OBJECTIVETo explore the mechanism of Alpha-fetoprotein (AFP) effects on hepatocellular carcinoma cells (HCC) resistances apoptosis induced by tumor necrosis factor-related apoptosis inducing-ligand (TRAIL).

METHODSThe expressed alteration of TRAIL receptor-2 (DR5) after the human hepatoma cells line Bel 7402 (AFP-producing) and HLE cells (non-AFP producing) were treated with all trans retinoic acid (ATRA) were determined by Western blot; Interaction of AFP with RAR-beta was analyzed by co-immunoprecipitation (Co-IP); Laser confocal microscopy was used to observe co-localization of AFP and RAR-beta; Short small RNA interfering (RNAi) was applied to knock down the expression of AFP in Bel 7402 cells; The full AFP gene cDNA was inserted into pcDNA3.1 vector and constructed the expressed vector of AFP (named pcDNA3.1-afp); The growth of hepatoma cells was analyzed by MTT.

RESULTSBel 7402 and HLE cells expressed DR5, lowed dosage of ATRA (40mumol/L) had no influence on the expression of DR5 in Bel 7402 cells, but ATRA (160mumol/L) could inhibit the expression of AFP and promote the expression of DR5 significantly; Co-IP indicated that AFP had a property for interacting with RAR-beta; The results also demonstrated AFP co-localization with RAR-beta in cytoplasm of Bel 7202 cells; The expression of DR5 was enhanced while the expression of AFP was knocked down by RNAi. pcDNA3.1-afp vector was transfected into HLE cells, the growth of HLE cells were stimulated and TRAIL cytotoxicity of HLE cells were reduced. But when the expression of AFP was knocked down the sensitivity of Bel 7402 cells to TRAIL was enhanced.

CONCLUSIONSThese data provided that AFP had a capability to interact with RAR-beta and suppressed the expression of DR5. AFP could play pivotal role on hepatoma cells resistance-induced apoptosis by TRAIL.

Apoptosis ; Cell Line, Tumor ; metabolism ; Humans ; Receptors, Retinoic Acid ; metabolism ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Tretinoin ; pharmacology ; alpha-Fetoproteins ; metabolism

PURPOSE: Current chemotherapeutics for treating locally advanced or metastatic soft tissue sarcomas (STS) are limited. Accordingly, the present in vitro study was conducted to evaluate the effects of treatment of STS cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) applied as a single agent or in combination with a proteasome inhibitor, MG132. MATERIALS AND METHODS: Sensitivity to TRAIL and activity of TRAIL-induced apoptotic pathways were analyzed in four STS cell lines: HTB-82 (rhabdomyosarcoma), HT-1080 (fibrosarcoma), HTB-93 (synovial sarcoma), and HTB-94 (chondrosarcoma). Reduction of the dye dimethylthiazolyl 2,5 diphenyltetrazolium bromide (MTT) was used to evaluate cytotoxic activity; western blots were used to evaluate TRAIL-induced apoptosis. RESULTS: TRAIL induced apoptosis in HTB-93 cells, but had little effect in HTB-82, HT-1080, or HTB-94 cells. Expression of TRAIL receptor-1 and -2 did not correlate with sensitivity to TRAIL. Co-incubation of cells with TRAIL and a proteasome inhibitor, MG132, augmented the apoptotic effect of TRAIL in both TRAIL-sensitive and TRAIL-resistant cells. This effect was due to up-regulation of TRAIL receptors and members of the pro-apoptotic BCL-2 family by MG132. CONCLUSION: These data show that combining TRAIL with MG132 enhances apoptosis and overcomes TRAIL resistance. This restoration of TRAIL sensitivity occurs through an increase in the expression of death receptor 5 and of pro-apoptotic BCL-2 family members such as BAX.
Apoptosis ; Blotting, Western ; Cell Line ; Humans ; Leupeptins ; Necrosis ; Proteasome Endopeptidase Complex ; Proteasome Inhibitors ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; Sarcoma ; TNF-Related Apoptosis-Inducing Ligand ; Up-Regulation

This study was aimed to investigate the mechanism of leukemia cell apoptosis induced by histone deacetylase inhibitor (HDACI). Flow cytometry was used to detect the apoptosis of leukemia cell lines NB4, U937 and Jurkat, and the changes of mRNA and protein expressions of TRAIL, DR4 and DR5 were detected by Western blot and RT-PCR respectively. The results showed that both TRAIL and DR5 protein and mRNA expressions in NB4, U937 and Jurkat cells increased after treated with sodium butyrate (SB) and in time-dependent manner. However, DR4 mRNA in leukemia cells was not significantly changed after treated with SB. It is concluded that the apoptosis mechanism of leukemic cell lines NB4, U937 and Jurkat induced by SB is closely related to the protein and mRNA expressions up-regulating TRAIL and DR5, but the DR4 may not participate in the apoptosis induced by SB.
Apoptosis ; drug effects ; Cell Line, Tumor ; Histone Deacetylase Inhibitors ; pharmacology ; Humans ; Isobutyrates ; pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; metabolism

OBJECTIVE: To investigate the effect of lycium barbarum polysaccharide (LBP) alone or combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the apoptosis of leukemia cell lines with MLL gene-rearrangement, and to explore the cell apoptotic pathway after the combined action. METHODS: MLL-ALL cell line KOCL44 and KOCL45 were selected as the research object, then the control and experimental groups were set up. The cell survival rate was measured by the trypan blue dye exclusion method, the cell early apoptosis and expression of death receptors on the cell surface were detected by flow cytometry with Annexin-V/PI double staining. The protein level of caspase-8, BID, caspase-3, caspase-9, BAD, BCL-2, as well as mitochondrial and cytosol Cyto-C were detected by Western blot. RESULTS: LBF combined with TRAIL inhibited the growth of KOCL44 and KOCL-45 cells and showed the synergistic effect, the results of flow cytometry with Amnexiu V/PI double staining were consistent with above-mentioned results. After treatment of KOCL44 and KOCL45 cells with LBF plus TRAIL, the significant expression of DR4 on cell surface was not found, while the expression of DR4 receptor was enhanced significantly, the pro-apoptotic proteins including caspase-8, BID, caspase-3, caspase-9 and BAD were activated significantly and BCL-2 was suppressed significantly with time-dependent manner. The expression of mitochondria cyto-C in KOCL44 and KOCL45 decreased along with prolonging of treatment time (r=-0.95, r=-0.866), while the expression of cytosol cyto-C in KOCL44 and KOCL45 increased along with prolonging of treatment time (r=0.883, r=0.903). CONCLUSION The combination of LBP and TRAIL significantly increases the apoptosis of KOCL44 and KOCL45, and the LBP and TRAIL can up-regulate the expression of TRAIL death receptor-DR5 on the cell surface, activate the pathway of caspase and mito-chrondia mitachondria, thus enhance the sensitivity of KOCL44 and KOCL45 to TRAIL induced apoptosis through both mitochondrial and apoptotic pathway.
Apoptosis ; Caspase 8 ; Drugs, Chinese Herbal ; Leukemia, Myeloid, Acute ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha

OBJECTIVE: To investigate the effect of tumor necrosis factor death receptor (DR) 4 demethylation to the proliferation and apoptosis of myeloid leukemia K562 cells. METHODS: The logarithmic phase of K562 cells were treated by desitabine (DCA) at 0, 0.8, 1.6 and 3.2 μmol/L, and the cells were divided into control group, DCA low dose group, DCA medium dose group and DCA high dose group respectively. The cells in control group were treated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 0.5 μg/ml for 24 h, and the cells were divided into TRAIL group. The cells in DCA high dose group were treated by TRAIL 0.5 μg/ml for 24 h, and were divided into DCA high dose + TRAIL group. Methylation-specific polymerase chain reaction (MS-PCR) was used to measure the methylation status of the DR4 gene promoter in the control group and DCA low, medium and high dose groups. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to determine the relative expression of DR4 mRNA and protein in the control group and DCA low, medium and high dose groups. Dime- thylthiazole (MTT) method was used to determine the inhibition rate of cell proliferation of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group. Flow cytometry was used to determine the apoptotic rate of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group. RESULTS: The cells in the control group were methylation-positive, the brightness of the methylation bands of the cells in the DCA low, medium, and high dose groups was gradually decreased to disappear, and the DCA high dose group showed negative for methylation. The relative expression of DR4 mRNA and protein in the control group, DCA low, medium and high dose groups was increased sequentially (r=0.624, 0.704). The inhibition rate of cell proliferation of the cells in the control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group was increased sequentially (r=0.653, 0.754, 0.709, 0.725) at 24, 48 and 72 h. CONCLUSION DCA can reverse the methylation level of DR4 gene promoter in ML K562 cells and up-regulate the expression of DR4, which may enhance the proliferation inhibition and apoptosis promotion effects of TRAIL on K562 cells.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Demethylation ; Humans ; K562 Cells ; Leukemia, Myeloid ; Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism* ; TNF-Related Apoptosis-Inducing Ligand/metabolism*

Human uterus has been known as a immune privileged site for the product of conception. At the feto-maternal interface, Fas system is a underlying main mechanism of maternal immune acceptance. To date, the TRAIL (TNF-related apoptosis-inducing ligand) system is known to be another pivotal mechanism. OBJECTIVE: To clarify the protein expression of TRAIL ligand and receptors in the normal and pathologic (preeclampsia, hydatidiform mole) placenta, chorioamnion, decidua. METHODS: we investigated the expression of TRAIL system in the above-mentioned tissues by using Western Hybridization. RESULTS: All tissues expressed TRAIL ligand and only a DcR2 among TRAIL receptors (DR4, DR5, DcR1, DcR2). CONCLUSION: we demonstrated the expression of TRAIL ligand and DcR2 protein at the feto-maternal interface of the normal and pathologic pregnancies. Further study regarding the expression of other receptors and quantitative analysis between normal and pathologic pregnancies should be followed.
Apoptosis* ; Decidua ; Female ; Fertilization ; Gestational Trophoblastic Disease* ; Humans* ; Placenta ; Pregnancy ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; Uterus

BACKGROUND: Hispolon has been shown to possess antitumor effects in various cancer cells. However, the underlying mechanisms are not fully understood. In this study, we evaluated the sensitizing effect of hispolon on TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human renal carcinoma cells. METHODS: Apoptosis was analyzed by using cell-based cytometer. The mRNA levels were assessed by reverse transcription-PCR. Bax activation was determined by oligomerization and fluorescence-activated cell sorting with Bax-NT monoclonal antibody. The protein expression was measured by Western blotting. RESULTS: Hispolon induced up-regulation of Bim and death receptors expression at the post-translational level. CONCLUSIONS: Hispolon enhanced TRAIL-mediated apoptosis in renal carcinoma cells, but not in normal cells.
Apoptosis ; Blotting, Western ; Flow Cytometry ; Humans ; Receptors, Death Domain ; RNA, Messenger ; TNF-Related Apoptosis-Inducing Ligand ; Up-Regulation

BACKGROUND AND OBJECTIVES: Chronic impairment of beta-adrenergic receptor signaling increases cardiac apoptosis, hypertrophy and fibrosis. The aim of this study was to investigate whether isoproterenol (ISO), an agonist of the adrenergic receptor, can enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human embryonic kidney (HEK) 293 cells. MATERIALS AND METHODS: HEK 293 cells were treated with ISO and/or TRAIL for 24 hours. Cell viability was evaluated by microscopy and an established viability assay, and apoptotic cell death was analyzed by staining with fluorescein isothiocynate-annexin-V/propidium iodide (PI) and caspase activation. To confirm the mechanism of cell death induced by co-treatment with ISO and TRAIL, expression of TRAIL receptor 2 (death receptor 5, DR5) was evaluated by immunoblotting. RESULTS: Although ISO or TRAIL treatment decreased HEK 293 cell viability by 13% and 17%, respectively, co-treatment with ISO and TRAIL resulted in a markedly higher death rate of 35% after 24 hours. Increases were evident in early apoptotic cells (i.e., annexin-V positive/PI negative; 19.4%), late apoptotic cells (i.e., annexin-V positive/PI positive; 6.3%) and dead cells (i.e., annexin-V negative/PI positive; 1.1%) when cells were co-treated with ISO and TRAIL, compared to cells treated with either ISO or TRAIL. In addition, marked increases of cleaved cas-3, cleaved poly (adenosine diphosphate-ribose) polymerase and DR5 were observed in HEK 293 cells co-treated with ISO and TRAIL. CONCLUSION: Treatments combining ISO with TRAIL may be responsible for death of HEK 293 cells through DR5 up-regulation. Activation of adrenergic receptors is responsible for the synergistic cell death observed with TRAIL.
Apoptosis* ; Cell Death ; Cell Survival ; Fibrosis ; Fluorescein ; HEK293 Cells ; Humans* ; Hypertrophy ; Immunoblotting ; Isoproterenol* ; Kidney* ; Microscopy ; Mortality ; Necrosis* ; Receptors, Adrenergic ; Receptors, TNF-Related Apoptosis-Inducing Ligand* ; TNF-Related Apoptosis-Inducing Ligand ; Up-Regulation*

TNF-related apoptosis-inducing ligand (TRAIL) is a newly identified member of the tumor necrosis factor (TNF) family. TRAIL induces apoptosis by activating caspase cascades, stimulating a loss of mitochondrial membrane potential (Delta Psim) and cytochrome C release in the FADD/caspase-8 dependent pathway. However, TRAIL can also trigger transcriptional activations of the pro-oncogene of c-fos, JNK, and NF-kappaB by other signaling pathways downstream of FADD/caspase-8. MAPK/ERK activation has a dominant protecting effect over apoptotic signaling from the death receptors. The functional expression of TRAIL by leukemic cells may be involved in tumor cells evasion of immunosurveillance. Somatic mutations of TRAIL-R1 and TRAIL-R2 genes may play a role in the pathogenesis of some tumors. TRAIL can induce apoptosis on various continuous transformed cell lines and primary tumor cells, including several of hematopoietic origin, displaying minimal toxic effects on normal tissues. Because of the abilities of induction of both cytotoxic (apoptosis) and cytostatic (cell cycle perturbation) effects on the leukemic cells, TRAIL is currently considered as a potential(co) therapeutic drug against tumors.
Animals ; Apoptosis Regulatory Proteins ; Hematologic Neoplasms ; etiology ; therapy ; Humans ; Membrane Glycoproteins ; physiology ; Mutation ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; Receptors, Tumor Necrosis Factor ; genetics ; physiology ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha ; physiology

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF super family found in recent years, which widely exists in the body tissues and participates in the immune regulation, immune stability, and immune surveillance of the human body. The TRAIL receptor is expressed in the surface of a variety of cells. Recent studies show that TRAIL induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. Its anti-tumor activity and safety have been widely recognized. The development of prostate cancer is regulated by the mechanisms of cell apoptosis. TRAIL can induce the apoptosis of prostate cancer cells, and therefore has a great application value in the treatment of prostate cancer.
Antineoplastic Agents ; therapeutic use ; Apoptosis ; Apoptosis Regulatory Proteins ; Humans ; Male ; Membrane Glycoproteins ; Prostatic Neoplasms ; drug therapy ; pathology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; physiology ; therapeutic use ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha