggest that the combination of WEE1 inhibitor and TRAIL could provide a novel approach to targeted therapy for TNBC.Although largely unknown, the abnormalities in DNA repair that underlie the genomic instability in sporadic cancers are potential therapeutic targets. Tobin and colleagues have characterized a novel defect in DNA double-strand break repair in hormone therapy–resistant breast cancer that can be identified in breast cancer cell lines and tumor biopsies and can be selectively targeted by a combination of PARP and DNA ligase inhibitors. These preclinical studies provide the rationale for the development of effective therapeutic strategies for hormone therapy–resistant breast cancer that may be applicable to other cancers, including leukemias, with similar DNA repair abnormalities.

TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3β pathway and activation of caspases

BackgroundThe development of new modulator possessing high efficacy, low toxicity and high selectivity is a pivotal approach to overcome P-glycoprotein (P-gp) mediated multidrug resistance (MDR) in cancer treatment. In this study, we suggest a new molecular mechanism that TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) down-regulates P-glycoprotein (P-gp) through inhibition of DNA-PKcs/Akt/GSK-3β pathway and activation of caspases and thereby sensitize MDR cells to MDR-related drugs.ResultsMDR variants, CEM/VLB10-2, CEM/VLB55-8 and CEM/VLB100 cells, with gradually increased levels of P-gp derived from human lymphoblastic leukemia CEM cells, were gradually more susceptible to TRAIL-induced apoptosis and cytotoxicity than parental CEM cells. The P-gp level of MDR variants was positively correlated with the levels of DNA-PKcs, pAkt, pGSK-3β and c-Myc as well as DR5 and negatively correlated with the level of c-FLIPs. Hypersensitivity of CEM/VLB100 cells to TRAIL was accompanied by the activation of mitochondrial apoptotic pathway as well as the activation of initiator caspases. In addition, TRAIL-induced down-regulation of DNA-PKcs/Akt/GSK-3β pathway and c-FLIP and up-regulation of cell surface expression of death receptors were associated with the increased susceptibility to TRAIL of MDR cells. Moreover, TRAIL inhibited P-gp efflux function via caspase-3-dependent degradation of P-gp as well as DNA-PKcs and subsequently sensitized MDR cells to MDR-related drugs such as vinblastine and doxorubicin. We also found that suppression of DNA-PKcs by siRNA enhanced the susceptibility of MDR cells to vincristine as well as TRAIL via down-regulation of c-FLIP and P-gp expression and up-regulation of DR5.ConclusionThis study showed for the first time that the MDR variant of CEM cells was hypersensitive to TRAIL due to up-regulation of DR5 and concomitant down-regulation of c-FLIP, and degradation of P-gp and DNA-PKcs by activation of caspase-3 might be important determinants of TRAIL-induced sensitization of MDR cells to MDR-related drugs. Therefore, combination of TRAIL and chemotherapeutic drugs may be a good strategy for treatment of cancer with multidrug resistance.

Bortezomib Sensitizes Human Esophageal Squamous Cell Carcinoma Cells to TRAIL–Mediated Apoptosis via Activation of Both Extrinsic and Intrinsic Apoptosis Pathways

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive human cancers, and novel treatment modalities are required. We investigated the therapeutic potential of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) in combination with the proteasome inhibitor bortezomib (Velcade) on human ESCC cell lines. Bortezomib enhanced the susceptibility to TRAIL in 12 of the 15 ESCC cell lines tested, although most showed low sensitivity to TRAIL as a single agent. The enhancement of TRAIL-induced apoptosis by bortezomib was caspase dependent. Increased processing of caspase-8 often accompanied enhancement of TRAIL-induced apoptosis by bortezomib. However, the increased cell surface expression of death receptors observed on bortezomib treatment did not seem to be crucial for this effect. For some ESCC, bortezomib treatment resulted in a more efficient recruitment of caspase-8 and the Fas-associated death domain to the death-inducing signaling complex. Additional downregulation of the cellular FLICE-inhibitory protein long isoform [c-FLIP(L)] could cooperate in the activation of the extrinsic pathway in some cases. For other ESCC, the crucial effect of bortezomib treatment seemed to be increased signaling via the intrinsic apoptotic pathway on subsequent exposure to TRAIL. Thus, bortezomib could sensitize ESCC to TRAIL apoptosis by multiple molecular mechanisms of action. Therefore, the combination of bortezomib and TRAIL might be a novel therapeutic strategy for ESCC patients who fail to respond to standard chemoradiotherapy that predominantly targets the mitochondrial apoptotic pathway.

Silencing of Fas-associated Death Domain Protects Mice from Septic Lung Inflammation and Apoptosis

A better understanding of the molecular mechanisms involved in the pathogenesis of sepsis and its resultant organ failure and new therapeutic approaches and targets are urgently needed. Accumulating evidence suggests that apoptosis plays an important role in the pathophysiology of sepsis and that apoptosis may be detrimental in septic acute lung injury (ALI). We tested the hypothesis that systemic administration of small interfering RNA (siRNA) targeting Fas-associated death domain (FADD), which recruits procaspase-8 into the death-inducing signaling complex, may be protective in septic ALI and mortality. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in BALB/c mice. In vivo delivery of siRNA was performed by using a transfection reagent at 10 hours after CLP. As a negative control, animals received nonsense (scrambled) siRNA. In CLP-induced septic mice, surface expression of death receptors was up-regulated, and FADD was highly expressed. DNA fragmentation ladder and transferase-mediated dUTP nick end labeling assays showed that treatment with FADD siRNA suppressed apoptosis induction in septic lungs. This siRNA treatment prevented the ALI development in CLP mice, as indicated by the findings that blood-gas derangements, histologic lung damage, and increased pulmonary inflammatory cells were greatly improved. Finally, FADD siRNA administration dramatically improved the survival of CLP mice. These results indicate the pathophysiologic significance of the death receptor apoptotic pathway, including FADD, in septic ALI and the potential usefulness of FADD siRNA for gene therapy of the septic syndrome.

In Vitro-Expanded NK Cells Have Increased TRAIL and NKG2D Expression and Enhanced TRAIL-Mediated Tumor Cytotoxicity Compared to Non-Expanded NK Cells.

The activation of NK cell inhibitory receptors may limit the antitumor efficacy of adoptive autologous and allogeneic NK cell infusions. Recently, we found that exposing malignant cells to the proteosome inhibitor bortezomib upregulated surface expression of death receptors for TRAIL, resulting in significant enhancement of autologous NK cell tumor cytotoxicity in vitro and in vivo. Here we show that NK cells expanded in vitro in the presence of IL-2 and EBV-LCL feeder cells upregulate surface expression of TRAIL which significantly augments bortezomib-induced tumor sensitization to NK cell killing. CD56+/CD3– NK cells were isolated from normal donors by immuno-magnetic bead selection and were co-cultured with irradiated EBV-LCL feeder cells in X-VIVO 20, 10% human AB serum, and 500 IU/ml hrIL-2 for up to 21 days. Depending on culture conditions, a 300 to 10,000 fold increase in NK cell numbers was achieved. Non-expanded and expanded NK cells were analyzed by flow cytometry for the expression of CD56, CD16, TRAIL, FasL, NKG2D, LFA-1, perforin, and granzymes A and B at baseline and ≥ 10 days following in vitro expansion. Chromium release assays were performed to assess fresh vs. expanded NK cell cytotoxicity of renal cell carcinoma (RCC) tumor targets treated with 10 nM bortezomib for 18 hr vs. untreated RCC controls. Freshly-isolated NK cells did not express TRAIL or FasL; in contrast NKG2D, LFA-1, perforin and granzymes A and B were constitutively expressed in fresh NK cells. After expansion, there was a dramatic increase in surface expression of TRAIL and NKG2D; on fresh vs. expanded NK cells from 3 different donors, TRAIL expression increased from 0% to 80.8±15.4% (mean fluorescence intensity [MFI] of TRAIL increased from 6.0±5.1 to 37.9±3.2). The MFI of NKG2D surface expression also increased following NK cell expansion (432.0±70.9 from 48.3±16.3). Expression of LFA-1 and perforin did not change, although there was a small increase in surface and intracellular expression of FasL and granzymes A and B respectively. At a 1:1 effector to target ratio, fresh NK cells lysed 3.4± 2.1% and 5.0± 2.7% of untreated and bortezomib-treated RCC tumor cells respectively. In contrast, there was a dramatic increase in bortezomib-treated tumor susceptibility to killing by expanded NK cells; NK cells expanded for 12-18 days killed 27.6± 9.3% and 55.8± 8.3% of untreated vs. bortezomib-treated RCC tumor cells respectively.Conclusion: In vitro-expanded NK cells are phenotypically and functionally different from non-expanded NK cells. Expanded cells have increased NKG2D and TRAIL expression and greatly enhanced TRAIL-mediated tumor cytotoxicity compared to non-expanded NK cells. Based on these findings, a phase I trial investigating the safety and anti-tumor effects of escalating doses of adoptively-infused ex vivo-expanded autologous NK cells following bortezomib treatment in patients with advanced metastatic tumors and hematological malignancies has recently been initiated. [Display omitted]

Cytoskeleton-mediated Death Receptor and Ligand Concentration in Lipid Rafts Forms Apoptosis-promoting Clusters in Cancer Chemotherapy

While investigating the mechanism of action of the novel antitumor drug Aplidin, we have discovered a potent and novel cell-killing mechanism that involves the formation of Fas/CD95-driven scaffolds in membrane raft clusters housing death receptors and apoptosis-related molecules. Fas, tumor necrosis factor-receptor 1, and tumor necrosis factor-related apoptosis-inducing ligand receptor 2/death receptor 5 were clustered into lipid rafts in leukemic Jurkat cells following Aplidin treatment, the presence of Fas being essential for apoptosis. Preformed membrane-bound Fas ligand (FasL) as well as downstream signaling molecules, including Fas-associated death domain-containing protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid, were also translocated into lipid rafts, connecting death receptor extrinsic and mitochondrial intrinsic apoptotic pathways. Blocking Fas/FasL interaction partially inhibited Aplidin-induced apoptosis. Aplidin was rapidly incorporated into membrane rafts, and drug uptake was inhibited by lipid raft disruption. Actin-linking proteins ezrin, moesin, RhoA, and RhoGDI were conveyed into Fas-enriched rafts in drug-treated leukemic cells. Disruption of lipid rafts and interference with actin cytoskeleton prevented Fas clustering and apoptosis. Thus, Aplidin-induced apoptosis involves Fas activation in both a FasL-independent way and, following Fas/FasL interaction, an autocrine way through the concentration of Fas, membrane-bound FasL, and signaling molecules in membrane rafts. These data indicate a major role of actin cytoskeleton in the formation of Fas caps and highlight the crucial role of the clusters of apoptotic signaling molecule-enriched rafts in apoptosis, acting as concentrators of death receptors and downstream signaling molecules and as the linchpin from which a potent death signal is launched.

Mechanisms of resistance to TRAIL-induced apoptosis in primary B cell chronic lymphocytic leukaemia.

Primary B cells from B cell chronic lymphocytic leukaemia (B-CLL) were resistant to the novel selective cytotoxic agent, TNF-related apoptosis-inducing ligand (TRAIL). Low levels of the death-inducing TRAIL receptors, TRAIL-R1 and TRAIL-R2 but not the putative 'decoy' receptors, TRAIL-R3 and TRAIL-R4, were expressed on the surface of B-CLL cells. Resistance to TRAIL was upstream of caspase-8 activation, as little or no caspase-8 was processed in TRAIL-treated B-CLL cells. Low levels of a TRAIL death-inducing signalling complex (DISC) were formed in these cells, accompanied by the recruitment of endogenous FADD, caspase-8 and c-FLIP(L) but not c-FLIP(S). Both caspase-8 and c-FLIP(L) were cleaved to form two stable intermediates of approximately 43 kDa, which remained associated with the DISC. Caspase-8 was not further processed to its active heterotetramer. Thus the resistance of B-CLL cells to TRAIL may be due partly to low surface expression of the death receptors resulting in low levels of DISC formation and also to the high ratio of c-FLIP(L) to caspase-8 within the DISC, which would prevent further activation of caspase-8. Our results highlight the possibility of sensitising B-CLL cells to TRAIL by modulation of c-FLIP levels or by upregulation of surface expression of death receptors.