Lonicera japonica Thunb. Induces caspase-dependent apoptosis through death receptors and suppression of AKT in U937 human leukemic cells.

Decoctions obtained from the dried flowers of Lonicera japonica Thunb. (Indongcho) have been utilized in folk remedies against inflammatory diseases. Recently, many agents that have used for inflammatory diseases are showing anticancer effects. Here, we have isolated polyphenols extracted from lyophilized Lonicera japonica Thunb (PELJ) and investigated the anticancer effects of PELJ on U937 cells. Here, we demonstrated that PELJ induced apoptosis by upregulation of DR4 and Fas, and further it is augmented by suppression of XIAP. In addition, The PELJ-induced apoptosis is at least in part by blocking PI3K/Akt pathway. These findings suggest that PELJ may provide evidence of anticancer activities on U937 cells. Further study for detailed mechanism and the effects on animal models is warranted to determine whether PELJ provide more conclusive evidence that PELJ which may provide a beneficial effect for treating cancer.

Somatostatin receptors: from signaling to clinical practice.

Somatostatin is a peptide with a potent and broad antisecretory action, which makes it an invaluable drug target for the pharmacological management of pituitary adenomas and neuroendocrine tumors. Somatostatin receptors (SSTR1, 2A and B, 3, 4 and 5) belong to the G protein coupled receptor family and have a wide expression pattern in both normal tissues and solid tumors. Investigating the function of each SSTR in several tumor types has provided a wealth of information about the common but also distinct signaling cascades that suppress tumor cell proliferation, survival and angiogenesis. This provided the rationale for developing multireceptor-targeted somatostatin analogs and combination therapies with signaling-targeted agents such as inhibitors of the mammalian (or mechanistic) target of rapamycin (mTOR). The ability of SSTR to internalize and the development of rabiolabeled somatostatin analogs have improved the diagnosis and treatment of neuroendocrine tumors.

Relevance of signaling molecules for apoptosis induction on influenza A virus replication.

Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. The severity of influenza A virus (IAV) infection is also closely related to dysfunction of apoptosis regulation. In the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by IAV proteins to enable effective virus replication. The modulation of the intracellular signaling pathway inducing apoptosis by the IAV infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of IAV infections. This review focuses on apoptosis related molecules involved in IAV replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed.

Antihepatoma peptide, scolopentide, derived from the centipede scolopendra subspinipes mutilans.

BACKGROUND: Centipedes have been used to treat tumors for hundreds of years in China. However, current studies focus on antimicrobial and anticoagulation agents rather than tumors. The molecular identities of antihepatoma bioactive components in centipedes have not yet been extensively investigated. It is a challenge to isolate and characterize the effective components of centipedes due to limited peptide purification technologies for animal-derived medicines. AIM: To purify, characterize, and synthesize the bioactive components with the strongest antihepatoma activity from centipedes and determine the antihepatoma mechanism. METHODS: An antihepatoma peptide (scolopentide) was isolated and identified from the centipede scolopendra subspinipes mutilans using a combination of enzymatic hydrolysis, a Sephadex G-25 column, and two steps of high-performance liquid chromatography (HPLC). Additionally, the CCK8 assay was used to select the extracted fraction with the strongest antihepatoma activity. The molecular weight of the extracted scolopentide was characterized by quadrupole time of flight mass spectrometry (QTOF MS), and the sequence was matched by using the Mascot search engine. Based on the sequence and molecular weight, scolopentide was synthesized using solid-phase peptide synthesis methods. The synthetic scolopentide was confirmed by MS and HPLC. The antineoplastic effect of extracted scolopentide was confirmed by CCK8 assay and morphological changes again in vitro. The antihepatoma effect of synthetic scolopentide was assessed by the CCK8 assay and Hoechst staining in vitro and tumor volume and tumor weight in vivo. In the tumor xenograft experiments, qualified model mice (male 5-week-old BALB/c nude mice) were randomly divided into 2 groups (n = 6): The scolopentide group (0.15 mL/d, via intraperitoneal injection of synthetic scolopentide, 500 mg/kg/d) and the vehicle group (0.15 mL/d, via intraperitoneal injection of normal saline). The mice were euthanized by cervical dislocation after 14 d of continuous treatment. Mechanistically, flow cytometry was conducted to evaluate the apoptosis rate of HepG2 cells after treatment with extracted scolopentide in vitro. A Hoechst staining assay was also used to observe apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. CCK8 assays and morphological changes were used to compare the cytotoxicity of synthetic scolopentide to liver cancer cells and normal liver cells in vitro. Molecular docking was performed to clarify whether scolopentide tightly bound to death receptor 4 (DR4) and DR5. qRT-PCR was used to measure the mRNA expression of DR4, DR5, fas-associated death domain protein (FADD), Caspase-8, Caspase-3, cytochrome c (Cyto-C), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), x-chromosome linked inhibitor-of-apoptosis protein and Cellular fas-associated death domain-like interleukin-1ß converting enzyme inhibitory protein in hepatocarcinoma subcutaneous xenograft tumors from mice. Western blot assays were used to measure the protein expression of DR4, DR5, FADD, Caspase-8, Caspase-3, and Cyto-C in the tumor tissues. The reactive oxygen species (ROS) of tumor tissues were tested. RESULTS: In the process of purification, characterization and synthesis of scolopentide, the optimal enzymatic hydrolysis conditions (extract ratio: 5.86%, IC50: 0.310 mg/mL) were as follows: Trypsin at 0.1 g (300 U/g, centipede-trypsin ratio of 20:1), enzymolysis temperature of 46 °C, and enzymolysis time of 4 h, which was superior to freeze-thawing with liquid nitrogen (IC50: 3.07 mg/mL). A peptide with the strongest antihepatoma activity (scolopentide) was further purified through a Sephadex G-25 column (obtained A2) and two steps of HPLC (obtained B5 and C3). The molecular weight of the extracted scolopentide was 1018.997 Da, and the peptide sequence was RAQNHYCK, as characterized by QTOF MS and Mascot. Scolopentide was synthesized in vitro with a qualified molecular weight (1018.8 Da) and purity (98.014%), which was characterized by MS and HPLC. Extracted scolopentide still had an antineoplastic effect in vitro, which inhibited the proliferation of Eca-109 (IC50: 76.27 µg/mL), HepG2 (IC50: 22.06 µg/mL), and A549 (IC50: 35.13 µg/mL) cells, especially HepG2 cells. Synthetic scolopentide inhibited the proliferation of HepG2 cells (treated 6, 12, and 24 h) in a concentration-dependent manner in vitro, and the inhibitory effects were the strongest at 12 h (IC50: 208.11 µg/mL). Synthetic scolopentide also inhibited the tumor volume (Vehicle vs Scolopentide, P = 0.0003) and weight (Vehicle vs Scolopentide, P = 0.0022) in the tumor xenograft experiment. Mechanistically, flow cytometry suggested that the apoptosis ratios of HepG2 cells after treatment with extracted scolopentide were 5.01% (0 µg/mL), 12.13% (10 µg/mL), 16.52% (20 µg/mL), and 23.20% (40 µg/mL). Hoechst staining revealed apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. The CCK8 assay and morphological changes indicated that synthetic scolopentide was cytotoxic and was significantly stronger in HepG2 cells than in L02 cells. Molecular docking suggested that scolopentide tightly bound to DR4 and DR5, and the binding free energies were-10.4 kcal/mol and-7.1 kcal/mol, respectively. In subcutaneous xenograft tumors from mice, quantitative real-time polymerase chain reaction and western blotting suggested that scolopentide activated DR4 and DR5 and induced apoptosis in SMMC-7721 Liver cancer cells by promoting the expression of FADD, caspase-8 and caspase-3 through a mitochondria-independent pathway. CONCLUSION: Scolopentide, an antihepatoma peptide purified from centipedes, may inspire new antihepatoma agents. Scolopentide activates DR4 and DR5 and induces apoptosis in liver cancer cells through a mitochondria-independent pathway.

ALK inhibitors downregulate the expression of death receptor 4 in ALK-mutant lung cancer cells via facilitating Fra-1 and c-Jun degradation and subsequent AP-1 suppression.

The successful treatment of patients with advanced non-small cell lung cancer (NSCLC) harboring chromosomal rearrangements of anaplastic lymphoma kinase (ALK) with ALK tyrosine kinase inhibitors (ALK-TKIs) represents a promising targeted therapy. As a result, various ALK-TKIs have been rapidly developed, some of which are approved while some are being tested in clinical trials. Death receptor 4 (DR4; also called TNFRSF10A or TRAIL-R1) is a cell surface protein, which functions as a pro-apoptotic protein that transduces TRAIL death signaling to trigger apoptosis. DR4 expression is positively regulated by MEK/ERK signaling and thus can be downregulated by MEK/ERK inhibition. This study thus focused on determining the effects of AKL-TKIs on DR4 expression and the underlying mechanisms. Three tested ALK-TKIs including APG-2449, brigatinib and alectinib effectively and preferentially inhibited Akt/mTOR as well as MEK/ERK signaling and decreased cell survival in ALK-mutant (ALKm) NSCLC cells with induction of apoptosis. This was also true for DR4 downregulation, which occurred even at 2 h post treatment. These ALK-TKIs did not affect DR4 protein stability, rather decreased DR4 mRNA expression. In parallel, they promoted degradation and reduced the levels of Fra-1 and c-Jun, two critical components of AP-1, and suppressed AP-1 (Fra-1/c-Jun)-dependent transcription/expression of DR4. Hence, it appears that ALK-TKIs downregulate DR4 expression in ALKm NSCLC cells via facilitating Fra-1 and c-Jun degradation and subsequent AP-1 suppression. Our findings thus warrant further investigation of the biological significance of DR4 downregulation in ALK-targeted cancer therapy.

Decitabine enhances the tumoricidal potential of TRAIL via the epigenetic regulation of death receptor 4 in gastric cancer.

Background: Deoxyribonucleic acid (DNA) methyltransferase inhibitors, such as decitabine, have made great advances in cancer therapy as combinational drugs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has an obvious anti-tumor effect; however, some gastric cancer (GC) cells are resistant to TRAIL-induced cell death. This study sought to explore the synergistic anti-tumor effect of TRAIL and decitabine, and the potential synergetic mechanism. Methods: The cell growth inhibition effect was monitored by the IncuCyte ZOOM Live-Cell Analysis System, and cell viability was determined by Cell Counting Kit-8 assays. Apoptosis was detected by Annexin V/Propidium Iodide double staining. Death receptor 4 (DR4) was knocked down by ribonucleic acid (RNA) interference, and the effect of DR4 deletion on TRAIL sensitivity was analyzed. Methylation-specific polymerase chain reaction (PCR) was applied to determine the methylation status of DR4. The messenger RNA (mRNA) and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The expression of the DRs on the cell membrane surfaces was analyzed by flow cytometry. Results: The combined use of decitabine and TRAIL synergistically inhibited cell growth in 2 TRAIL-resistant cell lines. Further, decitabine augmented TRAIL-induced apoptosis in a caspase-dependent manner. The co-application of decitabine and TRAIL facilitated the activation of caspase-7, -8, -9, and poly ADP-ribose polymerase (PARP). Notably, decitabine increased the expression of DR4 at the transcriptional and post-transcriptional levels. DR4 expression on the cell membrane surfaces was also upregulated after decitabine exposure. The depletion of DR4 by specific inhibitors attenuated TRAIL-induced apoptosis and weakened the synergistic effects of decitabine and TRAIL. In addition, DR4 gene presented methylation status in SNU-1 cells. The low mRNA and protein expression of DR4 were also detected in SNU-1 cells. Conclusions: Decitabine enhances the effect of TRAIL by inhibiting the growth and inducing the apoptosis of GC cells. This is achieved by the epigenetic modification of decitabine, which upregulates DR4. Decitabine may act as a sensitizing agent of TRAIL. The combined use of decitabine and TRAIL may provide a novel idea for GC treatment.

Downregulation of death receptor 4 is tightly associated with positive response of EGFR mutant lung cancer to EGFR-targeted therapy and improved prognosis.

Death receptor 4 (DR4), a cell surface receptor, mediates apoptosis or induces inflammatory cytokine secretion upon binding to its ligand depending on cell contexts. Its prognostic impact in lung cancer and connection between EGFR-targeted therapy and DR4 modulation has not been reported and thus was the focus of this study. Methods: Intracellular protein alterations were measured by Western blotting. Cell surface protein was detected with antibody staining and flow cytometry. mRNA expression was monitored with qRT-PCR. Gene transactivation was analyzed with promoter reporter assay. Drug dynamic effects in vivo were evaluated using xenografts. Gene modulations were achieved with gene overexpression and knockdown. Proteins in human archived tissues were stained with immunohistochemistry. Results: EGFR inhibitors (e.g., osimertinib) decreased DR4 levels only in EGFR mutant NSCLC cells and tumors, being tightly associated with induction of apoptosis. This modulation was lost once cells became resistant to these inhibitors. Increased levels of DR4 were detected in cell lines with acquired osimertinib resistance and in NSCLC tissues relapsed from EGFR-targeted therapy. DR4 knockdown induced apoptosis and augmented apoptosis when combined with osimertinib in both sensitive and resistant cell lines, whereas enforced DR4 expression significantly attenuated osimertinib-induced apoptosis. Mechanistically, osimertinib induced MARCH8-mediated DR4 proteasomal degradation and suppressed MEK/ERK/AP-1-dependent DR4 transcription, resulting in DR4 downregulation. Moreover, we found that DR4 positive expression in human lung adenocarcinoma was significantly associated with poor patient survival. Conclusions: Collectively, we suggest that DR4 downregulation is coupled to therapeutic efficacy of EGFR-targeted therapy and predicts improved prognosis, revealing a previously undiscovered connection between EGFR-targeted therapy and DR4 modulation.

Sensitizing TRAIL­resistant A549 lung cancer cells and enhancing TRAIL­induced apoptosis with the antidepressant amitriptyline.

Tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) is a cytokine with the potential to induce cancer cell­specific apoptosis with minimal toxicity to normal cells. Therefore, the resistance of certain cancer cells to TRAIL is a major concern and agents that can either enhance TRAIL capabilities or overcome TRAIL resistance are necessary for the development of cancer treatments. The present study investigated whether the antidepressant drug amitriptyline could sensitize TRAIL­resistant A549 lung cancer cells and enhance TRAIL­induced apoptosis. Antidepressants are usually prescribed to cancer patients to relieve emotional distress, such as depression or dysthymia. The present study revealed for the first time, to the best of our knowledge, that amitriptyline increased death receptor (DR) 4 and 5 expression, a requirement for TRAIL­induced cell death. Genetic inhibitors of DR4 and DR5 significantly reduced amitriptyline­enhanced TRAIL­mediated apoptosis. Additionally, the present study explored whether blocking autophagy increased DR4 and DR5 expression. Blocking autophagy flux with the final stage autophagy inhibitor chloroquine (CQ) also upregulated DR4 and DR5 expression. TRAIL in combination with amitriptyline or CQ significantly increased the expression of apoptosis­indicator proteins cleaved caspase­8 and caspase­3. The expression levels of LC3­II and p62 were significantly higher in amitriptyline­treated cells, which confirmed that amitriptyline blocks autophagy by inhibiting the fusion of autophagosomes with lysosomes. Overall, the present results contributed to understanding the mechanism responsible for the synergistic anticancer effect of amitriptyline and TRAIL and also presented a novel mechanism involved in DR4 and DR5 upregulation.

Core 2 ß1,6-N-acetylglucosaminyltransferases accelerate the escape of choriocarcinoma from natural killer cell immunity.

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted from choriocarcinoma and contains a core2 O-glycan formed by core2 ß1,6-N-acetylglucosaminyl transferase (C2GnT). Choriocarcinoma is considered immunogenic as it is gestational and contains paternal chromosomal components. Here we examined the function of C2GnT in the evasion of choriocarcinoma cells from natural killer (NK) cell-mediating killing. We determined that C2GnT is highly expressed in malignant gestational trophoblastic neoplasms. C2GnT KO downregulates core2 O-glycan expression in choriocarcinoma cells, which are more efficiently killed by NK cells than control cells. C2GnT KO cell containing tumor necrosis factor-related apoptosis inducing ligand have lower viability than control cells. Additionally, poly-N-acetyllactosamine in core2 branched oligosaccharides on MHC class I-related chain A (MICA) and mucin1 (MUC1) is significantly reduced in C2GnT KO cells. Meanwhile, the cumulative survival rate of nude mice inoculated with C2GnT KO tumors was higher than that of the control group. These findings suggest that choriocarcinoma cells may escape NK cell-mediated killing via glycosylation of MICA and MUC1.

Andrographolide sensitizes human renal carcinoma cells to TRAIL­induced apoptosis through upregulation of death receptor 4.

Tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) selectively induces apoptosis in cancer cells, with minimal toxicity to normal tissues. However, accumulating evidence suggests that certain cancer types are insensitive to TRAIL signaling. The aim of this study was to identify an effective combination regimen, which can overcome TRAIL resistance in renal cancer cell. Herein, we found that human renal carcinoma cells (RCCs) are widely resistant to TRAIL­mediated growth inhibition and subsequently identified that andrographolide (Andro), a major constituent of Andrographis paniculate, an annual herbaceous plant in the family Acanthaceae, counteracts TRAIL resistance in RCCs. Combined treatment with TRAIL and Andro suppressed cell viability as determined by MTS and proliferation as determined by EdU in a dose­dependent manner and inactivated the clonogenic and migration ability of RCCs. Andro significantly enhances TRAIL­mediated cell cycle arrest at the G2/M phase as determined by flow cytometry and senescence. Moreover, Andro restored TRAIL signaling, which in turns activated pro­apoptosis caspases as determined by immunoblot assay. The TRAIL receptor, death receptor (DR)4, but not DR5, was found to be significantly upregulated in Andro­treated RCC cells, which contributed to the role of Andro as a TRAIL sensitizer. The present study demonstrated that the combined treatment of Andro and TRAIL has potential therapeutic value against renal cancer.

Interleukin-4 Enhances the Sensitivity of Human Monocytes to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Through Upregulation of Death Receptor 4.

Interleukin (IL)-4 is generally thought to promote tumor cell growth and inhibit apoptosis. However, its role in characteristics of monocytic leukemia cells was rarely reported. In this study, we assessed the role of IL-4 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity of human monocytes. After incubation with IL-4 for 24 h, death receptor 4 (DR4) was significantly increased without downregulation of TRAIL decoy receptors and antiapoptotic proteins in THP-1 monocytes, and human primary monocytes and U-937 cells also exhibited increased TRAIL-induced apoptosis compared with control. Enhancement of TRAIL-mediated apoptosis by IL-4 was blocked by anti-DR4-neutralizing antibodies. Both upregulation of DR4 and enhancement of TRAIL-mediated apoptosis by IL-4 could be blocked by inhibitors of Janus kinase (JAK)/signal transducer and activator of transcription (STAT), phosphoinositol 3-kinase (PI3K)/Akt, and extracellular signal-regulated kinase to varying degrees. Thus, our data demonstrated a novel effect on TRAIL sensitivity on monocytes and monocytic leukemia cells of IL-4 and suggested that it may be necessary to reconsider the impact of current therapies against IL-4, JAK/STAT, and PI3K/Akt pathways with regard to TRAIL sensitivity.

Sphingomyelin synthase 2 overexpression promotes cisplatin-induced apoptosis of HepG2 cells.

Hepatoblastoma (HB) is the most type of common pediatric liver cancer. The primary chemotherapy drug for HB is cisplatin (DDP). However, patients readily develop intrinsic and acquired resistance, and severe side effects to treatment. Sphingomyelin synthase 2 (SMS2) is a key enzyme involved in the generation of sphingomyelin (SM), which is able to regulate cell proliferation, apoptosis and differentiation. The death receptors (DRs) have important functions in DDP-induced apoptosis. However, whether SMS2 is able to modulate cell apoptosis through the DR signaling pathway remains unknown. To investigate this question, SMS2 was overexpressed in HepG2 cells and treated with 3.5 mg/l cisplatin in the present study. After 24 h, the expression of SMS2, avian myelocytomatosis viral oncogene homolog (c-Myc), DR4, DR5 and caspase-3 was analyzed. Furthermore, cell viability was quantified, and apoptosis was assessed by western blot and flow cytometry analysis as well as Cell Counting kit-8. The results of the present study revealed that overexpression of SMS2 was able to increase the expression of c-Myc, cleaved caspase-3, DR4 and DR5 compared with the control group (P<0.05, n=3), and increase the levels of apoptosis in the SMS2 + DDP group, compared with the control (P<0.001, n=3). These results indicate that overexpression of SMS2 is able to improve sensitivity of HepG2 cells to DDP by increasing the expression of c-Myc, DR4 and DR5 in HepG2 cells. This increased sensitivity may decrease intrinsic and acquired resistance of chemotherapy in HB, and reduce the associated severe side effects in pediatric patients.

Artesunate acts as fuel to fire in sensitizing HepG2 cells towards TRAIL mediated apoptosis via STAT3 inhibition and DR4 augmentation.

In the present study, we investigated in vitro, the role of artesunate (ATS) with comparable potency to oxaliplatin (OXP) in sensitizing tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) resistant HepG2 cells towards apoptosis. ATS in consistency with OXP was found to reverse TRAIL resistant HepG2 cells towards TRAIL mediated apoptosis by enhancing caspase-3 and cleavage of PARP. Additionally, ATS also suppressed the nuclear translocation of activated signal transducers and activators of transcription 3 (STAT3) thereby sensitizing the HepG2 cells towards only death receptor 4 (DR4) mediated apoptosis. Furthermore, ATS exposure in TRAIL resistant cells resulted in significant increase of both DR4/DR5 expression and STAT3 inhibition thereby arbitrating TRAIL mediated apoptosis in HepG2 cells. The increase in expression was comparable to that of STAT3 silenced cells. From all the above observations, we conclude that ATS up-regulated DR4 expression by targeting STAT3, which in turn sensitized HepG2 cells to TRAIL mediated apoptosis.

BAY61-3606 potentiates the anti-tumor effects of TRAIL against colon cancer through up-regulating DR4 and down-regulating NF-κB.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is well known for its ability to preferentially induce apoptosis in malignant cells without causing damage to most normal cells. However, inherent and acquired resistance of tumor to TRAIL-induced apoptosis limits its therapeutic applicability. Here we show that the orally available tyrosine kinase inhibitor, BAY61-3606, enhances the sensitivity of human colon cancer cells, especially those harboring active mutations in Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) gene, to TRAIL-induced apoptosis in vitro and in vivo. The sensitization was achieved by up-regulating death receptor 4 (DR4) and the tumor suppressor p53. BAY61-3606-induced the up-regulation of DR4 is p53-dependent. Knockout of p53 decreased BAY61-3606-induced DR4 expression and inhibited the effect of BAY61-3606 on TRAIL-induced apoptosis. In addition, BAY61-3606 suppressed activity of NF-κB and regulated its gene products, which might also contribute to TRAIL-induced apoptosis. In conclusion, our results showed that BAY61-3606 sensitizes colon cancer cells to TRAIL-induced apoptosis via up-regulating DR4 expression in p53-dependent manner and inhibiting NF-κB activity, suggesting that the combination of TRAIL and BAY61-3606 may be a promising therapeutic approach in the treatment of colon cancer.

The structure of the death receptor 4-TNF-related apoptosis-inducing ligand (DR4-TRAIL) complex.

The structure of death receptor 4 (DR4) in complex with TNF-related apoptosis-inducing ligand (TRAIL) has been determined at 3 Šresolution and compared with those of previously determined DR5-TRAIL complexes. Consistent with the high sequence similarity between DR4 and DR5, the overall arrangement of the DR4-TRAIL complex does not differ substantially from that of the DR5-TRAIL complex. However, subtle differences are apparent. In addition, solution interaction studies were carried out that show differences in the thermodynamics of binding DR4 or DR5 with TRAIL.

ABC294640, a sphingosine kinase 2 inhibitor, enhances the antitumor effects of TRAIL in non-small cell lung cancer.

Evidences suggest that tumor microenvironment may play an important role in cancer drug resistance. Sphingosine kinase 2 (SphK2) is proposed to be the key regulator of sphingolipid signaling. This study is aimed to investigate whether the combination of molecular targeting therapy using a specific inhibitor of SphK2 (ABC294640), with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can enhance the apoptosis of non-small cell lung cancer (NSCLC) cells. Our results revealed that NSCLC cells' sensitivity to TRAIL is correlated with the level of SphK2. Compared with TRAIL alone, the combination therapy enhanced the apoptosis induced by TRAIL, and knockdown of SphK2 by siRNA presented a similar effect. Combination therapy with ABC294640 increased the activity of caspase-3/8 and up-regulated the expression of death receptors (DR). Additional investigations revealed that translocation of DR4/5 to the cell membrane surface was promoted by adding ABC294640. However, expression of anti-apoptosis proteins such as Bcl(-)2 and IAPs was not significantly modified by this SphK2 inhibitor. Overall, this work demonstrates that SphK2 may contribute to the apoptosis resistance in NSCLC, thus indicating a new therapeutic target for resistant NSCLC cells.

A Study of the Impact of Death Receptor 4 (DR4) Gene Polymorphisms in Alzheimer's Disease.

BACKGROUND: Excessive apoptosis is believed to play a role in many degenerative and non-degenerative neurological diseases including Alzheimer's disease (AD). Much recent data suggest that apoptotic mechanisms may represent the missing link between Aß deposition and proteolysis of tau protein. However, there is emerging evidence that apoptotic mechanisms may play a role in Alzheimer's Disease pathogenesis in the absence of overt apoptosis. TNF-related apoptosis inducing ligand receptor 1 (Death Receptor 4, DR4) might impair the apoptotic signal transduction and lead to dysregulation of the homeostasis between cell survival and cell death. AIMS: The aim of our study was to further investigate the relationship between genetic variants of DR4 and Alzheimer's Disease. STUDY DESIGN: Case control study. METHODS: Sixty-eight patients with AD were included in the study. The control group comprised 72 subjects without signs of neurodegenerative diseases, as evidenced by the examination.DNA was extracted from whole blood using the salting-out procedure. Genotypes were identified by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR-RFLP) products. RESULTS: We observed significant differences in the genotypic distribution of the rs6557634 polymorphism in AD patients compared with controls (p<0.05); our data suggest that the GA genotype in rs6557634 could be protective against AD (p<0.05). However, there were no significant differences between AD patients and control groups in terms of the DR4 rs20575 polymorphism (p>0.05) and the DR4 rs20576 polymorphism (p>0.05). According to haplotype analysis of the DR4 gene for rs6557634, rs20575 and rs20576 polymorphisms, GCA and GCC haplotypes might be a risk factor for AD. Also, we have shown that ACA, GGC and GGA haplotypes might be protective factors against AD. CONCLUSION: The present results indicate for the first time the possible contribution of the DR4 gene rs6557634, rs20575, rs20576 polymorphisms in Alzheimer's Disease, which may influence susceptibility to Alzheimer's Disease.

AY4, an agonistic anti-death receptor 4 MAB, induces apoptotic cell death in anaplastic thyroid cancer cells via downregulation of Bcl-xL with reactive oxygen species generation.

Anaplastic thyroid carcinoma (ATC) is an aggressive human tumor with a median survival of 6 months. We previously developed an agonistic anti-death receptor 4 MAB, AY4, and demonstrated the antitumor effects of AY4 in head and neck cancer cells. Presently, we show that ATC cells are sensitive to AY4 and that the sensitivity correlates with the reduced expression level of Bcl-xL and reactive oxygen species (ROS) generation. AY4 induced death of C-643, U-HTH 7, HTH83, and SW1736 cells. To elucidate the role of ROS generation in AY4-induced apoptosis of ATC cells, U-HTH 7 and SW1736 cells were pretreated with an antioxidant (N-acetyl cysteine, NAC) followed by AY4 treatment. The cell death was blocked by NAC. AY4-induced cell death was accompanied by the downregulation of the anti-apoptotic protein, Bcl-xL (BCL2L1). To examine the link between the apoptotic response and Bcl-xL protein expression, U-HTH 7 cells were transfected with Bcl-xL plasmid. The consequence of the overexpression of Bcl-xL appeared to decrease AY4-mediated cell death by blocking ROS generation in U-HTH 7 cells. By contrast, Bcl-xL knockdown using small interfering RNA of Bcl-xL enhanced AY4 sensitivity in HTH83 and C-643 cells and rendered the cells sensitive to AY4-induced cell death. The results support the conclusion that the expression level of Bcl-xL is important in the AY4-induced apoptosis of ATC cells through ROS generation. AY4 may be a promising tool for ATC therapy.

TRAIL mRNA expression in peripheral blood mononuclear cells of Egyptian SLE patients.

Although the definite etiopathogenesis of systemic lupus erythematosus (SLE) remains unclear, many different mechanisms may contribute to its pathogenesis. Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family with pro-apoptotic activity. The accumulation of apoptotic cell debris has been hypothesized to induce the autoimmune inflammation in SLE, and TRAIL may trigger this programmed cell death. We investigated TRAIL mRNA expression levels in peripheral blood mononuclear cells (PBMCs) from 60 SLE patients and 40 controls using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), and we studied the association between the results and clinical and laboratory parameters of the patients. Expression levels of TRAIL mRNAs in SLE patients were significantly higher than in controls (p<0.001). A statistically significant association was detected between TRAIL mRNA expression and SLE activity (p=0.001).