Tumor Necrosis Factor-related Apoptosis-inducing Ligand Induction 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.

TRAIL-R3 Expression on Myeloid Leukemic Blasts Is Related to a Shortened Overall Survival.

Since chemotherapy and transplantation can cure only around 35% of patients with acute myeloid leukemia (AML), there is still need for complementary and targeted treatment modalities. One of them could be the use of TNF-related apoptosis-inducing ligand (TRAIL). TRAIL is expressed by effector T cells and induces apoptosis via the death receptor intrinsic pathway. Activation of this pathway in addition to the mitochondrial pathway (by chemotherapy) has synergistic effects in vitro. In human, 4 membrane bound receptors have been identified: two of them (R1 and R2) contain a functional death domain and are capable of starting the apoptotic cascade, and two others (R3 and R4) lack a functional death domain and function as decoy receptors. Most normal cells express R3 and R4, where many tumor cells express R1 and R2. This makes soluble recombinant TRAIL an attractive candidate for targeted therapy; phase 1 clinical studies for solid tumors are launched. Until now, sparse data on TRAIL sensitivity of myeloid leukemic cells have demonstrated low TRAIL sensitivity. We investigated blood and bone-marrow samples of 113 patients with AML for TRAIL receptor expression by flow-cytometry. In contrast with published data, we found presumably (pro-apoptotic) R1 and R2 expression (mean percentage positive cells 16% and 34%, range 0–79% and 0–97% respectively) versus R3 and R4 expression (mean 9% and 10%, range 0–71% an 0–45%) indicating a TRAIL sensitive profile for myeloid blasts. Surprisingly, the expression of the anti-apoptotic R3 strongly correlated to survival. Expression of > 25% blasts positive for R3 resulted in shortened overall survival (p=0.0051), see figure 1. In multivariate analysis R3 expression remained a significant prognostic factor next to cytogenetics (p=0.03 and p= 0.015 respectively). In vitro studies on 4 myeloid leukemic cell lines confirmed TRAIL sensitivity in cell lines that expressed R1 and R2. Furthermore, simultaneous expression of R3 clearly reduced the amount of apoptosis, suggesting that TRAIL effects are inhibited by binding to R3. TRAIL induction studies on fresh leukemic samples are currently underway in our laboratory.In conclusion, our data suggest that, in contrast to earlier reports, there might be a role for TRAIL in apoptosis induction of AML blasts. R3 expression is a strong predictor for overall survival and modulation of R3 might yield additional new therapeutic options for AML patients. [Display omitted]

Silencing of Mcl-1 overcomes resistance of melanoma cells against TRAIL-armed oncolytic adenovirus by enhancement of apoptosis

Arming of oncolytic viruses with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown as a viable approach to increase the antitumor efficacy in melanoma. However, melanoma cells may be partially or completely resistant to TRAIL or develop TRAIL resistance, thus counteracting the antitumor efficiency of TRAIL-armed oncolytic viruses. Recently, we found that TRAIL resistance in melanoma cells can be overcome by inhibition of antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1). Here, we investigated whether the cytotoxicity of AdV-TRAIL, an oncolytic adenovirus, which expresses TRAIL after induction by doxycycline (Dox), can be improved in melanoma cells by silencing of Mcl-1. Two melanoma cell lines, the TRAIL-resistant MeWo and the TRAIL-sensitive Mel-HO were investigated. Treatment of both cell lines with AdV-TRAIL resulted in a decrease of cell viability, which was caused by an increase of apoptosis and necrosis. The proapoptotic effects were dependent on induction of TRAIL by Dox and were more pronounced in Mel-HO than in MeWo cells. SiRNA-mediated silencing of Mcl-1 resulted in a further significant decrease of cell viability and a further increase of apoptosis and necrosis in AdV-TRAIL-infected MeWo and Mel-HO cells. However, while in absolute terms, the effects were more pronounced in Mel-HO cells, in relative terms, they were stronger in MeWo cells. These results show that silencing of Mcl-1 represents a suitable approach to increase the cytotoxicity of a TRAIL-armed oncolytic adenovirus in melanoma cells.Key messages• Cytotoxicity of TRAIL-expressing adenovirus can be enhanced by silencing of Mcl-1.• The effect occurs in TRAIL-sensitive and TRAIL-resistant melanoma cells.• Increase of apoptosis is the main mechanism induced by Mcl-1 silencing.

Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows promising antitumor activity in preclinical studies. However, the efficacy of recombinant TRAIL in clinical trials is compromised by its short serum half-life and low in vivo stability. Induction of endogenous TRAIL may overcome the limitations and become a new strategy for cancer treatment. Here, we discovered that metformin increased TRAIL expression and induced apoptosis in triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) cells. Metformin did not alter the expression of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Metformin-upregulated TRAIL was secreted into conditioned medium (CM) and found to be functional, since the CM promoted TNBC cells undergoing apoptosis, which was abrogated by a recombinant TRAIL-R2-Fc chimera. Moreover, blockade of TRAIL binding to DR4/DR5 or specific knockdown of TRAIL expression significantly attenuated metformin-induced apoptosis. Studies with a tumor xenograft model revealed that metformin not only significantly inhibited tumor growth but also elicited apoptosis and enhanced TRAIL expression in vivo. Collectively, we have demonstrated that upregulation of TRAIL and activation of death receptor signaling are pivotal for metformin-induced apoptosis in TNBC and NSCLC cells. Our studies identify a novel mechanism of action of metformin exhibiting potent antitumor activity via induction of endogenous TRAIL.

Abstract PS19-18: Metformin exhibits cytotoxicity effects on triple-negative cancer cells via TRAIL-mediated apoptosis

Abstract Introduction: Triple-negative breast cancer (TNBC) does not respond to conventional targeted therapy, necessitating novel treatment options. Metformin possesses unique anti-proliferative and pro-apoptotic properties in TNBC cells. However, the molecular mechanism through which metformin may induce apoptosis is incompletely understood. In the current study, we aim to determine whether tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptotic signaling is involved in metformin-induced cytotoxicity on TNBC cells. Methods: TNBC cell lines HCC70, MDA-MB-468, BT549 and MDA-MB-231 were used in the study. A cell death detection ELISA kit was used to quantitatively measure cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes). Western blot analyses were carried out to assess the expression of TRAIL, TRAIL receptors DR5 and DR4, and apoptosis-related proteins (PARP, Caspase-8 and Caspase-3). Flow cytometric analyses were performed to define the expression of cell surface TRAIL receptors DR5 and DR4. Secreted TRAIL protein level in the conditioned medium (CM) of cell culture was measured by using a highly specific ELISA and western blot. MDA-MB-231 cells were treated with concentrated CM from metformin-treated cells to test the activity of secreted TRAIL in inducing cell apoptosis. A soluble recombinant TRAIL decoy receptor, which contains a normal extracellular domain of death receptor 5 (DR5) but a truncated intracellular domain and thus is unable to transduce death signals, was used to block TRAIL function. Infection with lentivirus containing specific shRNAs was performed to knockdown TRAIL expression. Results: Metformin induced apoptosis in a panel of TNBC cell lines in a dose-dependent manner, as evidenced by the increase of cytoplasmic histone-associated DNA fragments and the cleavage of PARP, caspase-8 and caspase-3. Interestingly, during the process of TNBC cells undergoing apoptosis, metformin elicited a dose-dependent upregulation of TRAIL protein levels (both cellular TRAIL and secreted TRAIL) without altering the expression of TRAIL receptors DR5 and DR4. The secreted TRAIL from concentrated CM of metformin-treated cells showed activity in inducing apoptosis of MDA-MB-231 cells. To determine if the induction of TRAIL was essential for metformin-induced cytotoxicity effects on TNBC cells, we examined whether inhibition of TRAIL function by a decoy receptor or specific knockdown of TRAIL expression with shRNAs would affect metformin-induced apoptosis. In all three cell lines (HCC70, MDA-MB-468, BT549) we tested, both the TRAIL decoy receptor and TRAIL-specific shRNAs significantly attenuated metformin-induced DNA fragmentation and cleavage of PARP, caspase-8, and caspase-3. Conclusion: We demonstrate that metformin promotes TNBC cells undergoing apoptosis via induction of TRAIL expression. Our data suggest that TRAIL-mediated apoptosis critically contributes to metformin's antitumor activity against TNBC. Keywords: Metformin, TRAIL, Apoptosis, triple-negative breast cancer Citation Format: Shuang Liu, Erik V Polsdofer, Lukun Zhou, Susan Edgerton, Ann Thor, Bolin Liu. Metformin exhibits cytotoxicity effects on triple-negative cancer cells via TRAIL-mediated apoptosis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-18.

Autophagic Vacuole Secretion Triggered by Chidamide Participates in TRAIL Apoptosis Effect in Breast Cancer Cells.

Breast cancer is one of the most prevalent diseases threatening women's health today. Indepth research on breast cancer (BC) pathogenesis and prevention and treatment methods are gradually receiving attention. Chidamide is a novel histone deacetylase inhibitor (HDACi) that depresses the function of histone deacetylase, consequently affecting the growth of BC cells through epigenetic modification. However, preclinical and clinical studies show that chidamide is ineffective in long-term treatment. We demonstrated in previous experiments that TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in BC cells and is significantly less non-toxic to normal cells than chidamide. Therefore, in this study, we treated BC cells with chidamide and TRAIL to explore a novel option to reduce the clinical toxicity through augmenting the sensitivity for BC cells. Results from the MTT and cell viability assays indicated that the combination of chidamide and TRAIL in MCF-7 and MDA-MB-231 cells induced BC cell death, while maintaining a reduced concentration of chidamide. Autophagy assay and annexin V analysis showed that the autophagosome microtubuleassociated protein1light chain3-II (LC3-II) was abnormally increased and much more early and late phase of apoptotic cells appeared during chidamide and TRAIL induction. Anti-tumor assays in a BC tumor xenograft model displayed that the mixture of chidamide and TRAIL exhibited stronger effects on inhibiting tumor growth. The data from real-time PCR and western blotting showed that the cytotoxic effect correlated with the expressions of related apoptosis and autophagy factors. Our data are the first to demonstrate the synergistic effects of chidamide and TRAIL in BC cells, specifically, the pharmacological effects on cell death induction. These results lay a solid experimental and theoretical basis to solve the clinical resistance of chidamide.

Tannic Acid Promotes TRAIL-Induced Extrinsic Apoptosis by Regulating Mitochondrial ROS in Human Embryonic Carcinoma Cells.

Human embryonic carcinoma (EC; NCCIT) cells have self-renewal ability and pluripotency. Cancer stem cell markers are highly expressed in NCCIT cells, imparting them with the pluripotent nature to differentiate into other cancer types, including breast cancer. As one of the main cancer stem cell pathways, Wnt/β-catenin is also overexpressed in NCCIT cells. Thus, inhibition of these pathways defines the ability of a drug to target cancer stem cells. Tannic acid (TA) is a natural polyphenol present in foods, fruits, and vegetables that has anti-cancer activity. Through Western blotting and PCR, we demonstrate that TA inhibits cancer stem cell markers and the Wnt/β-catenin signaling pathway in NCCIT cells and through a fluorescence-activated cell sorting analysis we demonstrated that TA induces sub-G1 cell cycle arrest and apoptosis. The mechanism underlying this is the induction of mitochondrial reactive oxygen species (ROS) (mROS), which then induce the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated extrinsic apoptosis pathway instead of intrinsic mitochondrial apoptosis pathway. Moreover, ribonucleic acid sequencing data with TA in NCCIT cells show an elevation in TRAIL-induced extrinsic apoptosis, which we confirm by Western blotting and real-time PCR. The induction of human TRAIL also proves that TA can induce extrinsic apoptosis in NCCIT cells by regulating mROS.

Axl Inhibitor R428 Enhances TRAIL-Mediated Apoptosis Through Downregulation of c-FLIP and Survivin Expression in Renal Carcinoma.

R428, a selective small molecule Axl inhibitor, is known to have anti-cancer effects, such as inhibition of invasion and proliferation and induction of cell death in cancer cells. The Axl receptor tyrosine kinase is highly expressed in cancer cells and the level of Axl expression is associated with survival, metastasis, and drug resistance of many cancer cells. However, the effect of Axl inhibition on overcoming anti-cancer drugs resistance is unclear. Therefore, we investigated the capability of Axl inhibition as a therapeutic agent for the induction of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) sensitivity. In this study, R428 markedly sensitized cancer cells to TRAIL-induced apoptotic cell death, but not in normal human skin fibroblast (HSF) and human umbilical vein cells (EA.hy926). Moreover, knockdown of Axl by siRNA also increased TRAIL-induced apoptosis. R428 decreased c-FLIP proteins levels via induction of miR-708 expression and survivin protein levels at the post-translational level, and we found that knockdown of Axl also decreased both c-FLIP and survivin protein expression. Overexpression of c-FLIP and survivin markedly inhibited R428 plus TRAIL-induced apoptosis. Furthermore, R428 sensitized cancer cells to multiple anti-cancer drugs-mediated cell death. Our results provide that inhibition of Axl could improve sensitivity to TRAIL through downregulation of c-FLIP and survivin expression in renal carcinoma cells. Taken together, Axl may be a tempting target to overcome TRAIL resistance.

Abstract 258: Recombinant human TRAIL or a DR5 agonistic antibody convert the response of non-triple negative breast cancer cells to ONC201 from anti-proliferative to apoptotic

Abstract ONC201 was initially identified as an inducer of cell death through the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. The compound is being tested in patients with a variety of tumor types, including those of the breast. Both triple negative breast cancer (TNBC) cells and non-TNBC cells are sensitive to ONC201. In a subset of TRAIL-sensitive TNBC cells, ONC201 treatment leads to the induction of apoptosis in a TRAIL-dependent manner. In the majority of TRAIL-resistant non-TNBC cells, ONC201’s effects are anti-proliferative rather than apoptotic. In vivo, the apoptotic response to ONC201 leads to efficacy of the compound while the anti-proliferative response does not (Ralff et al., Mol Cancer Ther., 2017). Here we worked to identify strategies to convert the response of cancer cells to ONC201 from anti-proliferative to apoptotic. ONC201 treatment primed TRAIL-resistant non-TNBC cells to undergo TRAIL-dependent cell death. Increases in the mRNA and surface protein levels of death receptor 5 (DR5) as well as decreases in the expression of multiple anti-apoptotic proteins occured in ONC201-treated cells. We also observed small but significant increases in TRAIL mRNA and surface protein in non-TNBC cells following treatment with ONC201. Despite apoptotic priming and the induction of TRAIL, the effect of the compound in the non-TNBC cells remains anti-proliferative but not apoptotic. We hypothesized that the level of TRAIL induced by ONC201 in these cells, known to be TRAIL-resistant, was insufficient. Remarkably, the addition of exogenous recombinant human TRAIL (rhTRAIL) converted the response of TRAIL-resistant non-TNBC cells to ONC201 from anti-proliferative to apoptotic. Cleaved PARP, caspase-3, caspase-9, and caspase-8 were observed in cells treated with ONC201 followed by rhTRAIL, but not in cells treated with ONC201 or rhTRAIL alone. Propidium iodide staining and quantification of the subG1 population via flow cytometry further confirmed these findings. Similar observations about the ability of rhTRAIL to convert the cellular response to ONC201 from anti-proliferative to apoptotic were made in cancer cells from other tumor types. Importantly, rhTRAIL did not convert the response of normal fibroblasts to ONC201 from anti-proliferative to apoptotic. Addition of a DR5 agonistic antibody to ONC201 treated non-TNBC cells also converted the response of the non-TNBC cells to ONC201 from anti-proliferative to apoptotic. Ongoing experiments are currently testing the combination of ONC201 and rhTRAIL in vivo. These findings describe a strategy for potently converting the response of a cancer cell to ONC201 from anti-proliferative to apoptotic. Clinical application of this combination therapy may result in tumor regressions in patients with decreased sensitivity to ONC201 as a single agent. Citation Format: Marie D. Ralff, Jocelyn E. Ray, Avital Lev, Lanlan Zhou, David T. Dicker, Wafik S. El-Deiry. Recombinant human TRAIL or a DR5 agonistic antibody convert the response of non-triple negative breast cancer cells to ONC201 from anti-proliferative to apoptotic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 258.

Non-replicating Newcastle Disease Virus as an adjuvant for DNA vaccine enhances antitumor efficacy through the induction of TRAIL and granzyme B expression

The potential of non-replicating Newcastle Disease Virus (NDV) as an adjuvant for DNA vaccination remains to be elucidated. To assess the therapeutic effects of DNA vaccine (HPV-16 E7 gene) adjuvanted with NDV, female C57/BL6 mice were inoculated with murine TC-1 cells of human papillomavirus (HPV)-related carcinoma, expressing human papillomavirus 16 (HPV-16) E6/E7 antigens, and immunized with DNA vaccine alone or pretreated with NDV. One week after third immunization, Cytotoxic T lymphocytes (CTLs), splenocyte proliferation, cytokine balance (IFN-γ, IL-4 and IL-12 secretions) and intratumoral expression of cytotoxicity related proteins in tumor lysates were investigated.The results showed that treatment with non-replicating NDV prior to DNA vaccine induced tumor-specific cytolytic and splenocyte proliferation responses. The levels of cytokines IL-12, IL-4 and IFN–γ after treating with combined E7-DNA -non-replicating NDV (NDV-DNA Vaccine) were significantly higher than those of control groups. The intratumoral granzyme B and Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL)-mediated apoptosis was also significantly increased. Tumor therapeutic experiments showed that the NDV pretreatment could reduce the tumor progression of established E7-expressing TC-tumors. Taken together these data suggest that the significant antitumor responses evidenced during treatment with non-replicating NDV prior to DNA vaccine are due, in part, to strong E7-induced cellular immunity and enhanced expression of cytotoxicity related proteins in the tumor microenvironment. These observations indicated the potential of non-replicating NDV as an adjuvant for enhancing therapeutic DNA vaccines -induced immunity and antitumor responses.

Pro-apoptotic signaling induced by Retinoic acid and dsRNA is under the control of Interferon Regulatory Factor-3 in breast cancer cells.

Breast cancer is one of the most lethal malignancies for women. Retinoic acid (RA) and double-stranded RNA (dsRNA) are considered signaling molecules with potential anticancer activity. RA, co-administered with the dsRNA mimic polyinosinic-polycytidylic acid (poly(I:C)), synergizes to induce a TRAIL (Tumor-Necrosis-Factor Related Apoptosis-Inducing Ligand)- dependent apoptotic program in breast cancer cells. Here, we report that RA/poly(I:C) co-treatment, synergically, induce the activation of Interferon Regulatory Factor-3 (IRF3) in breast cancer cells. IRF3 activation is mediated by a member of the pathogen recognition receptors, Toll-like receptor-3 (TLR3), since its depletion abrogates IRF3 activation by RA/poly(I:C) co-treatment. Besides induction of TRAIL, apoptosis induced by RA/poly(I:C) correlates with the increased expression of pro-apoptotic TRAIL receptors, TRAIL-R1/2, and the inhibition of the antagonistic receptors TRAIL-R3/4. IRF3 plays an important role in RA/poly(I:C)-induced apoptosis since IRF3 depletion suppresses caspase-8 and caspase-3 activation, TRAIL expression upregulation and apoptosis. Interestingly, RA/poly(I:C) combination synergizes to induce a bioactive autocrine/paracrine loop of type-I Interferons (IFNs) which is ultimately responsible for TRAIL and TRAIL-R1/2 expression upregulation, while inhibition of TRAIL-R3/4 expression is type-I IFN-independent. Our results highlight the importance of IRF3 and type-I IFNs signaling for the pro-apoptotic effects induced by RA and synthetic dsRNA in breast cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates caspases in human prostate cancer cells through sigma 1 receptor

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-based therapy is currently evaluated in clinical studies as a tumor cell-selective pro-apoptotic approach. Unfortunately, many clinical studies have shown that cancer cells acquire TRAIL resistance and finally avoid TRAIL-induced apoptosis. Therefore, defining the mechanisms that permit TRAIL to activate apoptosis is critical for the development of strategies that maximize the potential effectiveness of TRAIL in clinical applications. This study aims at understanding the molecular mechanisms underlying TRAIL-induced apoptosis and unraveling signaling pathways that could revert sensitivity to apoptosis stimuli. Our current study demonstrates for the first time that Sigma 1 Receptor (Sig1R), a ligand-regulated protein chaperone, contributes to TRAIL induction of apoptosis. We show that Sig1R agonist (+)-SKF10047 action or increasing Sig1R expression, significantly reduced apoptosis by TRAIL in prostate cell lines, indicating the importance of Sig1R and signifying that higher levels of Sig1R in prostate cancer cells make them more resistant to TRAIL treatment. Here we show that Sig1R is critically involved in TRAIL-induced caspase activation. Furthermore, we show that Sig1R protein is degraded upon TRAIL treatment. Knockdown of Sig1R, by siRNA transfection increased the sensitivity of breast cancer cells to TRAIL. These results indicate that Sig1R could represent a promising molecule to sensitize human breast cancers to TRAIL. Collectively, these studies define Sig1R as a key mediator of TRAIL induction of cancer-specific killing.

Expression of TRAIL and its receptor DR5 and their significance in acute leukemia cells.

We investigated the roles of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor death receptor 5 (DR5) in the onset of acute leukemia and changes in their expression during chemotherapy. Bone marrow samples from 16 patients newly diagnosed with acute leukemia were collected before chemotherapy. Bone marrow samples from patients with non-hematologic malignancies served as the control group. Peripheral blood samples of patients with acute leukemia were also collected before chemotherapy and at 1 and 3 days after chemotherapy. Mononuclear cells in the bone marrow and peripheral blood were isolated and used to detect the expression of TRAIL and DR5 by flow cytometry. Compared with mononuclear cells from the control group, mononuclear cells from newly diagnosed patients with acute leukemia showed no significant difference in the expression of TRAIL (P > 0.05) but showed significantly increased expression of DR5 (P < 0.05). TRAIL and DR5 expression in peripheral blood mononuclear cells after chemotherapy was significantly increased compared to expression before chemotherapy (P < 0.05). Patients showing high expression of DR5 had a higher remission rate. One of the mechanisms underlying the treatment of leukemia with chemotherapy drugs may be the induction of TRAIL and DR5, which may promote apoptosis in leukemia cells. TRAIL-mediated apoptosis is regulated by DR5 expression.

Geldanamycin induces apoptosis in human gastric carcinomas by affecting multiple oncogenic kinases that have synergic effects with TNF-related apoptosis-inducing ligand.

The aim of the present study was to evaluate the effect of geldanamycin (GA) on the treatment of human gastric carcinomas and to investigate the molecular mechanism that provides the basis for the combination of GA with the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induction strategy. The expression of target proteins at the mRNA level was determined using reverse transcription-polymerase chain reaction (RT-PCR), and apoptosis was evaluated with the terminal deoxynucleotidyl transferase mediated digoxigenin-dUTP nick-end labeling and Annexin V/propidium iodide (PI) staining methods. Phosphorylation of targeted kinases was studied using immunocytochemistry methods, and malignant phenotypes were studied using in vitro assays. GA treatment inhibits proliferation, migration and invasion, and induces apoptosis in human gastric cancer SGC-7901 cells, most likely by decreasing the expression of B-RAF and by phosphorylation of protein kinase B (AKT) and ERK. The inhibitory role of AKT in TRAIL regulation holds considerable potential for achieving a synergic effect in clinical therapy, using a combination of GA treatment and TRAIL induction. The present study provides a basis for the future application of heat shock protein 90 (Hsp90) inhibitors, such as GA, in the clinical treatment of gastric cancer, particularly in combination therapies with TRAIL inducers.

An Lnc RNA (GAS5)/SnoRNA-derived piRNA induces activation of TRAIL gene by site-specifically recruiting MLL/COMPASS-like complexes.

PIWI-interacting RNA (piRNA) silences the transposons in germlines or induces epigenetic modifications in the invertebrates. However, its function in the mammalian somatic cells remains unknown. Here we demonstrate that a piRNA derived from Growth Arrest Specific 5, a tumor-suppressive long non-coding RNA, potently upregulates the transcription of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a proapoptotic protein, by inducing H3K4 methylation/H3K27 demethylation. Interestingly, the PIWIL1/4 proteins, which bind with this piRNA, directly interact with WDR5, resulting in a site-specific recruitment of the hCOMPASS-like complexes containing at least MLL3 and UTX (KDM6A). We have indicated a novel pathway for piRNAs to specially activate gene expression. Given that MLL3 or UTX are frequently mutated in various tumors, the piRNA/MLL3/UTX complex mediates the induction of TRAIL, and consequently leads to the inhibition of tumor growth.

Expression of TRAIL and its receptor DR5 and their significance in acute leukemia cells

We investigated the roles of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor death receptor 5 (DR5) in the onset of acute leukemia and changes in their expression during chemotherapy. Bone marrow samples from 16 patients newly diagnosed with acute leukemia were collected before chemotherapy. Bone marrow samples from patients with non-hematologic malignancies served as the control group. Peripheral blood samples of patients with acute leukemia were also collected before chemotherapy and at 1 and 3 days after chemotherapy. Mononuclear cells in the bone marrow and peripheral blood were isolated and used to detect the expression of TRAIL and DR5 by flow cytometry. Compared with mononuclear cells from the control group, mononuclear cells from newly diagnosed patients with acute leukemia showed no significant difference in the expression of TRAIL (P > 0.05) but showed significantly increased expression of DR5 (P < 0.05). TRAIL and DR5 expression in peripheral blood mononuclear cells after chemotherapy was significantly increased compared to expression before chemotherapy (P < 0.05). Patients showing high expression of DR5 had a higher remission rate. One of the mechanisms underlying the treatment of leukemia with chemotherapy drugs may be the induction of TRAIL and DR5, which may promote apoptosis in leukemia cells. TRAIL-mediated apoptosis is regulated by DR5 expression.

FOXL1, a Novel Candidate Tumor Suppressor, Inhibits Tumor Aggressiveness and Predicts Outcome in Human Pancreatic Cancer

The forkhead box L1 (FOXL1) transcription factor regulates epithelial proliferation and development of gastrointestinal tract and has been implicated in gastrointestinal tumorigenesis in mouse models. However, the role of FOXL1 in pancreatic cancer development and progression remains to be elucidated. Here, we report that higher expression of FOXL1 is significantly associated with better clinical outcome in human pancreatic ductal adenocarcinoma (PDAC). A lower FOXL1 expression is correlated with metastasis and advanced pathologic stage of pancreatic cancer. Mechanistic analyses showed that overexpression of FOXL1 induces apoptosis and inhibits proliferation and invasion in pancreatic cancer cells, whereas silencing of FOXL1 by siRNA inhibits apoptosis and enhances tumor cell growth and invasion. Furthermore, FOXL1 overexpression significantly suppressed the growth of tumor xenografts in nude mice. FOXL1 promoted apoptosis partly through the induction of TNF-related apoptosis-inducing ligand (TRAIL) in pancreatic cancer cells. In addition, FOXL1 suppressed the transcription of zinc finger E-box-binding homeobox 1 (ZEB1), an activator of epithelial-mesenchymal transition, and the negative regulation of ZEB1 contributed to the inhibitory effect of FOXL1 on tumor cell invasion. Taken together, our findings suggest that FOXL1 expression is a candidate predictor of clinical outcome in patients with resected PDAC and it plays an inhibitory role in pancreatic tumor progression.

Wild-type Measles Virus Infection Upregulates Poliovirus Receptor-Related 4 and Causes Apoptosis in Brain Endothelial Cells by Induction of Tumor Necrosis Factor-Related Apoptosis-lnducing Ligand

Small numbers of brain endothelial cells (BECs) are infected in children with neurologic complications of measles virus (MV) infection. This may provide a mechanism for virus entry into the central nervous system, but the mechanisms are unclear. Both in vitro culture systems and animal models are required to elucidate events in the endothelium. We compared the ability of wild-type (WT), vaccine, and rodent-adapted MV strains to infect, replicate, and induce apoptosis in human and murine brain endothelial cells (HBECs and MBECs, respectively). Mice also were infected intracerebrally. All MV stains productively infected HBECs and induced the MV receptor PVRL4. Efficient WT MV production also occurred in MBECs. Extensive monolayer destruction associated with activated caspase 3 staining was observed in HBECs and MBECs, most markedly with WT MV. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not Fas ligand, was induced by MV infection. Treatment of MBECs with supernatants from MV-infected MBEC cultures with an anti-TRAIL antibody blocked caspase 3 expression and monolayer destruction. TRAIL was also expressed in the endothelium and other cell types in infected murine brains. This is the first demonstration that infection of low numbers of BECs with WT MV allows efficient virus production, induction of TRAIL, and subsequent widespread apoptosis.

A SILAC-based Approach Identifies Substrates of Caspase-dependent Cleavage upon TRAIL-induced Apoptosis

The extracellular ligand-induced extrinsic pathway of apoptosis is executed via caspase protease cascades that activate downstream effectors by means of site-directed proteolysis. Here we identify proteome changes upon the induction of apoptosis by the cytokine tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in a Jurkat T cell line. We detected caspase-dependent cleavage substrates by quantifying protein intensities before and after TRAIL induction in SDS gel slices. Apoptotic protein cleavage events are identified by a characteristic stable isotope labeling with amino acids in cell culture (SILAC) ratio pattern across gel slices that results from differential migration of the cleaved and uncleaved proteins. We applied a statistical test to define apoptotic substrates in the proteome. Our approach identified more than 650 of these cleaved proteins in response to TRAIL-induced apoptosis, including many previously unknown substrates and cleavage sites. Inhibitor treatment combined with triple SILAC demonstrated that the detected cleavage events were caspase dependent. Proteins located in the lumina of organelles such as mitochondria and endoplasmic reticulum were significantly underrepresented in the substrate population. Interestingly, caspase cleavage is generally observed in not only one but several members of stable complexes, but often with lower stoichiometry. For instance, all five proteins of the condensin I complex were cleaved upon TRAIL treatment. The apoptotic substrate proteome data can be accessed and visualized in the MaxQB database and might prove useful for basic and clinical research into TRAIL-induced apoptosis. The technology described here is extensible to a wide range of other proteolytic cleavage events.

Homoharringtonine and SAHA synergistically enhance apoptosis in human acute myeloid leukemia cells through upregulation of TRAIL and death receptors

Single‑agent histone deacetylase (HDAC) inhibitors have exhibited marked antileukemic activity in preclinical and clinical studies and have undergone trials in combination with standard chemotherapeutics. However, the mechanisms of action of combination therapies are not completely understood. In the present study, a novel strategy for treatment of acute myeloid leukemia (AML) was identified, in which the chemotherapeutic agent, homoharringtonine (HHT), was combined with suberoylanilide hydroxamic acid (SAHA), a pan‑HDAC inhibitor. A synergistic effect was observed when HHT was added to SAHA to induce apoptosis in Kasumi‑1 and THP‑1 leukemia cells. This combination was found to significantly enhance the activation of caspase‑8 and ‑9 compared with treatment with each drug separately. Notably, while SAHA induced upregulation of death receptor 4 (DR4) and DR5, HHT upregulated tumor necrosis factor‑related apoptosis-inducing ligand (TRAIL) expression in a dose‑dependent manner. In addition, the synergistic effect between HHT and SAHA was blocked partially using a specific anti‑TRAIL antibody. The combination therapy was also found to significantly inhibit the growth of leukemia xenografts in vivo with enhanced apoptosis. These results indicate that, by regulating the induction of TRAIL and activation of the TRAIL apoptotic pathway, it is possible to administer HHT at low concentrations in combination with SAHA as an effective therapeutic approach for the treatment of AML.