Tumor Necrosis Factor-related Apoptosis-inducing Ligand Sensitivity Research Articles

Hypoxia regulates TRAIL sensitivity of colorectal cancer cells through mitochondrial autophagy.

The capacity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to selectively induce cell death in malignant cells triggered numerous attempts for therapeutic exploitation. In clinical trials, however, TRAIL did not live up to the expectations, as tumors exhibit high rates of TRAIL resistance in vivo. Response to anti-cancer therapy is determined not only by cancer cell intrinsic factors (e.g. oncogenic mutations), but also modulated by extrinsic factors such as the hypoxic tumor microenvironment.Here, we address the effect of hypoxia on pro-apoptotic TRAIL signaling in colorectal cancer cells. We show that oxygen levels modulate susceptibility to TRAIL-induced cell death, which is severely impaired under hypoxia (0.5% O2). Mechanistically, this is attributable to hypoxia-induced mitochondrial autophagy. Loss of mitochondria under hypoxia restricts the availability of mitochondria-derived pro-apoptotic molecules such as second mitochondria-derived activator of caspase (SMAC), thereby disrupting amplification of the apoptotic signal emanating from the TRAIL death receptors and efficiently blocking cell death in type-II cells. Moreover, we identify strategies to overcome TRAIL resistance in low oxygen environments. Counteracting hypoxia-induced loss of endogenous SMAC by exogenous substitution of SMAC mimetics fully restores TRAIL sensitivity in colorectal cancer cells. Alternatively, enforcing a mitochondria-independent type-I mode of cell death by targeting the type-II phenotype gatekeeper X-linked inhibitor of apoptosis protein (XIAP) is equally effective.Together, our results indicate that tumor hypoxia impairs TRAIL efficacy but this limitation can be overcome by combining TRAIL with SMAC mimetics or XIAP-targeting drugs. Our findings may help to exploit the potential of TRAIL in cancer therapy.

Inhibition of PI3K-Akt pathway reverses LMP1 induced TRAIL resistance in nasopharyngeal carcinoma cell

Objective:To observe the role of PI3K-Akt pathway in epsterin barr virus latent membrane protein 1(LMP1) induced TNF-related apoptosis inducing ligand(TRAIL) resistance in nasopharyngeal carcinoma(NPC) cell. Method:Expressions of Akt and p-Akt were detected after LMP1 overexpression by western blot. Colocation of LMP1 and p-Akt was detected by immunofluorescence. PI3K-Akt pathway was inhibited by LY294002 in CNE 1 and CNE 1+ LMP1. Then TRAIL sensitivity of CNE 1+ LMP1 and CNE 1 was analyzed by MTT and flow cytometric analysis, respectively. Activation of cell extrinsic apoptotic pathway was analyzed by western blot. Result:The expression of p-Akt in CNE 1+ LMP1 was higher than CNE 1. Immunofluorescence showed that after LMP1 overexpression in CNE 1, p-Akt was translocated from cytoplasm to membrane. With 1 μmol/ml LY294002 pretreatment for 8 hours, p-Akt expression was both downregulated in CNE 1 and CNE 1+ LMP1, but there was no significant difference between them(P >0.05). Moreover, the cytotoxicity and apoptosis induced by TRAIL in CNE 1 were enhanced after LMP1 overexpression and there was no significant difference between CNE 1 and CNE 1+ LMP1(P >0.05). Conclusion:LMP1 induced TRAIL-resistance in NPC cell through activeting PI3K-Akt pathway, sug-gesting that therapy targeting PI3K-Akt pathway could reverse TRAIL-resistance in NPC.

Tanshinone IIA induces TRAIL sensitization of human lung cancer cells through selective ER stress induction.

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promised anticancer medicine targeting only the tumor, most cancers show resistance to TRAIL-induced apoptosis. For this reason, new therapeutic strategies to overcome the TRAIL resistance are required for more effective tumor treatment. In the present study, potential of tanshinone IIA as a TRAIL sensitizer was evaluated in human non-small cell lung cancer (NSCLC) cells. NSCLC cells showed resistance to TRAIL-mediated cell death, but combination treatment of Tanshinone IIA and TRAIL synergistically decreased cell viability and increased apoptosis in TRAIL-resistant NSCLC cells. Tanshinone IIA greatly induced death receptor 5 (DR5), but not death receptor 4 (DR4). Furthermore, DR5 knockdown attenuated the combination treatment of tanshinone IIA with TRAIL-mediated cell death in human NSCLC cells. Tanshinone IIA also increased CHOP and activated the PERK-ATF4 pathway suggesting that tanshinone IIA increased DR5 and CHOP by activating the PERK-ATF4 pathway. Tanshinone IIA also downregulated phosphorylation of STAT3 and expression of survivin. Taken together, these results indicate that tanshinone IIA increases TRAIL-induced cell death via upregulating DR5 and downregulating survivin mediated by, respectively, selective activation of PERK/ATF4 and inhibition of STAT3, suggesting combinatorial intervention of tanshinone IIA and TRAIL as a new therapeutic strategy for human NSCLC.

Paclitaxel enhances tumoricidal potential of TRAIL via inhibition of MAPK in resistant gastric cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds promise for cancer therapy due to its unique capacity to selectively trigger apoptosis in cancer cells. However, TRAIL therapy is greatly hampered by its resistance. A preclinical successful strategy is to identify combination treatments that sensitize resistant cancers to TRAIL. In the present study, we fully assessed TRAIL sensitivity in 9 gastric cancer cell lines. We found combined administration of paclitaxel (PTX) markedly enhanced TRAIL-induced apoptosis in resistant cancer cells both in vitro and in vivo. The sensitization to TRAIL was accompanied by activation of mitochondrial apoptotic pathway, upregulation of TRAIL receptors and downregulation of anti-apoptotic proteins including C-IAP1, C-IAP2, Livin and Mcl-1. Noticeably, we found PTX could suppress the activation of mitogen-activated protein kinases (MAPKs). Inhibition of MAPKs using specific inhibitors (ERK inhibitor U0126, JNK inhibitor SP600125 and P38 inhibitor SB202190) facilitated TRAIL-mediated apoptosis and cytotoxicity. Additionally, SP600125 upregulated TRAL receptors as well as downregulated C-IAP2 and Mcl-1 suggesting the anti-apoptotic role of JNK. Thus, PTX-induced suppression of MAPKs may contribute to restoring TRAIL senstitivity. Collectively, our comprehensive analyses gave new insight into the role of PTX on enhancing TRAIL sensitivity, and provided theoretical references on the development of combination treatment in TRAIL-resistant gastric cancer.

Decursin enhances TRAIL-induced apoptosis through oxidative stress mediated- endoplasmic reticulum stress signalling in non-small cell lung cancers.

The TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. However, because most tumours exhibit resistance to TRAIL-induced apoptosis, the development of combination therapies to overcome resistance to TRAIL is required for effective cancer therapy. Cell viability and possible synergy between the plant pyranocoumarin decursin and TRAIL was measured by MTT assay and calcusyn software. Reactive oxygen species (ROS) and apoptosis were measured using dichlorodihydrofluorescein and annexin/propidium iodide in cell flow cytometry. Changes in protein levels were assessed with Western blotting. Combining decursin and TRAIL markedly decreased cell viability and increased apoptosis in TRAIL-resistant non-small-cell lung cancer (NSCLC) cell lines. Decursin induced expression of the death receptor 5 (DR5). Inhibition of DR5 attenuated apoptotic cell death in decursin + TRAIL treated NSCLC cell lines. Interestingly, induction of DR5 and CCAAT/enhancer-binding protein homologues protein by decursin was mediated through selective induction of the pancreatic endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) branch of the endoplasmic reticulum stress response pathway. Furthermore, enhancement of PERK/ATF4 signalling by decursin was mediated by ROS generation in NSCLC cell lines, but not in normal human lung cells. Decursin also markedly down-regulated expression of survivin and Bcl-xL in TRAIL-resistant NSCLC cells. ROS generation by decursin selectively activated the PERK/ATF4 axis of the endoplasmic reticulum stress signalling pathway, leading to enhanced TRAIL sensitivity in TRAIL-resistant NSCLC cell lines, partly via up-regulation of DR5.

Morusin Induces TRAIL Sensitization by Regulating EGFR and DR5 in Human Glioblastoma Cells.

Glioblastoma is one of the most malignant primary tumors, and the prognosis for glioblastoma patients remains poor. Tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) is considered a promising anticancer agent due to its remarkable ability to selectively kill tumor cells. However, since many cancers are resistant to TRAIL, strategies to overcome resistance are required for the successful use of TRAIL in the clinic. In the present study, the potential of morusin as a TRAIL sensitizer in human glioblastoma cells was evaluated. Treatment with TRAIL or morusin alone showed weak cytotoxicity in human glioblastoma cells. However, combination treatment of TRAIL with morusin synergistically decreased cell viability and increased apoptosis compared with single treatment. Morusin induced expression of death receptor 5 (DR5), but not DR4 or decoy receptors (DcR1 and DcR2). Furthermore, morusin significantly decreased anti-apoptotic molecules survivin and XIAP. In addition, morusin reduced expression of EGFR and PDFGR as well as phosphorylation of STAT3, possibly mediating down-regulation of survivin and XIAP. Together these results suggest that morusin enhances TRAIL sensitivity in human glioblastoma cells through regulating expression of DR5 and EGFR. Therefore, the combination treatment of TRAIL and morusin may be a new therapeutic strategy for malignant glioma patients.

Trichostatin A potentiates TRAIL-induced antitumor effects via inhibition of ERK/FOXM1 pathway in gastric cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an ideal apoptosis inducer and believed to have promise in cancer therapy, yet part of cancer cells exhibit resistance to TRAIL-mediated apoptosis. This necessitates the exploration of agents that resensitizes cancer cells to TRAIL. In our study, we found that Trichostatin A (TSA), an histone deacetylase (HDAC) inhibitor, augmented TRAIL-induced apoptosis in gastric cancer cells in a caspase-dependent manner. Besides, upregulation of DR5 and downregulation of anti-apoptotic proteins including XIAP, Mcl-1, Bcl-2 and Survivin also contributed to this synergism. Noticeably, TSA treatment inhibited Forkhead boxM1 (FOXM1), which expression level showed negative correlation with TRAIL sensitivity. Similarly, silencing of FOXM1 by small interfering RNA (siRNA) resensitized cancer cells to TRAIL and strengthened the TRAIL-augmenting effect of TSA. In addition, we demonstrated the depletion of FOXM1 was a consequence of the inactivation of ERK mediated by TSA. Collectively, it was first shown that TSA potentiated TRAIL sensitivity via ERK/FOXM1 pathway in gastric cancer cells. FOXM1 might serve as a biomarker for predicting sensitivity to TRAIL.

Modulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by Helicobacter pylori in immune pathogenesis of gastric mucosal damage.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer, gastric carcinoma, and gastric mucosa-associated lymphoid tissue lymphomas. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Enhanced gastric epithelial cell apoptosis during H. pylori infection was suggested to play an important role in the pathogenesis of chronic gastritis and gastric pathology. In addition to directly triggering apoptosis, H. pylori induces sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in gastric epithelial cells. Human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death-receptor signaling. The induction of TRAIL sensitivity by H. pylori is dependent upon the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex through downregulation of cellular FLICE-inhibitory protein. Moreover, H. pylori infection induces infiltration of T lymphocytes and triggers inflammation to augment apoptosis. In H. pylori infection, significant increases in CCR6+ CD3+ Tcell infiltration in the gastric mucosa was observed, and the CCR6 ligand, CCL20 chemokine, was selectively expressed in inflamed gastric tissues. These mechanisms initiate chemokine-mediated T lymphocyte trafficking into inflamed epithelium and induce mucosal injury during Helicobacter infection. This article will review recent findings on the interactions of H. pylori with host-epithelial signaling pathways and events involved in the initiation of gastric pathology, including gastric inflammation and mucosal damage.

Choline kinase inhibitors synergize with TRAIL in the treatment of colorectal tumors and overcomes TRAIL resistance

Aim: The aim of this study was to investigate the effects of the combination of choline kinase inhibitor MN58b and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) against colon cancer cells. Methods: TRAIL-sensitive (DLD-1) and TRAIL-resistant (SW620) cells were treated with MN58b and/or TRAIL. Cell viability and induction of apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide and flow cytometry. Posttreatment expression levels of different proteins (PARP, caspase-3, X-linked inhibitor of apoptosis protein [XIAP], CHOP, DR5, DR4, CHOP) were analyzed by quantitative reverse transcription polymerase chain reaction, Western blot, and flow cytometry.In vivo antitumoral activity was assessed by xenograft models. Results: A strong synergistic effect of TRAIL and MN58b was observed in both TRAIL-sensitive and resistant cells. The combinatory treatment induced an increase in PARP and active-caspase 3 fragments along with a decrease in XIAP, enhancing TRAIL sensitivity. Reduced cellular viability and increased cell death correlated with increased DR5 expression and membrane surface recruitment, an effect that was concomitant with CHOP expression. Conclusion: MN58b, which alone exhibits anticancer activities against a wide variety of tumor-derived cell lines, synergizes with TRAIL through a mechanism that involves DR5 upregulation. This study supports the use of MN58b in combination with TRAIL on colorectal tumors, including those that develop TRAIL resistance.

A promising "TRAIL" of tanshinones for cancer therapy.

An ideal cancer therapy specifically targets cancer cells while sparing normal tissues. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) elicits apoptosis by engaging its cognate death receptors (DRs—namely, DR4 and DR5. The cancer cell-selective proapoptotic action of TRAIL is highly attractive for cancer therapy, but clinical application of TRAIL is rather limited due to tumors’ inherent or acquired TRAIL resistance. Combining TRAIL with agents that reverse resistance to it has proved promising in the sensitization of TRAIL-induced apoptosis. Noteworthy, natural compounds have already been validated as potential resources for TRAIL sensitizers. In this review, we focus on the recently identified TRAILsensitizing effect of tanshinones, the anticancer ingredients of the medicinal plant Salvia miltiorrhiza (Danshen in Chinese). Research from our laboratories and others have revealed the synergy of a tanshinones-TRAIL combination in diverse types of cancer cells through up-regulation of DR5 and/or down-regulation of antiapoptotic proteins such as survivin. Thus, in addition to their anticancer mechanisms, tanshinones as TRAIL sensitizers hold great potential to be translated to TRAIL-based therapeutic modalities for combatting cancer.

Hypoxia-mediated activation of autophagic flux inhibits apoptosis of keratinocytes via blocking tumor necrosis factor-related apoptosis-inducing ligand

Tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) is toxic against transformed tumor cells. Cornification is the terminal differentiation of keratinocytes and a specific form of programmed cell death caused by TRAIL that occurs in keratinocytes. Apoptosis can also be triggered when TRAIL induces expression of keratinocyte differentiation markers. The present study reported that hypoxia inhibits TRAIL‑induced apoptosis due to autophagic flux. It is well known that hypoxia activates autophagy in keratinocytes and reduces p62 protein levels. The present study demonstrated that hypoxia inhibited TRAIL‑mediated apoptosis and induced autophagic flux in HaCaT cells. In addition, autophagic flux‑inactivating reagents, including 3‑methyladenine and chloroquine, increased the TRAIL sensitivity of HaCaT cells exposed to hypoxia. In conclusion, these results indicated that inactivating autophagy increased TRAIL sensitivity in hypoxic HaCaT cells. Autophagy inhibitors may be beneficial in therapies using TRAIL against skin cancers.

An added dimension to tumour TRAIL sensitivity.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF cytokine family and a selective inducer of apoptosis in a range of tumour cells, but not in healthy normal, untransformed cells. It is expressed by natural killer cells and natural killer-T cells when they encounter malignantly transformed cells and it is a key effector molecule in tumour immune surveillance. TRAIL has 5 receptors, which is the highest receptor promiscuity in the TNF ligand family. It binds to death receptor 4 (DR4) or DR5 on the surface of target cells [1] and initiates a conformational change which promotes association of the receptors with FADD facilitating pro-caspase-8 and/or pro-caspase-10 recruitment which then activates effector caspases to execute cell death [2]. Signalling through DR4 and DR5 can also activate pro-inflammatory intracellular molecules such as MAPK, PKB and NF-κB and overexpression of DR4 or DR5 has been shown to stimulate the release of inflammatory cytokines [3]. However, TRAIL also has three regulatory receptors. Two of these, decoy receptor 1(DcR1) and DcR2 are membrane bound and the third regulatory receptor, osteoprotegerin is a secreted protein. DcRs regulate TRAIL-induced apoptosis by either sequestering TRAIL from the death receptors or by forming inactive, heteromeric DcR1/2–DR4/5 complexes [1]. Indeed, DcRs have been shown to be highly expressed in a number of tumour tissues such as acute myeloid leukaemia, prostate cancer and breast cancer and their expression is linked with poor prognosis [4]. However DcR expression in tumour cells does not correlate with TRAIL sensitivity and non-transformed cells do not require DcRs to be protected from TRAIL-induced apoptosis, suggesting that the in vivo role of the DcRs may be more complex than originally thought [5] The tumor-specific cytotoxicity of TRAIL has been exploited as a therapeutic strategy by utilizing recombinant versions of TRAIL and agonistic antibodies against DR4 and DR5 [6]. While recombinant soluble human TRAIL was highly potent against a broad range of tumours in vitro and in pre-clinical studies, in clinical trials TRAIL has failed to exhibit the same potency [6]. One of the major shortcomings of the preclinical models was the lack of assessment of the contribution of the tumour microenvironment (TME). The TME consists of various cell types, soluble factors and signals from the extracellular matrix, and is in a reciprocal interaction with the tumour cells. It is thus important to understand the interplay of different cell types in the tumour microenvironment and the effect of the factors they express and secrete on tumour growth, development and resistance to therapy. Figure 1 Cell autonomous and supracellular regulation of TRAIL-sensitivity by decoy receptor 1 (DcR1) and -2 The study by O'Leary and colleagues explored the hypothesis that DcRs exerted a ‘supracellular level control’ of TRAIL-sensitivity rather than simply regulating TRAIL resistance at a cell-autonomous level [7]. They firstly examined the expression of the decoy receptors in tumour cells, tumour stroma and in non-malignant, tumour adjacent tissues. Interestingly they found that DcR1 and DcR2 are commonly expressed in tissues but tissue stroma only express DcR1. To determine whether DcR-expressing stromal cells influence the TRAIL sensitivity of tumour cells sharing the same microenvironment they generated and characterized DcR-insensitive TRAIL mutants and used a combination of mathematical modelling, cell based assays and a stroma/tumour co-culture system allowing them to model the effect of DcRs expressed by adjacent stromal cells on DR4/5 activation in tumour cells. They found that stromal DcRs profoundly reduced TRAIL-induced DR4/DR5 activation and protected tumour cells against TRAIL. The authors confirmed this finding in a 3D mixed-cell type (stroma-tumour) spheroid tumour model. Interestingly they also observed that TNF increased surface expression of the DcRs in fibroblasts raising the possibility that an inflammatory environment may induce DcR expression and promote TRAIL resistance. This study clearly demonstrates that stromal DcRs in the tumour microenvironment can exert trans-cellular regulation affecting tumour cells and it highlights the importance of developing therapeutic TRAIL variants that can selectively activate the two death inducing TRAIL receptors but are not mopped up by the decoy receptors present on stromal tissues. Future studies will hopefully establish a feed forward signalling loop that drives overall tissue sensitivity in order to achieve tumour eradication.

Sensitization of Melanoma Cells for Death Ligand TRAIL Is Based on Cell Cycle Arrest, ROS Production, and Activation of Proapoptotic Bcl-2 Proteins

The death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) represents a promising strategy for melanoma due to significant expression of TRAIL receptor 1 in melanoma metastases and high TRAIL sensitivity through this receptor. However, prevalent and inducible resistance are limiting its clinical use. In previous work, we and others have described multiple strategies leading to TRAIL sensitization; however, the common principles of these strategies remained elusive. Here, we demonstrate in melanoma cell lines (TRAIL-sensitive, TRAIL-resistant, and TRAIL-selected cells with acquired resistance) that cell cycle arrest clearly correlates with enhanced TRAIL sensitivity. Cell cycle arrest was induced by high cell confluence, serum starvation, or cyclin-dependent kinase (CDK) 4/6 inhibition. Addressing the signaling pathways revealed disruption of mitochondrial membrane potential and production of reactive oxygen species (ROS) in response to antiproliferative conditions alone. Activation of the proapoptotic Bcl-2 protein Bax and inhibition of apoptosis by Bcl-2 overexpression or by the antioxidant N-acetyl cysteine underlined the critical involvement of mitochondrial apoptosis pathways and of ROS, respectively. Most pronounced was the upregulation of small proapoptotic Bcl-2 proteins (Puma and Bcl-xS). These data provide a general understanding on TRAIL sensitization as well as an alternative view on CDK inhibitors and may suggest selective targeting of melanoma cells by cell cycle inhibition and TRAIL.

CBIO-08IGFBP2 IS A NOVEL MOLECULAR DETERMINANT IN TRAIL-RESISTANT SUBPOPULATIONS OF GBM CELL LINES

Glioblastoma Multiforme (GBM) is the most common and aggressive brain tumor, as survival rate is less than 12 months after diagnosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in tumor cells; therefore, it has appeared as a strong candidate for cancer treatment. Studies have shown that many cancer cell lines are resistant to TRAIL, however the mechanisms regulating this resistance are not well understood. Moreover, a number of cancer cells exhibit partial sensitivity to TRAIL. In this study, we describe TRAIL-resistant cell line models. Firstly, we selected TRAIL-resistant subpopulations of a GBM cell line, which displays partial TRAIL sensitivity. U87MG, mid-TRAIL sensitive cell line, was treated with different, but fixed concentrations of TRAIL for several weeks, after which the surviving cell fractions were expanded in TRAIL-free conditions. Cell viability and caspase assays revealed that these cells exhibited sustained TRAIL-resistance. To examine the molecular differences in subpopulations, we examined the expression of apoptosis-related genes with qPCR and observed that Bcl-xL was significantly upregulated in TRAIL-resistant populations. We used genome wide RNA sequencing for understanding the gene expression changes and observed upregulation of IGFBP2 gene in resistant subpopulations compared with control cells. Our future efforts are directed at understanding the role of IGFBP2 gene for resistance mechanisms Our results will provide further understanding of molecular mechanisms of tumor heterogeneity in GBM cells, and help with the novel treatment strategies.

Bcl-xL inhibition by molecular-targeting drugs sensitizes human pancreatic cancer cells to TRAIL.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various types of cancer cells without damaging normal cells. However, in terms of pancreatic cancer, not all cancer cells are sensitive to TRAIL. In this study, we examined a panel of human pancreatic cancer cell lines for TRAIL sensitivity and investigated the effects of Bcl-2 family inhibitors on their response to TRAIL. Both ABT-263 and ABT-737 inhibited the function of Bcl-2, Bcl-xL, and Bcl-w. Of the nine pancreatic cancer cell lines tested, six showed no or low sensitivity to TRAIL, which correlated with protein expression of Bcl-xL. ABT-263 significantly sensitized four cell lines (AsPC-1, Panc-1, CFPAC-1, and Panc10.05) to TRAIL, with reduced cell viability and increased apoptosis. Knockdown of Bcl-xL, but not Bcl-2, by siRNA transfection increased the sensitivity of AsPC-1 and Panc-1 cells to TRAIL. ABT-263 treatment had no effect on protein expression of Bcl-2, Bcl-xL, or c-FLIPs. In Panc-1 cells, ABT-263 increased the surface expression of death receptor (DR) 5; the NF-κB pathway, but not endoplasmic reticulum stress, participated in the increase. In xenograft mouse models, the combination of TRAIL and ATB-737 suppressed the in vivo tumor growth of AsPC-1 and Panc-1 cells. These results indicate that Bcl-xL is responsible for TRAIL resistance in human pancreatic cancer cells, and that Bcl-2 family inhibitors could represent promising reagents to sensitize human pancreatic cancers in DR-targeting therapy.

Delivery of tumor-homing TRAIL sensitizer with long-acting TRAIL as a therapy for TRAIL-resistant tumors

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has attracted great interest as a cancer therapy because it selectively induces death receptor (DR)-mediated apoptosis in cancer cells while sparing normal tissue. However, recombinant human TRAIL demonstrates limited therapeutic efficacy in clinical trials, possibly due to TRAIL-resistance of primary cancers and its inherent short half-life. Here we introduce drug delivery approaches to maximize in vivo potency of TRAIL in TRAIL-resistant tumor xenografts by (1) extending the half-life of the ligand with PEGylated TRAIL (TRAILPEG) and (2) concentrating a TRAIL sensitizer, selected from in vitro screening, in tumors via tumor-homing nanoparticles. Antitumor efficacy of TRAILPEG with tumor-homing sensitizer was evaluated in HCT116 and HT-29 colon xenografts. Western blot, real-time PCR, immunohistochemistry and cell viability assays were employed to investigate mechanisms of action and antitumor efficacy of the combination. We discovered that doxorubicin (DOX) sensitizes TRAIL-resistant HT-29 colon cancer cells to TRAIL by upregulating mRNA expression of DR5 by 60% in vitro. Intravenously administered free DOX does not effectively upregulate DR5 in tumor tissues nor demonstrate synergy with TRAILPEG in HT-29 xenografts, but rather introduces significant systemic toxicity. Alternatively, when DOX was encapsulated in hyaluronic acid–based nanoparticles (HAC/DOX) and intravenously administered with TRAILPEG, DR-mediated apoptosis was potentiated in HT-29 tumors by upregulating DR5 protein expression by 70% and initiating both extrinsic and intrinsic apoptotic pathways with reduced systemic toxicity compared to HAC/DOX or free DOX combined with TRAILPEG (80% vs. 40% survival rate; 75% vs. 34% tumor growth inhibition). This study demonstrates a unique approach to overcome TRAIL-based therapy drawbacks using sequential administration of a tumor-homing TRAIL sensitizer and long-acting TRAILPEG.

Temozolomide sensitizes stem-like cells of glioma spheres to TRAIL-induced apoptosis via upregulation of casitas B-lineage lymphoma (c-Cbl) protein.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has potent antitumor effects in glioma cell lines but has shown little clinical benefit for patients. We investigated whether the widely used chemotherapeutic agent temozolomide (TMZ) can sensitize glioma stem-like cells (GSCs) from human glioblastoma multiforme (GBM) to TRAIL-induced apoptosis. GSCs were isolated from GBM, and stem cell properties were confirmed by immunocytochemistry and in vivo tumorigenicity. Primary GSCs (PGCs) were produced by serum treatment of GBM-derived cells. Changes in expression levels of various TRAIL-related signaling factors before and after TRAIL or TRAIL + TMZ treatment were measured by Western blotting. Overexpression vectors and siRNAs were used to investigate mechanism of TRAIL sensitivity. GSCs showed greater resistance to TRAIL-induced apoptosis than PGCs and had lower basal caspase activity. Caspase knockdown in PGCs reduced TRAIL sensitivity. Expression levels of c-Fas-associated death domain-like interleukin 1-converting enzyme-like inhibitory protein long and short isoforms (c-FLIPL and c-FLIPS) were significantly higher in GSCs than PGCs, and siRNA-mediated c-FLIP knockdown in GSCs enhanced TRAIL-induced apoptosis. TMZ enhanced TRAIL-induced apoptosis in GSCs and downregulated c-FLIP expression. Add of TMZ also upregulated the expression of the E3 ubiquitin ligase casitas B-lineage lymphoma (c-Cbl). Moreover, overexpression of c-Cbl alone reduced c-FLIP expression, and c-Cbl knockdown both enhanced c-FLIP expression and reduced the potentiating effect of TMZ on TRAIL-induced apoptosis. The result indicated that TMZ may overcome TRAIL resistance in GSCs by suppressing c-FLIP expression through c-Cbl-mediated ubiquitination and degradation.

Decoy receptors block TRAIL sensitivity at a supracellular level: the role of stromal cells in controlling tumour TRAIL sensitivity.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand cytokine known for its cytotoxic activity against malignantly transformed cells. TRAIL induces cell death through binding to death receptors DR4 and DR5. The inhibitory decoy receptors (DcR1 and DcR2) co-expressed with death receptor 4 (DR4)/DR5 on the same cell can block the transmission of the apoptotic signal. Here, we show that DcRs also regulate TRAIL sensitivity at a supracellular level and thus represent a mechanism by which the microenvironment can diminish tumour TRAIL sensitivity. Mathematical modelling and layered or spheroid stroma-extracellular matrix-tumour cultures were used to model the tumour microenvironment. By engineering TRAIL to escape binding by DcRs, we found that DcRs do not only act in a cell-autonomous or cis-regulatory manner, but also exert trans-cellular regulation originating from stromal cells and affect tumour cells, highlighting the potent inhibitory effect of DcRs in the tumour tissue and the necessity of selective targeting of the two death-inducing TRAIL receptors to maximise efficacy.

Notch4 Signaling Confers Susceptibility to TRAIL-Induced Apoptosis in Breast Cancer Cells.

Notch signaling has been established as a key regulator of cell fate in development, differentiation, and homeostasis. In breast cancers, increased Notch1 and Notch4 activity have been implicated in tumor progression and, accumulation of the intracellular domain of Notch4 (ICN4), reported in basal breast cancer cells. While, TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists have demonstrated selectively in targeting tumor cells, the majority of primary tumors are resistant to TRAIL. This necessitates the identification of factors that might regulate TRAIL sensitivity. Here we investigate TRAIL sensitivity in tumor cells following the modulation of Notch (1 and 4) activity using siRNA-mediated depletions or ectopic expression of GFP-tagged constructs of the intracellular domains of Notch1 (ICN1) or Notch4 (ICN4). Our findings suggest that Notch4, but not Notch1 signaling, sensitizes breast tumor cells to TRAIL-induced apoptosis. ICN4-induced sensitization to TRAIL is characterized by CBF1-dependence. Apoptosis was mediated via caspase-8 activation and regulated by the Bcl-2 family pro-apoptotic proteins Bak and Bid. Finally, we present evidence that endogenous Notch4 activity regulates susceptibility to TRAIL in basal-like breast cancer cells but not in cell lines of luminal origin. These experiments reveal a hitherto unexplored Notch4-TRAIL signaling axis in breast cancer cells.

Metformin Causes G1-Phase Arrest via Down-Regulation of MiR-221 and Enhances TRAIL Sensitivity through DR5 Up-Regulation in Pancreatic Cancer Cells.

Although many chemotherapeutic strategies against cancer have been developed, pancreatic cancer is one of the most aggressive and intractable types of malignancies. Therefore, new strategies and anti-cancer agents are necessary to treat this disease. Metformin is a widely used drug for type-2 diabetes, and is also known as a promising candidate anti-cancer agent from recent studies in vitro and in vivo. However, the mechanisms of metformin’s anti-cancer effects have not been elucidated. We demonstrated that metformin suppressed the expression of miR-221, one of the most well-known oncogenic microRNAs, in human pancreatic cancer PANC-1 cells. Moreover, we showed that the down-regulation of miR-221 by metformin caused G1-phase arrest via the up-regulation of p27, one of the direct targets of miR-221. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also a promising agent for cancer treatment. While recent studies showed that treatment with only TRAIL was not effective against pancreatic cancer cells, the present data showed that metformin sensitized p53-mutated pancreatic cancer cells to TRAIL. Metformin induced the expressions of death receptor 5 (DR5), a receptor for TRAIL, and Bim with a pro-apoptotic function in the downstream of TRAIL-DR5 pathway. We suggest that the up-regulation of these proteins may contribute to sensitization of TRAIL-induced apoptosis. The combination therapy of metformin and TRAIL could therefore be effective in the treatment of pancreatic cancer.