OBJECTIVETo investigate the effect of tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its receptor on apoptosis in thymus during early post-burn stage in rat with severe burns.

METHODSFifty Wistar rats were randomly divided into sham scald group (SS, n = 10) and burn group (n = 40). The apoptosis in thymus in rats was detected with annexin V/FITC-PI double staining at 4, 12, 24, 48 post-burn hours (PBH). The expression of TRAIL death receptor DR5, DR4 and its decoy receptor DcR1, DcR2 in thymus were detected by RT-PCR and Western blot at above time-points.

RESULTSCompared with that in SS group (6.7 +/- 0.8)%, the apoptosis in the thymus in burn group started to increase at 4 PBH [(17.1 +/- 0.4)%], peaked at 12 PBH [(25.2 +/- 1.1)%], and it was still evidently higher than that in SS group at 48 PBH (P < 0.05). There was no obvious difference in the apoptosis rate in rats in burn group among all the time-points. The expression of DR5 in burn group at each time-points was significantly higher than those in SS group, while that of DcR2 shown an opposite tendency (P < 0.05). The expression of DR4, DcR1 was similar in both groups.

CONCLUSIONThe marked increase in apoptosis rate in rat thymus at early post-burn stage, and the significant change in the expression of DR5 and DcR2 show that TRAIL pathway may participate in apoptosis.

Animals ; Apoptosis ; Burns ; metabolism ; Disease Models, Animal ; Female ; Male ; Rats ; Rats, Wistar ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Thymus Gland ; metabolism

OBJECTIVETo investigate the molecular mechanism of doxorubicin enhancement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) inducing apoptotic effect on multiple myeloma cell line KM3.

METHODSApoptosis was studied independently through flow cytometry analysis and TUNEL staining. The expression of death receptor 5 (DR5) and nuclear factor P65 in nuclear was examined by Western blot.

RESULTSThe apoptosis ratio of KM3 cells was 20.88%, 40.03%, 57.87%, 60.82% respectively when treated with different concentration of TRAIL (10, 20, 50, 100 ng/ml) combining with doxorubicin. It is markedly higher than the group treated with TRAIL or doxorubicin alone. DR5 expression increased while P65 decreased as the doses of doxorubicin increased when KM3 cells treated with doxorubicin (0.5, 1.0, 2.0 and 4.0 microg/ml) plus 20 ng/ml TRAIL.

CONCLUSIONIncreasing the expression of DR5 and nuclear transferring of P65 are the important molecular mechanism by which doxorubicin enhances TRAIL-inducing apoptosis of KM3 cells.

Apoptosis ; drug effects ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Interactions ; Humans ; Multiple Myeloma ; metabolism ; pathology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Transcription Factor RelA ; genetics ; metabolism

OBJECTIVETo clone and identify novel proteins binding to the death domain of the death receptor 4 (DR4).

METHODSThe yeast two-hybrid system was used for this study. Automatic sequencing was carried out for DNA sequencing. The sequence homology and the functional domains were analyzed by BLAST and the ScanProsite Tool softwares, respectively. Co-immunoprecipitate method was used to confirm human formyl peptide receptor-like 1 (FPRL1) binding specifically with DR4CD (the cytoplasmic domain of DR4) in HEK293T cells.

RESULTSTwo positive clones, named as pADB1 and pADB2, were obtained. BLAST searching showed that the homology of the insert sequence of pADB1 with the mRNA of FPRL1 was 97%. The insert of pADB2 shared no homology with any known peptides in GeneBank. Co-immunoprecipitate analysis further confirmed that FPRL1 could bind to DR4CD in vivo specifically.

CONCLUSIONSFPRL1 may associate with DR4CD in vivo specifically. The functional studies of FPRL1 in signaling pathway mediated by TNF-related apoptosis inducing ligand (TRAIL) are in active progress in our laboratory.

Amino Acid Sequence ; Apoptosis ; Apoptosis Regulatory Proteins ; Base Sequence ; Carrier Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Humans ; Membrane Glycoproteins ; metabolism ; Molecular Sequence Data ; Protein Structure, Tertiary ; Receptors, Formyl Peptide ; metabolism ; Receptors, Lipoxin ; metabolism ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; Receptors, Tumor Necrosis Factor ; genetics ; metabolism ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha ; metabolism

There exist two major apoptotic signaling pathways: the intrinsic mitochondria-mediated pathway, and the extrinsic death receptor-induced pathway. TNF-related apoptosis-inducing ligand (TRAIL), which is the ligand for death receptor 4 (DR4) and death receptor 5 (DR5) and induces apoptosis by ligation with DR4 or DR5. We review the characteristic of TRAIL and its receptors, the mechanism of apoptosis induced by TRAIL, the distribution of death receptors in cancer, and applications and prospects of TRAIL signaling pathway in the treatment of cancer.
Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Drug Delivery Systems ; GPI-Linked Proteins ; genetics ; metabolism ; Genetic Therapy ; Humans ; Neoplasms ; metabolism ; pathology ; therapy ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; classification ; genetics ; metabolism ; Receptors, Tumor Necrosis Factor, Member 10c ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; pharmacology ; Tumor Necrosis Factor Decoy Receptors ; genetics ; metabolism

OBJECTIVETo investigate the structure and function of the N-terminal region (NTR) of death receptor 5 (DR5).

METHODSA series of deletions of the DR5 extracellular domain (DR5-ECD) proteins were expressed in E.coli. and purified by affinity chromatography. The binding ability of these deletant proteins to AD5-10, a mouse anti-human DR5 monoclonal antibody, was evaluated by immunoblotting and ELISA.

RESULTSRecombinant DR5-ECD proteins containing the NTR were recognized and bound by AD5-10, while the other deletant proteins without the NTR failed to interact with AD5-10.

CONCLUSIONThere is an AD5-10 targeting site in the NTR of DR5, which may play a role in developing novel immunotherapies for cancers.

Animals ; Antibodies, Monoclonal ; chemistry ; Binding Sites ; Gene Deletion ; Genetic Engineering ; Genetic Vectors ; Humans ; Mice ; Protein Binding ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; chemistry ; genetics ; metabolism

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. Here, we report that paxilline, an indole alkaloid from Penicillium paxilli, can sensitize various glioma cells to TRAIL-mediated apoptosis. While treatment with TRAIL alone caused partial processing of caspase-3 to its p20 intermediate in TRAIL-resistant glioma cell lines, co-treatment with TRAIL and subtoxic doses of paxilline caused complete processing of caspase-3 into its active subunits. Paxilline treatment markedly upregulated DR5, a receptor of TRAIL, through a CHOP/GADD153-mediated process. In addition, paxilline treatment markedly downregulated the protein levels of the short form of the cellular FLICE-inhibitory protein (c-FLIPS) and the caspase inhibitor, survivin, through proteasome-mediated degradation. Taken together, these results show that paxilline effectively sensitizes glioma cells to TRAIL-mediated apoptosis by modulating multiple components of the death receptor-mediated apoptotic pathway. Interestingly, paxilline/TRAIL co-treatment did not induce apoptosis in normal astrocytes, nor did it affect the protein levels of CHOP, DR5 or survivin in these cells. Thus, combined treatment regimens involving paxilline and TRAIL may offer an attractive strategy for safely treating resistant gliomas.
Antineoplastic Agents/*pharmacology ; Apoptosis/*drug effects ; Astrocytes/metabolism ; CASP8 and FADD-Like Apoptosis Regulating Protein/genetics/*metabolism ; Caspase 3/metabolism ; Cell Line, Tumor ; Drug Discovery ; Flow Cytometry ; Glioma/*metabolism/pathology ; Humans ; Indoles/*pharmacology ; Inhibitor of Apoptosis Proteins/metabolism ; RNA, Small Interfering ; Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; TNF-Related Apoptosis-Inducing Ligand/metabolism/*pharmacology ; Transcription Factor CHOP/analysis

OBJECTIVETo investigate the antitumor efficacy of death receptor 5, its ligand (TRAIL) and DR5mAb in human hepatocellular carcinoma.

METHODSExpression of DR5 in the HCC cell lines HepG2, SMMC 7721 and normal human liver cell line LO2 was measured at mRNA and protein level by semi-quantitative RT-PCR and Western blot, respectively. MTT method was used to measure the cell viability and flow cytometry assay was used to detect apoptosis so as to observe the inhibitory effect of TRAIL and DR5mAb on HCC cells.

RESULTSDeath receptor 5 was highly expressed in the HCC cell lines, but rarely expressed in normal human liver cell line (P < 0.01). With the increase of TRAIL concentration, the cell viability of HCC cells decreased gradually. However, when the concentration of TRAIL was above 1000 ng/ml, HCC cells were resistant to TRAIL, but still sensitive to DR5mAb. After incubation with DR5mAb (1000 ng/ml) for 24 h, the rate of apoptosis in HCC cells reached to 52.45% +/- 0.57%, which was higher than that incubated with TRAIL under the same condition (14.74% +/- 0.48%) (P < 0.05). The cell viability of normal human liver cell line treated with TRAIL tended to decline with the increase of the concentration, which was significantly different from that of matched control group. But DR5mAb had little effect on normal human liver cell line.

CONCLUSIONDeath receptor 5 as a target plays an important role in the course of HCC apoptosis induction. Agonistic monoclonal antibody specific for human DR5 can selectively and effectively kill hepatocellular carcinoma cells in vitro, while is not harmful to normal human hepatocytes. It reveals that DR5mAb might provide a new direction in hepatocellular carcinoma treatment research.

Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line ; Cell Line, Tumor ; Cell Survival ; drug effects ; Humans ; Liver Neoplasms ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics ; immunology ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis

To investigate the combined effect of human recombinant soluble TNF-related apoptosis induced ligand (hrsTRAIL) with Ara-C or alone on HL-60 leukemia cell lines and its mechanism, human leukemia cell lines HL-60 were cultured in vitro. HL-60 cells were divided into 5 groups: control group, Ara-C group, rsTRAIL group, Ara-C + rsTRAIL simultaneously given group, Ara-C + rsTRAIL tandem given group (Ara-C followed by rsTRAIL group). The cytotoxic effect was measured by MTT assay; cell apoptosis rate was determined by flow cytometry after Annexin V/PI staining; the expression level of DR5 on surface of HL-60 cells treated with Ara-C at different concentrations for 24 hours was determined by flow cytometry. The expression level of DR5 on surface of HL-60 cells and caspase-8 activity in HL-60 cells of rsTRAIL group and Ara-C + rsTRAIL tandem group was determined by flow cytometry. The result showed that rsTRAIL could inhibit the proliferation of HL-60 cells and induce apoptosis of HL-60 cells in a concentration-dependent manner. The apoptosis rate of HL-60 cells in Ara-C + rsTRAIL tandem given group was higher than that in Ara-C + rsTRAIL simultaneously given group, the expression level of DR5 on surface of HL-60 cells and intracellular activity of caspase-8 in Ara-C + rsTRAIL tandem given group were higher than those in rsTRAIL group. When HL-60 cells treated with 5 and 10 mg/L of Ara-C for 24 hours, the expression level of DR5 on surface of HL-60 cells was higher than that in control group. It is concluded that rsTRAIL can inhibit the proliferation of HL-60 cells, and induce apoptosis of HL-60 cells. Ara-C can upregulate DR5 expression on the surface of HL-60 cells and enhance the effect of rsTRAIL-inducing apoptosis. Tandem treatment of HL-60 cells with Ara-C followed by rsTRAIL induce more apoptosis than that of co-treatment with rsTRAIL and Ara-C. Ara-C and rsTRAIL has a synergistic inhibitory effect on growth of HL-60 cells. The mechanism may correlate with up-regulation of the expression level of DR5 and/or caspase-8 in HL-60 cells by Ara-C.
Apoptosis ; drug effects ; Caspase 8 ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cytarabine ; pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; HL-60 Cells ; Humans ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Recombinant Proteins ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Up-Regulation

This study was aimed to detect the expression of TNF related apoptosis-inducing ligand (TRAIL) and its receptors on acute myeloid leukemic (AML) cells, and explore its possible role in leukemia therapy. RT-PCR and flow cytometry were used to detect the expression of TRAIL and its receptors on AML cells of 39 cases (patient group), AML cells of 18 cases with complete remission (CR group) and BMMNC or PBMNC of 21 normal persons (control group). The results showed that (1) TRAIL, DR4 and DR5 were highly expressed in both patient group and CR group, while the DcR1 and DcR2 were poorly expressed. (2) The level of DR5 expression in CR group was higher than that in patient group. (3) The level of DR5 was higher than DR4 in both patient group and CR group. (4) TRAIL and its receptors were expressed similarly in different subtypes of AML. In conclusion, there are differences between the expressions of TRAIL and its receptors in AML cells. DR5 may play an important role in TRAIL-inducing apoptosis of AML cells.
Adult ; Female ; Flow Cytometry ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics

OBJECTIVETo detect the expression profile of NK ligands in acute leukemia cell lines and investigate the differential expression pattern between acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

METHODSUsing quantitative real-time PCR, 23 NK ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, HLA-E, HLA-G, CD48, NBTA, HLA-F, LLT-1, PVR, Nectin2, CD72, CD80, ICAM-1, LFA-3, CRACC, Fas, DR4, DR5, TNFR1) were detected in 6 acute leukemia cell lines, including 3 ALL cell lines (CEM, Jurkat T, Reh) and 3 AML cell lines (HL-60, KG-1a, NB4), respectively. Independent-samples t test analysis was performed to determine statistical significance.

RESULTSUsing β-actin as reference gene, the relative expression results showed that the expression of 4 NK ligands between ALL and AML is significantly different. Specifically, the level of ULBP-2 is higher in ALL (CEM: 1, Jurkat T: 0.617, Reh: 0.246) than that in AML (HL-60: 0.000, KG-1a: 0.003, NB4: 0.000)(P = 0.047). However, the expressions of CD48, PVR(PVR-1, PVR-2) and DR4 is higher in AML (HL-60: 13.987, 4.403, 10.334, 8.711; KG-1a: 5.387, 2.900, 7.315, 4.512; NB4: 7.763, 3.248, 7.049, 6.127) than that in ALL (CEM: 1, 1, 1, 1; Jurkat T: 2.035, 1.553, 3.888, 0.449; Reh: 1.559, 0.000, 0.000, 1.304) (P = 0.044, 0.014, 0.014, 0.011). And there're no significant differences between the rest 19 NK ligands.

CONCLUSIONSULBP-2, CD48, PVR and DR4 might play an important role in the distinct mechanisms in leukemogenesis between ALL and AML and could be potential targets for diagnosis and treatment.

Acute Disease ; Antigens, CD ; genetics ; metabolism ; CD48 Antigen ; Cell Line, Tumor ; GPI-Linked Proteins ; genetics ; metabolism ; HL-60 Cells ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Leukemia ; genetics ; metabolism ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Ligands ; Membrane Proteins ; genetics ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Receptors, Virus ; genetics ; metabolism