Expression Of Death Receptor Research Articles

Abstract IA07: Improving CAR T cell therapy efficacy in hematologic malignancies with low-dose total body irradiation

Abstract Chimeric Antigen Receptor (CAR) T cell therapy represents a significant advance in the treatment of hematologic malignancies, as demonstrated by the high response rates and improved survival in diseases such as acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), Diffuse large cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). The FDA has approved several CAR T cell therapies, including some approaches targeting CD19 (CART19) for adult and pediatric patients with B-cell malignancies. Response rates for CD19 CART therapy have been quite promising, reaching 80% overall response and 60% complete response; however, relapses are observed at an estimate of 30-40% within the first year. Thus, finding clinically translatable approaches to improve response rates for patients receiving CART19 therapies, such as the potential of LD-TBI, is critical. Multiple mechanisms of CAR T cell therapy resistance include limited persistence of the CAR T cell product upon infusion, CAR T cell exhaustion, and reduced or complete loss of target expression. Here, we will describe the significant role of radiation therapy, particularly low-dose total body irradiation (LD-TBI), in enhancing the long-term response of CAR T cell therapy. LD-TBI can modulate antigen stability, augment the expression of death receptors in tumor cells, and improve the persistence of CAR T cells. Moreover, LD-TBI also enhances lymphodepletion and the trafficking of CAR T cells to the tumor-infiltrated organs. As a result, LD-TBI significantly prolongs the survival of mice bearing lymphoma or B-ALL in combination with anti-CD19 CAR T cells, thereby improving patient outcomes and offering a promising future for cancer treatment. Citation Format: Monica L. Guzman. Improving CAR T cell therapy efficacy in hematologic malignancies with low-dose total body irradiation. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr IA07

A randomized controlled study of neoadjuvant metformin with chemotherapy in nondiabetic breast cancer women: The METNEO study.

Clinical data demonstrate that metformin exhibits antiproliferative, proapoptotic and antimetastatic actions. Here, correlative molecular studies were undertaken to determine the roles of transmembrane tumour necrosis factor-related apoptosis-inducing ligand death receptors (DRs) and CD133, a glycoprotein biomarker of breast cancer (BC) stem cells, in the advantageous action of metformin on pathological and clinical outcomes in BC patients on neoadjuvant chemotherapy. We randomly assigned 70 nondiabetic BC patients in a 1:1 ratio to either neoadjuvant AC-T chemotherapy (4cycles of adriamycin 60 mg/m2 and cyclophosphamide 600 mg/m2, followed by 12 cycles of weekly paclitaxel 80 mg/m2) or AC-T with adjunct metformin (850 mg twice/day). The expressions of DR4, DR5 and CD133 were quantified in excised tissue samples with residual tumour cells. The overall clinical response (odds ratio: 22.67 [2.77-185.18], P= .004), breast-conserving surgery (odds ratio: 3.67 [1.303-10.321], P= .014) and pathological complete response (β =2.49 ±1.13 [0.274-4.712], P= .028) rates were significantly improved in the metformin arm. Tissues obtained from the metformin arm had upregulated mRNA expression of DR4 (Mean delta cycle thresholds ± standard error of the mean: 2.68 ±0.25 vs. 4.87 ±0.53, P= .0003) and DR5 (0.21 ±0.25 vs. 4.29 ±0.95, P= .0004) compared to control arm. The enhanced DR expression negatively correlated with that of CD133 + BC stem cells, which was significantly reduced by metformin at both cytoplasmic/membranous (43.48 vs. 100.00%, P< .0001) and nuclear sites (4.35 vs. 95.00%, P< .0001). Metformin improves clinical and pathological responses to neoadjuvant AC-T chemotherapy in BC via prompting directionally opposite changes in DRs (increments) and CD133 + (decrements) expressions. This study was registered in ClinicalTrials.gov (registration number: NCT04170465, https://clinicaltrials.gov/ct2/show/NCT04170465).

MOTS-c relieves hepatocellular carcinoma resistance to TRAIL-induced apoptosis under hypoxic conditions by activating MEF2A

BackgroundMitochondrial ORF of the 12S rRNA type-c (MOTS-c) as an AMPK agonist can regulate the expression of adaptive nuclear genes to promote cell homeostasis. However, the investigation of MOTS-c in hepatocellular carcinoma (HCC) is insufficient. This study aims to reveal the role of MOTS-c on HCC cell apoptosis. MethodsHuh7 and HCCLM3 cells were incubated with MOTS-c under a hypoxic condition. MOTS-c levels were quantified by enzyme-linked immunosorbent assay in the peripheral blood of HCC patients and healthy controls. Cell viability was detected by 3-(4,5-Dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was investigated by flow cytometry and Tunel assay. Protein expression was detected by western blotting or immunohistochemistry assay. Dual-luciferase reporter assay and chromatin immunoprecipitation assay were performed to identify the association among myocyte enhancer factor 2A (MEF2A), death receptor 4 (DR4) and DR5. A tumor-bearing nude mouse model was conducted to assess the effect of MOTS-c on HCC tumor formation in vivo. ResultsMOTS-c levels in the peripheral blood of HCC patients were significantly lower compared to healthy individuals. MOTS-c promoted HCC cell apoptosis under hypoxia conditions. Hypoxia stimulation decreased the protein expression of MEF2A, DR4, DR5, fas-associating via death domain (FADD) and caspase-8, while these effects were attenuated after MOTS-c treatment. MOTS-c induced TRAIL-induced apoptosis of HCC cells by activating MEF2A through the phosphorylation of AMPK under hypoxia treatment. In addition, MEF2A transcriptionally up-regulated DR4 and DR5. MOTS-c activated MEF2A to regulate DR4 and DR5 expression, further mediating TRAIL-induced apoptosis. Further, MOTS-c treatment relieved hypoxia-induced tumor growth in vivo. ConclusionMOTS-c relieved hypoxia-induced HCC cell resistance to TRAIL-caused apoptosis by activating MEF2A.

TRAIL induces podocyte PANoptosis via death receptor 5 in diabetic kidney disease

Podocytes can undergo PANoptosis (apoptosis, pyroptosis, and necroptosis). Diabetic kidney disease (DKD) is the leading cause of kidney failure, and podocyte loss is a major event leading to the progression of DKD. Here, we compared single cell RNA sequencing (scRNA-seq) data between three normal and three DKD human kidney samples and found a significant increase of TNFSF10 and TNFRSF10B expression in podocytes of patients with DKD. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), coded by TNFSF10, belongs to the TNF superfamily members and TNFRSF10B codes for death receptor 5 (DR5). We confirmed that expression of TRAIL and DR5 increased in podocytes of patients with DKD and correlated with the severity of DKD. Invitro, TNF-α stimulated TRAIL and DR5 expression in cultured human podocytes. Silence of TRAIL or DR5 by small interfering RNA alleviated TNF-α-stimulated podocytes PANoptosis, while overexpression of TRAIL, treatment with recombinant human TRAIL (rh-TRAIL) or the DR5 activator (Bioymifi) enhanced podocytes PANoptosis. Invivo, podocyte-specific deletion of TNFSF10 or TNFRSF10B alleviated podocyte and glomerular injury in high fat diet and streptozotocin-induced obese diabetic mice and was associated with decreased podocyte PANoptosis. Conversely, the induction of TNFSF10 overexpression specifically in podocytes exacerbated albuminuria and kidney injury in diabetic mice with increased podocyte PANoptosis. Additionally, administration of soluble DR5-Fc, an inhibitor of DR5, resulted in a marked reduction in albuminuria and glomerular injury in BTBR ob/ob mice. Our findings suggest a critical autocrine role of TRAIL/DR5 in inducing podocyte injury in DKD via activation of PANoptosis.

Loss of p53 Impairs Death Receptor Expression and Confers Resistance to CD19 CAR T Cell Therapy in BCP-ALL

Loss of p53 function predicts a dismal outcome in relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Chimeric antigen receptor T cell (CAR T) therapy was recently approved to salvage relapsed/refractory BCP-ALL. We observed a significantly worse overall survival after CD19-targeting CAR T therapy in children with TP53-mutated (TP53Mut) compared to TP53-wildtype (TP53WT) BCP-ALL. To investigate the effect of p53 loss on CAR T therapy response, we modelled TP53 mutations in two BCP-ALL cell lines and observed resistance to CAR T upon p53 loss. Moreover, expression analysis in cell lines and xenografts demonstrated that loss of p53 abrogates expression of the death receptors Fas and DR5, both implicated in CAR T cytotoxicity. Conversely, ectopic expression of Fas improved CAR T cytotoxicity. Furthermore, p53 stabilization induced expression of both Fas and DR5, accompanied by increased CAR T-mediated killing. While these findings provide mechanistic insight as to why CAR T therapy fails against TP53Mut BCP-ALL, they may also provide opportunities to enhance the efficacy of CAR T treatment both in patients with TP53Mut and TP53WT BCP-ALL. Furthermore, these data underscore the need of alternative curative therapies for this very high-risk patient group.

Unlocking Apoptotic Pathways: Overcoming Tumor Resistance in CAR-T-Cell Therapy.

Chimeric antigen receptor (CAR)-T-cell therapy has transformed cancer treatment, leading to remarkable clinical outcomes. However, resistance continues to be a major obstacle, significantly limiting its efficacy in numerous patients. This review critically examines the challenges associated with CAR-T-cell therapy, with a particular focus on the role of apoptotic pathways in overcoming resistance. We explore various strategies to sensitize tumor cells to CAR-T-cell-mediated apoptosis, including the use of combination therapies with BH3 mimetics, Mcl-1 inhibitors, IAP inhibitors, and HDAC inhibitors. These agents inhibit anti-apoptotic proteins and activate intrinsic mitochondrial pathways, enhancing the susceptibility of tumor cells to apoptosis. Moreover, targeting the extrinsic pathway can increase the expression of death receptors on tumor cells, further promoting their apoptosis. The review also discusses the development of novel CAR constructs that enhance anti-apoptotic protein expression, such as Bcl-2, which may counteract CAR-T cell exhaustion and improve antitumor efficacy. We assess the impact of the tumor microenvironment (TME) on CAR-T cell function and propose dual-targeting CAR-T cells to simultaneously address both myeloid-derived suppressor cells (MDSCs) and tumor cells. Furthermore, we explore the potential of combining agents like PPAR inhibitors to activate the cGAS-STING pathway, thereby improving CAR-T cell infiltration into the tumor. This review highlights that enhancing tumor cell sensitivity to apoptosis and increasing CAR-T cell cytotoxicity through apoptotic pathways could significantly improve therapeutic outcomes. Targeting apoptotic proteins, particularly those involved in the intrinsic mitochondrial pathway, constitutes a novel approach to overcoming resistance. The insights presented herein lay a robust foundation for future research and clinical applications aimed at optimizing CAR-T cell therapies.

Enhancing cancer immunotherapy using cordycepin and Cordyceps militaris extract to sensitize cancer cells and modulate immune responses

Integrating immunotherapy with natural compounds holds promise in enhancing the immune system’s ability to eliminate cancer cells. Cordyceps militaris, a traditional Chinese medicine, emerges as a promising candidate in this regard. This study investigates the effects of cordycepin and C. militaris ethanolic extract (Cm-EE) on sensitizing cancer cells and regulating immune responses against breast cancer (BC) and hepatocellular carcinoma (HCC) cells. Cordycepin, pentostatin and adenosine were identified in Cm-EE. Cordycepin treatment decreased HLA-ABC-positive cells in pre-treated cancer cells, while Cm-EE increased NKG2D ligand and death receptor expression. Additionally, cordycepin enhanced NKG2D receptor and death ligand expression on CD3-negative effector immune cells, particularly on natural killer (NK) cells, while Cm-EE pre-treatment stimulated IL-2, IL-6, and IL-10 production. Co-culturing cancer cells with effector immune cells during cordycepin or Cm-EE incubation resulted in elevated cancer cell death. These findings highlight the potential of cordycepin and Cm-EE in improving the efficacy of cancer immunotherapy for BC and HCC.

Apoptosis of Pancreatic Cancer Cells after Co-Treatment with Eugenol and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

Pancreatic cancer is a refractory cancer with limited treatment options. Various cancer types are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Eugenol, the main component of clove oil, exhibits anticancer, anti-inflammatory, and antioxidant effects. However, no studies have reported that eugenol increases TRAIL sensitivity by upregulating death receptor (DR) expression. Here, we aimed to investigate eugenol as a potent TRAIL sensitizer. Increased apoptosis and inhibition of cell proliferation was observed in pancreatic cancer cells treated with eugenol and TRAIL compared with those treated with eugenol alone. Eugenol upregulated the expression of DR5, inhibited the FLICE-inhibitory protein (FLIP), an anti-apoptotic protein, and increased p53, a tumor suppressor protein. In addition, eugenol induced the generation of reactive oxygen species (ROS) and caused endoplasmic reticulum (ER) stress. C/EBP-homologous protein (CHOP) knockdown using siRNA decreased the expression of DR5 and reduced the combined effects of eugenol and TRAIL. These results demonstrate that eugenol enhances TRAIL-induced apoptosis by upregulating DR5 through the ROS-mediated ER stress-CHOP pathway, which enhances ER stress by inducing p53 and downregulating FLIP expression. This suggests that eugenol has the potential to treat pancreatic cancer by increasing cell sensitivity to TRAIL.

The Association of Death Receptors and TGF-β1 Expression in Urothelial Bladder Cancer and Their Prognostic Significance.

Death receptor signalization that triggers the extrinsic apoptotic pathway and TGF-β1 have important roles in urothelial carcinogenesis, with a complex interplay between them. The aim of this research was to assess the association of death receptors DR4, DR5, and FAS as well as TGF-β1 immunohistochemical expression with the clinicopathological characteristics of urothelial bladder cancer (UBC) and to evaluate their prognostic significance. The decrease or loss of death receptors' expression was significantly associated with muscle-invasive tumors, while non-invasive UBC often retains the expression of death receptors, which are mutually strongly linked. High DR4 expression is a marker of low-grade tumors and UBC associated with exposition to known carcinogens. Conversely, TGF-β1 was significantly associated with high tumor grade and advanced stage. High expression of DR4 and FAS indicates longer overall survival. High TGF-β1 signifies an inferior outcome and is an independent predictor of adverse prognosis in UBC patients. This study reveals the expression profile of death receptors in UBC and their possible interconnection with TGF-β1 and indicates independent prognostic significance of high FAS and TGF-β1 expression in UBC, which may contribute to deciphering the enigma of UBC heterogeneity in light of the rapid development of novel and effective therapeutic approaches, including targeting of the TRAIL-induced apoptotic pathway.

Rolapitant treats lung cancer by targeting deubiquitinase OTUD3

BackgroundLung cancer is cancer with the highest morbidity and mortality in the world and poses a serious threat to human health. Therefore, discovering new treatments is urgently needed to improve lung cancer prognosis. Small molecule inhibitors targeting the ubiquitin-proteasome system have achieved great success, in which deubiquitinase inhibitors have broad clinical applications. The deubiquitylase OTUD3 was reported to promote lung tumorigenesis by stabilizing oncoprotein GRP78, implying that inhibition of OTUD3 may be a therapeutic strategy for lung cancer.ResultsIn this study, we identified a small molecule inhibitor of OTUD3, Rolapitant, by computer-aided virtual screening and biological experimental verification from FDA-approved drugs library. Rolapitant inhibited the proliferation of lung cancer cells by inhibiting deubiquitinating activity of OTUD3. Quantitative proteomic profiling indicated that Rolapitant significantly upregulated the expression of death receptor 5 (DR5). Rolapitant also promoted lung cancer cell apoptosis through upregulating cell surface expression of DR5 and enhanced TRAIL-induced apoptosis. Mechanistically, Rolapitant directly targeted the OTUD3-GRP78 axis to trigger endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP)-DR5 signaling, sensitizing lung cancer cells to TRAIL-induced apoptosis. In the vivo assays, Rolapitant suppressed the growth of lung cancer xenografts in immunocompromised mice at suitable dosages without apparent toxicity.ConclusionIn summary, the present study identifies Rolapitant as a novel inhibitor of deubiquitinase OTUD3 and establishes that the OTUD3-GRP78 axis is a potential therapeutic target for lung cancer.

Abstract 1899: Novel combinations of aplitabart, a DR5 agonist IgM antibody, with ADCs or chemotherapeutic agents lead to robust anti-tumor responses in solid tumor models

Abstract Agonism of death receptor 5 (DR5), a tumor necrosis factor receptor superfamily member, induces the killing of tumor cells via the extrinsic apoptotic pathway upon receptor multimerization. We have engineered a multivalent IgM DR5 agonist antibody, aplitabart (IGM-8444), that induces robust anti-tumor activity in both in vitro and in vivo preclinical models. We also demonstrated potent synergistic in vitro activity of aplitabart with different classes of chemotherapeutic agents including microtubule inhibitors, nucleoside analogs and topoisomerase inhibitors in a range of solid cancer cell lines. Preliminary results from an ongoing phase 1a/ab clinical study show that aplitabart has an encouraging safety profile and promising activity in combination with FOLFIRI ± bevacizumab in heavily pretreated metastatic colorectal cancer patients. To further explore the combination therapies for aplitabart in lung and breast cancers, we evaluated standard of care chemotherapies, such as paclitaxel, carboplatin, and antibody drug conjugates (ADCs) in vitro on a range of human lung and breast cancer cell lines. We observed dose-dependent synergistic cytotoxic effects of aplitabart on tumor cells when combined with these agents. Additionally, these agents increased the expression of DR5 on tumor cells and could explain their synergistic combinatorial activity with aplitabart. Representative cancer cell lines were selected to evaluate these combinations in vivo using xenograft mouse models where robust anti-tumor responses were demonstrated in the xenograft lung and breast cancer models, resulting in enhanced tumor growth inhibition and extended survival. Our findings demonstrate potent in vitro synergistic cytotoxicity and enhanced anti-tumor response in preclinical models in vivo by combining aplitabart with ADCs, or carboplatin and paclitaxel. These results support the combination of DR5 agonist IgM antibodies with agents known to upregulate DR5 expression on solid tumors. Aplitabart is currently under evaluation in a randomized clinical study in combination with FOLFIRI + bevacizumab in comparison with FOLFIRI + bevacizumab in patients with colorectal cancer (NCT04553692). Citation Format: Mélanie Desbois, Susan E. Calhoun, Kevin C. Hart, Carolyn R. Denson, Poonam Yakkundi, Thomas J. Matthew, Maya K. Leabman, Sanela Bilic, Genevive Hernandez, Eric W. Humke, Albert F. Candia, Bruce Keyt, Angus M. Sinclair, Maya F. Kotturi, Beatrice T. Wang. Novel combinations of aplitabart, a DR5 agonist IgM antibody, with ADCs or chemotherapeutic agents lead to robust anti-tumor responses in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1899.

Abstract 3602: TRAIL-NK-92 MI as a novel adoptive therapy for castration-resistant prostate cancer (CRPC): Preliminary in vitro results

Abstract Introduction: Natural killer (NK) cell tumor infiltration and higher expression of activating receptors NKp30 and NKp46 are associated with improved clinical outcomes in prostate cancer (PC). Activation of NK cells by androgen receptor inhibitors enhanced the killing of PC cell lines (Schwermann 2023). These findings support the therapeutic potential of strategies inducing NK cell activation to treat CRPC. NK cells induce apoptosis of cancer cells via TNF-related apoptosis-inducing ligand) (TRAIL) binding of death receptor 5 (DR5), which is overexpressed in PC cells. This is the first report of adoptive therapy utilizing high expression of TRAIL as a novel therapeutic strategy. Methods: We transduced NK-92-MI and CD4+ (Jurkat) cell lines with human TRAIL (pLVX-EF1alpha-TRAIL-IRES-eGFP).Transduced cells were pre-treated with simvastatin or atorvastatin (4uM) for 36h, followed by stimulation (IL-2 100 IU/ml and IL-12 100 ng/ml) for 2h. The eGFP-expressing cells were then sorted by FACS. PC (PC3, DU145, LNCaP, and 22Rv1), fibroblast (IMR-90), and brain endothelial (hCMEC/D3) cell lines were used in co-culture experiments with non-transduced and transduced NK cells and CD4+ cells. These experiments were quantified using the ImageXpress Confocal HT instrument. Viability assays were performed using Annexin-PI and flow cytometry staining for caspases 3/7. Results: Transduced NK-92 MI cells with TRAIL displayed increased surface expression of TRAIL (control[C]: 4.38%±1; TRAIL-overexpression[NK-TRAIL]: 78.54%±3.76; p&amp;lt;0.001). Co-culture of PC cell lines with TRAIL-NK-92 MI cells resulted in significant tumor-induced apoptosis compared to co-culture with non-transduced NK cells after 48 hours (LNCaP + NK [C]: 16.52%±5, LNCaP + NK-TRAIL: 73.19%±6; PC [C]: 14.4%±3, PC3 + NK-TRAIL: 79%±8; 22Rv1 [C]: 16.78%±1.7, 22Rv1 + NK-TRAIL: 76%±8; DU145 [C]: 13%±4, DU145 + NK-TRAIL: 59.1%±10; p&amp;lt;0.001). This enhanced cytotoxic function was also observed in CD4+ cells expressing high levels of TRAIL. Annexin-PI staining as well as Caspase 3 and 7 showed increased apoptosis in PC co-cultured with NK-TRAIL. Co-culture of NK-TRAIL cells did not affect the viability of the non-malignant cells (IMR-90 and hCMEC/D3) over 48h (IMR-90 + NK: 4.6%±1, IMR-90 + NK-TRAIL: 4.47±2, p=0.9; hCMEC/D3 + NK: 2.7%±0.8, hCMEC/D3 + NK-TRAIL: 2.2±0.5, p&amp;gt;0.99). These results align with the lower expression of DR5 on non-malignant cells compared to PC cell lines (IMR-90: 1.46%±0.5, hCMEC/D3: 1.9%±0.8, LNCaP: 92.3%±4.5). Conclusion: Transduced NK-92 MI cells with human TRAIL-induced higher levels of apoptosis of PC cell lines compared with non-transduced NK cells in co-culture experiments. The results highlight the potential of TRAIL-expressing adoptive cell therapy for CRPC. Engaging adoptive therapies with specific targets (anti-PSMA CAR) might increase the therapeutic potential of this strategy. Citation Format: Maximilian Schwermann, Praveen Srinivasan, William MacDonald, Vida Tajiknia, Andrea Schmidt, Shengliang Zhang, Lindsey Carlsen, Andrew George, Connor Purcell, Shaolei Lu, Matthew Hadfield, Anthony Mega, Benedito Carneiro, Wafik El-Deiry. TRAIL-NK-92 MI as a novel adoptive therapy for castration-resistant prostate cancer (CRPC): Preliminary in vitro results [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3602.

METTL16 deficiency attenuates apoptosis through translational control of extrinsic death receptor during nutrient deprivation

METTL16 is a well-characterized m6A methyltransferase that has been reported to contribute to tumorigenesis in various types of cancer. However, the effect of METTL16 on tumor progression under restricted nutrient conditions, which commonly occur in tumor microenvironment, has yet to be elucidated. Herein, our study initially reported the inhibitory effect of METTL16 depletion on apoptosis under amino acid starvation conditions. Mechanistically, we determined that the METTL16 knockdown represses the expression of extrinsic death receptors at both transcription and translation levels. Depletion of METTL16 prevented protein synthesis of GCN2, resulting in diminished ATF4 expression in a GCN2-eIF2α-dependent manner. Reduction of ATF4 further declined the expression of apoptotic receptor protein DR5. Meanwhile, METTL16 deficiency directly hampered protein synthesis of FADD and DR5, thereby impairing apoptosis and promoting cancer cell survival. Taken together, our study provides novel evidence for the involvement of METTL16 in regulating cancer progression, suggesting that METTL16 as a potential therapeutic target for cancer treatment.

Cyclizine induces cytotoxicity and apoptosis in macrophages through the extrinsic and intrinsic apoptotic pathways.

Cyclizine, an over-the-counter and prescription antihistamine, finds widespread application in the prevention and treatment of motion sickness, encompassing symptoms such as nausea, vomiting, dizziness, along with its effectiveness in managing vertigo. However, the overuse or misuse of cyclizine may lead to hallucinations, confusion, tachycardia, and hypertension. The molecular mechanisms underlying cyclizine-induced cytotoxicity and apoptosis remain unclear. During the 24 h incubation duration, RAW264.7 macrophages were exposed to different concentrations of cyclizine. Cytotoxicity was assessed through the lactate dehydrogenase assay. Flow cytometry employing annexin V-fluorescein isothiocyanate and propidium iodide was utilized to evaluate apoptosis and necrosis. Caspase activity and mitochondrial dysfunction were evaluated through a fluorogenic substrate assay and JC-1 dye, respectively. Flow cytometry employing fluorogenic antibodies was utilized to evaluate the release of cytochrome c and expression of death receptor, including tumor necrosis factor-α receptor and Fas receptor. Western blotting was utilized to evaluate the expression of the Bcl2 and Bad apoptotic regulatory proteins. The findings unveiled from the present study demonstrated that cyclizine exerted a concentration-dependent effect on RAW264.7 macrophages, leading to the induction of cytotoxicity, apoptosis, and necrosis. This compound further activated the intrinsic apoptotic pathway by inducing mitochondrial dysfunction, Bcl2/Bad exchange expression, cytochrome c liberation, and activation of caspases contained caspase 3, 8, and 9. Moreover, the activation of the extrinsic apoptotic pathway was observed as cyclizine induced the upregulation of death receptors and increased caspase activities. Based on our investigations, it can be inferred that cyclizine prompts cytotoxicity and apoptosis in RAW264.7 macrophages in a concentration-dependent manner by triggering both the intrinsic and extrinsic apoptotic pathways.

Wogonin induces apoptosis in macrophages by exhibiting cytotoxic and genotoxic effects.

Macrophages play an important role in defending the body against invading pathogens. In the face of pathogens, macrophages become activated and release toxic materials that disrupt the pathogens. Macrophage overactivation can lead to severe illness and inflammation. Wogonin has several therapeutic effects, including anti-inflammatory, anticancer, antioxidant, and neuroprotective effects. No studies have investigated the cytotoxic effects of wogonin at concentrations of more than 0.1 mM in RAW264.7 cells. In this study, RAW 264.7 cells were treated with wogonin, which, at concentrations of more than 0.1 mM, had cytotoxic and genotoxic effects in the RAW264.7 cells, leading to apoptosis and necrosis. Further, wogonin at concentrations of more than 0.1 mM induced caspase-3, caspase-8, and caspase-9 activation and mitochondrial dysfunction and death receptor expression. These results suggest that wogonin induces apoptosis through upstream intrinsic and extrinsic pathways by exhibiting cytotoxic and genotoxic effects.

Bacteriophage-based particles carrying the TNF-related apoptosis-inducing ligand (TRAIL) gene for targeted delivery in hepatocellular carcinoma.

The TRAIL (Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand) is a promising candidate for cancer treatment due to its unique ability to selectively induce programmed cell death, or apoptosis, in cancer cells while sparing healthy ones. This selectivity arises from the preferential binding of the TRAIL to death receptors on cancer cells, triggering a cascade of events that lead to their demise. However, significant limitations in using the TRAIL for cancer treatment are the administration of the TRAIL protein that can potentially lead to tissue toxicity (off-target) and the short half-life of the TRAIL in the body which may necessitate frequent and sustained administration; these can pose logistical challenges for long-term treatment regimens. We have devised a novel approach for surmounting these limitations by introducing the TRAIL gene directly into cancer cells, enabling them to produce the TRAIL locally and subsequently trigger apoptosis. A novel gene delivery system such as a bacteriophage-based particle TPA (transmorphic phage/AAV) was utilized to address these limitations. TPA is a hybrid M13 filamentous bacteriophage particle encapsulating a therapeutic gene cassette with inverted terminal repeats (ITRs) from adeno-associated viruses (AAVs). The particle also showed a tumour targeting ligand, CDCRGDCFC (RGD4C), on its capsid (RGD4C.TPA) to target the particle to cancer cells. RGD4C selectively binds to αvβ3 and αvβ5 integrins overexpressed on the surface of most of the cancer cells but is barely present on normal cells. Hepatocellular carcinoma (HCC) was chosen as a model because it has one of the lowest survival rates among cancers. We demonstrated that human HCC cell lines (Huh-7 and HepG2) express αvβ5 integrin receptors on their surface. These HCC cells also express death receptors and TRAIL-binding receptors. We showed that the targeted TPA particle carrying the transmembrane TRAIL gene (RGD4C.TPA-tmTRAIL) selectively and efficiently delivered the tmTRAIL gene to HCC cells resulting in the production of tmTRAIL from transduced cells and subsequently induced apoptotic death of HCC cells. This tumour-targeted particle can be an excellent candidate for the targeted gene therapy of HCC.

Sec1 regulates intestinal mucosal immunity in a mouse model of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a common immune-mediated condition with its molecular pathogenesis remaining to be fully elucidated. This study aimed to deepen our understanding of the role of FUT2 in human IBD, by studying a new surrogate gene Sec1, a neighboring gene of Fut2 and Fut1 that co-encodes the α 1,2 fucosyltransferase in mice. CRISPR/Cas9 was used to prepare Sec1 knockout (Sec1−/−) mice. IBD was induced in mice using 3% w/v dextran sulphate sodium. Small interfering RNA (siRNA) was employed to silence Sec1 in murine colon cancer cell lines CT26.WT and CMT93. IBD-related symptoms, colonic immune responses, proliferation and apoptosis of colon epithelial cells were assessed respectively to determine the role of Sec1 in mouse IBD. Impact of Sec1 on the expression of death receptor 5 (DR5) and other apoptosis-associated proteins were determined. Sec1 knockout was found to be associated with deterioration of IBD in mice and elevated immune responses in the colonic mucosa. Silencing Sec1 in CT26.WT and CMT93 cells led to greater secretion of inflammatory cytokines IL-1β, IL-6 and TNF-α. Cell counting kit 8 (CCK8) assay, flow cytometry and TUNEL detection suggested that Sec1 expression promoted the proliferation of colon epithelial cells, inhibited cell apoptosis, reduced cell arrest in G0/G1 phase and facilitated repair of inflammatory injury. Over-expression of DR5 and several apoptosis-related effector proteins was noticed in Sec1−/− mice and Sec1-silenced CT26.WT and CMT93 cells, supporting a suppressive role of Sec1 in cell apoptosis. Our results depicted important regulatory roles of Sec1 in mouse IBD, further reflecting the importance of FUT2 in the pathogenesis of human IBD.

An Antidepressant Drug Increased TRAIL Receptor-2 Expression and Sensitized Lung Cancer Cells to TRAIL-induced Apoptosis.

TRAIL has emerged as a promising therapeutic target due to its ability to selectively induce apoptosis in cancer cells while sparing normal cells. Autophagy, a highly regulated cellular recycling mechanism, is known to play a cell survival role by providing a required environment for the cell. Recent studies suggest that autophagy plays a significant role in increasing TRAIL resistance in certain cancer cells. Thus, regulating autophagy in TRAIL-mediated cancer therapy is crucial for its role in cancer treatment. Our study explored whether the antidepressant drug desipramine could enhance the ability of TRAIL to kill cancer cells by inhibiting autophagy. The effect of desipramine on TRAIL sensitivity was examined in various lung cancer cell lines. Cell viability was measured by morphological analysis, trypan blue exclusion, and crystal violet staining. Flow cytometry analysis was carried out to measure apoptosis with annexin V-PI stained cells. Western blotting, rtPCR, and immunocytochemistry were carried out to measure autophagy and death receptor expression. TEM was carried out to detect autophagy inhibition. Desipramine treatment increased the TRAIL sensitivity in all lung cancer cell lines. Mechanistically, desipramine treatment induced death receptor expression to increase TRAIL sensitivity. This effect was confirmed when the genetic blockade of DR5 reduced the effect of desipramine in enhanced TRAIL-mediated cell death. Further investigation revealed that desipramine treatment increased the LC3 and p62 levels, indicating the inhibition of lysosomal degradation of autophagy. Notably, TRAIL, in combination with either desipramine or the autophagy inhibitor chloroquine, exhibited enhanced cytotoxicity compared to TRAIL treatment alone. Our findings revealed the potential of desipramine to induce TRAIL-mediated cell death by autophagy impairment. This discovery suggests its therapeutic potential for inducing TRAIL-mediated cell death by increasing the expression of death receptors, which is caused by impairing autophagy.

AICAR Induces AMPK-Independent Cell Death in Adult T-Cell Leukemia/Lymphoma and Has Anti-Tumor Activity

Introduction: Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell leukemia/lymphoma caused by human T-cell lymphotropic virus type I (HTLV-1) and has a poor outcome. Recently, we reported that AMPKα, the catalytic unit of AMP-activated protein kinase (AMPK), is highly expressed in cells from patients with ATL and that an AMPK inhibitor had anti-tumor activity in ATL mice 1. On the other hand, acadesine or 5-aminoimidazole-4-carboxamide riboside (AICAR) is anAMPK activator, and was recently shown to have tumor suppressive effects on B cell chronic lymphocytic leukemia via the activation of AMPK. However, the effect of AICAR on the death of ATL-related cell lines is unknown. This study evaluated the effects of AICAR on death in ATL-related cell lines and its anti-tumor activity. Results: We demonstrated that AICAR inhibited the proliferation of ATL-related cell lines (S1T, MT-1 and MT-2) but not non-HTLV-1-infected Jurkat cells and peripheral blood mononuclear cells from healthy individuals. However, AICAR did not increase the phosphorylation levels of AMPKα. We found that AICAR increased annexin V-positive cell numbers in the sub-G0/G1 phase, and decreased the mitochondrial membrane potential and caspase-3/8/9 activation, which are hallmarks of apoptosis. However, AICAR did not increase the phosphorylation levels of AMPKα. On the other hand, HTLV-1 Tax, an HTLV-1-encoded oncogenic factor, expression did not affect AICAR-induced cell death. Moreover, AICAR induced the expression of death receptors (DR) DR4 and DR5 in vitro. Furthermore, AICAR inhibited the growth and infiltration of S1T cells transplanted subcutaneously in NOD/SCID/gamma mice. Conclusion: This is the first evidence to demonstrate the AMPK-independent effects of AICAR on death in ATL-related cell lines and its anti-tumor activity. Thus, AICAR might be a candidate for the treatment of ATL. Disclosures : No relevant conflicts of interest to declare. 1. Aikawa A, Kozako T, Uchida Y, et al. Cell death induced by dorsomorphin in adult T-cell leukemia/lymphoma is AMPK-independent. Febs j. 2020;287(18):4005-4015.

A novel phthalazinone derivative as a capsid assembly modulator inhibits hepatitis B virus expression

Development of new anti-hepatitis B virus (HBV) drugs that target viral capsid assembly is a very active research field. We identify a novel phthalazinone derivative, compound 5832, as a potent HBV inhibitor. In this study, we intend to elaborate the antiviral effect and mechanism of 5832 against HBV in vitro and in vivo. Compound 5832 treatment induces the formation of genome-free empty capsid by interfering with the core protein assembly domain, which significantly decreases the extracellular and intracellular HBV DNA. In the AAV-HBV transduced mouse model, 5832 suppresses serum HBV DNA after 4-week treatment, and decreases HBsAg and HBeAg levels. 5832 treatment also reduces intrahepatic HBV RNA, DNA and HBcAg levels. During the follow-up period after treatment withdrawal, serum antigen levels demonstrated no increase. We demonstrate 5832 treatment could active apoptotic signaling by elevating the expression of death receptor 5 (DR5), which participated in corresponding HBcAg-positive hepatocyte eradication. Phthalazinone derivative 5832 may serve as a promising anti-HBV drug candidate to improve the treatment options for chronic HBV infection.