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

Pretreatment of umbilical cord derived MSCs with IFN-γ and TNF-α enhances the tumor-suppressive effect on acute myeloid leukemia

Currently, the standard therapeutic approach of AML consists of chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT). However, these strategies are usually associated with adverse side effects and high risk of relapse following HSCT. Thus, it is imperative to find an alternative way against AML progression. Here, we showed that treatment with umbilical cord-derived mesenchymal stem cells (UC-MSCs) could efficiently induce apoptosis in both primary AML patient-derived leukemic cells and AML cell lines. Mechanistically, tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) in UC-MSCs mediated the proapoptotic effect in AML cells. Besides, indoleamine 2,3-dioxygenase (IDO) secreted by UC-MSCs blocked the cell cycle progression and inhibited the proliferation of AML cells. Importantly, we found that incubation of UC-MSCs with IFN-γ and TNF-α could upregulate the expression of TRAIL and IDO, resulting in an intensive pro-apoptotic efficacy. UC-MSCs pre-treatment could not only relieve the AML burden but also eliminate AML cells in a xenograft AML model. Our findings have shed light on an effective pre-activated approach to aggravating the anti-leukemia effect of MSC. Furthermore, a novel and safe stem cell-based therapy approach for AML treatment.

Appearance of Signs of Differentiation and Pro-inflammatory Phenotype in Acute Myeloid Leukemia Cells THP-1 with an Increase in Their TRAIL Resistance in Cell Aggregates in Vitro

Previously, we found a significant increase in TRAIL (TNFα-related apoptosis-inducing ligand) resistance of acute myeloid leukemia (AML) cells in multicellular aggregates in vitro that simulate the location of leukemia cells in the bone marrow. In this study, we tried to assess whether the increased resistance of the AML cells to the cytotoxic effect of TRAIL in multicellular aggregates in vitro can be associated with the launch of mechanisms mediated by cell differentiation or the acquisition of a pro-inflammatory phenotype. The phenotype of THP-1 cells in aggregated and disaggregated cultures was studied in comparison with the phenotype of a disaggregated THP-1 cell culture exposed to factors that induce monocyte-like differentiation or pro-inflammatory phenotype. It was found that an increase in the TRAIL resistance of the THP-1 cells in the aggregated culture was accompanied by the appearance of signs characteristic of monocyte-like differentiation and a pro-inflammatory phenotype. The results are of interest for developing approaches for suppressing TRAIL resistance of AML cells in human bone marrow.

Developing Bioinspired Three-Dimensional Models of Brain Cancer to Evaluate Tumor-Homing Neural Stem Cell Therapy.

Engineered neural stem cells (NSCs) have recently emerged as a promising therapy. Acting as a tumor-homing drug-delivery system, NSCs migrate through brain tissue to seek out primary and invasive tumor foci. NSCs can deliver therapeutic agents, such as TNFα-related apoptosis-inducing ligand, directly to the tumor and suppress glioblastoma (GBM) in murine models. While the mainstays for evaluating NSC migration and efficacy have been two-dimensional chemotaxis assays and mouse models, these low-throughput and small-scale systems limit our ability to implant and track these cells for human translation. To circumvent these challenges, we developed a three-dimensional culture system using a matrix of poly-l-lactic acid 6100 microfibers suspended in agar. These bioinspired brain matrices were used to model tumor growth, NSC migration, and efficacy of NSC therapy at small and human scale. Kinetic fluorescent imaging confirmed growth of tumors in both small and human-sized bioinspired brain matrix. Tumors proliferated 50-fold and 3-fold for GBM and human metastatic breast cancer, respectively, over 7 days. We next explored the impact of tumor location on NSC migration. When NSCs were implanted 2 mm lateral from the tumor foci, NSCs colocalized with the GBM within 7 days. In models of multifocal disease, NSCs were found to colocalize with multiple tumors, preferentially migrating to tumor foci closest to the site of NSC implantation. Lastly, therapeutic NSCs were implanted at increasing distances (0, 2, 5, or 10 mm) laterally from GBM foci to investigate the effects of distance on NSC efficacy. Serial imaging showed reduced fluorescence at tumor sites, implicating GBM apoptosis across all distances. NSCs coinjected with tumor induced a near-complete response in <10 days, while NSCs implanted 10 mm laterally from the tumor induced a near-complete response by day 30. Lastly, GBM foci were established in each hemisphere of the model and control or therapeutic NSCs were implanted adjacent to tumor cells in the right hemisphere. Kinetic imaging showed that NSC therapy attenuated progression of GBM foci, while GBM cells treated with control NSC expanded rapidly over 21 days. In conclusion, we developed a new bioinspired model that supports growth of human brain cancer cells and enables rapid tracking of NSC therapy. Impact statement Tumor-homing and tumor-killing-engineered neural stem cell (NSC) therapies have shown immense promise in both preclinical and clinical trials. However, as cell therapies continue to evolve, cost-effective and high-throughput screening assays are needed to assess the proliferation, migration, and efficacy of these cells. In this study, we developed a bioinspired brain matrix for the evaluation of engineered NSCs. Importantly, this matrix is easy to fabricate, scalable, and allows for sterile real-time, noninvasive imaging using our custom bioreactor. We then utilized the bioinspired brain matrix system to answer key questions around the tumor-homing migration and efficacy of engineered NSC therapies that are challenging to address with traditional models.

Abstract LB-069: TRAILing chemoresistance: Oxaliplatin resistance sensitizes colorectal cancer to TRAIL via death receptor localization

Abstract Colorectal cancer (CRC) remains the second leading cause of cancer related death in the United States. Clinical studies show combination chemotherapy cocktails increase Stage IV patient progression free survival when containing 5-fluorouracil, leucovorin and oxaliplatin (FOLFOX). Despite an initial response rate of 40-50%, patients undergoing FOLFOX have a low median overall survival of 18-months. Treatment induced chemoresistance is in part responsible for this high morbidity, as subpopulations of surviving cells develop resistance mechanisms to induce a more aggressive, metastatic phenotype. Consequently, there exists an urgent need to develop new therapies for metastatic cancers that have become chemoresistant. We hypothesized that Tumor Necrosis factor - α Related Apoptosis Inducing Ligand (TRAIL) based therapeutics would be efficacious in treating oxaliplatin resistant CRC, providing an adjuvant for patients who have exhausted conventional treatment modalities. Four different CRC cell lines were chosen for this study: SW620, SW480, HCT116 and HT29 cells as they all have varying sensitivities to TRAIL. Briefly, oxaliplatin resistant (OxR) derivatives of these cell lines were developed by treating with oxaliplatin and subjecting surviving subpopulations to many rounds of increasing concentrations of chemotherapy. Resistant cells show increased expression of mesenchymal markers along with increased invasiveness. Metastatic CRC patient blood samples were obtained from Guthrie Clinic. Interestingly, SW620 OxR and HCT116 OxR cells show increased sensitization to soluble TRAIL compared to their parental counterparts. Flow cytometry and confocal microscopy demonstrate these sensitized cell lines have increased expression of death receptor 4 (Dr4). Previous studies have demonstrated that combination treatment with oxaliplatin and TRAIL sensitize cancer cells via localization of death receptors into lipid rafts. In our study, we observe a similar phenomenon where oxaliplatin resistant cells have a higher degree of lipid raft-Dr4 colocalization. Induction of death receptor-lipid raft complexes using resveratrol enhances parental SW620 cell sensitivity to TRAIL by 70%, whereas inhibition of lipid rafts via cholesterol sequestration using nystatin decreases sensitization of SW620 OxR cells by 30%. Circulating tumor cells (CTCs) from metastatic CRC patients who have undergone FOLOFX or oxaliplatin based chemotherapy exhibited colocalization of Dr4 and Dr5 into lipid rafts. Additionally, treating whole blood samples from patients with TRAIL-conjugated nanoparticles decreased the number of viable CTCs in circulation 4-fold compared to oxaliplatin monotherapy. These results demonstrate the utility of TRAIL based therapeutics to treat chemoresistant and metastatic CRC. Additionally, this mechanism of apoptosis signal propagation via lipid raft-death receptor complexes can be explored further to probe possible drug targets to augment receptor localization. Citation Format: Joshua Dale Greenlee, Nerymar Ortiz-Otero, Tejas Subramanian, Kevin Liu, Michael King. TRAILing chemoresistance: Oxaliplatin resistance sensitizes colorectal cancer to TRAIL via death receptor localization [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 LB-069.

Personalized-induced neural stem cell therapy: Generation, transplant, and safety in a large animal model.

In this study, we take an important step toward clinical translation by generating the first canine‐induced neural stem cells (iNSCs). We explore key aspects of scale‐up, persistence, and safety of personalized iNSC therapy in autologous canine surgery models. iNSCs are a promising new approach to treat aggressive cancers of the brain, including the deadly glioblastoma. Created by direct transdifferentiation of fibroblasts, iNSCs are known to migrate through the brain, track down invasive cancer foci, and deliver anticancer payloads that significantly reduce tumor burden and extend survival of tumor‐bearing mice. Here, skin biopsies were collected from canines and converted into the first personalized canine iNSCs engineered to carry TNFα‐related apoptosis‐inducing ligand (TRAIL) and thymidine kinase (TK), as well as magnetic resonance imaging (MRI) contrast agents for in vivo tracking. Time‐lapse analysis showed canine iNSCs efficiently migrate to human tumor cells, and cell viability assays showed both TRAIL and TK monotherapy markedly reduced tumor growth. Using intraoperative navigation and two delivery methods to closely mimic human therapy, canines received autologous iNSCs either within postsurgical cavities in a biocompatible matrix or via a catheter placed in the lateral ventricle. Both strategies were well tolerated, and serial MRI showed hypointense regions at the implant sites that remained stable through 86 days postimplant. Serial fluid sample testing following iNSC delivery showed the bimodal personalized therapy was well tolerated, with no iNSC‐induced abnormal tissue pathology. Overall, this study lays an important foundation as this promising personalized cell therapy advances toward human patient testing.

TRAIL-induced variation of cell signaling states provides nonheritable resistance to apoptosis.

TNFα-related apoptosis-inducing ligand (TRAIL), specifically initiates programmed cell death, but often fails to eradicate all cells, making it an ineffective therapy for cancer. This fractional killing is linked to cellular variation that bulk assays cannot capture. Here, we quantify the diversity in cellular signaling responses to TRAIL, linking it to apoptotic frequency across numerous cell systems with single-cell mass cytometry (CyTOF). Although all cells respond to TRAIL, a variable fraction persists without apoptotic progression. This cell-specific behavior is nonheritable where both the TRAIL-induced signaling responses and frequency of apoptotic resistance remain unaffected by prior exposure. The diversity of signaling states upon exposure is correlated to TRAIL resistance. Concomitantly, constricting the variation in signaling response with kinase inhibitors proportionally decreases TRAIL resistance. Simultaneously, TRAIL-induced de novo translation in resistant cells, when blocked by cycloheximide, abrogated all TRAIL resistance. This work highlights how cell signaling diversity, and subsequent translation response, relates to nonheritable fractional escape from TRAIL-induced apoptosis. This refined view of TRAIL resistance provides new avenues to study death ligands in general.

LncRNA MEG3 inhibits trophoblast invasion and trophoblast‐mediated VSMC loss in uterine spiral artery remodeling

Extravillous trophoblasts (EVTs) migrate into uterine decidua and induce vascular smooth muscle cell (VSMC) loss through mechanisms thought to involve migration and apoptosis, achieving complete spiral artery remodeling. Long noncoding RNA maternally expressed gene 3 (MEG3) can regulate diverse cellular processes, such as proliferation and migration, and has been discovered highly expressed in human placenta tissues. However, little is known about the role of MEG3 in modulating EVT functions and EVT-induced VSMC loss. In this study, we first examined the location of MEG3 in human first-trimester placenta by in situ hybridization. Then, exogenous upregulation of MEG3 in HTR-8/SVneo cells was performed to investigate the effects of MEG3 on EVT motility and EVT capacity to displace VSMCs. Meanwhile, the molecules mediating EVT-induced VSMC loss, such as tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) were detected at transcriptional and translational levels. Finally, VSMCs were cocultured with MEG3-upregulated HTR-8/SVneo to explore the role of MEG3 on EVT-mediated VSMC migration and apoptosis. Results showed that MEG3 was expressed in trophoblasts in placental villi and decidua, and MEG3 enhancement inhibited HTR-8/SVneo migration and invasion. Meanwhile, the displacement of VSMCs by HTR-8/SVneo and the expression of TNF-α, FasL and TRAIL in HTR-8/SVneo were reduced following MEG3 overexpression in HTR-8/SVneo. Furthermore, HTR-8/SVneo with MEG3 upregulation impaired VSMC migration and apoptosis. The PI3K/Akt pathway, which is possibly downstream, was inactivated in MEG3-upregulated HTR-8/SVneo. These findings suggest that MEG3 might be a negative regulator of spiral artery remodeling via suppressing EVT invasion and EVT-mediated VSMC loss.

TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between members of the tumor necrosis factor (TNF) ligand superfamily and their receptors. Recent evidence indicated that TNF-α-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation via a TRAF6-dependent signaling pathway; but paradoxically, it inhibits RANK ligand (RANKL)-induced osteoclast differentiation. Although a number of signaling pathways were linked to the RANK and osteoclastogenesis, it is not known how TRAIL regulates RANK signaling. In this study, we demonstrate that TRAIL regulates RANK-induced osteoclastogenesis in terms of the assembly of lipid raft-associated signaling complexes. RANKL stimulation induced recruitment of TRAF6, c-Src, and DAP-12 into lipid rafts. However, the RANKL-induced assembly of lipid raft-associated signaling complexes and TRAF6 recruitment was abolished in the presence of TRAIL. TRAIL-induced dissociation of RANKL-induced lipid raft signaling complexes was reversed by treatment with TRAIL receptor (TRAIL-R) siRNA or an anti-TRAIL-R blocking antibody, indicating that TRAIL mediates suppression of RANKL-induced lipid raft signaling via interactions with TRAIL-R. Finally, we demonstrated that TRAIL suppressed inflammation-induced bone resorption and osteoclastogenesis in a collagen-induced arthritis (CIA) rat animal model. Our results provide a novel apoptosis-independent role of TRAIL in regulating RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment.

Improved Anticancer Effect of Recombinant Protein izTRAIL Combined with Sorafenib and Peptide iRGD.

One of the main problems in oncology is the development of drugs that cause the death of cancer cells without damaging normal cells. Another key problem to be solved is to suppress the drug resistance of cancer cells. The third important issue is to provide effective penetration of drug molecules to cancer cells. TRAIL (TNFα-related apoptosis inducing ligand)/Apo2L is a highly selective anticancer agent. However, the recombinant TRAIL protein having high efficiency against cancer cells in vitro was not effective in clinical trials. Recently we have discovered an acquisition of TRAIL resistance by cancer cells in confluent cultures, which is apparently a manifestation of the general phenomenon of multicellular resistance. The aim of this study was to evaluate whether the anticancer effect of the recombinant protein TRAIL in vivo can be improved by the suppression of multicellular TRAIL-resistance using sorafenib and a tumor-penetrating peptide iRGD, c(CRGDKGPDC). The results testified a great increase in the resistance of human fibrosarcoma HT-1080 cells to izTRAIL both in confluent cultures and in spheroids. Sorafenib administered at nontoxic concentration effectively suppressed confluent- or spheroid-mediated TRAIL-resistance of HT-1080 cells in vitro. Sorafenib combined with iRGD significantly improved the anticancer effect of the recombinant protein izTRAIL in HT-1080 human fibrosarcoma grafts in BALB/c nude mice. Consistent with this finding, multicellular TRAIL-resistance may be a reason of inefficacy of izTRAIL alone in vivo. The anticancer effect of the recombinant protein izTRAIL in vivo may be improved in combination with sorafenib, an inhibitor of multicellular TRAIL resistance and iRGD, the tumor-penetrating peptide.

Alogliptin abates memory injuries of hepatic encephalopathy induced by acute paracetamol intoxication via switching-off autophagy-related apoptosis

AimsHepatic encephalopathy (HE) is a complex neuropsychiatric syndrome. Paracetamol (APAP) causes, in high doses, a hepatic injury. Alogliptin (ALO), with its 100% oral bioavailability, may be able to reverse the acute hepatic injury and memory impairments. Materials and methodsForty rats were divided into four groups as follows; Normal Control Group, APAP intoxicated group, ALO and SIL groups. Behavioral tests (Morris water maze, Y-maze spontaneous alteration, and novel object recognition test) were performed together with evaluating HE score. Neurotransmitters (gamma-aminobutyric acid, glutamate, dopamine, serotonin, norepinephrine and acetylcholine), as well as acetylcholinesterase activity, were determined in the hippocampus. Also, hepatotoxicity markers (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and ammonia) were measured in blood. Additionally, transforming growth factor beta 1, tumor necrosis factor alpha, cytochrome c, granzyme B and caspase-3, coiled-coil Moesin-like BCL-interacting protein 1 “beclin-1”, cellular FLICE-like inhibitory protein, protein 53, TNF-α related apoptosis-inducing ligand, Fas-ligand and alpha-smooth muscle actin were measured in liver homogenate. Moreover, the histopathological investigation was performed. Key findingsAPAP was able to disturb neurotransmitters which were mirrored in the performance of rats in the behavioral test. Most hepatotoxicity, apoptosis and inflammation indicators were elevated after APAP administration, while beclin-1 (autophagy marker) was declined. The tested drugs, both, reversed most of the last mentioned parameters but ALO was more efficient in reducing TGF-β1, α-SMA, TNF-α and ALP as well as increasing % alteration. SignificanceALO and SIL elicited anti-apoptotic, anti-inflammatory and autophagic effects on paracetamol-damaged liver cells and improved memory impairments of HE.

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds.

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNFα-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Combinatorial effect of curcumin and tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) in induction of apoptosis via inhibition of nuclear factor kappa activity and enhancement of caspase-3 activity in chronic myeloid cells: An In-vitro study.

Nuclear factor kappa B (NFkB-light-chain-enhancer of activated B-cells) expression and its regulation is a key role in the development of number of malignancies, as NFkB mediates the balance between cell death and its survival. Therefore, NFkB regulation constitutes an attractive target to overcome the resistance to chemotherapeutic agents in anticancer therapy. Curcumin, as a chemopreventive agent, has a potential role in inhibiting cell growth in a variety of malignancies. Thus, this study was aimed to investigate the efficacy of curcumin along with tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) in KCL-22 myeloid cells along with an investigation of the mechanism by which both the agents exert their effects. KCL-22 cells were exposed to different doses of curcumin and TRAIL alone and in combination. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, caspase activity by fluorescent method, protein expression by western Blot, and NFkB activity by electrophoretic mobility shift assays, respectively. Cell viability assay revealed that when both the agents, curcumin and TRAIL, were used together, there was reduced cell viability in dose- and time-dependent manner as compared to each agent alone. Curcumin and TRAIL enhanced the caspase-3 activity as compared to caspase-8 and caspase-9. Both the agents induced apoptosis in KCL-22 cells by suppressing the IκB kinase and NFkB activity. Our results conclude that curcumin and TRAIL effectively induce the apoptosis through the inhibition of NFkB activity and by enhancing the caspase-3 activity. Thus, curcumin may prove as a potent inhibitor of NFkB by representing its role in cancer pathogenesis, especially in chronic myeloid leukemia cells.

Abstract 20: Oncolytic virotherapy enhances Smac mimetic treatment of acute myeloid leukemia

Abstract This study demonstrates that the combination of Smac mimetic and oncolytic virotherapy (OVT) induces cell death in acute myeloid leukemia (AML) cell lines and primary patient samples; this is the first time the efficacy of this combination approach has been investigated in a human cancer model and for the treatment of AML. Methods: The Smac mimetic LCL161 and several oncolytic viruses (OVs) were tested for in vitro cytotoxicity against a panel of diverse AML cell lines. Bystander killing of LCL161-treated AML cells was explored using conditioned media from OV-treated PBMCs. Multiplex immunoassays were used to detect potential mediators of bystander cytotoxicity produced following OV treatment. Several upregulated cytokines were tested for their ability to enhance LCL161 toxicity in AML cell lines by MTS assay. The activation of healthy donor peripheral blood mononuclear cells (PBMC) was investigated by flow cytometry and immune cell-mediated death of AML cells was measured using chromium release assays. In vitro direct and bystander toxicity was further verified using fresh blood samples from AML patients. Results: A rhabdovirus, MG1, showed the greatest cytotoxicity across a panel of AML cell lines compared to several other OVs. In resistant cell lines the addition of LCL161 treatment led to increased cell death, indicating the potential merits of a combination strategy. Primary AML samples exposed to both LCL161 and MG1 showed greater levels of cell death than either treatment alone, confirming combinational efficacy. Treating healthy donor PBMCs with virus induced the activation of innate immune effector cells, as indicated by an increase in CD69 on NK cells as well as stimulating CD14+ monocytes to produce membrane-bound TNFα-related apoptosis-inducing ligand (TRAIL). Immune cell-mediated death was increased following activation by MG1, and LCL161 sensitized AML cell lines to death by the membrane mimicking KillerTRAIL. Conditioned media from MG1-treated healthy and patient-derived PBMCs displayed an inflammatory milieu that was toxic to AML cell lines. Further analysis identified several cytokines relevant to AML therapy, including the anti-viral interferon alpha (IFNα), soluble TRAIL, and tumor necrosis factor alpha (TNFα); LCL161 was able to increase the sensitivity of AML cells to MG1-conditioned media and recombinant versions of IFNα and TNFα. Discussion: OVT has shown positive clinical efficacy in many solid tumors; however, it is underexplored in the context of hematologic malignancies. Many cases of spontaneous remission following viral infection, the correlation of cytomegalovirus reactivation and positive prognosis following hematologic stem cell transplant, along with the identification of OVs which target leukemic cells without harming healthy hematologic stem cells, suggest that AML patients may be suitable for OVT. Indeed, data from our lab suggest that PBMCs from AML patients are able to recognize and respond to OV, to induce antitumor cytotoxicity. MG1 is cytotoxic to AML cells and acts as an immunogenic agent to induce the release of soluble inflammatory mediators by PBMCs that lead to the death of bystander cells without the need for direct infection. Recent studies into the use of Smac mimetics in hematologic malignancies have demonstrated the synergistic action of combining treatment with inflammatory cytokines, suggesting that combination with an immunogenic agent may be beneficial. Indeed, LCL161 is able to sensitize both AML cell lines and patient-derived AML blasts to MG1 treatment. The release of inflammatory mediators following MG1 treatment also stimulates the activation of innate immune effector cells to induce AML cell death, indicating the ability to initiate a cell-mediated innate immune attack on AML cells using this regime. In conclusion, these data suggest that MG1 should be considered in combination with the Smac mimetic, LCL161, for the treatment of AML. Citation Format: Joanne L. Hopper, Louise ME Müller, Victoria A. Jennings, Gina B. Scott, Stewart McConnell, Richard Kelly, Fiona Errington-Mais. Oncolytic virotherapy enhances Smac mimetic treatment of acute myeloid leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 20.

Neuroprotective effect of (-)-epigallocatechin-3-gallate on autoimmune thyroiditis in a rat model by an anti-inflammation effect, anti-apoptosis and inhibition of TRAIL signaling pathway.

(-)-Epigallocatechin-3-gallate (EGCG) is a polyphenol monomer compound extracted and separated from green tea, and is a key catechin in green tea. Recent research has identified that EGCG is equipped with important biological activities, including antitumor, antioxidant, anti-inflammation, blood fat reduction and radiation protection abilities. In the current study, it was investigated whether EGCG exerts a neuroprotective effect on AIT and examined the possible underlying mechanism. The present study sought to establish an experimental autoimmune thyroiditis (AIT) rat model and to investigate the neuroprotective effect of EGCG in this model. EGCG was demonstrated to inhibit urinary iodine values and thyroid pathological features in AIT model rats. Treatment with EGCG significantly reduced interleukin-1β, interferon-γ (INF-γ) and tumor necrosis factor-α (TNF-α) levels in the AIT rats through suppression of nuclear factor-κB (NF-κB) pathway. In addition, pretreatment with EGCG significantly increased B-cell lymphoma-2 protein expression, and suppressed caspase-3 activity and TNF-α-related apoptosis-inducing ligand (TRAIL) protein expression levels in the AIT model rats. In conclusion, these results suggested that the neuroprotective effect of EGCG protects against AIT through its anti-inflammatory ability, anti-apoptosis and TRAIL signaling pathway in model rats, and it may be used as a therapeutic agent against AIT caused by inflammation.

Overcoming resistance to anti-PD immunotherapy in a syngeneic mouse lung cancer model using locoregional virotherapy

ABSTRACTAnti-PD-1 and anti-PD-L1 immunotherapy has provided a new therapeutic opportunity for treatment of advanced-stage non-small cell lung cancer (NSCLC). However, overall objective response rates are approximately 15%–25% in all NSCLC patients who receive anti-PD therapy. Therefore, strategies to overcome primary resistance to anti-PD immunotherapy are urgently needed. We hypothesized that the barrier to the success of anti-PD therapy in most NSCLC patients can be overcome by stimulating the lymphocyte infiltration at cancer sites through locoregional virotherapy. To this end, in this study, we determined combination effects of anti-PD immunotherapy and oncolytic adenoviral vector-mediated tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) gene therapy (Ad/E1-TRAIL) or adenoviral-mediated TP53 (Ad/CMV-TP53) gene therapy in syngeneic mice bearing subcutaneous tumors derived from M109 lung cancer cells. Both anti–PD-1 and anti–PD-L1 antibodies failed to elicit obvious therapeutic effects in the M109 tumors. Intratumoral administration of Ad/E1-TRAIL or Ad/CMV-TP53 alone suppressed tumor growth in animals preexposed to an adenovector and bearing subcutaneous tumors derived from M109 cells. However, combining either anti–PD-1 or anti–PD-L1 antibody with these two adenoviral vectors elicited the strongest anticancer activity in mice with existing immunity to adenoviral vectors. Dramatically enhanced intratumoral immune response was detected in this group of combination therapy based on infiltrations of CD4+ and CD8+ lymphocytes and macrophages in tumors. Our results demonstrate that resistance to anti–PD-1 immunotherapy in syngeneic mouse lung cancer can be overcome by locoregional virotherapy.

N-terminal gelsolin fragment potentiates TRAIL mediated death in resistant hepatoma cells

TNF-α related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells, without damaging normal cells. TRAIL receptors facilitate induction of apoptosis for selective elimination of malignant cells. However, some cancer cells have developed resistances to TRAIL which limits anticancer potential. Gelsolin, a multifunctional actin-binding protein, mediates cell death involving the TRAIL receptors in the hepatic stellate cell line, LX2. Here, we have shown that conditioned medium (CM) containing gelsolin fragments or an N-terminal gelsolin fragment (amino acid residues 1–70) in the presence of TRAIL impairs cell viability of TRAIL resistant transformed human hepatocytes (HepG2). Cell growth regulation by CM and TRAIL was associated with the modulation of p53/Mdm2, Erk and Akt phosphorylation status. The use of N-terminal gelsolin peptide1–70 alone or in combination with TRAIL, induced inhibition of Akt phosphorylation and key survival factors, Mdm2 and Survivin. Treatment of cells with an Akt activator SC79 or p53 siRNA reduced the effects of the N-terminal gelsolin fragment and TRAIL. Together, our study suggests that the N-terminal gelsolin fragment enhances TRAIL-induced loss of cell viability by inhibiting phosphorylation of Akt and promoting p53 function, effecting cell survival.

Effects of the Notch1 signaling pathway on human lung cancer A549 cells

ABSTRACTPurpose: To evaluate the effects of the Notch1 signaling pathway on human lung cancer A549 cells. Materials and Methods: A549 cells were transfected with recombinant plasmids. Cell proliferation was detected by MTT assay. A tumor-bearing mouse model was established for intratumoral gene injection. Apoptosis-related factors were detected by immunohistochemical assay. Caspase-8, caspase-3, caspase-9, PI3K, pAkt and pSTAT3 expressions were detected by Western blotting. Results: Compared with A549-GFP and A549 cells, A549-ICN cell growth in mice decelerated, tumor volume significantly reduced (p < 0.01), and survival time significantly increased (p < 0.05). Cyclin E and phosphorylated Rb protein expressions were significantly down-regulated. Compared with control, apoptosis-related protein Bcl-2 expression in tumors injected with Notch1 gene was significantly inhibited. After Deltex1 transfection, A549 cell proliferation decelerated, growth was significantly inhibited (p < 0.05), and survival time was significantly extended (p < 0.05). Cyclin E and mutant p53 protein expressions in tumors were down-regulated, phosphorylated Rb expression was almost completely inhibited, and Bcl-2 expression was significantly inhibited. TNF-α-related apoptosis-inducing ligand (TRAIL) inhibited A549-ICN cell growth time- and dose-dependently. After treatment for 24 h or longer, TRAIL induced apoptosis of more A549-ICN cells. Cleaved caspase-3 and cleaved caspase-9 were detected only in A549-ICN cells after 6 h of 40 ng/mL TRAIL treatment, but cleaved caspase-8 was not detected. Combining Notch1 signal with TRAIL inhibited PI3K, phosphorylated Akt and phosphorylated STAT3 expressions. Conclusion: The Notch1 signaling pathway may inhibit A549 cell growth in vitro and in vivo by regulating cell cycle-related and anti-apoptotic protein expressions. Notch1 activation also suppressed A549 cell apoptosis by inhibiting the PI3K/pAkt pathway and activating the caspase-3 pathway in cooperation with TRAIL.

Quinacrine Mediated Sensitization of Glioblastoma (GBM) Cells to TRAIL through MMP-Sensitive PEG Hydrogel Carriers.

Overcoming drug resistance is a major challenge for cancer therapy. Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is a potent therapeutic as an activator of apoptosis, particularly in tumor but not in healthy cells. However, its efficacy is limited by the resistance of tumor cell populations to the therapeutic substance. Here, we have addressed this limitation through the development of a controlled release system, matrix-metalloproteinase (MMP)-sensitive and arg-gly-asp-ser (RGDS) peptide functionalized poly (ethylene-glycol) (PEG) particles which are synthesized via visible-light-induced water-in-water emulsion polymerization. Quinacrine (QC), a recently discovered TRAIL sensitizer drug, is loaded into the hydrogel carriers and the influence of this system on the apoptosis of a malignant type of brain cancer, glioblastoma multiforme (GBM), has been investigated in detail. The results suggest that MMP-sensitive particles are cytocompatible and superior to promote TRAIL-induced apoptosis in GBM cells when loaded with QC. Compared to QC and TRAIL alone, combination of QC-loaded PEG hydrogel and TRAIL demonstrates synergistic apoptotic inducing behavior. Furthermore, QC-loaded particles, but not QC or PEG-hydrogels alone, enhance apoptosis as is measured through expression of apoptosis-related genes. This system is promising to significantly improve the efficacy of chemotherapeutic drugs and suggests a combination treatment for GBM therapy.

ChemInform Abstract: Bioactive Secondary Metabolites with Unique Aromatic and Heterocyclic Structures Obtained from Terrestrial Actinomycetes Species

AbstractReview: 50 refs.

Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β.

Our recent study showed that human mesenchymal stem/stromal cells (hMSCs) are activated to express tumor necrosis factor (TNF)-α-related apoptosis-inducing ligand (TRAIL) by exposure to TNF-α and these activated hMSCs effectively induce apoptosis in triple-negative breast cancer MDA-MB-231 (MDA) cells in vitro and in vivo. Here, we further demonstrated that activated hMSCs not only induced apoptosis of MDA cells but also reduced metastatic features in MDA cells. These activated hMSC-exposed MDA cells showed reduced tumorigenicity and suppressed formation of lung metastasis when implanted in the mammary fat pad. Surprisingly, the activated hMSC-exposed MDA cells increased TRAIL expression, resulting in apoptosis in MDA cells. Interestingly, upregulation of TRAIL in MDA cells was mediated by interferon-beta (IFN-β) secreted from activated hMSCs. Furthermore, IFN-β in activated hMSCs was induced by RNA and DNA released from apoptotic MDA cells in absent in melanoma 2 (AIM2) and IFN induced with helicase C domain 1 (IFIH1)-dependent manners. These observations were only seen in the TRAIL-sensitive breast cancer cell lines but not in the TRAIL-resistant breast cancer cell lines. Consistent with these results, Kaplan–Meier survival analysis also showed that lack of innate sensors detecting DNA or RNA is strongly associated with poor survival in estrogen receptor-negative breast cancer patients. In addition, cancer-associated fibroblasts (CAF) isolated from a breast cancer patient were also able to express TRAIL and IFN-β upon DNA and RNA stimulation. Therefore, our results suggest that the crosstalk between TRAIL-sensitive cancer cells and stromal cells creates a tumor-suppressive microenvironment and further provide a novel therapeutic approach to target stromal cells within cancer microenvironment for TRAIL sensitive cancer treatment.