Recombinant Human Tumor Necrosis Factor-related Apoptosis-inducing Ligand Research Articles

Novel Structurally Related Flavones Augment Cell Death Induced by rhsTRAIL.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) was identified as a powerful activator of apoptosis in tumor cells and one of the most promising candidates for cancer therapy with no toxicity against normal tissues. However, many tumor cells are resistant to TRAIL-induced apoptosis. The aim of this work was to analyze the improvement of the anticancer effect of rhsTRAIL (recombinant human soluble TRAIL) by nine flavones: 5-Hydroxyflavone, 6-Hydroxyflavone, 7-Hydroxyflavone and their new synthetic derivatives 5-acetoxyflavone, 5-butyryloxyflavone, 6-acetoxyflavone, 6-butyryloxyflavone, 7-acetoxyflavone and 7-butyryloxyflavone. We examined the cytotoxic and apoptotic effects of rhsTRAIL enhanced by novel structurally-related flavones on SW480 and SW620 colon cancer cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test, the lactate dehydrogenase assay and annexin V-FITC fluorescence staining. We observed a slight difference in the activities of the flavones that was dependent on their chemical structure. Our study indicates that all nine flavones significantly augment cell death by rhsTRAIL (cytotoxicity range 36.8 ± 1.7%–91.4 ± 1.7%; apoptosis increase of 33.0 ± 0.7%–78.5 ± 0.9%). Our study demonstrates the potential use of tested flavones in TRAIL-based anticancer therapy and prevention.

Effects of A.marina-Derived Isoquercitrin on TNF-Related Apoptosis-Inducing Ligand Receptor (TRAIL-R) Expression and Apoptosis Induction in Cervical Cancer Cells.

TNF-related apoptosis-inducing ligand (TRAIL) is an anticancer agent, which has greater apoptosis inducing capacity, but most of the cancer cells become resistant to TRAIL-induced apoptosis. The combined treatment of TRAIL with natural products could restore the cancer cell sensitivity to recombinant human TRAIL (rhTRAIL) protein and might enhance the TNF-related apoptosis-inducing ligand receptor (TRAIL-R) expression. This investigation was aimed to isolate flavonoids from leaves of Avicennia marina and evaluate their potential for sensitization of rhTRAIL in human cervical cancer cells (SiHa). The methanolic extract of A.marina leaves were purified and structure was elucidated as isoquercitrin by NMR and LC-MS analysis. Isolated isoquercitrin showed cytotoxicity against SiHa cell line at IC50 of 980μM. Messenger RNA (mRNA) expression of TRAIL-Rs was quantified by qRT-PCR, combination of isoquercitrin, and/or rhTRAIL increased TRAIL-R1 and TRAIL-R2 gene expression by 7 folds and 4 folds, respectively. Also, FACS assay revealed that combined treatment has increased the early apoptosis up to 7.24%. In the present study, we found that isoquercitrin enhances the mRNA expression of TRAIL-Rs, but the percentage of apoptosis was meager, possibly due to the influence of other anti-apoptotic proteins.

Involvement of DR4/JNK pathway-mediated autophagy in acquired TRAIL resistance in HepG2 cells

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, a number of cancer cells demonstrate TRAIL resistance. To date, various molecular targets leading to TRAIL resistance have been elucidated by many researchers, but the mechanisms involved are still not fully understood. In the present study, we obtained TRAIL-resistant cells from the human hepatocellular carcinoma cell line HepG2 by exposing cells to recombinant human TRAIL (rhTRAIL), and determined a mechanism for TRAIL resistance. The selected TRAIL-resistant cells (HepG2-TR) were insensitive to rhTRAIL and triggered autophagy in response to rhTRAIL. The inhibition of autophagy by 3-methyladenine or the knockdown of ATG5 partially restored rhTRAIL-induced apoptosis and cytotoxicity, indicating that protective autophagy occurred in the cells. Notably, rhTRAIL-induced autophagy was mediated through DR4 in HepG2-TR cells, but not in parental HepG2 cells. In addition, the c-Jun N-terminal kinase was involved in DR4-mediated autophagy in HepG2-TR cells. Our results suggest a novel mechanism of TRAIL resistance which is regulated through alterations in DR4 function, which may extend our understanding of the mechanisms of TRAIL resistance.

Fed-batch production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in soluble form in Escherichia coli and its purification and characterization

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent. The aim of this study is to produce large quantities of highly pure and bioactive recombinant human TRAIL. Here, TRAIL was expressed in soluble form by pH-stat fed-batch cultivation and purified using a rapid and simple two-step chromatographic procedure. To improve the soluble yield, expression of TRAIL in Escherichia coli was induced with low IPTG concentration (0.1 mM) at low temperature (28 °C) supplemented with ZnSO4 (0.5 mM), using glycerol as carbon source. Under the optimized conditions, 4.14 ± 0.19 g/L of TRAIL in soluble form was achieved at 19 h without pure oxygen. To purify the recombinant TRAIL, we developed an efficient two-step chromatographic procedure including affinity chromatography and cation-exchange chromatography, especially improved the cation-exchange chromatography using a combination of pH and NaCl gradients strategy. Consequently, 4313.5 mg of target protein with high purity (98.1%) was obtained from 2.3 L of cell broth. Our results also showed that the purified TRAIL was with ordered secondary and tertiary structures, in homogeneous form and with strong cytotoxicity.

Onto better TRAILs for cancer treatment.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand (Apo2L), is a member of the TNF cytokine superfamily. By cross-linking TRAIL-Receptor (TRAIL-R) 1 or TRAIL-R2, also known as death receptors 4 and 5 (DR4 and DR5), TRAIL has the capability to induce apoptosis in a wide variety of tumor cells while sparing vital normal cells. The discovery of this unique property among TNF superfamily members laid the foundation for testing the clinical potential of TRAIL-R-targeting therapies in the cancer clinic. To date, two of these therapeutic strategies have been tested clinically: (i) recombinant human TRAIL and (ii) antibodies directed against TRAIL-R1 or TRAIL-R2. Unfortunately, however, these TRAIL-R agonists have basically failed as most human tumors are resistant to apoptosis induction by them. It recently emerged that this is largely due to the poor agonistic activity of these agents. Consequently, novel TRAIL-R-targeting agents with increased bioactivity are currently being developed with the aim of rendering TRAIL-based therapies more active. This review summarizes these second-generation novel formulations of TRAIL and other TRAIL-R agonists, which exhibit enhanced cytotoxic capacity toward cancer cells, thereby providing the potential of being more effective when applied clinically than first-generation TRAIL-R agonists.

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.

Abstract A28: TRAIL mediated reduction of an immunogenic cell death protein, heat shock protein 70, in glioblastoma

Abstract There are various Damage-Associated Molecular Patterns (DAMPs) that are recognizable by the immune system in the cancer microenvironment. DAMPs are revealed under a specific type of cell death termed immunogenic cell death in which stimuli force the cancer cells to die and leave antigen traces such as heat shock proteins that can be used against the disease. The stimulus of interest in this study was recombinant human Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) which was known to selectively kill cancer cells such as glioblastoma cells. This study examined the role of TRAIL in immunogenic cell death for brain cancer. High density T98G glioblastoma cells were cultured with or without 50 ng/ml recombinant human TRAIL for four days. The trypan blue exclusion assay revealed that the average death rate for the TRAIL treated glioblastoma cells was 42%. Furthermore, western blot analysis showed that TRAIL treated cells had a reduction in heat shock protein 70 expression. Previous studies in the literature showed heat shock protein 70 mediated apoptosis resistance in cancer cells. The current study suggests that, even in the absence of any other inhibitors, heat shock protein 70 down-regulation via TRAIL may cause the chemoresistant glioblastoma cells to become susceptible to TRAIL-mediated cell death. Future studies will further investigate the relationship between DAMPs and TRAIL and the extent to which TRAIL generates sufficient apoptotic glioblastoma cells that would serve as the antigenic source for T cell immunotherapy regimens. Disclaimer: This research was supported in part by the James A. Haley Veterans' Hospital. The contents of this publication do not represent the views of the Department of Veterans Affairs or the United States Government. Citation Format: Andrea N. McCray. TRAIL mediated reduction of an immunogenic cell death protein, heat shock protein 70, in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A28.

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.

Gefitinib upregulates death receptor 5 expression to mediate rmhTRAIL-induced apoptosis in Gefitinib-sensitive NSCLC cell line.

BackgroundTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in tumor cells, but when used alone, it is not effective in the treatment of TRAIL-resistant tumors. Some studies have shown that gefitinib interacts with recombinant mutant human TRAIL (rmhTRAIL) to induce high levels of apoptosis in gefitinib-responsive bladder cancer cell lines; however, the molecular mechanisms underlying the anticancer effects are not fully understood. Several reports have shown that the death receptor 5 (DR5) plays an important role in sensitizing cancer cells to apoptosis induced by TRAIL. Therefore, we investigated the effects of the combination of drugs and the expression of the DR5 to analyze the growth of a gefitinib-responsive non-small cell lung cancer cell line PC9, which was treated with rmhTRAIL and gefitinib individually or in combination.MethodsHuman PC9 non-small cell lung cancer cells harboring an epidermal growth factor receptor mutation were used as a model for the identification of the therapeutic effects of gefitinib alone or in combination with rmhTRAIL, and cytotoxicity was assessed by MTT assays. Cell cycle and apoptosis were investigated using flow cytometry. Moreover, the effects of drugs on DR5, BAX, FLIP, and cleaved-caspase3 proteins expressions were analyzed using Western blot analyses. Finally, quantitative polymerase chain reaction analysis was carried out to assess whether rmhTRAIL and gefitinib modulate the expression of genes related to drug activity.ResultsGefitinib and rmhTRAIL synergistically interact to inhibit cell proliferation, and apoptosis assessment demonstrated that associations of drug increased the apoptotic index. rmhTRAIL when used alone downregulated DR5 and upregulated BAX, FLIP, and cleaved-caspase3 proteins expressions. However, results obtained in Western blot analyses demonstrated that the combined treatment-induced cell apoptosis was achieved involving upregulated DR5, cleaved-caspase3, and BAX proteins expression and downregulated FLIP protein expression. Moreover, quantitative polymerase chain reaction showed that gefitinib modulated the expression of targets related to rmhTRAIL activity.ConclusionThese results indicate that epidermal growth factor receptor inhibitors enhance rmhTRAIL antitumor activity in the gefitinib-responsive PC9 cell line, and upregulated DR5 expression plays a critical role in activating caspase-signaling apoptotic pathway.

MP55-07 FLAVONOIDS ENHANCE TRAIL SENSITIVITY IN PROSTATE CANCER CELLS BY TARGETING ADENINE NUCLEOTIDE TRANSLOCASE-2

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2015MP55-07 FLAVONOIDS ENHANCE TRAIL SENSITIVITY IN PROSTATE CANCER CELLS BY TARGETING ADENINE NUCLEOTIDE TRANSLOCASE-2 Masakatsu Oishi, Takashi Ueda, Terukazu Nakamura, Yoshio Naya, Fumiya Hongo, Kazumi Kamoi, Koji Okihara, and Tsuneharu Miki Masakatsu OishiMasakatsu Oishi More articles by this author , Takashi UedaTakashi Ueda More articles by this author , Terukazu NakamuraTerukazu Nakamura More articles by this author , Yoshio NayaYoshio Naya More articles by this author , Fumiya HongoFumiya Hongo More articles by this author , Kazumi KamoiKazumi Kamoi More articles by this author , Koji OkiharaKoji Okihara More articles by this author , and Tsuneharu MikiTsuneharu Miki More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2050AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent. Recombinant human TRAIL has been under clinical trials, whereas various kinds of malignant tumors have resistance to TRAIL. It has been shown that several anticancer agents and flavonoids (apigenin, quercetin etc.) overcome resistance to TRAIL by upregulating death receptor 5 (DR5) in malignant tumor cells. However, the mechanisms by which these compounds induce DR5 expression remain unknown. On the other hand, although docetaxel plus prednisone is effective in hormone-refractory prostate cancer, the outcome of this treatment is still insufficient. Therefore, new strategies are needed to treat this cancer. Thus we investigated the mechanism in a hormone-refractory prostate cancer cell line DU145. METHODS To elucidate the mechanisms by which flavonoids induce DR5 expression in DU145 cells, we explored the proteins binding to flavonoids using FG beads with epoxy linkers. We fixed genistein, which enhances TRAIL sensitivity without upregulating DR5, apigenin, and quercetin onto these beads (Figure 1A). The binding proteins of apigenin, quercetin, and genistein were purified from the DU145 whole cell extracts and were identified by MALDI-TOF MS analysis. Then, function of binding proteins was analized. RESULTS Comparing the binding proteins, we discovered that ANT2 was a target of apigenin and quercetin, but not genistein in DU145 cells (Figure 1B). Similarly to treatment of apigenin and quercetin, knockdown of ANT2 enhanced TRAIL-induced apoptosis by upregulating DR5 expression. Moreover, silencing of ANT2 attenuated the enhancement of TRAIL-induced apoptosis by apigenin or quercetin. CONCLUSIONS These results suggest that flavonoids upregulate DR5 and enhance TRAIL-induced apoptosis in hormone-refractory prostate cancer cells by binding and inhibiting ANT2 (Figure 1C). We propose that ANT2 inhibitors may contribute to TRAIL therapy in hormone-refractory prostate cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e675 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Masakatsu Oishi More articles by this author Takashi Ueda More articles by this author Terukazu Nakamura More articles by this author Yoshio Naya More articles by this author Fumiya Hongo More articles by this author Kazumi Kamoi More articles by this author Koji Okihara More articles by this author Tsuneharu Miki More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Spatial dynamics of TRAIL death receptors in cancer cells.

TNF-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in cancer cells without harming most normal cells. Currently, multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL (rhTRAIL) and agonistic antibodies that target death receptors (DRs) 4 or 5. It is encouraging that these products have shown a tolerated safety profile in early phase studies. However, their therapeutic potential is likely limited by the emergence of tumor drug resistance phenomena. Increasing evidence indicates that TRAIL DRs are deficient on the plasma membrane of some cancer cells despite their total protein expression. Notably, the lack of surface DR4/DR5 is sufficient to render cancers resistant to TRAIL-induced apoptosis, regardless of the status of other apoptosis signaling components. The current review highlights recent findings on the dynamic expression of TRAIL death receptors, including the regulatory roles of endocytosis, autophagy, and Ras GTPase-mediated signaling events. This information could aid in the identification of novel predictive biomarkers of tumor response as well as the development of combinational drugs to overcome or bypass tumor drug resistance to TRAIL receptor-targeted therapies.

Parthenolide enhances sensitivity of colorectal cancer cells to TRAIL by inducing death receptor 5 and promotes TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent. Recombinant human TRAIL has been evaluated in clinical trials, however, various malignant tumors are resistant to TRAIL. Parthenolide (PT) has recently been demonstrated as a highly effective anticancer agent and has been suggested to be used for combination therapy with other anticancer agents. In this study, we investigate the molecular mechanisms by which PT sensitizes colorectal cancer (CRC) cells to TRAIL-induced apoptosis. HT-29 (TRAIL-resistant) and HCT116 (TRAIL-sensitive) cells were treated with PT and/or TRAIL. The results demonstrated that combined treatment induced apoptosis which was determined using MTT, cell cycle analysis, Annexin V assay and Hoechst 33258 staining. Interestingly, we confirmed that HCT116 cells have much higher death receptor (DR) 5 than HT-29 cells and PT upregulates DR5 protein level and surface expression in both cell lines. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, p53 cytochrome C release, and caspase cascades. These results suggest that PT sensitizes TRAIL-induced apoptosis via upregulation of DR5 and mitochondria-dependent pathway. Combination treatment using PT and TRAIL may offer an effective strategy to overcome TRAIL resistance of certain CRC cells.

Abstract 2289: Effects of TRAIL and ellagic acid on malignant melanoma A375 in vitro and in vivo

Abstract Cancer is a widespread disease characterized by unregulated cell growth and evasion of apoptotic stimuli. Most conventional therapies lack cancer cell specificity and induce cell death indirectly through cellular damage in a p53-dependent manner. A direct and selective p53-independent cancer therapy is the application of Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL). Recombinant human TRAIL (rhTRAIL) can induce apoptosis in a broad range of transformed human cells while showing minimal toxicity towards non-malignant cells. However, some cancers are resistant to rhTRAIL-induced apoptosis as a direct result of overexpressed antiapoptotic proteins. One key driver mediating the expression of such antiapoptotic proteins is Casein Kinase 2 (CK2). CK2 is upregulated in the nucleus of all cancer cells and promotes the expression of inhibitors of apoptosis resulting in resistance to cell death. Therefore, by inhibiting CK2, TRAIL-resistant cells may become more sensitive to TRAIL-induced apoptosis. We utilized cotreatment with Ellagic Acid (EA), a natural CK2 inhibitor, and TRAIL against the malignant melanoma cell line A375, which is resistant to TRAIL. Our in vitro analysis showed that TRAIL and EA, as single-agents, can inhibit cell growth in a dose-dependent manner, and the combination treatment of TRAIL plus EA synergistically enhanced the cytostatic effects of the single-agent treatments. We show that TRAIL reduces the viability of A375 cells; whereas, EA although it can inhibit growth, cannot reduce the viability of A375 as determined by Trypan Blue exclusion assays. In combination treatments, the cytotoxic effects of TRAIL were not enhanced. Annexin-V Apoptosis assays and Western Blot analysis show that TRAIL can induce apoptosis in A375 cells characterized by the formation of Annexin-V positive cells, PARP fragmentation and the activation of caspase 3. Additionally, TRAIL initiated both the extrinsic and intrinsic pathways of apoptosis marked by caspase 8 activation and the mitochondrial release of cytochrome c, respectively. EA treatment alone did not induce apoptosis and did not induce formation of Annexin-V positive cells or PARP fragmentation. Combination treatments show that EA does not potentiate TRAIL-induced apoptosis. In vivo, TRAIL-treated nude mice bearing A375 xenografts exhibited ∼80% tumor growth inhibition and a prolonged survival rate compared to the untreated control group. EA was not able to inhibit the growth of established A375 tumors and did not augment the in vivo antiproliferative effects of TRAIL. These data demonstrate that EA can enhance the cytostatic but not the cytotoxic or proapoptotic effects of TRAIL in vitro. In vivo, TRAIL can inhibit the growth of established tumors as a single-agent but is not augmented by cotreatment with EA. Citation Format: Katherine Turner, Daniel Lindner, Michael Kalafatis. Effects of TRAIL and ellagic acid on malignant melanoma A375 in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2289. doi:10.1158/1538-7445.AM2014-2289

H-Ras regulation of TRAIL death receptor mediated apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide mRNA expression data of the NCI60 cancer cell lines, we found that H-Ras expression was consistently upregulated in TRAIL-resistant cell lines. By contrast, no correlation was found between TRAIL sensitivity and K-Ras expression levels or their mutational profiles. Notably, H-Ras upregulation associated with a surface deficiency of TRAIL death receptors. Selective inhibition of H-Ras activity in TRAIL-resistant cells restored the surface expression of both DR4 and DR5 without changing their total protein levels. The resulting cells became highly susceptible to both TRAIL and agonistic DR5 antibody, whereas K-Ras inhibition had little or no effect on TRAIL-induced apoptosis, indicating H-Ras plays a distinct role in the regulation of TRAIL death receptors. Further studies are warranted to determine the therapeutic potential of H-Ras-specific inhibitors in combination with TRAIL receptor agonists.

Surrounding pancreatic adenocarcinoma by killer mesenchymal stromal/stem cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic malignancy still associated with an unacceptably poor prognosis. In addition to neoplastic tissue, PDAC is characterized by a prominent desmoplastic reaction infiltrating and surrounding cancer cells (Chu et al., 2007). This fibrotic microenvironment is generated predominantly by stromal cells known as tumor-associated fibroblasts, the origin of which has been also attributed to mesenchymal stromal/stem cells (MSCs) deriving from either bone marrow (BM) or adipose tissue (AD) (Grisendi et al., 2011). Because MSCs can be induced to produce anticancer molecules, such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Grisendi et al., 2010), we here show the impact of gene-modified human MSCs producing TRAIL against PDAC. The BxPC3 cell line (Fig. 1, left) was selected on the basis of its high expression of functional TRAIL receptors associated with recombinant human TRAIL sensitivity confirmed in a dose–response assay (data not shown). FIG. 1. Pancreatic adenocarcinoma cell line BxPC3 interplays with adipose-derived mesenchymal stem cells expressing green fluorescent protein (GFP AD-MSCs) only or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/GFP AD-MSCs). Scale bar, 50 μm. ... Microscopic examination of cocultures (Fig. 1, middle) of BxPC3 cells with adipose-derived MSCs expressing green fluorescent protein (GFP AD-MSCs), as control, revealed a peculiar interaction between MSCs and tumor cells, with BxPC3 cells arranged as islets surrounded by green AD-MSC bundles, in parallel to what is described for the PDAC-associated desmoplastic reaction in vivo (Chu et al., 2007). Having proved the interaction between GFP AD-MSCs and BxPC3 cells, coculture experiments were then performed for both 24 and 48 hr, testing various target-to-effector (T:E) ratios (1:1, 1:2, and 1:5), using TRAIL/GFP AD-MSCs. Tumor cell death was detected in all tested conditions. The interplay between TRAIL/GFP AD-MSCs and BxPC3 cells was followed by a breakdown of tumor islets together with the disruption of adherent BxPC3 cells, resulting in clusters of dead floating elements in culture medium above an established layer of TRAIL/GFP AD-MSCs (Fig. 1, right; T:E ratio, 1:2 at 48 hr; scale bar, 50 μm). These morphological findings were quantified by flow cytometric detection (data not shown) of apoptotic propidium iodide-positive cells, reaching 69±3% (average±SD) for TRAIL/GFP AD-MSCs versus 14±1% for GFP AD-MSCs (p=0.001 by two-tailed t test). Similar features were observed in cocultures of TRAIL/GFP AD-MSCs and an additional PDAC cell line (MIA PaCa-2 cells; data not shown). Depicting the strong interaction with genetically modified AD-MSCs and PDAC lines, we here suggest MSCs as suitable vectors for anticancer agent delivery, opening novel therapeutic opportunities for this still incurable cancer.

Effect of Naive and Radiolabeled rhTRAIL on the Cervical Cancer Xenografts in Mice

There is a need for novel treatments of advanced cervical cancer. We investigated the utility of recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL), a molecule capable of inducing apoptosis in cancer cells, for the therapy of CasKi cervical cancer xenografts in nude mice. CasKi cells proved to be sensitive in vitro to rhTRAIL with an IC50 of 120 ng/ml. (125)I-tagged rhTRAIL specifically accumulated in CasKi tumors in mice with the highest uptake of 9.4% ID/g at 2 h post-injection. Both naive and 200 µCi (188)Re-tagged rhTRAIL administered in the amount of 0.35 mg/kg body weight significantly retarded CasKi tumor growth to the same extent in mice without the side effects of cisplatin chemotherapeutic control. rhTRAIL is a promising novel agent for treatment of advanced cervical cancer.

Regulation of TRAIL receptor expression by -catenin in colorectal tumours

Tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) is being investigated as a targeted cancer therapeutic and the expression of its pro-apoptotic receptors, DR4 and DR5, increases during colorectal carcinogenesis. This study investigated the role of β-catenin in the regulation of these receptors. In human colorectal adenoma and carcinoma cell lines, downregulation of β-catenin resulted in lower total DR4 and DR5 protein levels. Similarly, cell membrane expression of DR4 and DR5 was reduced after downregulation of β-catenin in colon carcinoma cells, whereas induction of β-catenin in HeLa cells led to increased cell membrane expression of DR4 and DR5. Downregulation of β-catenin decreased the recombinant human TRAIL sensitivity of human colon carcinoma cells. Activation of the transcription factor T-cell factor-4 (TCF-4) is an important function of β-catenin. Dominant-negative TCF-4 overexpression, however, did not significantly affect TRAIL receptor expression or recombinant human TRAIL sensitivity. Human colorectal adenomas (N = 158) with aberrant (cytoplasmic and nuclear) β-catenin expression had a higher percentage of immunohistochemical DR4 and DR5 staining per tumour (mean: 73 and 88%, respectively) than those with membranous β-catenin staining only (mean: 50 and 70%, respectively, P < 0.01 for both). Furthermore, aberrant β-catenin staining co-localized with DR4 and DR5 expression in 92% of adenomas. In 53 human colorectal carcinomas, aberrant β-catenin expression was present in most cases and DR4/5 expression was largely homogenous. Similarly, in adenomas from APC(min) mice, cytoplasmic β-catenin staining co-localized with staining for the murine TRAIL death receptor. In conclusion, the gradual increase in TRAIL receptor expression during colorectal carcinogenesis is at least partially mediated through increased β-catenin expression, independently of TCF-4-signalling.

Nutlin-3 preferentially sensitises wild-type p53-expressing cancer cells to DR5-selective TRAIL over rhTRAIL

Background:Tumour cell-selective activation of apoptosis by recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL) is enhanced through co-activation of p53 by chemotherapeutic drugs. The novel anticancer agent nutlin-3 provides a promising alternative for p53 activation by disrupting the interaction between p53 and its negative feedback regulator MDM2.Methods:We examined whether nutlin-3 enhances apoptosis induction by rhTRAIL and the DR5-selective TRAIL variant D269H/E195R in wild-type p53-expressing ovarian, colon and lung cancer cell lines and in an ex vivo model of human ovarian cancer.Results:Nutlin-3 enhanced p53, p21, MDM2 and DR5 surface expression. Although nutlin-3 did not induce apoptosis, it preferentially enhanced D269H/E195R-induced apoptosis over rhTRAIL. Combination treatment potentiated the cleavage of caspases 8, 9, 3 and PARP. P53 and MDM2 siRNA experiments showed that this enhanced apoptotic effect was mediated by wild-type p53. Indeed, nutlin-3 did not enhance rhTRAIL-induced apoptosis in OVCAR-3 cells harbouring mutant p53. Addition of the chemotherapeutic drug cisplatin to the combination further increased p53 and DR5 levels and rhTRAIL- and D269H/E195R-induced apoptosis. As a proof of concept, we show that the combination of D269H/E195R, nutlin-3 and cisplatin induced massive apoptosis in ex vivo tissue slices of primary human ovarian cancers.Conclusion:Nutlin-3 is a potent enhancer of D269H/E195R-induced apoptosis in wild-type p53-expressing cancer cells. Addition of DNA-damaging agents such as cisplatin further enhances DR5-mediated apoptosis.

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.

TRAIL induces apoptosis in oral squamous carcinoma cells: a crosstalk with oncogenic Ras regulated cell surface expression of death receptor 5

TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis through its death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. The selectivity of TRAIL towards cancer cells has promoted clinical evaluation of recombinant human TRAIL (rhTRAIL) and its agonistic antibodies in treating several major human cancers including colon and non-Hodgkin's lymphoma. However, little is known about their ability in killing oral squamous cell carcinoma (OSCC) cells. In this study, we tested the apoptotic responses of a panel of seven human OSCC cell lines (HN31, HN30, HN12, HN6, HN4, Cal27, and OSCC3) to rhTRAIL and monoclonal antibodies against DR4 or DR5. We found that rhTRAIL is a potent inducer of apoptosis in most of the oral cancer cell lines tested both in vitro and in vivo. We also showed that DR5 was expressed on the surface of the tested cell lines which correlated with the cellular susceptibility to apoptosis induced by rhTRAIL and anti-DR5 antibody. By contrast, little or no DR4 was detected on the surface of OSCC3 and HN6 cells rendering cellular resistance to DR4 antibody and a reduced sensitivity to rhTRAIL. Notably, the overall TRAIL sensitivity correlated well with the levels of endogenous active Ras in the cell lines tested. Expression of a constitutively active Ras mutant (RasV12) in OSCC3 cells selectively upregulated surface expression of DR5, but not DR4, and restored TRAIL sensitivity. Our findings could have implications for the use of TRAIL receptor targeted therapies in the treatment of human OSCC tumors particularly the ones harboring constitutively active Ras mutant.