TRAIL Protein Expression Research Articles

Cytoprotective effect of genistein against dexamethasone-induced pancreatic β-cell apoptosis

Steroid-induced diabetes is a well-known metabolic side effect of long-term use of glucocorticoid (GC). Our group recently demonstrated dexamethasone-induced pancreatic β-cell apoptosis via upregulation of TRAIL and TRAIL death receptor (DR5). Genistein protects against pancreatic β-cell apoptosis induced by toxic agents. This study aimed to investigate the cytoprotective effect of genistein against dexamethasone-induced pancreatic β-cell apoptosis in cultured rat insulinoma (INS-1) cell line and in isolated mouse islets. In the absence of genistein, dexamethasone-induced pancreatic β-cell apoptosis was associated with upregulation of TRAIL, DR5, and superoxide production, but downregulation of TRAIL decoy receptor (DcR1). Dexamethasone also activated the expression of extrinsic and intrinsic apoptotic proteins, including Bax, NF-κB, caspase-8, and caspase-3, but suppressed the expression of the anti-apoptotic Bcl-2 protein. Combination treatment with dexamethasone and genistein protected against pancreatic β-cell apoptosis, and reduced the effects of dexamethasone on the expressions of TRAIL, DR5, DcR1, superoxide production, Bax, Bcl-2, NF-κB, caspase-8, and caspase-3. Moreover, combination treatment with dexamethasone and genistein reduced the expressions of TRAIL and DR5 in isolated mouse islets. The results of this study demonstrate the cytoprotective effect of genistein against dexamethasone-induced pancreatic β-cell apoptosis in both cell line and islets via reduced TRAIL and DR5 protein expression.

Arginine-tocopherol bioconjugated lipid vesicles for selective pTRAIL delivery and subsequent apoptosis induction in glioblastoma cells

The incorporation of specific therapeutic gene into glioblastoma offers potent therapeutic strategy to treat the disease. Non-viral gene delivery vectors are of particular interest due to their tuneable transfection efficiency and easy scale-up. Herein, we demonstrate successful delivery of plasmid encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (pTRAIL) using arginine-conjugated tocopherol lipid (AT) nanovesicles into glioblastoma cell lines. Another cationic lipid, glycine-conjugated tocopherol lipid (GT) having glycine in the head group region is also synthesized as a control lipid. Both lipid-derived liposomes effectively condensed the pDNA and the corresponding biomacromolecular assemblies (lipoplexes) are efficiently transfected into different cell lines. AT-based liposomes exhibit higher transfection efficacy in various cell lines, particularly selective in glioma cell lines. At an optimized N/P ratio, both the liposomal formulations show low cytotoxicity. AT-based lipoplexes have superior cellular uptake in U87 than the control lipid GT. The expression of TRAIL protein regulated death receptor and apoptosis signaling pathway is assayed by western blot using transfection of AT-based/pTRAIL into U87 cell lines. Induction of apoptosis in U87 cells exposed to AT-based/pTRAIL plasmid is evaluated by MTT assay as well as Annexin V-propidium iodide dual-staining assay. All results indicate that the developed AT-based/pTRAIL system offers a potentially safe and efficient therapeutic strategy for glioblastoma gene therapy.

The impact of TRAIL on proliferation of secretory prostate cancer PC-3 cell and LMO2 gene expression.

To determine the expressions of TRAIL protein and LMO2 gene in prostate cancer tissues with different differentiation degree and identify the influence of TRAIL on prostate cancer PC-3 cell proliferation. Surgical specimens from a total of 30 prostate cancer patients with radical prostatectomy were collected. The subjects were divided into three groups according to the different degrees of differentiation. TRAIL positive rate was detected by immunohistochemistry (IHC). LMO2 expression was assessed by Real-time PCR and Western-blot. PC-3 cell proliferation was determined by CCK-8 assay. The positive rate of TRAIL protein was significantly higher in moderately differentiated group (80%) and well differentiated group (100%) compared with that in poorly differentiated group (54.55%), respectively (χ2 = 27.33, p < 0.05; χ2 = 40.12, p < 0.01). Streptavidin-peroxidase (SP) assay showed that TRAIL protein expression in well-differentiated group was significantly higher than that in moderately differentiated group and poorly differentiated group. qRT-PCR result demonstrated that LMO2 mRNA levels in moderately and well-differentiated group were significantly increased compared to that in poorly differentiated group (p < 0.001). Also, the proliferation rate of PC-3 cells in well-differentiated group was significantly higher than that in well-differentiated and moderately differentiated groups (p < 0.05). Our data indicated that the positive rate of TRAIL protein increased in a prostate cancer differentiation dependent manner.

The Murine Natural Cytotoxic Receptor NKp46/NCR1 Controls TRAIL Protein Expression in NK Cells and ILC1s

SummaryTRAIL is an apoptosis-inducing ligand constitutively expressed on liver-resident type 1 innate lymphoid cells (ILC1s) and a subset of natural killer (NK) cells, where it contributes to NK cell anti-tumor, anti-viral, and immunoregulatory functions. However, the intrinsic pathways involved in TRAIL expression in ILCs remain unclear. Here, we demonstrate that the murine natural cytotoxic receptor mNKp46/NCR1, expressed on ILC1s and NK cells, controls TRAIL protein expression. Using NKp46-deficient mice, we show that ILC1s lack constitutive expression of TRAIL protein and that NK cells activated in vitro and in vivo fail to upregulate cell surface TRAIL in the absence of NKp46. We show that NKp46 regulates TRAIL expression in a dose-dependent manner and that the reintroduction of NKp46 in mature NK cells deficient for NKp46 is sufficient to restore TRAIL surface expression. These studies uncover a link between NKp46 and TRAIL expression in ILCs with potential implications in pathologies involving NKp46-expressing cells.

Multifunctionalized polyethyleneimine-based nanocarriers for gene and chemotherapeutic drug combination therapy through one-step assembly strategy.

Gene therapy combined with chemotherapy to achieve synergistic therapeutic effects has been a hot topic in recent years. In this project, the human tumor necrosis factor-related apoptosis-inducing ligand-encoding plasmid gene (TRAIL) and doxorubicin (Dox)-coloaded multi-functional nanocarrier was constructed based on the theory of circulation, accumulation, internalization, and release. Briefly, polyethyleneimine (PEI) was selected as skeleton material to synthesize PEI–polyethylene glycol (PEG)–TAT (PPT). Dox was conjugated to PEI using C6-succinimidyl 6-hydrazinonicotinate acetone hydrazone (C6-SANH), and a pH-sensitive Dox-PEI (DP) conjugate was obtained. Then, intracellular cationic pH-sensitive cellular assistant PPT and DP were mixed to condense TRAIL, and TRAIL–Dox coloaded PPT/DP/TRAIL (PDT) nanocarriers were obtained by one-step assembly. TRAIL was completely condensed by DP or PPT when mass ratios (DP/PPT to TRAIL) were up to 100:64, which indicated that DP and PPT could be mixed at any ratio for TRAIL condensation. The intracellular uptake rate of PDT was enhanced (P<0.05) when the contents of PPT in PPT+DP increased from 0 to 30%. Free Dox and TRAIL-loaded nanocarriers (PPT/C6-SANH-PEI/TRAIL [PCT]) were selected as controls to verify the synergistic antitumor effects of PDT. Compared with free TRAIL, TRAIL-protein expression was upregulated by PDT and PCT on Western blotting assays. The in vitro cytotoxicity of PDT was significantly enhanced compared to free Dox and PCT (P<0.01). Furthermore, murine PDT nanocarriers showed higher in vivo antitumor ability than both the Dox group (P<0.05) and the murine PCT group (P<0.05). These results indicated that the TRAIL + Dox synergistic antitumor effect could be achieved by PDT, which paves the way to gene–drug combination therapy for cancer.

Systemic delivery and activation of the TRAIL gene in lungs, with magnetic nanoparticles of chitosan controlled by an external magnetic field.

Recently, functional therapies targeting a specific organ without affecting normal tissues have been designed. The use of magnetic force to reach this goal is studied in this work. Previously, we demonstrated that nanocarriers based on magnetic nanoparticles could be directed and retained in the lungs, with their gene expression under the control of a promoter activated by a magnetic field. Magnetic nanoparticles containing the TRAIL gene and chitosan were constructed using the ionic gelation method as a nanosystem for magnetofection and were characterized by microscopy, ζ-potential, and retention analysis. Magnetofection in the mouse melanoma cell line B16F10 in vitro induced TRAIL-protein expression and was associated with morphological changes indicative of apoptosis. Systemic administration of the nanosystem in the tail vein of mice with melanoma B16F10 at the lungs produced a very significant increase in apoptosis in tumoral cells that correlated with the number of melanoma tumor foci observed in the lungs. The high levels of apoptosis detected in the lungs were partially related to mouse survival. The data presented demonstrate that the magnetofection nanosystem described here efficiently induces apoptosis and growth inhibition of melanoma B16F10 in the lungs. This new approach for systemic delivery and activation of a gene based in a nanocomplex offers a potential application in magnetic gene delivery for cancer.

Cooperative TRAIL production mediates IFNα/Smac mimetic-induced cell death in TNFα-resistant solid cancer cells.

Smac mimetics antagonize IAP proteins, which are highly expressed in several cancers. Recent reports indicate that Smac mimetics trigger a broad cytokine response and synergize with immune modulators to induce cell death. Here, we identify a differential requirement of TRAIL or TNFα as mediators of IFNα/Smac mimetic-induced cell death depending on the cellular context. Subtoxic concentrations of Smac mimetics cooperate with IFNα to induce cell death in various solid tumor cell lines in a highly synergistic manner as determined by combination index. Mechanistic studies show that IFNα/BV6 cotreatment promotes the formation of a caspase-8-activating complex together with the adaptor protein FADD and RIP1. Assembly of this RIP1/FADD/caspase-8 complex represents a critical event, since RIP1 silencing inhibits IFNα/BV6-induced cell death. Strikingly, pharmacological inhibition of paracrine/autocrine TNFα signaling by the TNFα scavenger Enbrel rescues HT-29 colon carcinoma cells, but not A172 glioblastoma cells from IFNα/BV6-induced cell death. By comparison, A172 cells are significantly protected against IFNα/BV6 treatment by blockage of TRAIL signaling through genetic silencing of TRAIL or its cognate receptor TRAIL receptor 2 (DR5). Despite this differential requirement of TNFα and TRAIL signaling, mRNA and protein expression is increased by IFNα/BV6 cotreatment in both cell lines. Interestingly, A172 cells turn out to be resistant to exogenously added recombinant TNFα even in the presence of BV6, whereas they display a high sensitivity towards TRAIL/BV6. In contrast, BV6 efficiently sensitizes HT-29 cells to TNFα while TRAIL only had limited efficacy. This demonstrates that a differential sensitivity towards TRAIL or TNFα determines the dependency on either death receptor ligand for IFNα/Smac mimetic-induced cell death. Thus, by concomitant stimulation of both death receptor systems IFNα/Smac mimetic combination treatment is an effective strategy to induce cell death in TNFα- or TRAIL-responsive cancers.

Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL

Multidrug resistance (MDR) has been associated with expression of ABC transporter genes including P-glycoprotein (Pgp, MDR1, ABCB1). However, deregulation of apoptotic pathways also renders cells resistant to chemotherapy. To discover apoptosis-related genes affected by Pgp expression, we used the HeLa MDR-off system. We found that using doxycycline to control Pgp expression has a significant advantage over tetracycline, in that doxycycline caused less endogenous gene expression modification/perturbation, and was more potent than tetracycline in suppressing Pgp expression. Cells overexpressing Pgp have lower TNFSF10 (TRAIL) expression than their parental cells. Controlled downregulation of Pgp increased endogenous TRAIL protein expression. Also, ectopic overexpression of TRAIL in Pgp-positive cells was associated with a reduction in Pgp levels. However, cells expressing a functionally defective mutant Pgp showed an increase in TRAIL expression, suggesting that Pgp function is required for TRAIL suppression. Cells in which Pgp is knocked down by upregulation of TRAIL expression are less susceptible to TRAIL ligand (sTRAIL)-induced apoptosis. Our findings reveal an inverse correlation between functional Pgp and endogenous TRAIL expression. Pgp function plays an important role in the TRAIL-mediated apoptosis pathway by regulating endogenous TRAIL expression and the TRAIL-mediated apoptosis pathway in MDR cancer cells.

Effect of the combined use of oncolytic adenovirus carrying tumor necrosis factor-related apoptosis-inducing ligand with epirubicin on proliferation and apoptosis of bladder cancer T24 cells

Objective To observe the effects of the combined use of oncolytic adenovirus carrying tumor necrosis factor-related apoptosis- inducing ligand(ZD55-TRAIL) with epirubicin(EPB) on the growth and apoptosis of bladder cancer T24 cells. Methods T24 cells were divided into 5 groups: ZD55-TRAIL[multiple of infection(MOI)= 10.0] plus EPB(1 mg/L) group, ZD55- TRAIL(MOI= 10.0) group, EPB(1 mg/L)group, ZD55- enhanced green fluorescent protein(EGFP, MOI= 10.0)group, and PBS group. The expression of TRAIL was detected by reverse transcriptase- polymerase chain reaction(RT - PCR)and Western blotting. The cytotoxicity of cells was assessed by cell counting kit- 8(CCK- 8)assay. Cell apoptosis was determined by annexin V- fluoresceine isothiocyanate(FITC)assay. Results RT-PCR analysis confirmed that T24 cells treated with ZD55- TRAIL plus EPB and ZD55- TRAIL could express TRAIL mRNA with high efficiency and there was no significant difference between two groups(P> 0.05). Western blotting analysis showed that ZD55- TRAIL could effectively promote the expression of TRAIL protein in T24 cells.CCK- 8 assay revealed the survival rate after 48 h in the ZD55- TRAIL(MOI= 10.0)plus EPB(1 mg/L)group[(18.44±2.38)%] was significantly higher than in ZD55- TRAIL group[(40.75± 4.12)%], EPB group[(53.62±1.41)%]and ZD55- EGFP group[(61.65±4.53)%,P<0.01]. The single use of EPBat the 2nd day achieved the same effect as MOI= 10.0 ZD55- TRAIL+ 1 mg/L EPBat 2 mg/ L,and at 3rd day,a higher concentration of 4 mg/L was needed. The results of cell apoptosis revealed the number of apoptosis cells in ZD55- TRAIL plus EPB group was 2.01 times,2.21 times, and 2.95 times of that in ZD55- TRAIL group, EPB group and ZD55- EGFP group respectively(P<0.01). Conclusion ZD55 - TRAIL conjugated with EPB exhibited a remarkably increased apoptosis- inducing effect and great antitumor effects in bladder cancer T24 cells. Key words: Oncolytic adenovirus; Tumor necrosis factor-related apoptosis-inducing ligand; Epirubicin

Enhanced effects of TRAIL-endostatin-based double-gene-radiotherapy on suppressing growth, promoting apoptosis and inducing cell cycle arrest in vascular endothelial cells

This study examined the effects of TRAIL-endostatin-based gene-radiotherapy on cellular growth, apoptosis and cell cycle progression in human vascular endothelial cells ECV304 in vitro. The expression of TRAIL and endostatin protein in ECV304 cells was detected by ELISA after the transfection of recombinant plasmid pshuttle-Egr1-shTRAIL-shES and X-ray irradiation. Then MTT assay was used for determining the cellular proliferation, and flow cytometry (FCM) plus Annexin V and propidium iodide (PI) double-staining or PI single-staining were employed for the detection of apoptosis and cell cycle progression. The results showed that expression of TRAIL and endostatin protein exhibited a time- and dose-dependent change in ECV304 cells after pshuttle-Egr1-shTRAIL-shES transfection in conjunction with irradiation. In the TRAIL-endostatin-based single- or double-gene-radiotherapy, the cell viability declined in a time- and dose-dependent manner, the percentage of cells at G(2)/M phase and apoptotic rate was increased, and the percentage of cells at G(0)/G(1) phase was lowered as compared with those receiving radiotherapy alone. Moreover, TRAIL-endostatin-based double-gene-radiotherapy demonstrated better effects on growth inhibition, promotion of apoptosis and induction of cell cycle arrest in ECV304 cells than single-gene-radiotherapy.

NFATc1 Regulation of TRAIL Expression in Human Intestinal Cells

TNF-related apoptosis-inducing ligand (TRAIL; Apo2) has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT) participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187 (Io) increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA), an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter.

Effects of modified Haizao Yuhu Decoction in experimental autoimmune thyroiditis rats

Modified Haizao Yuhu Decoction (MHYD), based on clinical experience, has been used for approximately 500 years and famous for its efficiency in treating thyroid-related diseases. To investigate therapeutic effects of MHYD on thyroid-related autoantibodies and thyroid hormone in experimental autoimmune thyroiditis (EAT) rats, and to provide evidence for the immunomodulatory potential during antigenmediated immunostimulation. The effect of MHYD was investigated in EAT rats induced by subcutaneous injection of porcine thyroglobulin with Freund's adjuvant (complete and incomplete, CFA and IFA). MHYD was fed to EAT rats daily at a dose of 100mg/kg for 10 weeks. The presence of mononuclear cell infiltration in thyroid tissues were examined to assess the severity of EAT. The levels of serum T3, T4, TgAb and TPOAb were determined by radioimmunoassay and protein expressions of TRAIL were detected by immunohistochemistry. MHYD exhibited a significant protective effects by reversing serum T3, T4, TgAb and TPOAb levels, histopathological changes and TRAIL protein expression of thyroid in EAT model rats. Sargassum fusiforme and Radix Glycyrrhiza in the formulation could be responsible for the immunomodulatory effects of MHYD. These results showed MHYD playing a role as an immunomodulator and TRAIL inhibitor during antigenmediated immunostimulation, may warrant further evaluation as a possible agent for the treatment of Hashimoto's thyroiditis.

Expression of Human Soluble Trail Protein in Transgenic Tobacco Nc89

Plant bioreactors have been considered as an efficient system for biopharmaceutical production. It offers many advantages, such as low cost of production, eukaryotic expression way and absence of human pathogens. In this study, we tried to express the soluble extracellular domain of human tumor necrosis factor related apoptosis-inducing ligand (sTRAIL) in transgenic plants of NC89, a tobacco variety that was planted widely. Using the chloroplast-targeted expression vector pGTPsT, which was constructed previously, we transformed NC89 tobacco leaf explants with the mediation of Agrobacterium tumefaciens LBA4404. We used kanamycin resistance to select transformants and PCR analysis to confirm the presence of sTRAIL gene. Then, we performed RT-PCR analysis to detect the transcription of sTRAIL gene and Western blot to analyze sTRAIL protein accumulation in the leaves of the transformed tobacco plants. The results showed that sTRAIL was successfully inserted into NC89 genome of 8 independent transgenic lines and was expressed in all tested lines. However, no significant protein accumulation was detected at present analysis conditions. This suggests that the accumulation of sTRAIL in transgenic plants of NC89 is much lower than that in transgenic Petit Havana, another variety, which we have investigated previously. NC89, therefore, may not be a suitable plant variety used as plant bioreactor for foreign protein expression and accumulation. A strong protein degradation system may exist in the chloroplast of this variety. However, this point should by experimently tested in the future study.

Proanthocyanidins (Vaccinium macrocarpon) induce apoptosis in DU145 human prostate cancer cells

Cranberry can slow the growth of DU145 cells by inhibiting cell proliferation and also by apoptosis induction. Apoptosis may occur either via the mitochondrial‐mediated intrinsic pathway or death‐receptor mediated extrinsic pathway. This study investigated how a proanthocyanidin‐enriched fraction (PAC) from cranberry may regulate this complex process. PAC contained proanthocyanidin oligomers (major ones appear to be dimers, trimers &amp; tetramers) (with 2–8 epicatechin units, with at least one A‐type linkage between units). PAC treatment resulted in a dose dependent inhibition of Bcl‐2, Bax, and Bid protein levels coupled with an induction of tBid protein. PAC also inhibits Smac/DIABLO levels. PAR‐4 protein levels increased in response to PAC. PAC resulted in a dose dependent induction of cytochrome ‐C in total cellular extract and cytochrome‐C protein expression also increased in mitochondrial extracts. Increased PARP protein expression occurred in response to PAC (25 ug/mL). PAC inhibited Fas‐L, FADD, and TRAIL protein expression levels whereas Fas protein levels were not affected by PAC. These results suggest that PAC is inducing apoptosis in DU145 cells via the intrinsic pathway and ensures this occurs by specifically inhibiting the extrinsic pathway of apoptosis.(N.C.I.C‐ Canadian Cancer Society, P.E.I. Heath Research Program, Cranberry Institute (Wisconsin Board) funded)

FasL Gene–Deficient Mice Display a Limited Disruption in Spermatogenesis and Inhibition of Mono-(2-ethylhexyl) Phthalate–Induced Germ Cell Apoptosis

FasL (TNFSF6, CD95L) is hypothesized to trigger testicular germ cell apoptosis that normally occurs during a distinct peripubertal period as well as in response to toxicant-induced Sertoli cell injury. To test this hypothesis, we evaluated the testis of FasL gene–deficient mice (FasL−/−) at two distinct developmental ages (postnatal day [PND] 28 and 44) and after toxicant-induced Sertoli cell injury. Testicular cross sections from peripubertal (PND 28) FasL−/− mice showed significant increases in the basal germ cell apoptotic index (AI; 20.58 ± 4.59) as compared to the testis of C57BL/6J wild-type mice (5.16 ± 0.08) and closely correlated with increased expression of TRAIL protein in the testis of FasL−/− mice. A limited, but significant, number of seminiferous tubules in the testis of PND 28 FasL−/− mice showed a severe loss of germ cells with only Sertoli cells present. In contrast, no apparent gross histological changes were observed in the testis of adult (PND 44) FasL−/− mice. However, PND 44 FasL−/− mice did show a 51% reduction in homogenization-resistant elongate spermatids as compared to age-matched C57BL/6J mice. Exposure of PND 28 FasL−/− mice to mono-(2-ethylhexyl) phthalate (MEHP), a well-described Sertoli cell toxicant, unexpectedly caused a rapid decrease in the germ cell AI that paralleled increased levels of the CFLAR (c-FLIP) protein, a known inhibitor of death receptor signaling. In contrast, MEHP treatment did not decrease c-FLIP levels in PND 28 C57BL/6J mice. Taken together, these findings indicate that FasL protein expression is required during the peripubertal period for the proper regulation of germ cell apoptosis that occurs normally during this period. The influence of FasL on the cellular regulation of c-FLIP protein levels appears to be a unique mechanism for modulating germ cell apoptosis after toxicant-induced Sertoli cell injury.

TRAIL protein expression in breast cancer cells correlates with nuclear grade

IntroductionTRAIL protein may serve as an escape mechanism for cancer cells from the immune response. The aim of the study was to assess whether the presence of TRAIL protein correlates with unfavourable prognostic factors in breast carcinoma.Material and methodsThe study group was composed of breast cancer patients treated surgically in the Department of Surgical Oncology, Medical University of Lodz, Poland, from January to December 2003. Inclusion criteria for the study were fulfilled by 117 women. The immunohistochemical study of TRAIL protein expression was performed in 118 breast carcinomas diagnosed in the study group. TRAIL protein expression was correlated with other variables: tumour size, lymph node status, grade, histological type of carcinoma, oestrogen and progesterone receptor status, HER2 expression, presence of lymphovascular invasion and age of the patient.ResultsExpression of TRAIL protein was present in 73% of breast carcinomas. The percentage of TRAIL-expressing breast carcinoma cells correlated with the nuclear grade (τ = 0.26, p < 0.05; Tau Kendall test). The intensity of TRAIL expression (intensity of staining) in breast carcinoma cells correlated with the nuclear grade (τ = 0.15, p < 0.05; Tau Kendall test). TRAIL expression in breast carcinoma did not correlate with other studied variables.ConclusionsOur analysis revealed that expression of TRAIL protein in breast carcinoma cells correlates with nuclear grade of carcinoma.

TNFSF10 (TRAIL), a p53 target gene that mediates p53-dependent cell death

We have identified TNFSF10 (TRAIL) as a p53-transcriptional target gene. There are two p53 DNA-binding sites in the human TNFSF10 promoter region, at 346 and 625 bp upstream of the transcription start site. A human p53-expressing adenovirus (Ad-p53) induced TRAIL mRNA and protein expression in HCT116 p53-/- human colon cancer cells. A human TRAIL-promoter reporter assay showed increased luciferase activity with the promoter vector that contains two p53 DNA-binding motifs, following Ad-p53 infection, compared to the control adenovirus infection. Using HCT116 cells, gene silencing of TNFSF10 by siRNA suppressed caspase 3 and 7 activity, even after treatment with the DNA-damaging chemotherapeutic agent adriamycin. TRAIL protein expression was elevated in adriamycin-treated breast cancer cells. In vivo, TRAIL expression was induced in mouse natural killer cells at 24 hours after systemic treatment with 5-Fluorouracil. p53-dependent TRAIL induction in natural killer cells after chemotherapy exposure provides a link between the tumor suppressor p53 and the host immune response during cancer therapy as well as a paracrine-mediated cell-extrinsic death response. Our findings provide new mechanistic insights into the signaling of p53-dependent cell death and tumor suppression, including the involvement of the host immune system and natural killer cells in vivo in the anti-tumor efficacy of chemotherapy.

Induction of TRAIL- and TNF-α-Dependent Apoptosis in Human Monocyte-Derived Dendritic Cells by Microfilariae of Brugia malayi

Dysregulation of professional APC has been postulated as a major mechanism underlying Ag-specific T cell hyporesponsiveness in patients with patent filarial infection. To address the nature of this dysregulation, dendritic cells (DC) and macrophages generated from elutriated monocytes were exposed to live microfilariae (mf), the parasite stage that circulates in blood and is responsible for most immune dysregulation in filarial infections. DC exposed to mf for 24-96 h showed a marked increase in cell death and caspase-positive cells compared with unexposed DC, whereas mf exposure did not induce apoptosis in macrophages. Interestingly, 48-h exposure of DC to mf induced mRNA expression of the proapoptotic gene TRAIL and both mRNA and protein expression of TNF-alpha. mAb to TRAIL-R2, TNF-R1, or TNF-alpha partially reversed mf-induced cell death in DC, as did knocking down the receptor for TRAIL-R2 using small interfering RNA. The mf also induced gene expression of BH3-interacting domain death agonist and protein expression of cytochrome c in DC; mf-induced cleavage of BH3-interacting domain death agonist could be shown to induce release of cytochrome c, leading to activation of caspase 9. Our data suggest that mf induce DC apoptosis in a TRAIL- and TNF-alpha-dependent fashion.

The release of soluble forms of TRAIL and DR5 by neutrophils of oral cavity cancer patients.

In the present study we examined the release of the soluble form of TRAIL by neutrophils (PMN) derived from patients with oral cavity cancer. Simultaneously, we estimated the ability of PMNs of these patients to release the soluble form of DR5 receptor, a natural regulatory protein of TRAIL. The obtained results were confronted with the serum levels of sTRAIL and sDR5. The cells were isolated from 21 patients with squamous cell carcinoma of oral cavity at diagnosis and three weeks after surgery treatment. For comparative purposes we performed similar examinations in autologous peripheral blood mononuclear cells (PBMC). Cytoplasmic protein fractions of the cells were analyzed for the presence of TRAIL and DR5 by western blotting. Soluble TRAIL and soluble DR5 concentrations in the culture supernatants of cells were confronted with their serum levels using ELISA kit. PMN and PBMC of the whole cancer patient group expressed decreased TRAIL protein and unchanged expression of DR5 receptor in comparison with the control group. Unchanged release of sTRAIL by PMNs of patients in Stage II was accompanying the decrease of the ability of PBMC to secrete this protein. In patients in Stage IV the secretion of sTRAIL by PMNs and PBMC was impaired. In contrast to changes in sTRAIL secretion by PMN and PBMC of oral cavity cancer patients, the secretion of sDR5 by these cells was unchanged. The serum levels of sTRAIL were increased in patients in Stage II before treatment and decreased in the same patients after treatment. The altered ability of PMN of PBMC to secrete sTRAIL may have different implications for the immune response of patients with oral cavity cancer cells at different stages of disease.

ORIGINAL ARTICLE: Decidual Natural Killer Cell Tuning by Autologous Dendritic Cells

Problem Dendritic cells (DC)/natural killer (NK) cells interactions in the deciduas of early human pregnancies were analyzed in vitro.Method of study Phenotype, cytokine expression and/or cytolytic mediators’ expression were measured by flow cytometry in NK and DC from the freshly isolated decidual mononuclear cells or after their purification and co‐culture in vitro. Proliferation of 5(6)‐Carboxyfluorescein diacetate N‐succinimidyl ester (CFSE)‐labeled CD56+ cells was analyzed by flow cytometry after the co‐culture with CD1a+ or CD83+ DC.Results Decidual CD1a+ cells show less mature phenotype with no expression of CD197, lower expression of CD80 and CD86 and higher expression of CD206 and CD195 in comparison to CD83+ cells. Interleukin (IL)‐15, interferon‐γ and tumor necrosis factor‐α productions were higher in immature than mature DC, whereas IL‐10 and IL‐18 were equally produced in both subpopulations. Immature DC increase perforin, FasL and TRAIL protein expression and proliferation of NK cells, but decrease their intracellular IL‐15 production. Mature DC caused less efficient proliferation of NK cells, and did not affect cytokine and cytolytic mediator expression.Conclusion These results suggest that decidual CD1a+ cells regulate and shape NK cell function more profoundly than CD83+ cells in decidua.