Upregulation Of TNF-related Apoptosis-inducing Ligand Research Articles

Association of circulating cytokine levels and tissue-infiltrating myeloid cells with achalasia: results from Mendelian randomization and validation through clinical characteristics and single-cell RNA sequencing.

Achalasia is a rare motility disorder of the esophagus often accompanied by immune dysregulation, yet specific underlying mechanisms remain poorly understood. We utilized Mendelian randomization (MR) to explore the causal effects of cytokine levels on achalasia, with cis-expression/protein quantitative trait loci (cis-eQTLs/pQTLs) for 47 cytokines selected from a genome-wide association study (GWAS) meta-analysis and GWAS data for achalasia obtained from FinnGen. For cytokines significantly linked to achalasia, we analyzed their plasma concentrations and expression differences in the lower esophageal sphincter (LES) using enzyme-linked immunosorbent assay and single-cell RNA sequencing (scRNA-seq) profiling, respectively. We further employed bioinformatics approaches to investigate underlying mechanisms. We revealed positive associations of circulating Eotaxin, macrophage inflammatory protein-1b (MIP1b), soluble E-selectin (SeSelectin) and TNF-related apoptosis-inducing ligand (TRAIL) with achalasia. When combining MR findings with scRNA-seq data, we observed upregulation of TRAIL (OR = 2.70, 95% CI, 1.20-6.07), encoded by TNFSF10, in monocytes and downregulation of interleukin-1 receptor antagonist (IL-1ra) (OR = 0.70, 95% CI 0.59-0.84), encoded by IL1RN, in FOS_macrophages in achalasia. TNFSF10high monocytes in achalasia displayed activated type I interferon signaling, and IL1RNlow FOS_macrophages exhibited increased intercellular communications with various lymphocytes, together shaping the proinflammatory microenvironment of achalasia. We identified circulating Eotaxin, MIP1b, SeSelectin and TRAIL as potential drug targets for achalasia. TNFSF10high monocytes and IL1RNlow macrophages may play a role in the pathogenesis of achalasia.

Enhancer Clusters Drive Type I Interferon-Induced TRAIL Overexpression in Cancer, and Its Intracellular Protein Accumulation Fails to Induce Apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine produced and secreted by immune cells in response to an infection, often in response to interferon (IFN) stimulation. In cancer, it has also been shown that IFN stimulates the production of TRAIL, and it has been proposed that this TRAIL can induce apoptosis in an autocrine or paracrine manner in different cancer cells. Yet, the mechanism mediating TRAIL upregulation and the implications of TRAIL as an apoptotic molecule in cancer cells are still poorly understood. We show here that in certain cancer cells, TRAIL is upregulated by enhancer clusters, potent genomic regulatory regions containing densely packed enhancers that have combinatorial and additive activity and that are usually found to be associated with cancer-promoting genes. Moreover, we found that TRAIL upregulation by IFNα is mediated by these enhancer clusters in breast and lung cancer cells. Surprisingly, IFNα stimulation leads to the intracellular accumulation of TRAIL protein in these cancer cells. Consequently, this TRAIL is not capable of inducing apoptosis. Our study provides novel insights into the mechanism behind the interferon-mediated upregulation of TRAIL and its protein accumulation in cancer cells. Further investigation is required to understand the role of intracellular TRAIL or depict the mechanisms mediating its apoptosis impairment in cancer cells.

Unspecific CTL Killing Is Enhanced by High Glucose via TNF-Related Apoptosis-Inducing Ligand.

TNF-related apoptosis inducing ligand (TRAIL) is expressed on cytotoxic T lymphocytes (CTLs) and TRAIL is linked to progression of diabetes. However, the impact of high glucose on TRAIL expression and its related killing function in CTLs still remains largely elusive. Here, we report that TRAIL is substantially up-regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. Non-mitochondrial reactive oxygen species, NFκB and PI3K/Akt are essential in HG-induced TRAIL upregulation in CTLs. TRAILhigh CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Treatment with metformin and vitamin D reduces HG-enhanced expression of TRAIL in CTLs and coherently protects 1.4E7 cells from TRAIL-mediated apoptosis. Our work suggests that HG-induced TRAILhigh CTLs might contribute to the destruction of pancreatic beta cells in a hyperglycemia condition.

Abstract 1044: ONC201 as a novel anti-cancer therapeutic via modulation of inhibitors of apoptosis and up-regulation of DR5 in gastric adenocarcinoma

Abstract Gastric adenocarcinoma is commonly asymptomatic early in its course, leading to late-stage diagnosis at a time when patients are inoperable. Currently available chemotherapy options are non-targeted and highly toxic, leading to a poor 5-year survival of only 31.5%. ONC201 is a small molecule imipridone anti-cancer therapy discovered by our lab that induces cell death in a variety of malignant tumors via a multitude of mechanisms including activation of the integrated stress response (ISR) leading to up-regulation of TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis after binding to the death receptor 5 (DR5). We investigated the ability of ONC201 to induce cell death in gastric adenocarcinoma cells when used in combination with recombinant human TRAIL (rhTRAIL), and its affect on DR5 cell surface expression and expression of inhibitors of apoptosis (IAP) in order to further establish mechanism of action. AGS (caspase 8, KRAS, PIK3CA mutant, HER2 amplified), SNU-1 (KRAS and MLH1 mutant, microsatellite instable), SNU-5 (p53 mutant) and SNU-16 (p53 mutant) gastric adenocarcinoma cell lines were treated with ONC201 and rhTRAIL and viability assays were performed to determine cell line sensitivities. All lines were then treated with combination therapy based on sensitivities and viability assays were performed in order to determine combination indices. Cell death was verified with sub-G1 analysis via flow cytometry and protein expression was established via western blotting. All cell lines exhibited strong synergy in response to dual therapy with ONC201 and rhTRAIL, with combination indices <0.6 at doses that did not induce cell death in normal fibroblast cells. Synergy was confirmed via flow cytometry analysis with increased cells in the sub-G1 phase of the cell cycle with dual therapy in excess of what would be expected from an additive effect. On western blot analysis, apoptosis was confirmed with increased cleavage of PARP, caspase 8 and caspase 3 after dual treatment with ONC201 and rhTRAIL. Flow cytometry revealed increased cell surface expression of DR5 with ONC201 therapy, and western blot analysis showed that ONC201 lead to up-regulation of ATF-4 and CHOP, indicating activation of the ISR, and down-regulation of anti-apoptotic CIAP-2 and XIAP in all cell lines except AGS, as well as FLIP in all cell lines except SNU-16. These in vitro results suggest that ONC201 in combination with rhTRAIL may be an effective and non-toxic option for the treatment of gastric adenocarcinoma by inducing apoptosis via activation of the ISR, increased cell surface expression of DR5 and down-regulation of inhibitors of apoptosis. We are currently applying this regimen to an organoid model, as well as a murine sub-cutaneous xenograft model using AGS and SNU-1 cells. Citation Format: Cassandra Susan Parker, Lanlan Zhou, Varun Prabhu, Josh Allen, Wafik El-Deiry. ONC201 as a novel anti-cancer therapeutic via modulation of inhibitors of apoptosis and up-regulation of DR5 in gastric adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1044.

Efficient and precise delivery of microRNA by photoacoustic force generated from semiconducting polymer-based nanocarriers

One major challenge in miRNA-based therapy is to explore facile delivery strategies, which can facilitate the efficient and precise accumulation of intrinsically instable microRNAs (miRNAs) at targeted tumor sites. To address this critical issue, for the first time we demonstrate that a near-infrared (NIR) pulse laser can guide efficient delivery of miRNAs mediated by a NIR-absorbing and photoacoustic active semiconducting polymer (SP) nanocarrier, which can generate photoacoustic radiation force to intravascularly overcome the endothelial barriers. Importantly, we demonstrate an ultrafast delivery of miRNA (miR-7) to tumor tissues under the irradiation of pulse laser in 20 min, showing a 5-fold boosted efficiency in comparison to the traditional passive targeting strategy. The delivered miR-7 acts as a sensitizer of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and synergizes with TRAIL-inducing compound (TIC), leading to sustained TRAIL upregulation for effective tumor suppression in mice. As such, our results indicate that the NIR-absorbing semiconducting polymer-mediated nanocarrier platform can significantly enhance the targeted delivery efficiency of therapeutic miRNAs to tumors, resulting in potent tumor growth inhibition.

Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows promising antitumor activity in preclinical studies. However, the efficacy of recombinant TRAIL in clinical trials is compromised by its short serum half-life and low in vivo stability. Induction of endogenous TRAIL may overcome the limitations and become a new strategy for cancer treatment. Here, we discovered that metformin increased TRAIL expression and induced apoptosis in triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) cells. Metformin did not alter the expression of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Metformin-upregulated TRAIL was secreted into conditioned medium (CM) and found to be functional, since the CM promoted TNBC cells undergoing apoptosis, which was abrogated by a recombinant TRAIL-R2-Fc chimera. Moreover, blockade of TRAIL binding to DR4/DR5 or specific knockdown of TRAIL expression significantly attenuated metformin-induced apoptosis. Studies with a tumor xenograft model revealed that metformin not only significantly inhibited tumor growth but also elicited apoptosis and enhanced TRAIL expression in vivo. Collectively, we have demonstrated that upregulation of TRAIL and activation of death receptor signaling are pivotal for metformin-induced apoptosis in TNBC and NSCLC cells. Our studies identify a novel mechanism of action of metformin exhibiting potent antitumor activity via induction of endogenous TRAIL.

Erlotinib Activates Different Cell Death Pathways in EGFR-mutant Lung Cancer Cells Grown in 3D Versus 2D Culture Systems.

Non-small cell lung cancer patients with epidermal growth factor receptor (EGFR) mutation have been shown to have a good response to erlotinib, a receptor tyrosine kinase inhibitor of EGFR. In this study, we found that the cell death pathways activated by erlotinib in 2D and 3D culture systems are different. The cell death pathways induced by erlotinib were evaluated by flow cytometry and immunoblotting in both 2D and 3D culture systems of EGFR mutant lung cancer cells. Treatment with erlotinib induced caspase 8 activation and up-regulation of TNF-related apoptosis-inducing ligand (TRAIL) expression only in 3D cultures. Knockdown of TRAIL attenuated both erlotinib-induced activation of caspase-8 and apoptosis in 3D cultures. Erlotinib also increased LC3, an autophagy marker, expression and c-Jun N terminal kinase (JNK) activation. Both 3-MA as an autophagy inhibitor and SP600125 as a JNK inhibitor, significantly inhibited erlotinib-induced cell death. Erlotinib induces apoptotic cell death in 3D cultures through an autophagy-TRAIL-JNK pathway.

Abstract 3012: ONC201 induces apoptosis in gastric adenocarcinoma and exhibits synergy with rhTRAIL

Abstract Gastric adenocarcinoma is highly lethal with a 5-year survival of only 31.5%, largely due to late presentation of disease and ineffective chemotherapy options. ONC201 is a first-in-class small molecule anti-cancer therapy that induces cell death in many different types of malignant tumors via a multitude of mechanisms including up-regulation of TNF-related apoptosis-inducing ligand (TRAIL) by Foxo3a and death receptor 5 (DR5) via activation of the integrated stress response. We investigated the efficacy of ONC201 in inducing cell death in gastric adenocarcinoma cells as monotherapy and in combination with recombinant human TRAIL (rhTRAIL). AGS (caspase 8 and PIK3CA mutant, HER2 amplified) and SNU-16 (p53 mutant) gastric adenocarcinoma cell lines were treated with ONC201 and rhTRAIL and viability assays were performed to determine cell line sensitivities. Both cell lines were then treated with combination therapy based on sensitivities and viability assays were performed in order to determine combination indices. Cell death was verified with sub-G1 analysis via flow cytometry and protein expression was established via western blotting. SNU-16 cells were sensitive to ONC201, while AGS cells were shown to be resistant to monotherapy. Both cell lines were sensitive to rhTRAIL and exhibited synergy when treated in combination with ONC201, resulting in combination indices of less than 0.5 at doses that did not induce cell death in fibroblasts. The proportion of cells in sub-G1 phase of the cell cycle increased in a dose-dependent manner with combination treatment. PARP cleavage increased in a dose-dependent manner after combination therapy, indicating apoptosis and ATF4 and CHOP expression also increased with increasing doses of combination therapy, indicating activation of the integrated stress response. These results suggest that ONC201 may be an effective therapeutic agent for gastric adenocarcinoma in combination with rhTRAIL by inducing apoptosis via the integrated stress response. We are currently expanding the in vitro model to include additional cell lines SNU-1(Ras mutant, microsatellite instability) and SNU-5 (p53 and CDH1 mutant). We are further exploring the role of ONC201 in priming cells via up-regulation of DR5 receptors such that they have increased sensitivity to rhTRAIL when pretreated with ONC201 as a potential source for the established synergy in gastric adenocarcinoma cells. We will also apply this study regimen to an organoid and in vivo model. Citation Format: Cassanda Susan Parker, Lanlan Zhou, Wafik S. El-Deiry. ONC201 induces apoptosis in gastric adenocarcinoma and exhibits synergy with rhTRAIL [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3012.

Imipridone enhances vascular relaxation via FOXO1 pathway.

Cardiovascular complications are a side effect of cancer therapy, potentially through reduced blood vessel function. ONC201 (TIC10) is currently used in phase 2 clinical trials to treat high-grade gliomas. TIC10 is a phosphatidylinositol 3-kinase (PI3K)/AKT/extracellular signal-regulated kinase (ERK) inhibitor that induces apoptosis via upregulation of TNF-related apoptosis-inducing ligand, which via stimulation of FOXO and death receptor could increase eNOS upregulation. This has the potential to improve vascular function through increased NO bioavailability. Our aim was to investigate the role of TIC10 on vascular function to determine if it would affect the risk of CVD. Excised abdominal aorta from White New Zealand male rabbits were cut into rings. Vessels were incubated with TIC10 and AS1842856 (FOXO1 inhibitor) followed by cumulative doses of acetylcholine (Ach) to assess vessel function. Vessels were then processed for immunohistochemistry. Incubation of blood vessels with TIC10 resulted in enhanced vasodilatory capacity. Combination treatment with the FOXO1 inhibitor and TIC10 resulted in reduced vascular function compared to control. Immunohistochemical analysis indicated a 3-fold increase in death receptor 5 (DR5) expression in the TIC10-treated blood vessels but the addition of the FOXO1 inhibitor downregulated DR5 expression. The expression of DR4 receptor was not significantly increased in the presence of TIC10; however, addition of the FOXO1 inhibitor downregulated expression. TIC10 has the capacity to improve the function of healthy vessels when stimulated with the vasodilator Ach. This highlights its therapeutic potential not only in cancer treatment without cardiovascular side effects, but also as a possible drug to treat established CVD.

A trifunctional bispecific antibody mediates efficient killing of B-cell lymphoma cells

Abstract Natural killer (NK) cells are a type of cytotoxic lymphocytes critical to immune surveillance. The mechanisms for NK-mediated tumor elimination including natural cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), and the secretion of interferon-γ (IFN-γ), which are essential for mediating the anti-tumor activity. CD19, a B-cell lineage-specific marker, is highly expressed in B-cell malignancies and an attractive target for therapy. Here, we generate a trifunctional bispecific antibody (TBAb) containing single chain antibody fragment (scFV) against CD16 and CD19 to create an immunologic synapse between NK cells and CD19+ lymphoma cells. X has been engineered with a modified human interleukin crosslinker for improving therapeutic efficacy. Specificity has been confirmed by investigating the ability of X to bind CD19+ tumor targets and CD16+ NK cells. We found that X TBAb mediated killing of human lymphoma B cell lines, including namalwa, daudi and raji, by human NK cells purified by microbeads or NK-cell amplification kit in vitro. X-mediated killing is accompanied by NK-cell activation, cytokine release and upregulation of TNF-related apoptosis-inducing ligand (TRAIL). Further, X TBAb demonstrated antitumor activity against lymphoma xenografts reconstituted with human PBMCs. These data provide a therapeutic candidate for the treatment of B-cell malignancies.

Everolimus enhances TRAIL-mediated anti-tumor activity of liver resident natural killer cells in mice.

In transplantation, innate immunity plays a pivotal role in immunosurveillance and host defence against microbes and neoplastic cells. Liver-resident NK cells express TNF-related apoptosis-inducing ligand (TRAIL), which distinguishes them from conventional NK cells. In this study, we investigated the impact of mTOR inhibition on liver-resident NK cells in comparison with that on splenic NK cells in a mouse model. In mice that received everolimus (EVR) for 7days (range: 0.0125-0.25mg/kg/day), the proportion of splenic NK cells was unchanged, whereas the number of liver NK cells including TRAIL+ NK subpopulation increased for all doses of EVR. Consistently, liver-resident NK cells from the EVR-treated mice displayed enhanced cytotoxicity against TRAIL-sensitive neoplastic cells. EVR treatment inhibited the transition of the immature subset of liver NK cells to a mature state. The negative regulator of NK cells FoxO1 was activated as a consequence of impaired mTORC2-dependent AKT phosphorylation. Activated FoxO1 both reduced T-bet expression and induced TRAIL expression, thereby inhibiting NK cell maturation and promoting the antitumour activity of the immature subset of liver NK cells in response to EVR treatment. These findings indicate that EVR treatment enhances the antitumour activity of immature liver-resident NK cells through TRAIL upregulation.

Screening of lactic acid bacteria feeding for pig, preparing of compound probiotics and its feeding effect on growing pigs

Newcastle disease virus (NDV) causes major economic losses in the poultry industry. Previous studies have shown that NDV utilizes different pathways to infect various cells, including dendritic cells (DCs). Here, we demonstrate that NDV gains entry into DCs mainly via macropinocytosis and clathrin-mediated endocytosis. The detection of cytokines interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), interleukin-4 (IL-4) and interleukin-10 (IL-10) indicates that NDV significantly induces Th1 responses and lowers Th2 responses. Furthermore, NDV entry into DCs resulted in the upregulation of TNF-related apoptosis-inducing ligand (TRAIL) and cleaved caspase-3 proteins, which in turn activated the extrinsic apoptosis pathway and induced DCs apoptosis. Transwell® co-culture demonstrated that direct contact between live NDV-stimulated DCs and T cells, rather than heated-inactivated NDV, inhibited CD4+ T cell proliferation. Taken together, these findings provide new insights into the mechanism underlying NDV infections, particularly in relation to antigen presentation cells and suppression of T cell proliferation.

Telomerase reverse transcriptase interference synergistically promotes tumor necrosis factor‑related apoptosis‑inducing ligand‑induced oral squamous cell carcinoma apoptosis and suppresses proliferation invitro and invivo.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to induce cell apoptosis in many types of cancer cells. However, some malignant cells still exhibit anti-apoptosis features induced by TRAIL; thus the underlying mechanisms that regulate sensitivity and resistance of tumor cells to TRAIL-induced apoptosis remain unclear. Human telomerase reverse transcriptase (hTERT) is overexpressed in most types of human tumors and is mostly inactive in somatic cells. The present study aimed to investigate the endogenous effects and mechanisms of hTERT inhibition and TRAIL overexpression on TRAIL-induced apoptosis of human oral squamous cell carcinoma (OSCC) cells. The effects of adeno-associated virus (AAV)-mediated TRAIL and hTERT gene silencing by RNA interference were investigated on the proliferation and apoptosis of human OSCC cells in vitro and in vivo. The present results suggest that knockdown of hTERT expression accelerated TRAIL-resistant OSCC cells to TRAIL-induced apoptosis and impaired OSCC cell proliferation. In addition, this process is accompanied by the upregulation of caspase-3, caspase-8 and caspase-9, and downregulation of B cell lymphoma-2. Additionally, the possible mechanisms underlying the association between TRAIL expression and hTERT silencing were explored. The results demonstrated that TRAIL expression levels were elevated when the hTERT gene was silenced, and notable anti-tumor effects were observed when TRAIL upregulation and hTERT gene silencing were carried out simultaneously. The present findings provide experimental evidence for the combined use of TRAIL and hTERT as a possible gene therapy strategy in oral cancer.

NDV entry into dendritic cells through macropinocytosis and suppression of T lymphocyte proliferation

Newcastle disease virus (NDV) causes major economic losses in the poultry industry. Previous studies have shown that NDV utilizes different pathways to infect various cells, including dendritic cells (DCs). Here, we demonstrate that NDV gains entry into DCs mainly via macropinocytosis and clathrin-mediated endocytosis. The detection of cytokines interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), interleukin-4 (IL-4) and interleukin-10 (IL-10) indicates that NDV significantly induces Th1 responses and lowers Th2 responses. Furthermore, NDV entry into DCs resulted in the upregulation of TNF-related apoptosis-inducing ligand (TRAIL) and cleaved caspase-3 proteins, which in turn activated the extrinsic apoptosis pathway and induced DCs apoptosis. Transwell® co-culture demonstrated that direct contact between live NDV-stimulated DCs and T cells, rather than heated-inactivated NDV, inhibited CD4+ T cell proliferation. Taken together, these findings provide new insights into the mechanism underlying NDV infections, particularly in relation to antigen presentation cells and suppression of T cell proliferation.

BAG3 promotes the phenotypic transformation of primary rat vascular smooth muscle cells via TRAIL.

Under normal physiological condition, the mature vascular smooth muscle cells(VSMCs) show differentiated phenotype. In response to various environmental stimuluses, VSMCs convert from the differentiated phenotype to dedifferentiated phenotype characterized by the increased ability of proliferation/migration and the reduction of contractile ability. The phenotypic transformation of VSMCs played an important role in atherosclerosis. Both Bcl-2-associated athanogene3(BAG3) and tumor necrosis factor-related apopt-osis inducing ligand(TRAIL) involved in apoptosis. The relationship between BAG3 and TRAIL and their effects the proliferation and migration in VSMCs are rarely reported. This study investigated the effects of BAG3 on the phenotypic modulation and the potential underlying mechanisms in primary rat VSMCs. Primary rat VSMCs were extracted and cultured invitro. Cell proliferation was detected by cell counting, real-time cell analyzer (RTCA) and EdU incorporation. Cell migration was detected by wound healing, Transwell and RTCA. BAG3 and TRAIL were detected using real-time PCR and western blotting and the secreted proteins in the cultured media by dot blot. The expression of BAG3 increased with continued passages in cultured primary VSMCs. BAG3 promoted the proliferation and migration of primary rat VSMC in a time-dependent manner. BAG3 significantly increased the expression of TRAIL while had no effects on its receptors. TRAIL knockdown or blocking by neutralizing antibody inhibited the proliferation of VSMCs induced by BAG3. TRAIL knockdown exerted no obvious influence on the migration of VSMCs. Based on this study, we report for the first time that BAG3 was expressed in cultured primary rat VSMCs and the expression of BAG3 increased with continued passages. Furthermore, BAG3 promoted the proliferation of VSMCs via increasing the expression of TRAIL. In addition, we also demonstrated that BAG3 promoted the migration of VSMCs independent of TRAIL upregulation.

Monocytes inhibit hepatitis C virus-induced TRAIL expression on CD56bright NK cells

Natural killer (NK) cells play an important role in the pathogenesis of hepatitis C virus (HCV) infection. We have previously shown that culture-derived HCV (HCVcc) enhance tumor necrosis-factor-related apoptosis-inducing ligand (TRAIL) expression on healthy NK cells, but not on those from patients infected with HCV, which was likely dependent on accessory cells. Here we sought to elucidate the mechanisms involved in altered TRAIL upregulation in this setting. Peripheral blood mononuclear cells (PBMC) from controls and patients infected with HCV were exposed to HCVcc. Cell depletions were performed to identify cells responsible for NK cell activation. Flow cytometry and ELISA were used to identify the cytokines involved in the NK activation process. In patients infected with HCV, soluble factors secreted by control PBMC restored the ability of NK cells to express TRAIL. Of note, CD14+ cell depletion had identical effects upon virus exposure and promoted increased degranulation. Moreover, increased concentrations of interleukin (IL)-18 binding protein a (IL-18BPa) and IL-36 receptor antagonist (IL-36RA) were observed after PBMC exposure to HCVcc in patients with HCV. HCVcc-induced NK cell TRAIL expression was inhibited by IL-18BPa and IL-36RA in control subjects. There were statistically significant correlations between IL-18BPa and indices of liver inflammation and fibrosis, supporting a role for this protein in the pathogenesis of chronic HCV infection. During chronic HCV infection, monocytes play a key role in negative regulation of NK cell activation, predominantly via secretion of inhibitors of IL-18 and IL-36. Coordination and collaboration between immune cells are essential to fight pathogens. Herein we show that during HCV infection monocytes secrete IL-18 and IL-36 inhibitory proteins, reducing NK cell activation, and consequently inhibiting their ability to express TRAIL and kill target cells.

Altered expression of TRAIL on mouse T cells via ERK phosphorylation by Rhodiola rosea L. and its marker compounds

Rhodiola rosea L. extracts have shown neuroprotective, anti-fatigue, anti-inflammatory and anti-tumor properties. However, the studies on their effect on T cell function are rather scarce. We examined the potential of R. rosea extract and its major constituents – salidroside, rosarin, rosavin and rosin to alter cell growth of human Jurkat T cells, apoptosis of splenic mouse CD3 T cells and expression of the surface markers and phosphorylation of extracellular signal-regulated kinase (ERK). The initial screening for cell viability in Jurkat T cells and for apoptosis of mouse T cells showed the strongest activity for rosavin and rosarin. Rosarin and rosavin did not alter significantly the dynamic of CD69 expression upon stimulation, but altered TNF-related apoptosis-inducing ligand (TRAIL) expression. Rosavin inhibited TRAIL up-regulation, while rosarin showed an opposite effect. Indeed, rosarin increased the frequencies of CD3+TRAIL+ T cells and the fold inhibition of ERK phosphorylation. Our data showed that different effects of rosarin and rosavin on TRAIL expression can involve distinct action on ERK signaling and hence highlighted their potential to manipulate TRAIL as a tool to rescue the resistance to apoptosis in autoimmune diseases and cancer.

Nanoparticle engineered TRAIL-overexpressing adipose-derived stem cells target and eradicate glioblastoma via intracranial delivery

Glioblastoma multiforme (GBM) is one of the most intractable of human cancers, principally because of the highly infiltrative nature of these neoplasms. Tracking and eradicating infiltrating GBM cells and tumor microsatellites is of utmost importance for the treatment of this devastating disease, yet effective strategies remain elusive. Here we report polymeric nanoparticle-engineered human adipose-derived stem cells (hADSCs) overexpressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as drug-delivery vehicles for targeting and eradicating GBM cells in vivo. Our results showed that polymeric nanoparticle-mediated transfection led to robust up-regulation of TRAIL in hADSCs, and that TRAIL-expressing hADSCs induced tumor-specific apoptosis. When transplanted in a mouse intracranial xenograft model of patient-derived glioblastoma cells, hADSCs exhibited long-range directional migration and infiltration toward GBM tumor. Importantly, TRAIL-overexpressing hADSCs inhibited GBM growth, extended survival, and reduced the occurrence of microsatellites. Repetitive injection of TRAIL-overexpressing hADSCs significantly prolonged animal survival compared with single injection of these cells. Taken together, our data suggest that nanoparticle-engineered TRAIL-expressing hADSCs exhibit the therapeutically relevant behavior of "seek-and-destroy" tumortropic migration and could be a promising therapeutic approach to improve the treatment outcomes of patients with malignant brain tumors.

Abstract 1294: Sensitization of malignant melanomas to TRAIL-induced apoptosis by quercetin

Abstract Skin cancer is among the most commonly-diagnosed cancers with malignant melanoma being associated with the highest rate of metastasis and mortality. In its early stage, melanoma is easily cured, but the prognosis associated with metastatic malignant melanoma remains very poor and is one of the most treatment-refractory malignancies. We propose the application of Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) as a potential therapeutic for malignant melanoma. TRAIL induces apoptosis in a broad range of transformed human cells while showing minimal toxicity towards non-malignant cells. However, some cancers are resistant to TRAIL, specifically certain melanomas, caused by lack of TRAIL receptors or upregulation of antiapoptotic proteins. Here we analyze the naturally-occurring flavonoid quercetin as a potential cotreatment with TRAIL to overcome the intrinsic resistance of melanoma. Found in a wide variety of sources from onions and apples to red wine, quercetin is a good candidate for TRAIL cotreatment due to its ability to upregulate TRAIL receptors and downregulate antiapoptotic proteins. We have evaluated our cotreatment of TRAIL plus quercetin on four malignant melanoma cells lines which harbor mutations in the MAPK pathway, namely A375 and WM164 (BRAF mutant), SK-Mel-2 (NRAS mutant) and MeWo (BRAF WT, NRAS WT). Numerous cell-based assays were utilized including antiproliferative SRB assay, apoptotic AnnexinV/PI assay and western blot analysis probing for key proteins of the apoptotic cascade. Out of the four melanoma cell lines evaluated, A375 and SK-Mel-2 were sensitive to TRAIL dose-dependently; whereas, MeWo and WM164 were resistant to TRAIL-induced apoptosis, even at the highest tested treatment concentration of 1μg/ml. Quercetin, as a single agent, was able to induce apoptosis in a dose-dependent manner in all four melanoma cell lines. To determine if the cotreatment, TRAIL plus quercetin, is able to sensitize melanoma cells to TRAIL-induced apoptosis, we treated resistant cell lines, MeWo and WM164 with 250 ng/ml TRAIL plus sub-cytotoxic concentrations of quercetin, 25 and 50 μM. Quercetin was able to sensitize both MeWo and WM164 to TRAIL-induced apoptosis marked by the fragmentation of PARP, a hallmark of apoptosis, and the activation of executioner caspases 3, 6 and 7. Specifically, quercetin was able to sensitize resistant melanomas to undergo TRAIL-induced apoptosis as evidenced by the cleavage of procaspase 8 to caspase 8, a marker for the initiation of TRAIL-induced apoptosis. Quercetin also promoted the TRAIL-mediated activation of the intrinsic pathway of apoptosis marked by the release of cytochrome C from the mitochondria. These preliminary data demonstrate that quercetin is a good potential cotreatment for TRAIL; however, further research is needed to reveal the mechanism of quercetin sensitization of TRAIL-resistant melanomas and its role in TRAIL receptor and antiapoptotic protein expression. Citation Format: Katherine Turner, Daniel Lindner, Michael Kalafatis. Sensitization of malignant melanomas to TRAIL-induced apoptosis by quercetin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1294.

Remote Limb Preconditioning Generates a Neuroprotective Effect by Modulating the Extrinsic Apoptotic Pathway and TRAIL-Receptors Expression.

As remote limb preconditioning (RPC) ameliorates brain damage after ischemic cerebral stroke (ICS), the purpose of the present study was to explore the molecular mechanisms in the course of RPC. Results of TUNEL staining and cleaved caspase-3 expression showed that ischemia-induced neuronal apoptosis was inhibited by RPC. The expression changes in cleaved caspase-8, cFLIP, Bid itself, and its truncated form represented that RPC suppressed the activation of extrinsic apoptotic pathway during ICS. Then, the level of cytoplasmic cytochrome c was also decreased by RPC. In addition, RPC might partially suppress TNF-related apoptosis-inducing ligand (TRAIL)-induced extrinsic apoptosis through downregulation of TRAIL death receptors and upregulation of TRAIL decoy receptors. As a counterproof, immunoneutralization of TRAIL in dMCAO rats resulted in significant restraint of tissue damage and in a marked functional recovery. Our data complemented the knowledge of RPC neuroprotective mechanism and provided new evidence for its clinical application.