Tumor Necrosis Factor-related Apoptosis-inducing Ligand Death Receptors Research Articles

Fisetin reduces the resistance of MOLT-4 and K562 cells to TRAIL-induced apoptosis through upregulation of TRAIL receptors.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that is capable of apoptosis induction selectively in tumor cells. Although TRAIL has been harnessed in numerous clinical trials, resistance to TRAIL-induced apoptosis is a major challenge ahead of this therapy in various cancer models as well as in leukemia. Since histone deacetylases (HDACs) are known to affect drug resistance in malignant cells, the present study aimed to evaluate the potential of fisetin for sensitization of MOLT-4 and K-562 leukemic cells to TRAIL-induced apoptosis. The MOLT-4 and K-562 cells were treated with increasing concentrations of fisetin and its impact on the growth inhibition and apoptosis induction of TRAIL were evaluated by MTT and Annexin V/7-AAD assays. The impact of fisetin on the mRNA and protein expression levels of apoptosis regulatory genes such as BIRC2/c-IAP1, CFLAR/cFLIP, CASP3, CASP7, CASPP9, TNFRSF10A/DR4, TNFRSF10B/DR5, and BID were examined by PCR array, qRT-PCR, and flow cytometry. Pre-treatment of MOLT-4 and K-562 cells with fisetin reduced the IC50 of TRAIL in growth inhibition along with an improvement in apoptosis induction by TRAIL. The expression of the BIRC2 gene encoding antiapoptotic protein c-IAP1 downregulated in the fisetin-treated cells while the expressions of TNFRSF10A and TNFRSF10B encoding TRAIL death receptors increased. Fisetin demonstrated a potential for alleviating the TRAIL resistance by modulating the apoptosis regulatory factors and improving the expressions of TRAIL receptors that could facilitate the application of TRAIL in cancer therapies.

Oxaliplatin Enhances the Apoptotic Effect of Mesenchymal Stem Cells, Delivering Soluble TRAIL in Chemoresistant Colorectal Cancer.

A key problem in colorectal cancer (CRC) is the development of resistance to current therapies due to the presence of cancer stem cells (CSC), which leads to poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a protein that activates apoptosis in cancer cells through union with TRAIL death receptors. Cell therapies as delivery systems can produce soluble TRAIL (sTRAIL) and full-length TRAIL (flTRAIL), showing a high capacity to produce apoptosis in vitro and in vivo assays. However, the apoptotic activity of TRAIL as monotherapy had limitations, so it is important to explore other ways to enhance susceptibility to TRAIL. This study evaluated the cytotoxic and proapoptotic activity of soluble TRAIL overexpressed by mesenchymal stem cells (MSC) in an oxaliplatin-resistant CRC cell line. Bone marrow-MSC were lentiviral transduced for soluble TRAIL expression. DR5 death receptor expression was determined in Caco-2 and CMT-93 CRC cell lines. Sensitivity to first-line chemotherapies and recombinant TRAIL was evaluated by half-maximal inhibitory concentrations. Cytotoxic and proapoptotic activity of soluble TRAIL-MSC alone and combined with chemotherapy pre-treatment was evaluated using co-cultures. Caco-2 and CMT-93 cell lines expressed 59.08 ± 5.071 and 51.65 ± 11.99 of DR5 receptor and had IC50 of 534.15 ng/mL and 581.34 ng/mL for recombinant murine TRAIL (rmTRAIL), respectively. This finding was classified as moderate resistance to TRAIL. The Caco-2 cell line showed resistance to oxaliplatin and irinotecan. MSC successfully overexpressed soluble TRAIL and induced cancer cell death at a 1:6 ratio in co-culture. Oxaliplatin pre-treatment in the Caco-2 cell line increased the cell death percentage (50%) and apoptosis by sTRAIL. This finding was statistically different from the negative control (p < 0.05), and activity was even higher with the oxaliplatin-flTRAIL combination. Thus, oxaliplatin increases apoptotic activity induced by soluble TRAIL in a chemoresistant CRC cell line.

Recent Advances in the Development of Nanodelivery Systems Targeting the TRAIL Death Receptor Pathway

The TRAIL (TNF-related apoptosis-inducing ligand) apoptotic pathway is extensively exploited in the development of targeted antitumor therapy due to TRAIL specificity towards its cognate receptors, namely death receptors DR4 and DR5. Although therapies targeting the TRAIL pathway have encountered many obstacles in attempts at clinical implementation for cancer treatment, the unique features of the TRAIL signaling pathway continue to attract the attention of researchers. Special attention is paid to the design of novel nanoscaled delivery systems, primarily aimed at increasing the valency of the ligand for improved death receptor clustering that enhances apoptotic signaling. Optionally, complex nanoformulations can allow the encapsulation of several therapeutic molecules for a combined synergistic effect, for example, chemotherapeutic agents or photosensitizers. Scaffolds for the developed nanodelivery systems are fabricated by a wide range of conventional clinically approved materials and innovative ones, including metals, carbon, lipids, polymers, nanogels, protein nanocages, virus-based nanoparticles, dendrimers, DNA origami nanostructures, and their complex combinations. Most nanotherapeutics targeting the TRAIL pathway are aimed at tumor therapy and theranostics. However, given the wide spectrum of action of TRAIL due to its natural role in immune system homeostasis, other therapeutic areas are also involved, such as liver fibrosis, rheumatoid arthritis, Alzheimer's disease, and inflammatory diseases caused by bacterial infections. This review summarizes the recent innovative developments in the design of nanodelivery systems modified with TRAIL pathway-targeting ligands.

Investigation of Apoptotic Effect of Klotho Protein on Human Colorectal Cancer Cells via TRAIL Death Receptors

Klotho is a transmembrane protein which is deficiency has pleiotropic effects in a number of aging-related disease processes and various cancers. In some recent studies about klotho protein disorders, it has been shown to klotho is effective in various cancers including lung, liver, breast, kidney, and colon. TNF-related apoptosis-inducing ligand (TRAIL), a TNF family molecule, is a cytokine that stimulates apoptosis through death receptors in many cancer types and therefore attracts attention for tumor therapies in various preclinical models. Interaction with TRAIL death receptors create an apoptotic effect in cancer treatments by reducing the proliferation of cancer cells. In this study, it was aimed to investigate the effects of exogen klotho administration on cell viability and apoptosis on TRAIL death receptors (TRAIL1 and TRAILR2) in human healthy colon cells (CCD 841 CoN) and TRAIL-resistant human colorectal cancer cells (caco2). For this purpose, cells were treated with different concentrations of klotho for 24 and 48 hours. To determine the cell viability, proliferation, and death receptors effects of klotho protein on cancer and healthy cells evaluations with WST-8 and Real-Time qRT-PCR analysis were performed. Our results showed that the increase in klotho protein concentration did not have a significant effect on cell viability in healthy colon cells, whereas it decreased cell viability and proliferation in apoptosis-resistant human colorectal cancer cells. Relative gene expression of TRAIL1 and TRAILR2 death receptors increased with klotho applied to the apoptosis-resistant colorectal cancer cell line caco-2. Therefore, targeting the ligand-receptors that induce TNF-related apoptosis with the klotho protein can be considered as a potential therapeutic approach for cancer therapy.

Targeting TRAIL Death Receptors in Triple-Negative Breast Cancers: Challenges and Strategies for Cancer Therapy

The tumor necrosis factor (TNF) superfamily member TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in cancer cells via death receptor (DR) activation with little toxicity to normal cells or tissues. The selectivity for activating apoptosis in cancer cells confers an ideal therapeutic characteristic to TRAIL, which has led to the development and clinical testing of many DR agonists. However, TRAIL/DR targeting therapies have been widely ineffective in clinical trials of various malignancies for reasons that remain poorly understood. Triple negative breast cancer (TNBC) has the worst prognosis among breast cancers. Targeting the TRAIL DR pathway has shown notable efficacy in a subset of TNBC in preclinical models but again has not shown appreciable activity in clinical trials. In this review, we will discuss the signaling components and mechanisms governing TRAIL pathway activation and clinical trial findings discussed with a focus on TNBC. Challenges and potential solutions for using DR agonists in the clinic are also discussed, including consideration of the pharmacokinetic and pharmacodynamic properties of DR agonists, patient selection by predictive biomarkers, and potential combination therapies. Moreover, recent findings on the impact of TRAIL treatment on the immune response, as well as novel strategies to address those challenges, are discussed.

A soluble DR5-Fc chimeric protein attenuates inflammatory responses induced by coronavirus MHV-A59 and SARS-CoV-2.

Mortality in coronavirus disease 2019 (COVID‐19) patients has been linked to the presence of a “cytokine storm” induced by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, which involves elevated levels of circulating cytokines and immune‐cell hyperactivation. Targeting cytokines during the management of COVID‐19 patients has the potential to improve survival rates and reduce mortality. Although cytokine blockers and immune‐host modulators are currently being tested in severely ill COVID‐19 patients to cope with the overwhelming systemic inflammation, there is not too many successful cases, thus finding new cytokine blockers to attenuate the cytokine storm syndrome is meaningful. In this paper, we significantly attenuated the inflammatory responses induced by mouse hepatitis viruses A59 and SARS‐CoV‐2 through a soluble DR5‐Fc (sDR5‐Fc) chimeric protein that blocked the TNF‐related apoptosis‐inducing ligand–death receptor 5 (TRAIL–DR5) interaction. Our findings indicates that blocking the TRAIL–DR5 pathway through the sDR5‐Fc chimeric protein is a promising strategy to treat COVID‐19 severe patients requiring intensive care unit admission or with chronic metabolic diseases.

Virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis.

Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.

Dexamethasone induces pancreatic β-cell apoptosis through upregulation of TRAIL death receptor.

Long-term medication with dexamethasone - a synthetic glucocorticoid (GC) drug - results in hyperglycemia, or steroid-induced diabetes. Although recent studies revealed that dexamethasone directly induces pancreatic β-cell apoptosis, its molecular mechanisms remain unclear. In our initial analysis of mRNA transcripts, we discovered the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway may be involved in dexamethasone-induced pancreatic β-cell apoptosis. In the present study, a mechanism of dexamethasone-induced pancreatic β-cell apoptosis through the TRAIL pathway was investigated in cultured cells and isolated mouse islets. INS-1 cells were cultured with and without dexamethasone in the presence or absence of a glucocorticoid receptor (GR) inhibitor, RU486. We found that dexamethasone induced pancreatic β-cell apoptosis in association with the upregulation of TNSF10 (TRAIL) mRNA and protein expression. Moreover, dexamethasone upregulated the TRAIL death receptor (DR5) protein but suppressed the decoy receptor (DcR1) protein. Similar findings were observed in mouse isolated islets: dexamethasone increased TRAIL and DR5 compared to that of control mice. Furthermore, dexamethasone stimulated pro-apoptotic signaling including superoxide production, caspase-8, -9, and -3 activities, NF-κB, and Bax but repressed the anti-apoptotic protein, Bcl-2. All these effects were inhibited by the GR-inhibitor, RU486. Furthermore, knock-down DR5 decreased dexamethasone-induced caspase 3 activity. Caspase-8 and caspase-9 inhibitors protected pancreatic β-cells from dexamethasone-induced apoptosis. Taken together, dexamethasone induced pancreatic β-cell apoptosis by binding to the GR and inducing DR5 and TRAIL pathway.

Behind the Adaptive and Resistance Mechanisms of Cancer Stem Cells to TRAIL.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand (Apo2L), is a member of the TNF cytokine superfamily. TRAIL has been widely studied as a novel strategy for tumor elimination, as cancer cells overexpress TRAIL death receptors, inducing apoptosis and inhibiting blood vessel formation. However, cancer stem cells (CSCs), which are the main culprits responsible for therapy resistance and cancer remission, can easily develop evasion mechanisms for TRAIL apoptosis. By further modifying their properties, they take advantage of this molecule to improve survival and angiogenesis. The molecular mechanisms that CSCs use for TRAIL resistance and angiogenesis development are not well elucidated. Recent research has shown that proteins and transcription factors from the cell cycle, survival, and invasion pathways are involved. This review summarizes the main mechanism of cell adaption by TRAIL to promote response angiogenic or pro-angiogenic intermediates that facilitate TRAIL resistance regulation and cancer progression by CSCs and novel strategies to induce apoptosis.

IL13Rα2‑ and EGFR‑targeted pseudomonas exotoxin potentiates the TRAIL‑mediated death of GBM cells.

Glioblastomas (GBMs) are refractory to current treatments and novel therapeutic approaches need to be explored. Pro‑apoptotic tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) is tumor‑specific and has been shown to induce apoptosis and subsequently kill GBM cells. However, approximately 50% of GBM cells are resistant to TRAIL and a combination of TRAIL with other therapeutics is necessary to induce mechanism‑based cell death in TRAIL‑resistant GBMs. The present study examined the ability of the tumor cell surface receptor, interleukin (IL)‑13 receptor α2 (IL13Rα2)‑ and epidermal growth factor receptor (EGFR)‑targeted pseudomonas exotoxin (PE) to sensitize TRAIL‑resistant GBM cells and assessed the dual effects of interleukin 13‑PE (IL13‑PE) or EGFR nanobody‑PE (ENb‑PE) and TRAIL for the treatment of a broad range of brain tumors with a distinct TRAIL therapeutic response. Receptor targeted toxins upregulated TRAIL death receptors (DR4 and DR5) and suppressed the expression of anti‑apoptotic FLICE‑inhibitory protein (FLIP) and X‑linked inhibitor of apoptosis protein (XIAP). This also led to the induction of the cleavage of caspase‑8 and caspase‑9 and resulted in the sensitization of highly resistant established GBM and patient‑derived GBM stem cell (GSC) lines to TRAIL‑mediated apoptosis. These findings provide a mechanism‑based strategy that may provide options for the cell‑mediated delivery of bi‑functional therapeutics to target a wide spectrum of TRAIL‑resistant GBMs.

Splicing reprogramming of TRAIL/DISC-components sensitizes lung cancer cells to TRAIL-mediated apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selective killing of cancer cells underlines its anticancer potential. However, poor tolerability and resistance underscores the need to identify cancer-selective TRAIL-sensitizing agents. Apigenin, a dietary flavonoid, sensitizes lung cancer cell lines to TRAIL. It remains unknown, however, whether apigenin sensitizes primary lung cancer cells to TRAIL and its underlying mechanisms. Here we show that apigenin reprograms alternative splicing of key TRAIL/death-inducing-signaling-complex (DISC) components: TRAIL Death Receptor 5 (DR5) and cellular-FLICE-inhibitory-protein (c-FLIP) by interacting with the RNA-binding proteins hnRNPA2 and MSI2, resulting in increased DR5 and decreased c-FLIPS protein levels, enhancing TRAIL-induced apoptosis of primary lung cancer cells. In addition, apigenin directly bound heat shock protein 70 (Hsp70), promoting TRAIL/DISC assembly and triggering apoptosis. Our findings reveal that apigenin directs alternative splicing and inhibits Hsp70 enhancing TRAIL anticancer activity. These findings underscore impactful synergies between diet and cancer treatments opening new avenues for improved cancer treatments.

BRD4 inhibition promotes TRAIL-induced apoptosis by suppressing the transcriptional activity of NF-κB in NSCLC.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and agonistic antibodies against TRAIL death receptors (DR) can induce apoptosis preferentially in tumor cells while causing virtually no damage to normal cells. However, their therapeutic potential is limited by occurring resistance in tumor cells, including non-small cell lung cancer (NSCLC). Thus, elucidation of the molecular targets and signaling pathways responsible for TRAIL resistance is imperative for devising effective therapeutic strategies for TRAIL resistant cancers. In the present study, we demonstrated that inhibition of Bromodomain-containing protein 4 (BRD4) or genetic knock-down of BRD4, an epigenetic reader and master transcription coactivator, can sensitize lung cancer cells to TRAIL. This sensitization is in a caspase-dependent manner. Inhibition of BRD4 by small molecule inhibitor (+)-JQ-1 and genetic knock-down of BRD4 can both recruit the FADD and activate caspases. The sensitization did not regulate the death receptors DR4 and DR5. Moreover, BRD4 inhibition can block TRAIL-induced IKK activation by suppressing the transcriptional activity of NF-κB. These findings indicate that targeting combination therapy with TRAIL and BRD4 inhibitors can be a promising strategy to overcome TRAIL resistance in NSCLC.

Membrane expression and significance of TRAIL death receptors DR4 and DR5 in Pancreatic cancer

Introduction: Tumor necrosis factor related-apoptosis-inducing ligand (TRAIL) is a powerful and selective activator of apoptosis in many cancer cells. We aim to investigate the expression and significance of TRAIL death receptor DR4 and DR5 in pancreatic cancer (PC) tissues.&#x0D; Method: Twenty-eight histologically verified samples of PC tissue were collected between 2018 and 2019. TRAIL death receptor expression profiles were determined by immunohistochemistry.&#x0D; Result: Death receptor DR4 and DR5 were expressed in the PC tissue and the adjacent non-cancerous pancreatic tissues, the expression of DR4 and DR5 in the PC tissue was significantly higher than that of the adjacent non-cancerous pancreatic tissues (p&lt;0.05). Additionally, in both the tissue group, the expression of DR4 was significantly stronger than the DR5 (p&lt;0.05). To assess the relationship between DR4 and DR5 expression, differentiation, and tumor staging of PC, the result reveals that the expression of DR4 and DR5 was significantly higher in stage I tumors than the stage II, III, IV tumors (p&lt;0.05). In contrast, the expression of DR4 and DR5 was decreased with a decrease in the degree of differentiation of tumors. However, the difference was not statistically significant.&#x0D; Conclusion: The membrane expression of TRAIL death receptor DR4 and DR5 is greater in PC than in the adjacent non-cancerous pancreatic tissues. Furthermore, increased membrane expression of TRAIL death receptor DR4 and DR5 in stage I PC and well-differentiated PC may predict the prognosis and feasibility of using TRAIL gene therapy as a treatment option for early PC.

Tristetraprolin Posttranscriptionally Downregulates TRAIL Death Receptors.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has attracted attention as a potential candidate for cancer therapy. However, many primary cancers are resistant to TRAIL, even when combined with standard chemotherapy. The mechanism of TRAIL resistance in cancer cells has not been fully elucidated. The TRAIL death receptor (DR) 3′-untranslated region (3′-UTR) is reported to contain AU-rich elements (AREs) that are important for regulating DR mRNA stability. However, the mechanisms by which DR mRNA stability is determined by its 3′-UTR are unknown. We demonstrate that tristetraprolin (TTP), an ARE-binding protein, has a critical function of regulating DR mRNA stability. DR4 mRNA contains three AREs and DR5 mRNA contains four AREs in 3′-UTR. TTP bound to all three AREs in DR4 and ARE3 in DR5 and enhanced decay of DR4/5 mRNA. TTP overexpression in colon cancer cells changed the TRAIL-sensitive cancer cells to TRAIL-resistant cells, and down-regulation of TTP increased TRAIL sensitivity via DR4/5 expression. Therefore, this study provides a molecular mechanism for enhanced levels of TRAIL DRs in cancer cells and a biological basis for posttranscriptional modification of TRAIL DRs. In addition, TTP status might be a biomarker for predicting TRAIL response when a TRAIL-based treatment is used for cancer.

Cytomegalovirus Evades TRAIL-Mediated Innate Lymphoid Cell 1 Defenses.

Cytomegalovirus (CMV) establishes a lifelong infection facilitated, in part, by circumventing immune defenses mediated by tumor necrosis factor (TNF)-family cytokines. An example of this is the mouse CMV (MCMV) m166 protein, which restricts expression of the TNF-related apoptosis-inducing ligand (TRAIL) death receptors, promoting early-phase replication. We show here that replication of an MCMV mutant lacking m166 is also severely attenuated during viral persistence in the salivary glands (SG). Depleting group I innate lymphoid cells (ILCs) or infecting Trail-/- mice completely restored persistent replication of this mutant. Group I ILCs are comprised of two subsets, conventional natural killer cells (cNK) and tissue-resident cells often referred to as innate lymphoid type I cells (ILC1). Using recently identified phenotypic markers to discriminate between these two cell types, their relative expression of TRAIL and gamma interferon (IFN-γ) was assessed during both early and persistent infection. ILC1 were found to be the major TRAIL expressers during both of these infection phases, with cNK expressing very little, indicating that it is ILC1 that curtail replication via TRAIL in the absence of m166-imposed countermeasures. Notably, despite high TRAIL expression by SG-resident ILC1, IFN-γ production by both ILC1 and cNK was minimal at this site of viral persistence. Together these results highlight TRAIL as a key ILC1-utilized effector molecule that can operate in defense against persistent infection at times when other innate control mechanisms may be muted and highlight the importance for the evolution of virus-employed countermeasures.IMPORTANCE Cytomegalovirus (a betaherpesvirus) is a master at manipulating immune responses to promote its lifelong persistence, a result of millions of years of coevolution with its host. Using a one-of-a-kind MCMV mutant unable to restrict expression of the TNF-related apoptosis-inducing ligand death receptors (TRAIL-DR), we show that TRAIL-DR signaling significantly restricts both early and persistent viral replication. Our results also reveal that these defenses are employed by TRAIL-expressing innate lymphoid type I cells (ILC1) but not conventional NK cells. Overall, our results are significant because they show the key importance of viral counterstrategies specifically neutralizing TRAIL effector functions mediated by a specific, tissue-resident subset of group I ILCs.

Molecular Mode of Action of TRAIL Receptor Agonists-Common Principles and Their Translational Exploitation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAILR1/death receptor 4 (DR4) and TRAILR2/DR5 trigger cell death in many cancer cells but rarely exert cytotoxic activity on non-transformed cells. Against this background, a variety of recombinant TRAIL variants and anti-TRAIL death receptor antibodies have been developed and tested in preclinical and clinical studies. Despite promising results from mice tumor models, TRAIL death receptor targeting has failed so far in clinical studies to show satisfying anti-tumor efficacy. These disappointing results can largely be explained by two issues: First, tumor cells can acquire TRAIL resistance by several mechanisms defining a need for combination therapies with appropriate sensitizing drugs. Second, there is now growing preclinical evidence that soluble TRAIL variants but also bivalent anti-TRAIL death receptor antibodies typically require oligomerization or plasma membrane anchoring to achieve maximum activity. This review discusses the need for oligomerization and plasma membrane attachment for the activity of TRAIL death receptor agonists in view of what is known about the molecular mechanisms of how TRAIL death receptors trigger intracellular cell death signaling. In particular, it will be highlighted which consequences this has for the development of next generation TRAIL death receptor agonists and their potential clinical application.

TRAIL and TRAIL Receptors as Prognostic Markers in Breast Cancer Patients

Background: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a key player in the extrinsic pathway of apoptosis; it selectively damages cancer cells through binding to its surface receptors, however, cancers can escape this pathway through expression of dysfunctional decoy receptors. Purpose: The present study directed mainly to elucidate the serum TRAIL levels in breast cancer patients and to explore the variation in gene expression of TRAIL death and decoy receptors in breast cancer tissues, and to explore their role as prognostic markers in breast cancer as well as to detect their correlation with Patients’ Clinical Characteristics. Subjects and Methods: TRAIL levels were assayed in the sera of 124 breast cancer patients and 150 healthy females. Moreover, the expression of TRAIL death and decoy receptors was determined in both malignant and adjacent normal breast tissues collected from patients. ER, PR and Her-2 expression in breast cancer tissue were performed using immunohistochemical method. Apoptotic index (AI) was analyzed using H&E stain under light microscopy. Results: Serum levels of TRAIL in breast cancer patients were significantly lower than controls (P < 0.001), additionally, the expression of DR4, DR5 and DcR1 were significantly up-regulated (p < 0.001, p < 0.001, p = 0.039, respectively), however, no significant difference was found in the expression of DcR2 in breast cancer tissues as compared to the corresponding normal tissues. Moreover, the apoptotic index in breast cancer tissues was significantly higher than the corresponding normal tissues. On the other side, decreased Serum TRAIL levels and increased DcR1 expression were associated with decreased overall patients’ survival. Conclusions: The expression of both DR4 and DR5 is required for TRAIL-induced apoptosis in breast cancer tissues; in addition, serum TRAIL and profiling of TRAIL receptors expression may serve as prognostic markers in breast cancer patients.

Hypertonicity-enforced BCL-2 addiction unleashes the cytotoxic potential of death receptors

Attempts to exploit the cytotoxic activity of death receptors (DR) for treating cancer have thus far been disappointing. DR activation in most malignant cells fails to trigger cell death and may even promote tumor growth by activating cell death-independent DR-associated signaling pathways. Overcoming apoptosis resistance is consequently a prerequisite for successful clinical exploitation of DR stimulation. Here we show that hyperosmotic stress in the tumor microenvironment unleashes the deadly potential of DRs by enforcing BCL-2 addiction of cancer cells. Hypertonicity robustly enhanced cytotoxicity of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and other DR ligands in various cancer entities. Initial events in TRAIL DR signaling remained unaffected, but hypertonic conditions unlocked activation of the mitochondrial death pathway and thus amplified the apoptotic signal. Mechanistically, we demonstrate that hyperosmotic stress imposed a BCL-2-addiction on cancer cells to safeguard the integrity of the outer mitochondrial membrane (OMM), essentially exhausting the protective capacity of BCL-2-like pro-survival proteins. Deprivation of these mitochondrial safeguards licensed DR-generated truncated BH3-interacting domain death agonist (tBID) to activate BCL-2-associated X protein (BAX) and initiated mitochondrial outer membrane permeabilization (MOMP). Our work highlights that hyperosmotic stress in the tumor environment primes mitochondria for death and lowers the threshold for DR-induced apoptosis. Beyond TRAIL-based therapies, our findings could help to strengthen the efficacy of other apoptosis-inducing cancer treatment regimens.

ONC201 kills breast cancer cells in vitro by targeting mitochondria.

We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

Juglanin inhibits lung cancer by regulation of apoptosis, ROS and autophagy induction.

Juglanin (Jug) is obtained from the crude extract of Polygonum aviculare, exerting suppressive activity against cancer cell progression in vitro and in vivo. Juglanin administration causes apoptosis and reactive oxygen species (ROS) in different types of cells through regulating various signaling pathways. In our study, the effects of juglanin on non-small cell lung cancer were investigated. A significant role of juglanin in suppressing lung cancer growth was observed. Juglanin promoted apoptosis in lung cancer cells through increasing Caspase-3 and poly ADP-ribose polymerase (PARP) cleavage, which is regulated by TNF-related apoptosis-inducing ligand/Death receptors (TRAIL/DRs) relied on p53 activation. Anti-apoptotic members Bcl-2 and Bcl-xl were reduced, and pro-apoptotic members Bax and Bad were enhanced in cells and animals receiving juglanin. Additionally, nuclear factor-κB (NF-κB), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinases (MAPKs) activation were inhibited by juglanin. Further, juglanin improved ROS and induced autophagy. ROS inhibitor N-acetyl-l-cysteine (NAC) reversed apoptosis induced by juglanin in cancer cells. The formation of autophagic vacoules and LC3/autophagy gene7 (ATG7)/Beclin1 (ATG6) over-expression were observed in juglanin-treated cells. Also, juglanin administration to mouse xenograft models inhibited lung cancer progression. Our study demonstrated that juglanin could be a promising candidate against human lung cancer progression.