Suppression of neointimal hyperplasia induced by arteriovenous anastomosis and balloon injury in rats by multimeric tumor necrosis factor-related apoptosis-inducing ligand.

Excessive blood vessel wall thickening, known as intimal hyperplasia, can result from injury or inflammation and increase the risk of vascular diseases. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays key roles in tumor surveillance, autoimmune diseases, and apoptosis; however, its role in vascular stenosis remains controversial. Treatment with recombinant isoleucine zipper hexamerization domain soluble TRAIL (ILz(6):TRAIL) significantly inhibited the progression of neointimal hyperplasia (NH) induced by anastomosis of the carotid artery and jugular vein dose dependently, and adenovirus expressing secretable ILz(6):TRAIL also inhibited NH induced by balloon injury in the femoral artery of rats. This study demonstrated the preventive and partial regressive effects of ILz(6):TRAIL on anastomosis of the carotid artery and jugular vein- or balloon-induced NH.

Licochalcone D exhibits cytotoxicity in breast cancer cells and enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis through upregulation of death receptor 5.

Anticancer strategies using natural products or derivatives are promising alternatives for cancer treatment. Here, we showed that licochalcone D (LCD), a natural flavonoid extracted from Glycyrrhiza uralensis Fisch, suppressed the growth of breast cancer cells, and was less toxic to MCF-10A normal breast cells. LCD-induced DNA damage, cell cycle arrest, and apoptosis in breast cancer cells. Furthermore, LCD potentiated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. Mechanistically, LCD was revealed to reduce survival protein expression and to upregulate death receptor 5 (DR5) expressions. Silencing DR5 blocked the ability of LCD to sensitize cells to TRAIL-mediated apoptosis. LCD increased CCAAT/enhancer-binding protein homologous protein (CHOP) expression in breast cancer cells. Knockdown of CHOP attenuated DR5 upregulation and apoptosis triggered by cotreatment with LCD and TRAIL. Furthermore, LCD suppressed the phosphorylation of extracellular signal-regulated kinase and promoted the phosphorylation of c-Jun amino-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Pretreatment with JNK inhibitor SP600125 or p38 MAPK inhibitor SB203580 abolished the upregulation of DR5 and CHOP, and also attenuated LCD plus TRAIL-induced cleavage of poly(ADP-ribose) polymerase. Overall, our results show that LCD exerts cytotoxic effects on breast cancer cells and arguments TRAIL-mediated apoptosis by inhibiting survival protein expression and upregulating DR5 in a JNK/p38 MAPK-CHOP-dependent manner.

Polyphyllin I enhances tumor necrosis factor-related apoptosis-inducing ligand-induced inhibition of human osteosarcoma cell growth downregulating the Wnt/ß-catenin pathway.

OBJECTIVE: To investigate the synergistic effects of polyphyllin I (PPI) combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the growth of osteosarcoma cells through downregulating the Wnt/ß-catenin signaling pathway. METHODS: Cell viability, apoptosis and cell cycle distribution were examined using cell counting kit-8 and flow cytometry assays. The morphology of cancer cells was observed with inverted phase contrast microscope. The migration and invasion abilities were examined by xCELLigence real time cell analysis DP system and transwell assays. The expressions of poly (adenosine diphosphate-ribose) polymerase, C-Myc, Cyclin B1, cyclin-dependent kinases 1, N-cadherin, Vimentin, Active-ß-catenin, ß-catenin, p-glycogen synthase kinase 3ß (GSK-3ß) and GSK-3ß were determined by Western blotting assay. RESULTS: PPI sensitized TRAIL-induced decrease of viability, migration and invasion, as well as increase of apoptosis and cell cycle arrest of MG-63 and U-2 OS osteosarcoma cells. The synergistic effect of PPI with TRAIL in inhibiting the growth of osteosarcoma cells was at least partially realized through the inactivation of Wnt/ß-catenin signaling pathway. CONCLUSION: The combination of PPI and TRAIL is potentially a novel treatment strategy of osteosarcoma.

Effect of Abdominal Aortic Calcification on Recurrence Following Initial Hepatectomy for Colorectal Liver Metastases.

BACKGROUND/AIM: The aim of the study was to analyze the association between abdominal aortic calcification (AAC) and patient prognosis following resection of colorectal liver metastases (CRLM). AAC potentially reflects intrahepatic immunity and is involved in tumor development and progression. However, the clinical effects of AAC on colorectal cancer (CRC) prognosis after curative-intent liver resection for CRLM remain unclear. PATIENTS AND METHODS: We evaluated the effect of AAC on the clinical prognosis and metastatic patterns in 99 patients who underwent hepatectomy for CRLM between 2010 and 2019. RESULTS: The high-AAC group had significantly worse overall survival (OS) and remnant liver recurrence rate (RR) after propensity score matching to adjust for differences in baseline characteristics of patients and tumors. In multivariate Cox regression analyses, high AAC volume was an independent risk factor for poor OS and liver RR, but not poor lung RR. The expression of tumor necrosis factor-related apoptosis-inducing ligand, known as an anti-tumor marker, in liver natural killer (NK) cells was lower in the high-AAC group than in the low-AAC group. CONCLUSION: High AAC volume showed a strong relationship with remnant liver RR after curative resection of CRLM. High AAC volume may be responsible for the suppression of anti-tumor activity of liver NK cells, which results in an increased risk of liver recurrence and poor prognosis.

Proteogenomic Characterization of Bladder Cancer Reveals Sensitivity to Apoptosis Induced by Tumor Necrosis Factor-related Apoptosis-inducing Ligand in FGFR3-mutated Tumors.

BACKGROUND: Molecular understanding of muscle-invasive (MIBC) and non-muscle-invasive (NMIBC) bladder cancer is currently based primarily on transcriptomic and genomic analyses. OBJECTIVE: To conduct proteogenomic analyses to gain insights into bladder cancer (BC) heterogeneity and identify underlying processes specific to tumor subgroups and therapeutic outcomes. DESIGN, SETTING, AND PARTICIPANTS: Proteomic data were obtained for 40 MIBC and 23 NMIBC cases for which transcriptomic and genomic data were already available. Four BC-derived cell lines harboring FGFR3 alterations were tested with interventions. INTERVENTION: Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), second mitochondrial-derived activator of caspases mimetic (birinapant), pan-FGFR inhibitor (erdafitinib), and FGFR3 knockdown. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Proteomic groups from unsupervised analyses (uPGs) were characterized using clinicopathological, proteomic, genomic, transcriptomic, and pathway enrichment analyses. Additional enrichment analyses were performed for FGFR3-mutated tumors. Treatment effects on cell viability for FGFR3-altered cell lines were evaluated. Synergistic treatment effects were evaluated using the zero interaction potency model. RESULTS AND LIMITATIONS: Five uPGs, covering both NMIBC and MIBC, were identified and bore coarse-grained similarity to transcriptomic subtypes underlying common features of these different entities; uPG-E was associated with the Ta pathway and enriched in FGFR3 mutations. Our analyses also highlighted enrichment of proteins involved in apoptosis in FGFR3-mutated tumors, not captured through transcriptomics. Genetic and pharmacological inhibition demonstrated that FGFR3 activation regulates TRAIL receptor expression and sensitizes cells to TRAIL-mediated apoptosis, further increased by combination with birinapant. CONCLUSIONS: This proteogenomic study provides a comprehensive resource for investigating NMIBC and MIBC heterogeneity and highlights the potential of TRAIL-induced apoptosis as a treatment option for FGFR3-mutated bladder tumors, warranting a clinical investigation. PATIENT SUMMARY: We integrated proteomics, genomics, and transcriptomics to refine molecular classification of bladder cancer, which, combined with clinical and pathological classification, should lead to more appropriate management of patients. Moreover, we identified new biological processes altered in FGFR3-mutated tumors and showed that inducing apoptosis represents a new potential therapeutic option.

Pharmacokinetics and immunogenicity of eftozanermin alfa in subjects with previously-treated solid tumors or hematologic malignancies: results from a phase 1 first-in-human study.

PURPOSE: Eftozanermin alfa is a second-generation tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor agonist that enhances death receptor 4/5 clustering on tumor cells to induce apoptosis. We report the pharmacokinetics and immunogenicity of eftozanermin alfa administered intravenously to 153 adults with previously-treated solid tumors or hematologic malignancies from the first-in-human, open-label, dose-escalation and dose-optimization study. METHODS: Dose escalation evaluated eftozanermin alfa monotherapy 2.5-15 mg/kg on Day 1 or Days 1/8 of a 21-day cycle. Dose optimization evaluated eftozanermin alfa monotherapy or combination therapy with either oral venetoclax 400-800 mg daily (eftozanermin alfa 1.25-7.5 mg/kg Days 1/8/15 of a 21-day cycle) or chemotherapy (eftozanermin alfa 3.75 or 7.5 mg/kg Days 1/8/15/22 of a 28-day cycle and FOLFIRI regimen [leucovorin, 5-fluorouracil, and irinotecan] with/without bevacizumab on Days 1/15 of a 28-day cycle). RESULTS: Systemic exposures (maximum observed concentration [Cmax] and area under the concentration-time curve [AUC]) of eftozanermin alfa were approximately dose-proportional across the entire dose escalation range with minimal to no accumulation in Cycle 3 versus Cycle 1 exposures. Comparable exposures and harmonic mean half-lives (35.1 h [solid tumors], 31.3 h [hematologic malignancies]) were observed between malignancy types. Exposures (dose-normalized Cmax and AUC) in Japanese subjects were similar to non-Japanese subjects. Furthermore, eftozanermin alfa/venetoclax combination therapy did not have an impact on the exposures of either agent. Treatment-emergent anti-drug antibodies were observed in 9.4% (13/138) of subjects. CONCLUSIONS: The study results, including a pharmacokinetic profile consistent with weekly dosing and low incidence of immunogenicity, support further investigation of eftozanermin alfa. TRIAL REGISTRATION ID: NCT03082209.

TRAIL-Sensitizing Effects of Flavonoids in Cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) represents a promising anticancer agent, as it selectively induces apoptosis in transformed cells without altering the cellular machinery of healthy cells. Unfortunately, the presence of TRAIL resistance mechanisms in a variety of cancer types represents a major hurdle, thus limiting the use of TRAIL as a single agent. Accumulating studies have shown that TRAIL-mediated apoptosis can be facilitated in resistant tumors by combined treatment with antitumor agents, ranging from synthetic molecules to natural products. Among the latter, flavonoids, the most prevalent polyphenols in plants, have shown remarkable competence in improving TRAIL-driven apoptosis in resistant cell lines as well as tumor-bearing mice with minimal side effects. Here, we summarize the molecular mechanisms, such as the upregulation of death receptor (DR)4 and DR5 and downregulation of key anti-apoptotic proteins [e.g., cellular FLICE-inhibitory protein (c-FLIP), X-linked inhibitor of apoptosis protein (XIAP), survivin], underlying the TRAIL-sensitizing properties of different classes of flavonoids (e.g., flavones, flavonols, isoflavones, chalcones, prenylflavonoids). Finally, we discuss limitations, mainly related to bioavailability issues, and future perspectives regarding the clinical use of flavonoids as adjuvant agents in TRAIL-based therapies.

Discovery of cancer-preventive juices reactivating RB functions.

BACKGROUND: Recent advances have been achieved in the genetic diagnosis and therapies against malignancies due to a better understanding of the molecular mechanisms underlying carcinogenesis. Since active preventive methods are currently insufficient, the further development of appropriate preventive strategies is desired. METHODS: We searched for drinks that reactivate the functions of tumor-suppressor retinoblastoma gene (RB) products and exert anti-inflammatory and antioxidant effects. We also examined whether lactic acid bacteria increased the production of the cancer-specific anti-tumor cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in human, and examined whether the RB-reactivating drinks with lactic acid bacteria decreased azoxymethane-induced rat colon aberrant crypt foci (ACF) and aberrant crypts (ACs) in vivo. RESULTS: Kakadu plum juice and pomegranate juice reactivated RB functions, which inhibited the growth of human colon cancer LIM1215 cells by G1 phase arrest. These juices also exerted anti-inflammatory and antioxidant effects. Lactiplantibacillus (L.) pentosus S-PT84 was administered to human volunteers and increased the production of TRAIL. In an in vivo study, Kakadu plum juice with or without pomegranate juice and S-PT84 significantly decreased azoxymethane-induced rat colon ACF and ACs. CONCLUSIONS: RB is one of the most important molecules suppressing carcinogenesis, and to the best of our knowledge, this is the first study to demonstrate that natural drinks reactivated the functions of RB. As expected, Kakadu plum juice and pomegranate juice suppressed the growth of LIM1215 cells by reactivating the functions of RB, and Kakadu plum juice with or without pomegranate juice and S-PT84 inhibited rat colon ACF and ACs. Therefore, this mixed juice has potential as a novel candidate for cancer prevention.

Anti-Cancer Effect of Neural Stem Cells Transfected with Carboxylesterase and sTRAIL Genes in Animals with Brain Lesions of Lung Cancer.

A metastatic brain tumor is the most common type of malignancy in the central nervous system, which is one of the leading causes of death in patients with lung cancer. The purpose of this study is to evaluate the efficacy of a novel treatment for metastatic brain tumors with lung cancer using neural stem cells (NSCs), which encode rabbit carboxylesterase (rCE) and the secretion form of tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). rCE and/or sTRAIL were transduced in immortalized human fetal NSCs, HB1.F3. The cytotoxic effects of the therapeutic cells on human lung cancer cells were evaluated in vitro with the ligands and decoy receptor expression for sTRAIL in the presence of CPT-11. Human NSCs encoding rCE (F3.CE and F3.CE.sTRAIL) significantly inhibited the growth of lung cancer cells in the presence of CPT-11 in vitro. Lung cancer cells were inoculated in immune-deficient mice, and therapeutic cells were transplanted systematically through intracardiac arterial injection and then treated with CPT-11. In resting state, DR4 expression in lung cancer cells and DcR1 in NSCs increased to 70% and 90% after CPT-11 addition, respectively. The volumes of the tumors in immune-deficient mice were reduced significantly in mice with F3.CE.sTRAIL transplantation and CPT-11 treatment. The survival was also significantly prolonged with treatment with F3.sTRAIL and F3.CE plus CPT-11 as well as F3.CE.sTRAIL plus CPT-11. NSCs transduced with rCE and sTRAIL genes showed a significant anti-cancer effect on brain metastatic lung cancer in vivo and in vitro, and the effect may be synergistic when rCE/CPT-11 and sTRAIL are combined. This stem-cell-based study using two therapeutic genes of different biological effects can be translatable to clinical application.

Snoopligase-catalyzed molecular glue enables efficient generation of hyperoligomerized TRAIL variant with enhanced antitumor effect.

Clinical application of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is predominantly limited by its inefficient apoptosis induction in tumor cells, which might be improved by using molecular superglue-mediated hyperoligomerization to increase its valency. Here, the minimal superglue peptide pairs, including Snoopligase-catalyzed SnoopTagJr/SnoopDogTag and SpyStapler-catalyzed SpyTag/SpyBDTag, were individually fused at the N- or C-terminus of the TRAIL promoter to produce superglue-fusion TRAIL variants. Similar to native trivalent TRAIL, these superglue-fusion TRAIL variants were highly expressed in Escherichia coli (E. coli) and spontaneously trimerized. In the presence of Snoopligase or SpyStapler, the trivalent superglue-fusion TRAIL variants were predominantly crosslinked into hexavalent TRAIL variants. Nevertheless, Snoopligase was more efficient than SpyStapler in the production of hexavalent TRAIL variants. In particular, Snoopligase-catalyzed trivalent TRAIL variants with N-terminal fusion of SnoopTagJr/SnoopDogTag produced hexavalent SnHexaTR with the highest yield (∼70%). The in vitro cytotoxicity of SnHexaTR was 10-40 times greater than that of TRAIL in several tumor cells. In addition, compared to trivalent TRAIL, hexavalent SnHexaTR showed a longer serum half-life and greater tumor uptake, which resulted in eradication of 50% of tumor xenografts of TRAIL-sensitive COLO 205. In mice bearing TRAIL-resistant HT-29 tumor xenografts, hexavalent SnHexaTR combined with bortezomib encapsulated in liposomes also showed robust tumor growth suppression, indicating that hyperoligomerization mediated by minimal molecular superglue significantly increased the cytotoxicity and antitumor effect of TRAIL. As a novel anticancer agent candidate, the hexavalent SnHexaTR has great potential for clinical application in cancer therapy.

Hyperthermia maintains death receptor expression and promotes TRAIL-induced apoptosis.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand activates apoptotic pathways and could potentially be used in anticancer treatments. However, oral squamous cell carcinoma cells are known to be resistant to tumor necrosis factor-related apoptosis-inducing ligand-induced cell death. It has been previously reported that hyperthermia upregulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in other cancers. As such, we evaluated whether hyperthermia upregulates tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in a tumor necrosis factor-related apoptosis-inducing ligand-resistant oral squamous cell carcinoma cell line. METHODS: The oral squamous cell carcinoma cell line HSC3 was cultured and divided into hyperthermia and control groups. We investigated the antitumor effects of recombinant human tumor necrosis factor-related apoptosis-inducing ligand using cell proliferation and apoptosis assays. Additionally, we measured death receptor 4 and 5 levels, and determined death receptor ubiquitination status, as well as E3 ubiquitin ligase targeting of death receptor in both hyperthermia and control groups before recombinant human tumor necrosis factor-related apoptosis-inducing ligand administration. RESULTS: Treatment with recombinant human tumor necrosis factor-related apoptosis-inducing ligand produced greater inhibitory effects in the hyperthermia group than in the control group. Moreover, death receptor protein expression in the hyperthermia group was upregulated on the cell surface (and overall), although death receptor mRNA was downregulated. The half-life of death receptor was several hours longer in the hyperthermia group; concomitantly, E3 ubiquitin ligase expression and death receptor ubiquitination were downregulated in this group. CONCLUSION: Our findings suggested that hyperthermia enhances apoptotic signaling by tumor necrosis factor-related apoptosis-inducing ligand via the suppression of death receptor ubiquitination, which upregulates death receptor expression. These data suggest that the combination of hyperthermia and tumor necrosis factor-related apoptosis-inducing ligand has implications in developing a novel treatment strategy for oral squamous cell carcinoma.

Current approaches in enhancing TRAIL therapies in glioblastoma.

Glioblastoma (GBM) is the most prevalent, aggressive, primary brain cancer in adults and continues to pose major medical challenges due in part to its high rate of recurrence. Extensive research is underway to discover new therapies that target GBM cells and prevent the inevitable recurrence in patients. The pro-apoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted attention as an ideal anticancer agent due to its ability to selectively kill cancer cells with minimal toxicity in normal cells. Although initial clinical evaluations of TRAIL therapies in several cancers were promising, later stages of clinical trial results indicated that TRAIL and TRAIL-based therapies failed to demonstrate robust efficacies due to poor pharmacokinetics, resulting in insufficient concentrations of TRAIL at the therapeutic site. However, recent studies have developed novel ways to prolong TRAIL bioavailability at the tumor site and efficiently deliver TRAIL and TRAIL-based therapies using cellular and nanoparticle vehicles as drug loading cargos. Additionally, novel techniques have been developed to address monotherapy resistance, including modulating biomarkers associated with TRAIL resistance in GBM cells. This review highlights the promising work to overcome the challenges of TRAIL-based therapies with the aim to facilitate improved TRAIL efficacy against GBM.

Anti-tumor Effects of IL-1ß Induced TRAIL-Expressing hUCMSCs on Embelin Treated Breast Cancer Cell Lines.

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUCMSCs) have high therapeutic value in cancer treatment. We have found that pre-activating hUCMSCs with IL-1ß promotes tumor necrosis factor-related apoptosis inducing ligand (TRAIL) expression and facilitates anti-tumor effect. Furthermore, embelin has been found to induce apoptosis of different cancer cell lines by upregulating the expression of TRAIL receptor 1 (DR4) and TRAIL receptor 2 (DR5). This study investigated whether IL-1ß induced TRAIL-expressing hUCMSCs, in combination with low-dose embelin, could further induce apoptosis in breast cancer cell lines. MATERIALS AND METHODS: MTT assay was used to examine the cytotoxicity of embelin in MDA-MB-231 and MCF-7. To detect the interested protein expression in cells, Western blot and cell immunofluorescence were used to double-confirm the observed results. Annexin V/PI apoptosis assay was detected by flow cytometry to analyze the apoptosis rate of embelin treated breast cancer cell lines and the effect of co-culturing with breast cancer cells and hUCMSCs. RESULTS: Using Western blot and immunofluorescence, we found that breast cancer cell lines treated with low-dose embelin (2.5-5 µM) increased the expression of apoptosis-related receptor DR4, DR5 and the cleaved caspase 8, 9 and 3. Moreover, TRAIL expression was enhanced in IL-1ß induced hUCMSCs. Combining these observations, we expected that coculturing IL-1ß induced hUCMSCs with low dose embelin treated MDA-MB-231 and MCF-7 cells might enhance the apoptosis of breast cancer cells. We confirmed via flow cytometry that coculture of IL-1ß induced TRAIL-expressing hUCMSCs and embelin treated MDA-MB-231 and MCF-7 cells enhances the apoptosis rate of these breast cancer cells. CONCLUSION: We found that embelin upregulated the expression of DR4 and DR5 to increase the TRAIL-mediated apoptosis in breast cancer cell lines. Low dose embelin treated breast cancer cell lines in combination with IL-1ß induced TRAIL-expressing hUCMSCs may become a potential anti-tumor therapy.

Natural killer cell-mimic nanoparticles can actively target and kill acute myeloid leukemia cells.

Natural killer (NK) cells play a crucial role in recognizing and killing emerging tumor cells. However, tumor cells develop mechanisms to inactivate NK cells or hide from them. Here, we engineered a modular nanoplatform that acts as NK cells (NK cell-mimics), carrying the tumor-recognition and death ligand-mediated tumor-killing properties of an NK cell, yet without being subject to tumor-mediated inactivation. NK cell mimic nanoparticles (NK.NPs) incorporate two key features of activated NK cells: cytotoxic activity via the death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and an adjustable tumor cell recognition feature based on functionalization with the NK cell Fc-binding receptor (CD16, FCGR3A) peptide, enabling the NK.NPs to bind antibodies targeting tumor antigens. NK.NPs showed potent in vitro cytotoxicity against a broad panel of cancer cell lines. Upon functionalizing the NK.NPs with an anti-CD38 antibody (Daratumumab), NK.NPs effectively targeted and eliminated CD38-positive patient-derived acute myeloid leukemia (AML) blasts ex vivo and were able to target and kill CD38-positive AML cells in vivo, in a disseminated AML xenograft system and reduced AML burden in the bone marrow compared to non-targeted, TRAIL-functionalized liposomes. Taken together, NK.NPs are able to mimicking key antitumorigenic functions of NK cells and warrant their development into nano-immunotherapeutic tools.

Molecular superglue-mediated higher-order assembly of TRAIL variants with superior apoptosis induction and antitumor activity.

Prompting higher-order death receptor (DR) clustering by increasing the valency of DR agonist is efficient to induce apoptosis of tumor cells. As an attractive DR agonist with superior biosafety, the trimeric tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts limited antitumor effect in patients, which is predominantly attributed to its low DR clustering ability and short serum half-life. Previous antibody scaffolds-based engineering strategies to increase the valency and/or prolong the serum half-life of TRAIL improve apoptosis induction, however, often produce large proteins with poor tumor penetration. Covalent protein ligation mediated by small molecular superglues such as SpyTag/SpyCatcher might be a novel strategy to assemble higher-order TRAIL variants. Upon fusion to TRAIL promotor, SpyTag/SpyCatcher molecular superglue preferentially ligated two trimeric TRAIL to produce a hexameric TRAIL variant, HexaTR, exhibiting a significantly increased apoptosis induction. In addition, an albumin-binding HexaTR, ABD-HexaTR, with a prolonged serum half-life by binding to endogenous albumin was also produced using the same strategy. Compared to the trimeric TRAIL, the hexameric HexaTR and ABD-HexaTR showed 20-50 times greater in vivo antitumor effect, resulting in eradication of several types of large (150-300 mm3) tumor xenografts. Combination with bortezomib carried by liposome further improved the antitumor effects of the hexavalent HexaTR and ABD-HexaTR in refractory cancer. Our results indicate that the superglue-mediated higher-order assembly is promising to improve the DR clustering and proapoptotic signaling of TRAIL, showing great advantages in constructing the next generation of DR agonists for cancer therapy.

Cytochalasin B-Induced Membrane Vesicles from TRAIL-Overexpressing Mesenchymal Stem Cells Induce Extrinsic Pathway of Apoptosis in Breast Cancer Mouse Model.

Tumor-necrosis-factor-associated apoptosis-inducing ligand (TRAIL) is one of the most promising therapeutic cytokines that selectively induce apoptosis in tumor cells. It is known that membrane vesicles (MVs) can carry the surface markers of parental cells. Therefore, MVs are of interest as a tool for cell-free cancer therapy. In this study, membrane vesicles were isolated from TRAIL-overexpressing mesenchymal stem cells using cytochalasin B treatment (CIMVs). To evaluate the antitumor effect of CIMVs-TRAIL in vivo, a breast cancer mouse model was produced. The animals were intratumorally injected with 50 µg of native CIMVs or CIMVs-TRAIL for 12 days with an interval of two days. Then, tumor growth rate, tumor necrotic area, the expression of the apoptosis-related genes CASP8, BCL-2, and BAX and the level of CASP8 protein were analyzed. A 1.8-fold increase in the CAS8 gene mRNA and a 1.7-fold increase in the CASP8 protein level were observed in the tumors injected with CIMVs-TRAIL. The expression of the anti-apoptotic BCL-2 gene in the CIMV-TRAIL group remained unchanged, while the mRNA level of the pro-apoptotic BAX gene was increased by 1.4 times, which indicated apoptosis activation in the tumor tissue. Thus, CIMVs-TRAIL were able to activate the extrinsic apoptosis pathway and induce tumor cell death in the breast cancer mouse model.

Exosomal delivery of TRAIL and miR­335 for the treatment of hepatocellular carcinoma (Review).

Liver cancer is the sixth most prevalent type of cancer worldwide and accounts for the third most frequent cause of cancer­associated mortality. Conventional anticancer drugs display limited efficacy owing to their short half­life, poor solubility and inefficient drug delivery. Despite advancements being made in drug discovery and development for the treatment of hepatocellular carcinoma (HCC), drug inefficacy and drug continue to pose significant obstacles to effective treatment. Therefore, it is imperative that novel treatment strategies be developed with the aim of developing anticancer treatments without any side­effects and with long­term durability. Extracellular vesicles, such as exosomes, intercellular communication agents which have the ability to carry heterogenous molecules with high penetrability, low immunogenicity and longer durability, may provide a versatile natural delivery system. The present review article illustrates the innovative treatment strategy using exosomes as a delivery agent for two distinct anticancer candidates, i.e., tumor necrosis factor­related apoptosis­inducing ligand and microRNA­335. The aim of the present review was to present a unique strategy for the development of an exceptional anticancer treatment therapy exploiting exosomes as a delivery vehicle which may be used for HCC.

Serum B7 homologous protein 2, tumor necrosis factor-related apoptosis-inducing ligand, interleukin-37, interleukin-17A levels in patients with primary immune thrombocytopenia and their clinical significance / 中国医师进修杂志

Objective:To explore the changes of serum B7 homolog 2 (B7-H2), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), interleukin(IL)-37 and IL-17A levels in patients with primary immune thrombocytopenia (ITP), and to analyze the clinical guiding significance of each index level in the diagnosis and treatment of ITP.Methods:A total of 90 patients with ITP treated in Jining Hospital of Traditional Chinese Medicine from September 2018 to September 2020 were retrospectively selected as the research group, and 90 healthy patients during the same period were selected as the normal control group. The levels of serum B7-H2, TRAIL, IL-37, IL-17A and platelet count (PLT) in the two groups were compared, and the clinical guidance significance of each index level in the diagnosis and treatment of ITP were analyzed by receiver operating characteristic(ROC) curve.Results:The serum levels of B7-H2, TRAIL, IL-37 and IL-17A in the research group were higher than those in the normal control group, and PLT was lower than that in the normal control group: (25.12 ± 4.29) μg/L vs. (12.26 ± 3.10) μg/L, (0.92 ± 0.20) μg/L vs.(0.46 ± 0.13) μg/L, (145.02 ± 43.18) ng/L vs. (50.23 ± 14.07) ng/L, (21.63 ± 5.06) ng/L vs. (7.71 ± 2.04) ng/L, (16.12 ± 4.61) × 10 9/L vs. (200.86 ± 61.4) × 10 9/L, there were statistical differences ( P<0.05). After treatment for 3 months, 73 patients obtained remission, and 13 patients were non-remission. The levels of B7-H2, TRAIL, IL-37, IL-17A in the non-remission group before and after treatment were higher than those in the remission group, and PLT was lower than that in the remission group, there were statistical differences ( P<0.05). Pearson correlation analysis showed that the levels of serum B7-H2, TRAIL, IL-37 and IL-17A were negatively correlated with PLT ( P<0.05). The ROC curve analysis showed that the area under the curve of serum B7-H2, TRAIL, IL-37 and IL-17A in prognostic prediction was 0.916, the sensitivity and the specificity were 94.12% and 86.30%. Conclusions:The serum levels of B7-H2, TRAIL, IL-37 and IL-17A in patients with ITP are significantly elevated, and are closely related to the level of PLT. Combined detection can help clinically predict the direction of disease outcome.

Performance Evaluation of Host Biomarker Combinations for the Diagnosis of Serious Bacterial Infection in Young Febrile Children: A Double-Blind, Multicentre, Observational Study.

The diagnosis of serious bacterial infection (SBI) in young febrile children remains challenging. This prospective, multicentre, observational study aimed to identify new protein marker combinations that can differentiate a bacterial infection from a viral infection in 983 children, aged 7 days-36 months, presenting with a suspected SBI at three French paediatric emergency departments. The blood levels of seven protein markers (CRP, PCT, IL-6, NGAL, MxA, TRAIL, IP-10) were measured at enrolment. The patients received the standard of care, blinded to the biomarker results. An independent adjudication committee assigned a bacterial vs. viral infection diagnosis based on clinical data, blinded to the biomarker results. Computational modelling was applied to the blood levels of the biomarkers using independent training and validation cohorts. Model performances (area under the curve (AUC), positive and negative likelihood ratios (LR+ and LR-)) were calculated and compared to those of the routine biomarkers CRP and PCT. The targeted performance for added value over CRP or PCT was LR+ ≥ 5.67 and LR- ≤ 0.5. Out of 652 analysed patients, several marker combinations outperformed CRP and PCT, although none achieved the targeted performance criteria in the 7 days-36 months population. The models seemed to perform better in younger (7-91 day-old) patients, with the CRP/MxA/TRAIL combination performing best (AUC 0.895, LR+ 10.46, LR- 0.16). Although computational modelling using combinations of bacterial- and viral-induced host-protein markers is promising, further optimisation is necessary to improve SBI diagnosis in young febrile children.

The inhibitory effect of human umbilical cord mesenchymal stem cells expressing anti-HAAH scFv-sTRAIL fusion protein on glioma.

Glioma is the most common malignant intracranial tumor with low 5-year survival rate. In this study, we constructed a plasmid expressing anti-HAAH single-chain antibody and sTRAIL fusion protein (scFv-sTRAIL), and explored the effects of the double gene modified human umbilical cord mesenchyreal stem cells (hucMSCs) on the growth of glioma in vitro and in vivo. The isolated hucMSCs were identified by detecting the adipogenic differentiation ability and the osteogenic differentiation ability. The phenotypes of hucMSCs were determined by the flow cytometry. The hucMSCs were infected with lentivirus expression scFv-sTRAIL fusion protein. The expression of sTRAIL in hucMSCs were detected by immunofluorescence staining, western blot and ELISA. The tropism of hucMSCs toward U87G cells was assessed by transwell assay. The inhibitory effect of hucMSCs on U87G cells were explored by CCK8 and apoptosis assay. The xenograft tumor was established by subcutaneously injection of U87G cells into the back of mice. The hucMSCs were injected via tail veins. The inhibitory effect of hucMSCs on glioma in vivo was assessed by TUNEL assay. The hucMSCs migrated into the xenograft tumor were revealed by detecting the green fluorescent. The results showed that the scFv-sTRAIL expression did not affect the phenotypes of hucMSCs. The scFv-sTRAIL expression promoted the tropism of hucMSCs toward U87G cells, enhanced the inhibitory effect and tumor killing effect of hucMSCs on U87G cells. The in vivo study showed that hucMSCs expressing scFv-sTRAIL demonstrated significantly higher inhibitory effect and tumor killing effect than hucMSCs expressing sTRAIL. The green fluorescence intensity in the mice injected with hucMSCs expressing scFv-sTRAIL was significantly higher than that injected with hucMSCs expressing sTRAIL. These data suggested that the scFv conferred the targeting effect of hucMSCs tropism towards the xenograft tumor. In conclusion, the hucMSCs expressing scFv-sTRAIL fusion protein gained the capability to target and kill gliomas cells in vitro and in vivo. These findings shed light on a potential therapy for glioma treatment.