Search

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. [Display omitted] • TRAIL variants with superglue fusion are highly expressed in E. coli. • Trivalent TRAIL were predominantly crosslinked into hexavalent TRAIL by both Snoopligase and SpyStapler. • Snoopligase is more effective than SpyStapler in hexavalent TRAIL crosslinking. • Hexavalent TRAIL exerts better pharmacokinetics and greater antitumor effect than trivalent TRAIL. [ABSTRACT FROM AUTHOR]

The objective was to evaluate the analytical performance of a new point-of-need platform for rapid and accurate measurement of a host-protein score that differentiates between bacterial and viral infection. The system comprises a dedicated test cartridge (MeMed BV®) and an analyzer (MeMed Key®). In each run, three host proteins (TRAIL, IP-10 and CRP) are measured quantitatively and a combinational score (0–100) computed that indicates the likelihood of Bacterial versus Viral infection (BV score). Serum samples collected from patients with acute infection representing viral (0 ≤ score < 35), equivocal (35 ≤ score ≤ 65), or bacterial (65 < score ≤ 100) scores based on pre-defined score cutoffs were employed for the analytical evaluation studies as well as samples from healthy individuals. To assess reproducibility, triplicate runs were conducted at 3 different sites, on 2 analyzers per site over 5 non-consecutive days. Lower limit of quantitation (LLoQ) and analytical measurement range were established utilizing recombinant proteins. Sample stability was evaluated using patient samples representative of BV score range (0–100). MeMed Key® and MeMed BV® passed the acceptance criteria for each study. In the reproducibility study, TRAIL, IP-10 and CRP measurements ranged with coefficient of variation from 9.7 to 12.7%, 4.6 to 6.2% and 5.0 to 11.6%, respectively. LLoQ concentrations were established as 15 pg/mL, 100 pg/mL and 1 mg/L for TRAIL, IP-10 and CRP, respectively. In summary, the analytical performance reported here, along with diagnostic accuracy established in the Apollo clinical validation study (NCT04690569), supports that MeMed BV® run on MeMed Key® can serve as a tool to assist clinicians in differentiating between bacterial and viral infection. [ABSTRACT FROM AUTHOR]

• Three biomarkers of infection, LCN2, TRAIL and IP-10, were evaluated in healthy paediatrics. • Reference intervals were established for LCN2, TRAIL and IP-10. • LCN2 and TRAIL levels continuously change with age. Differentiating bacterial from viral infections in children is a common clinical challenge. Novel host immune biomarkers have the potential to aid thediagnosis of infection aetiology and identify children who require antibiotics. Data on novel infection biomarkers gender and age-specific correlations, and reference intervals in healthy paediatrics is lacking. This study reports the plasma levels of three novel biomarkers that can aid in the differentiation of bacterial and viral infection in a healthy group of paediatrics. The levels of (Interferon-Gamma Inducible Protein 10 kDa (IP-10), Lipocalin-2 (LCN2) and TNF-Related Apoptosis-Inducing Ligand (TRAIL) were quantified in 199 plasma samples from healthy paediatrics aged 2 to 16 years old from across the UK. Reference intervals (2.5th and 97.5th) were determined, and biomarker levels were examined for sex and age associations. Reference intervals for IP-10, LCN2 and TRAIL for ages 2–16 years were 36.7–168.1 pg/ml, 14.2–123.3 ng/ml, 57.4–71.4 pg/ml respectively. No biomarker showed an association with sex and IP-10 did not show any association with age. TRAIL levels had a weak continuous negative correlation with age and LCN2 levels had a continuous positive correlation with age. Specific cut-offs for LCN2 in two age categories were identified, while TRAIL did not require age partitions. This study provides age-appropriate reference intervals for three biomarkers of infection in healthy children. These findings have the potential to improve the impact of future research on these biomarkers, the accuracy of clinical decision-making in children with infection, paediatric patient care and outcomes, and antimicrobial stewardship. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) demonstrates unique characteristics in anticancer therapies as it selectively induces apoptosis in cancer cells. However, most cancer cells are TRAIL-resistant. Odanacatib (ODN), a cathepsin K inhibitor, is considered a novel sensitizer for cancer treatment. Combination therapy between TRAIL and sensitizers is considered a potent platform that improves TRAIL-based anticancer therapies beyond TRAIL monotherapy. Herein, we developed ODN loaded poly(lactic- co -glycolic) nanoparticles conjugated to GST-TRAIL (TRAIL-ODN-PLGA-NPs) to target and treat TRAIL-resistant cancer. TRAIL-ODN-PLGA-NPs demonstrated a significant increase in cellular uptake via death receptors (DR5 and DR4) on surface of cancer cells. TRAIL-ODN-PLGA-NPs exposure destroyed more TRAIL-resistant cells compared to a single treatment with free drugs. The released ODN decreased the Raptor protein, thereby increasing damage to mitochondria by elevating reactive oxygen species (ROS) generation. Additionally, Bim protein stabilization improved TRAIL-resistant cell sensitization to TRAIL-induced apoptosis. The in vivo biodistribution study revealed that TRAIL-ODN-PLGA-NPs demonstrated high location and retention in tumor sites via the intravenous route. Furthermore, TRAIL-ODN-PLGA-NPs significantly inhibited xenograft tumor models of TRAIL-resistant Caki-1 and TRAIL-sensitive MDA-MB-231 cells.The inhibition was associated with apoptosis activation, Raptor protein stabilizing Bim protein downregulation, Bax accumulation, and mitochondrial ROS generation elevation. Additionally, TRAIL-ODN-PLGA-NPs affected the tumor microenvironment by increasing tumor necrosis factor-α and reducing interleukin-6. In conclusion, we evealed that our formulation demonstrated synergistic effects against TRAIL compared with the combination of free drug in vitro and in vivo models. Therefore, TRAIL-ODN-PLGA-NPs may be a novel candidate for TRAIL-induced apoptosis in cancer treatment. [Display omitted] • Success of loading and decorating ODN and TRAIL into a PLGA nanocarrier. • Efficient cellular uptake by TRAIL-mediated manner and controlled release kinetics. • Re-sensitization TRAIL-resistant cancer to TRAIL in vitro and in vivo models. • Stability and preservation target and apoptosis of TRAIL in vivo administration. Long retention and sysnergistic inhibition on TRAIL-resistant tumor growth. [ABSTRACT FROM AUTHOR]

• Biomarkers TNF-related apoptosis-inducing ligand (TRAIL), interferon gamma-induced protein 10 (IP-10), and C-reactive protein (CRP) are correlated with COVID-19 severity • TRAIL yielded lower levels and IP-10 yielded higher levels in patients with COVID-19 than in controls • Obesity, CRP, and high peak IP-10 were associated with intensive care unit mortality • Patients with low initial TRAIL levels more often required mechanical ventilation • Including TRAIL, IP-10, and CRP in risk assessment models seems promising Early prognostication of COVID-19 severity will potentially improve patient care. Biomarkers, such as TNF-related apoptosis-inducing ligand (TRAIL), interferon gamma-induced protein 10 (IP-10), and C-reactive protein (CRP), might represent possible tools for point-of-care testing and severity prediction. In this prospective cohort study, we analyzed serum levels of TRAIL, IP-10, and CRP in patients with COVID-19, compared them with control subjects, and investigated the association with disease severity. A total of 899 measurements were performed in 132 patients (mean age 64 years, 40.2% females). Among patients with COVID-19, TRAIL levels were lower (49.5 vs 87 pg/ml, P = 0.0142), whereas IP-10 and CRP showed higher levels (667.5 vs 127 pg/ml, P <0.001; 75.3 vs 1.6 mg/l, P <0.001) than healthy controls. TRAIL yielded an inverse correlation with length of hospital and intensive care unit (ICU) stay, Simplified Acute Physiology Score II, and National Early Warning Score, and IP-10 showed a positive correlation with disease severity. Multivariable regression revealed that obesity (adjusted odds ratio [aOR] 5.434, 95% confidence interval [CI] 1.005-29.38), CRP (aOR 1.014, 95% CI 1.002-1.027), and peak IP-10 (aOR 1.001, 95% CI 1.00-1.002) were independent predictors of in-ICU mortality. We demonstrated a correlation between COVID-19 severity and TRAIL, IP-10, and CRP. Multivariable regression showed a role for IP-10 in predicting unfavourable outcomes, such as in-ICU mortality. Clinicaltrials.gov, NCT04655521 [ABSTRACT FROM AUTHOR]

In this issue of Cancer Cell , Yang et al. report the third in a series of phase III trials that demonstrates the survival benefit of combining a PD-1 inhibitor with chemotherapy in nasopharyngeal cancer. A gene expression analysis identifies "hot" and "cold" tumor signatures with prognostic and predictive significance. [ABSTRACT FROM AUTHOR]

The aim of the investigation was to evaluate variations in blood TNF-related apoptosis-inducing ligand (TRAIL) levels between patients with viral and bacterial infections and the diagnostic performance of TRAIL for identifying viral and bacterial infections. The investigation included 169 adult (>18 years) patients presenting with medical signs of acute infections (inclusion criteria included a body temperature over 37.5 °C, an onset of symptoms no more than 12 days). Reference standard was based on a rigorous expert panel and the majority of the panel determined the infectious etiology. Finally, 104 patients with 78 bacterial and 26 viral reference standard outcomes were enrolled in this investigation (24 were eliminated depending on the exclusion criteria; 41 had indeterminate reference standard diagnosis). ELISA was employed to measure TRAIL levels in the group of 78 subjects with bacterial infections and 26 individuals with viral infections, and the diagnostic performance of TRAIL was identified by receiver operating characteristic (ROC) analysis. The TRAIL level in individuals with bacterial infections was significantly lower than that in subjects with viral infections (16.59 (2.61–32.6) pg/mL vs. 97.39 (36.18–127.74) pg/mL, P < 0.05). The area under the ROC curve (AUC) of TRAIL was 0.86 (95 %CI:0.79 to 0.94) for identifying bacterial and viral infections. Combining TRAIL with C-reactive protein (CRP), the AUC was 0.94 (95 %CI:0.89 to 1.00). TRAIL is diagnostic for discriminating between viral and bacterial infections. Combining TRAIL with CRP increases the AUC. [ABSTRACT FROM AUTHOR]

Oncology patients presenting to EDs with acute respiratory illnesses (ARI) require a broad workup and early treatment due to risk of underlying infection and immunocompromised status. Early antibiotic administration is often required as determining a nonbacterial source is time consuming. A novel host bio-array developed to rapidly differentiate bacterial from viral sources could improve ED resource utilization, reduce unnecessary antibiotics, and increase healthcare quality. An IRB approved subject on multiple immunosuppressants presents with ARI and positive SIRS criteria. The MeMed BV® (BV) is an FDA approved rapid immunoassay that reports a computationally integrated score (0-100) of three host proteins (TNF-related apoptosis-inducing ligand (TRAIL), interferon gamma-induced protein 10 (IP-10), and C-reactive protein (CRP). The BV score and ED standard of care tests were obtained in a modified ED sepsis protocol (mEDSP). Scores above 65 are associated with bacterial infections, while scores below 10 are associated with viral infections. Cost of laboratory, radiology, professional and facilities fees (room charges, nursing, and ancillaries) were summed for the admission. Investigators received three surveys to: (1) establish pretest probability of disease, (2) determine posttest probability after receiving BV results, (3) query what resources might be withheld or changed if presented with the BV test results early. The BV reported a '0' value (viral source) within 20 minutes. Further testing confirmed no bacterial source of infection. An adjudicated cost and resource review of the mEDSP and admission revealed a potential reduction of unnecessary antibiotics by 59%, lab testing by 24.2%, and overall costs by 23.2%. Admission time could have been reduced by 24 hours (43%). Furthermore, the ED physician was amenable to changing care based on this test. Significant improvement in resource utilization and greater quality was identified with early identification of nonbacterial sources in ARI in the ED. Adjunct biomarkers differentiating viral from other sources can improve quality and resource utilization when deployed early in the ED. Further trials in the Emergency Oncology realm are necessary. [ABSTRACT FROM AUTHOR]

Novel strategies in intratumoral injection and emerging immunotherapies have heralded a new era of precise cancer treatments. The affinity of SARS-CoV-2 to ACE2 receptors, a feature which facilitates virulent human infection, is leveraged in this research. Colon cancer cells, with their high ACE2 expression, provide a potentially strategic target for using this SARS-CoV-2 feature. While the highly expression of ACE2 is observed in several cancer types, the idea of using the viral spike protein for targeting colon cancer cells offers a novel approach in cancer treatment. Intratumoral delivery of nucleic acid-based drugs is a promising alternative to overcoming the limitations of existing therapies. The increasing importance of nucleic acids in this realm, and the use of Lipid Nanoparticles (LNPs) for local delivery of nucleic acid therapeutics, are important breakthroughs. LNPs protect nucleic acid drugs from degradation and enhance cellular uptake, making them a rapidly evolving nano delivery system with high precision and adaptability. Our study leveraged a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) combined with a receptor-binding domain from the SARS-CoV-2 spike protein, encapsulated in LNPs, to target colon cancer cells. Our results indicated that the TRAIL fusion-mRNA induced apoptosis in vitro and in vivo. Collectively, our findings highlight LNP-encapsulated TRAIL fusion-mRNA as a potential colon cancer therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

ERCC2 plays a pivotal role in DNA damage repair, however, its specific function in cancer remains elusive. In this study, we made a significant breakthrough by discovering a substantial upregulation of ERCC2 expression in glioblastoma (GBM) tumor tissue. Moreover, elevated levels of ERCC2 expression were closely associated with poor prognosis. Further investigation into the effects of ERCC2 on GBM revealed that suppressing its expression significantly inhibited malignant growth and migration of GBM cells, while overexpression of ERCC2 promoted tumor cell growth. Through mechanistic studies, we elucidated that inhibiting ERCC2 led to cell cycle arrest in the G0/G1 phase by blocking the CDK2/CDK4/CDK6/Cyclin D1/Cyclin D3 pathway. Notably, we also discovered a direct link between ERCC2 and CDK4, a critical protein in cell cycle regulation. Additionally, we explored the potential of TRAIL, a low-toxicity death ligand cytokine with anticancer properties. Despite the typical resistance of GBM cells to TRAIL, tumor cells undergoing cell cycle arrest exhibited significantly enhanced sensitivity to TRAIL. Therefore, we devised a combination strategy, employing TRAIL with the nanoparticle DMC-siERCC2, which effectively suppressed the GBM cell proliferation and induced apoptosis. In summary, our study suggests that targeting ERCC2 holds promise as a therapeutic approach to GBM treatment. [Display omitted] [ABSTRACT FROM AUTHOR]

Environmental arsenic (As) exposure is strongly related to the progression of chronic obstructive pulmonary disease (COPD). Pulmonary epithelial cells apoptosis is implicated in the pathophysiological mechanisms of COPD. However, the role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one biomarker of apoptosis, remains unclear in As-mediated pulmonary function alternations in COPD patients. This study included 239 COPD patients. The serum level of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was measured by enzyme-linked immunosorbent assay (ELISA). The blood As level was determined through inductively coupled plasma mass spectrometry (ICP-MS). Blood As levels exhibited a negative and dose-dependent correlation with pulmonary function. Per unit elevation of blood arsenic concentrations was related to reductions of 0.339 L in FEV1, 0.311 L in FVC, 1.171% in FEV1/FVC%, and 7.999% in FEV1% in COPD subjects. Additionally, a positive dose-response correlation of blood As with serum TRAIL was found in COPD subjects. Additionally, the level of serum TRAIL was negatively linked to lung function. Elevated TRAIL significantly mediated As-induced decreases of 11.05%, 13.35%, and 31.78% in FVC, FEV1, and FEV1%, respectively among the COPD patients. Blood As level is positively correlated with pulmonary function decline and serum TRAIL increase in individuals with COPD. Our findings suggest that elevated TRAIL levels may serve as a mediating mechanism through which As contributes to declining lung function in COPD patients. [Display omitted] • ·Blood As concentration are negatively correlated with lung function parameters in COPD patients. • ·Blood As concentration are positively associated with serum TRAIL level in COPD patients. • ·Serum TRAIL level is inversely associated with pulmonary function parameters in COPD patients. • ·TRAIL partially mediates As exposure-related pulmonary function decline in COPD patients. [ABSTRACT FROM AUTHOR]

Programmed cell death, in particular apoptosis, is essential during development and tissue homeostasis, and also is the primary strategy to induce cancer cell death by cytotoxic therapies. Precision therapeutics targeting TRAIL death receptors are being evaluated as novel anti-cancer agents, while in parallel highly specific proteasome inhibitors have gained approval as drugs. TRAIL-dependent signalling and proteasomal control of cellular proteostasis are intricate processes, and their interplay can be exploited to enhance therapeutic killing of cancer cells in combination therapies. This review provides detailed insights into the complex signalling of TRAIL-induced pathways and the activities of the proteasome. It explores their core mechanisms of action, pharmaceutical druggability, and describes how their interplay can be strategically leveraged to enhance cell death responses in cancer cells. Offering this comprehensive and timely overview will allow to navigate the complexity of the processes governing cell death mechanisms in TRAIL- and proteasome inhibitor-based treatment conditions. • TRAIL therapeutics and proteasome inhibitors are clinically tested or approved. • Proteasome inhibition can sensitize to TRAIL-based therapeutics. • We mechanistically describe and review the complex signalling interplay. [ABSTRACT FROM AUTHOR]

• MMBV is a host signature based on TRAIL, IP10, and CRP for infection diagnosis. • This is the first evaluation of MMBV in the ICU cohort. • MMBV is not useful in ICU patients, neither in diagnosing nor monitoring infection. • MMBV may be a negative prognostic factor. Infection complications are common in intensive care unit patients, and early detection remains a diagnostic challenge. Procalcitonin (PCT) and C-reactive protein (CRP) are commonly used biomarkers. A novel diagnostic approach focuses on the host immune response. One of the approaches, the MMBV index, is based on measuring in a blood sample three parameters: (i) tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), (ii) interferon-γ-induced protein-10 (IP10), and (iii) CRP. This study aimed to evaluate the usefulness of MMBV as an infection biomarker in an ICU cohort. Forty-six patients treated in the University Clinical Center in Gdansk ICU were enrolled in the study, and their clinical data were retrospectively analyzed. In total, 91 MMBV results were analyzed. Most of the patients had high MMBV values, suggesting bacterial etiology. A weak correlation between PCT and MMBV was observed, and no correlation between parameter changes was noted. There was a correlation between CRP/MMBV and between changes in CRP / changes in MMBV. It seems that MMBV is not valuable for ICU patients neither in diagnosing nor monitoring infection. Higher MMBV values may predict unfavorable treatment outcomes. [ABSTRACT FROM AUTHOR]

Chronic lung allograft dysfunction (CLAD) is the major barrier to long-term survival following lung transplantation and results from repeated acute injuries to the graft epithelium. Epithelial club cells are known to have protective, anti-inflammatory, and regenerative roles and are depleted in CLAD. Mechanisms driving club cell loss in CLAD are unknown. Our single cell RNA sequencing (scRNAseq) data showed that TRAIL transcript was upregulated in CLAD club cells compared to control donor lung club cells. TRAIL induces apoptotic cell death by binding to its receptors (TRAIL-R1 and TRAIL-R2). To validate the scRNAseq data, we here further investigate the expression of TRAIL and its receptors at the protein level. We hypothesized that CLAD develops as a consequence of club cell death induced by TRAIL signaling during episodes of acute lung allograft dysfunction (ALAD). Lung transplant recipients' (LTR) airway brushings were obtained 3-12 months post-LT and processed for flow cytometry. TRAIL, TRAIL-R1, TRAIL-R2 expression was examined in club cells in LTRs with or without ALAD, defined as a >=10% drop in forced expiratory volume in 1 second from prior. After exclusion of non-epithelial cells, EpCAM+ epithelial cells and EpCAM+CCSP+ club cells were identified. Our findings show that the frequencies of both TRAIL+ club cells and TRAIL+ non-club epithelial cells were significantly higher in ALAD LTRs compared to non-ALAD LTRs (Fig. 1A-C). Further, TRAIL-R1 expression on club and non-club epithelial cells tended to be higher in ALAD compared to non-ALAD LTRs (Fig. 1A, D-E). TRAIL-R2 expression did not differ between ALAD and non-ALAD LTRs. Our preliminary data show an upregulation of TRAIL within epithelial cells found in ALAD airway brushings compared to control. The co-localization of TRAIL and TRAIL-R1 in club cell suggests that autocrine or paracrine cell death might be potential mechanisms for club cell loss in CLAD. [ABSTRACT FROM AUTHOR]

The gut microbiota has been found to be altered in different inflammatory disorders, but its involvement in the regulation of inflammatory cytokines remains unclear. Therefore, this study aimed to investigate the impacts of gut microbiota on circulating inflammatory cytokines and their potential roles in host diseases. Two-sample Mendelian randomization (MR) analyses were conducted using summary-level data from genome-wide association studies (GWAS) to identify significant causal associations between 196 gut microbiota and 41 inflammatory cytokines. Meta-analysis was applied to test the robustness of the results. Enrichment analyses of identified cytokines were further utilized to infer the effects of gut microbiota on the host. The MR analyses and meta-analyses identified the following significant causal associations: phylum Euryarchaeota on interleukin-2 (IL-2) (β IVW = 0.085, P = 1.5 × 10−2) and interleukin-8 (IL-8) (β IVW = 0.065, P = 4.1 × 10−2), phylum Tenericutes and class Mollicutes on macrophage inflammatory protein 1a (MIP1a) (β IVW = −0.142, P = 7.0 × 10−3), class Bacilli on hepatocyte growth factor (HGF) (β IVW = −0.106, P = 2.5 × 10−2), order Enterobacteriales on monocyte chemoattractant protein-1 (MCP1) (β IVW = 0.182, P = 1.8 × 10−2), and genus Lachnospiraceae NC2004 group on TNF-related apoptosis-inducing ligand (TRAIL) (β IVW = −0.207, P = 6.0 × 10−4). Enrichment analyses suggested that phylum Euryarchaeota and order Enterobacteriales might be risk factors for certain autoimmune diseases and neoplasms, while the phylum Tenericutes may have a protective effect. This study represents the first evidence confirming the causal effect of specific gut microbial taxa on circulating inflammatory cytokines and sheds light on their potential roles in the development and progression of various host diseases. • There is still a lack of evidence regarding the causal relationship between gut microbiota and inflammatory cytokines.ss • Mendelian randomization and meta-analysis identified six taxa that had causal effect on inflammatory cytokines. • Enrichment analysis indicated that gut microbiota may contribute to certain inflammatory disorders through key cytokines. • The research yielded valuable information on the development of inflammatory diseases as related to gut microbiota. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In order to discover more effective and less toxic drugs in the field of anti-tumor, the backbone structure of 17 β -estradiol was modified, and 11 target compounds were synthesized. Compounds 5 and 10 , which exhibited better anti-tumor activity and higher selectivity (more than 10-fold), were chosen for further biological investigation. Flow cytometry results indicated that 5 and 10 could arrest MCF-7 cells in the G2 phase and induce apoptosis. Immunohistochemical analysis revealed that 5 and 10 could bind to the estradiol receptor alpha in MCF-7 cells. Western blotting and real-time PCR assays were performed to detect the effects of compounds on apoptosis-related targets at the protein and gene levels. These results showed that both 5 and 10 could dosed-dependently increase the expression of Apaf-1, Bax, caspase-3,8,9 and reduce the expression levels of the anti-apoptotic factors Bcl-2 and Bcl-xL. Besides, the Human apoptosis array assay demonstrated the expression level of death receptor Trail R2/DR5 was upregulated obviously while the expression of TNF R1, IAPs and Hsp27/60/70 were downregulated. On the whole, 5 induced MCF-7 cell death through the endogenous pathway in mitochondria and the exogenous pathway with death receptor Trail R2/DR5. • Eleven 17 β -estradiol derivatives from modification were synthesized. • 5 and 10 possessed much better antiproliferative activities and higher selectivity than 17 β -estradiol. • 5 and 10 could arrest cell cycle at the G2 phase in MCF-7 cells and partly bind with the estradiol receptor alpha. • 5 induced MCF-7 cells death through both endogenous and exogenous pathways. [ABSTRACT FROM AUTHOR]

The key to making a practicable hydrogel for pharmaceutical or medical purposes is to endow it with relevant properties, i.e., facile fabrication, gelation time-controllability, and in situ injectability given a firm basis for safety/biocompatibility. Here, the authors describe an in situ gelling, injectable, albumin-cross-linked polyethylene glycol (PEG) hydrogel that was produced using a thiol–maleimide reaction. This hydrogel consists of two biocompatible components, namely, thiolated human serum albumin and 4-arm PEG 20k -maleimide, and can be easily fabricated and gelled in situ within 60 s by simply mixing its two components. In addition, the gelation time of this system is controllable in the range 15 s to 5 min. This hydrogel hardly interacted with an apoptotic TRAIL protein, ensuring suitable release profiles that maximize therapeutic efficacy. Specifically, tumors (volume: 278.8 mm 3 ) in Mia Paca-2 cell-xenografted BALB/c nu / nu mice treated with the TRAIL-loaded HSA-PEG hydrogel were markedly smaller than mice treated with the hydrogel prepared via an amine-N-hydroxysuccinimide reaction or non-treated mice (1275.5 mm 3 and 1816.5 mm 3 , respectively). We believe that this hydrogel would be a new prototype of locally injectable sustained-release type anti-cancer agents, and furthermore offers practical convenience for a doctor and universal applicability for a variety of therapeutic proteins. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) remains a leading causes of cancer-related death in the world, mainly due to the lack of effective treatment of advanced disease. TNF-related apoptosis-inducing ligand (TRAIL)-driven cell death, a crucial event in the control of tumor growth, selectively targets malignant rather than non-transformed cells. However, the fact that cancer cells, including CRC cells, are either intrinsically resistant or acquire resistance to TRAIL, represents a major hurdle to the use of TRAIL-based strategies in the clinic. Agents able to overcome CRC cell resistance to TRAIL have thus great therapeutic potential and many researchers are making efforts to identify TRAIL sensitizers. The anthelmintic drug rafoxanide has recently emerged as a potent anti-tumor molecule for different cancer types and we recently reported that rafoxanide restrained the proliferation of CRC cells, but not of normal colonic epithelial cells, both in vitro and in a preclinical model mimicking sporadic CRC. As these findings were linked with the induction of endoplasmic reticulum stress, a phenomenon involved in the regulation of various components of the TRAIL-driven apoptotic pathway, we sought to determine whether rafoxanide could restore the sensitivity of CRC cells to TRAIL. Our data show that rafoxanide acts as a selective TRAIL sensitizer in vitro and in a syngeneic experimental model of CRC, by decreasing the levels of c-FLIP and survivin, two key molecules conferring TRAIL resistance. Collectively, our data suggest that rafoxanide could potentially be deployed as an anti-cancer drug in the combinatorial approaches aimed at overcoming CRC cell resistance to TRAIL-based therapies. [Display omitted] • TRAIL specifically induces apoptosis in malignant cells thus representing a safe and powerful candidate for cancer treatment. • TRAIL therapy is less effective in certain cancer types, including CRC, due to the presence of resistance mechanisms. • Accumulating evidence suggests the need of a combinatorial approach to overcome the CRC cell resistance to TRAIL. • Repositioning studies indicated the anthelmintic drug rafoxanide as a potent anti-tumor agent in different cancer types. • Rafoxanide acts as a selective TRAIL sensitizing agent against CRC cells in vitro and in vivo. [ABSTRACT FROM AUTHOR]

In spite of the extensive research for developing new therapies, prostate cancer is still one of the major human diseases with poor prognosis and high mortality. Therefore, with the aim of identifying novel agents with antigrowth and pro-apoptotic activity on prostate cancer cells, in the present study, we evaluated the effect of lichen secondary metabolite physodic acid on cell growth in human prostate cancer cells. In addition, we tested the apoptotic activity of physodic acid on TRAIL-resistant LNCaP cells in combination with TRAIL. The cell viability was measured using MTT assay. LDH release, a marker of membrane breakdown, was also measured. For the detection of apoptosis, the evaluation of DNA fragmentation and caspase-3 activity assay were employed. The expression of proteins was detected by Western blot analysis. It was observed that physodic acid showed a dose–response relationship in the range of 12.5–50 μM concentrations in LNCaP and DU-145 cells, activating an apoptotic process. In addition, physodic acid sensitizes LNCaP cells to TRAIL-induced apoptosis. The combination of physodic acid with other anti-prostate cancer therapies could be considered a promising strategy that warrants further investigations. • Physodic acid induce apoptosis in prostate cancer cells. • Physodic acid sensitizes LNCaP cells to TRAIL-induced apoptosis. • Physodic acid could avoid cell chemoresistance in prostate cancer. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a highly fatal disease recognized as a growing global health crisis. Traditional Chinese herbal medicines have been used to treat patients with cancer for many years in China. This study investigated the effects of licochalcone B (LCB), a flavonoid compound isolated from the root of Glycyrrhiza uralensis Fisch., on cell proliferation, DNA damage and TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in HCC cells. Our results showed that LCB inhibited cell proliferation and induced DNA damage, cell cycle arrest and apoptosis. Treatment with LCB significantly inhibited the Akt/mTOR pathway and activated endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling pathway. Moreover, combined treatment with LCB and TRAIL yielded evident enhancements in the viability reduction and apoptosis. LCB upregulated death receptor 4 (DR4) and death receptor 5 (DR5) protein in a concentration- and time-dependent manner. The knockdown of DR5 significantly suppressed TRAIL-induced cleavage of PARP, which was enhanced by LCB. Treatment with an extracellular-regulated kinase (ERK) inhibitor (PD98059) or c-Jun N-terminal kinase (JNK) inhibitor (SP600125) markedly reduced the LCB-induced upregulation of DR5 expression and attenuated LCB-mediated TRAIL sensitization. In summary, LCB exhibits cytotoxic activity through modulation of the Akt/mTOR, ER stress and MAPK pathways in HCC cells and effectively enhances TRAIL sensitivity through the upregulation of DR5 expression in ERK- and JNK-dependent manner. Combination therapy with LCB and TRAIL may be an alternative treatment strategy for HCC. [Display omitted] • LCB induces cell cycle arrest, DNA damage and apoptosis in HCC cells. • LCB regulates the Akt/mTOR, ER stress and MAPK pathways. • LCB upregulates DR5 and sensitizes TRAIL-induced apoptosis. [ABSTRACT FROM AUTHOR]

NT157, a small-molecule tyrosine kinase inhibitor, exhibits broad-spectrum anti-tumor activity. However, NT157-mediated inhibition against glioma has not been explored yet. Herein, the anticancer effects and underlying mechanism of NT157 against human giloma growth were evaluated. The results showed that NT157 alone significantly inhibited glioma cells growth in vitro by lunching cell cycle arrest through up-regulating p21 and p27, and down-regulating cell cycle-related factors. NT157 alone also induced significant glioma cells apoptosis, followed by PARP cleavage and caspase-3 activation. Our findings further revealed that NT157 triggered significant DNA damage and dysfunction of PI3K/AKT, MAPKs and EGFR-STAT3 signaling pathways. Addition of several kinases inhibitors effectively abrogated NT157-induced DR5 up-regulation, which further confirmed the significant role of DR5 pathway. Moreover, combined treatment of NT157 and TRAIL showed enhanced apoptosis against U251 and U87 cells. However, Knockdown of DR5 expression significantly attenuated combined treatment-induced PARP cleavage and caspase-3 activation. Importantly, combined administration of NT157 and TRAIL in vivo effectively inhibited glioma xenograft growth of nude mice by inhibiting cell proliferation and angiogenesis, and inducing DNA damage and apoptosis. Taken together, our findings validated the rational design that combined strategy of NT157 and TRAIL to trigger DNA damage and apoptosis by up-regulating DR5 could be a high efficient way to combat human glioma. [Display omitted] • NT157 induced glioma cell cycle arrest and apoptosis. • NT157 enhanced TRAIL's anticancer effect by activating ROS and DR5 expression. [ABSTRACT FROM AUTHOR]

Activation of the TRAIL proapoptotic pathway can promote cancer cell apoptosis. Histone deacetylases (HDACs) also are promising drug targets for cancers, and their synergistic effect with TRAIL can improve the inhibitory effect on cancer cells. Therefore, the development of highly TRAIL-sensitive HDAC inhibitors might be a promising strategy for the treatment of cancers. We synthesized a series of HDAC inhibitors by introducing effective fragments sensitive to TRAIL. Compound IIc showed good inhibitory activity against HDAC1 and HCT116 cells and showed higher sensitivity to activating the expression of the TRAIL protein and promoting the apoptosis of HCT-116 cells compared with ONC201. The inhibitory activity of compound IIc (25 mg/kg) in the HCT-116 xenograft model was significantly greater than those of the positive control drugs (ONC201, chidamide). These findings suggested that development of highly TRAIL-sensitive HDAC inhibitors as colorectal tumor cancer drugs. [Display omitted] • Compound IIc is a novel and effective highly TRAIL-sensitive HDAC inhibitors. • IIc mediated HCT-116 cell apoptosis through the TRAIL-induced apoptosis signaling pathway. • IIc showed good antitumor efficacy in HCT-116 xenograft model. [ABSTRACT FROM AUTHOR]

Aims: Cardiovascular disease (CVD) is the leading cause of mortality in type 2 diabetes mellitus (T2DM). Epidemiological studies suggest serum Osteoprotegrin (OPG)/Tumour-necrosis-factor-related-apoptosis-inducing- ligand (TRAIL) ratio may be a useful marker of cardiovascular risk. This study aimed to compare serum levels of TRAIL, OPG and OPG/TRAIL ratio in people with T2DM, with and without a history of CVD, and controls; and to determine which of these indices, if any, predict cardiovascular risk.Methods: In this single centre observational study of 133 participants, people with T2DM, with and without a history of a cardiovascular event in the last 5 years, were recruited along with a control cohort without T2DM or CVD. Demographic information and anthropometric measurements were recorded. Blood samples were taken and OPG and TRAIL were measured using ELISA.Results: People with T2DM and CVD had higher OPG/TRAIL ratios compared to controls or those with a new diagnosis of T2DM. After adjustment for potential confounding factors, OPG/TRAIL ratio was significantly associated with the presence of CVD in people with T2DM and an OPG/TRAIL ratio cut-off > 38.6 predicted the presence of CVD in this cohort with a sensitivity of 80% and specificity of 82%.Conclusion: This study suggests that OPG/TRAIL ratio may have a role as a biomarker of CVD in people with T2DM. [ABSTRACT FROM AUTHOR]

The immune status of the tumor microenvironment is a key indicator determining the antitumor effect of immunotherapy. Oncolytic viruses directly target tumor cells or indirectly modulate the tumor microenvironment (TME) especially when properly armed. It was previously demonstrated that conditionally replicating adenovirus serotype 5 (CRAd5) encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) had outstanding antitumor effects in different human cancer cells xenograft models; however, its antitumor immune mechanism has not been evaluated in immunocompetent preclinical mouse models. We first explored the antitumor activity of CRAd5-TRAIL in several murine tumor models and found that the expression of TRAIL induced increases or activation in tumor-infiltrating T cells. To further improve the antitumor effects, mouse CD40 ligand (mCD40L) as an immune activator expressed by recombinant Ad5 vector was firstly used in combination with CRAd5-TRAIL for tumor immunotherapy. Both in vitro and in vivo studies demonstrated that mCD40L effectively activated dendritic cells (DCs), B cells, and tumor-infiltrating T cells, and also promoted tumor cell apoptosis by increasing the expression of TRAIL receptors, thereby significantly enhancing the antitumor activity of oncolytic adenoviruses in CT26 and B16 tumor-bearing models. Although affected by the restriction of oncolytic adenovirus replication in mouse cells, the combination treatment failed to completely eliminate tumor cells, our research still provided a promising strategy for oncolytic adenovirus-mediated solid tumor immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract: The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors. [Copyright &y& Elsevier]

Abstract: The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4+ T cells is not known. Here we investigated whether primary CD4+ T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4+ T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images. [Copyright &y& Elsevier]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that kills various tumor cells without damaging normal tissues. However, many cancers remain resistant to TRAIL. To overcome TRAIL resistance, combination therapies using sensitizers of the TRAIL pathway would be an efficacious approach. To investigate potential sensitizers of TRAIL-induced apoptosis, we used TRAIL-resistant human T cell leukemia virus type 1 (HTLV-1)-associated adult T cell leukemia/lymphoma (ATL) cells as a model system. So far, HTLV-1-associated ATL is incurable by presently known therapies. Here, we show that wogonin and the structurally related natural flavones apigenin and chrysin break TRAIL resistance in HTLV-1-associated ATL by transcriptional down-regulation of c-FLIP, a key inhibitor of death receptor signaling, and by up-regulation of TRAIL receptor 2 (TRAIL-R2). This effect is mediated through transcriptional inhibition of the p53 antagonist murine double minute 2 (Mdm2), leading to an increase in p53 levels and, consequently, to up-regulation of the p53 target gene TRAIL-R2. We also show that these flavones can sensitize to TNFa- and CD95-mediated cell death. Furthermore, we show that wogonin, apigenin, and chrysin also enhance TRAIL-mediated apoptosis in other human cancer cell lines including breast cancer cell line MDA-MB-231, colon cancer cell line HT-29, hepatocellular carcinoma cell line HepG2, melanoma cell line SK-MEL-37, and pancreatic carcinoma cell line Capan-1 by the same mechanism. Thus, our study suggests the potential use of these flavones as an adjuvant for TRAIL-mediated anticancer therapy. [ABSTRACT FROM AUTHOR]

MADD plays an essential role in cancer cell survival. Abrogation of endogenous MADD expression results in significant spontaneous apoptosis and enhanced susceptibility to tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, the regulation of MADD function is largely unknown. Here, we demonstrate that endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing Fas-associated death domain recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and Fas-associated death domain association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis. Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting Akt-MADD axis is likely to increase efficacy of TRAIL-based therapies. [ABSTRACT FROM AUTHOR]

Abstract: Background: This study was designed to evaluate the biological significance of simultaneous changes in the circulating levels of osteoprotegerin (OPG) and TNF-related apoptosis inducing ligand (TRAIL) in patients with coronary artery disease (CAD), and, in particular, with acute myocardial infarction (AMI). Methods: Total levels of OPG and TRAIL were measured by ELISA in patients with AMI (n =113), unstable angina (UA, n =21) and healthy controls (n =120). Results: Since OPG was elevated during the acute phase (first 12–24–48h) after AMI and in patients with UA with respect to healthy controls, while TRAIL was decreased in acute AMI patients, CAD patients were characterized by an increased OPG/TRAIL ratio. Moreover, the OPG/TRAIL ratio was significantly (p <0.05) higher in the acute AMI patients who developed heart failure (HF) than in those who did not develop HF in the follow-up. Conclusions: An impaired OPG/TRAIL ratio after AMI is related to a higher risk of HF. [Copyright &y& Elsevier]

Whether celastrol, a triterpene from traditional Chinese medicine, can modulate the anticancer effects of TRAIL, the cytokine that is currently in clinical trial, was investigated. As indicated by assays that measure plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3, celastrol potentiated the TRAIL-induced apoptosis in human breast cancer cells, and converted TRAIL-resistant cells to TRAIL-sensitive cells. When examined for its mechanism, we found that the triterpene down-regulated the expression of cell survival proteins including cFLIP, lAP-1, Bcl-2, Bcl-xL, survivin, and XIAP and up-regulated Bax expression. In addition, we found that celastrol induced the cell surface expression of both the TRAIL receptors DR4 and DR5. This increase in receptors was noted in a wide variety of cancer cells including breast, lung, colorectal, prostate, esophageal, and pancreatic cancer cells, and myeloid and leukemia cells. Gene silencing of the death receptor abolished the effect of celastrol on TRAIL-induced apoptosis. Induction of the death receptor by the triterpenoid was found to be p53-independent but required the induction of CAAT/enhancer-binding protein homologous protein (CHOP), inasmuch as gene silencing of CHOP abolished the induction of DR5 expression by celastrol and associated enhancement of TRAIL-induced apoptosis. We found that celastrol also induced reactive oxygen species (ROS) generation, and ROS sequestration inhibited celastrol-induced expression of CHOP and DR5, and consequent sensitization to TRAIL. Overall, our results demonstrate that celastrol can potentiate the apoptotic effects of TRAIL through down-regulation of cell survival proteins and up-regulation of death receptors via the ROS-mediated up-regulation of CHOP pathway. [ABSTRACT FROM AUTHOR]

[Display omitted] Tumor necrosis factor-related apoptosis ligand (TRAIL) is a promising antitumor agent candidate for its selective proapoptotic activity to various tumor cells. However, TRAIL showed limited efficacy in clinical trials despite of good tolerability. One important reason might be attributed to the poor tumor-homing ability of TRAIL. Herein, we designed an EGFR-targeting TRAIL (Z-TRAIL) by genetically fusing an EGFR-antagonistic affibody (Z EGFR:1907) to the N-terminus of TRAIL. Z-TRAIL was produced as a soluble protein with high yield in E. coli and it maintained the trimeric state of active TRAIL. Under the EGFR-binding mediated by Z EGFR:1907 , Z-TRAIL showed a ∼5 to 20-fold enhancement of cytotoxicity compared to TRAIL on tumor cells in vitro. Furthermore, fusion to Z EGFR:1907 endowed TRAIL with a ∼1.8-fold increase of tumor uptake and a dramatical stronger apoptosis-inducing ability in the mice bearing EGFR-overexpressing A431 tumor xenografts. More importantly, Z-TRAIL exhibited significantly enhanced antitumor efficacy against whether EGFR high-expressing or low-expressing tumors than TRAIL in vivo. In addition, repeated injection of high-dose Z-TRAIL did not show obvious acute toxicity in mice. These results demonstrated that the newly engineered Z-TRAIL might be a promising agent for targeted therapy of EGFR-expressing tumors. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor α family of cytokines that preferentially induces apoptosis in transformed cells, making it a promising cancer therapy. However, many neoplasms are resistant to TRAIL-induced apoptosis by mechanisms that are poorly understood. We demonstrate that the expression of the small heat shock protein αB-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) correlates with TRAIL resistance in a panel of human cancer cell lines. Stable expression of wild-type αB-crystallin, but not a pseudophosphorylation mutant impaired in its assembly and chaperone function, protects cancer cells from TRAIL-induced caspase-3 activation and apoptosis in vitro. Furthermore, selective inhibition of αB-crystallin expression by RNA interference sensitizes cancer cells to TRAIL. In addition, wild-type αB-crystallin promotes xenograft tumor growth and inhibits TRAIL-induced apoptosis in vivo in nude mice, whereas a pseudophosphorylation αB-crystallin mutant impaired in its anti-apoptotic function inhibits xenograft tumor growth. Collectively, these findings indicate that αB-crystallin is a novel regulator of TRAIL-induced apoptosis and tumor growth. Moreover, these results demonstrate that targeted inhibition of αB-crystallin promotes TRAIL-induced apoptosis, thereby suggesting a novel strategy to overcome TRAIL resistance in cancer. [ABSTRACT FROM AUTHOR]

In many mammalian cell types, engagement of the TRAIL/Apo2L death receptors DR4 and DR5 alters mitochondrial physiology, thereby promoting the release of pro-apoptotic proteins normally contained within this organelle. A contemporary view of this process is that in so-called type II cells death receptor-activated caspase-8 cleaves the Bcl-2 family member Bid, which generates a truncated Bid fragment that collaborates with Bax, another Bcl-2 relative, to promote the release of mitochondrial factors necessary for activation of executioner caspases and apoptosis. Here we show that in some type II cells caspase-2 is necessary for optimal TRAIL-mediated cleavage of Bid. Downregulation of caspase-2 using RNA interference significantly inhibited TRAIL-induced apoptosis. Analysis of the TRAIL proteolytic cascade following gene silencing of specific pathway components revealed that caspase-2 is necessary for efficient cleavage of Bid; however, caspase-2 proteolytic processing, which occurs downstream of Bax, is not necessary for its role in Bid cleavage. [ABSTRACT FROM AUTHOR]

Neuroblastoma is a metastatic brain tumor particularly common in children. The cure rate is below 50% for patients of high-risk condition. Novel therapeutic agents and approaches are needed to improve the cure rate. Tumor necrosis factor-related and apoptosis-inducing ligand (TRAIL) is a promising proapoptotic factor that rapidly induces apoptosis preferentially in transformed and cancerous cells. Unfortunately, the common TRAIL resistance in cancers has hampered the clinical application of the ligand. Previously we prepared a novel TRAIL-armed ER derived nanosomal agent (ERN-T) that overcomes TRAIL resistance in some cancer lines when combined with a synthetic antagonist of inhibitors of apoptosis proteins (IAPs), AZD5582. However, how AZD5582 sensitizes cancer cells to ERN-T remains not well understood. In this study we continued to test the therapeutic efficacy of the combinatory therapy of ERN-T and AZD5582 on neuroblastoma, aiming to reveal the molecular mechanism underlying the synergism between AZD5582 and ERN-T. The obtained data revealed that ERN-Ts overcame TRAIL resistance and showed significant cytotoxicity on the resistant neuroblastoma line SH-SH5Y when combined with AZD5582 whilst sparing normal cells. The combination of low doses of ERN-Ts and AZD5582 induced intensive apoptosis in SH-SY5Y but not in normal skin fibroblasts (NSFs). Importantly we discovered that TRAIL sensitization in SH-SY5Y was associated with the concomitant downregulation of antiapoptotic factors cFLIP, MCL-1 and IAPs and upregulation of proapoptotic protein BAX and the death receptor 5 (DR5) by the cotreatment of ERN-T and AZD5582. In vivo study demonstrated that the combination of ERN-T and AZD5582 constituted a highly effective and safe therapy for subcutaneous SH-SY5Y xenograft neuroblastoma in nude mice. In conclusion, we identified that the concomitant regulation of both antiapoptotic and proapoptotic factors and DR5 is an essential molecular mechanism for overcoming TRAIL resistance in SH-SY5Y and the combination of ERN-T and AZD5582 potentially constitutes a novel therapeutic strategy, which is highly effective and safe for neuroblastoma. [ABSTRACT FROM AUTHOR]