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

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 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*

OBJECTIVETo explore the effects of mitochondrial pathways on apoptosis in colon carcinoma cells induced by Tumor necrosis factor related apoptosis inducing ligand and offer evidences for TRAIL application in clinic.

METHODSApoptosis, integration of mitochondria (including DeltaPsim, cardiolipin), activity of Caspase-9 and release of cytochrome c in colon carcinoma cells SW1116 treated with TRAIL, were detected by means of flowcytometry, fluorometer method and western-blot at the different time point.

RESULTSAfter treated with TRAIL for 4 hours, the apoptosis index was 32.98%, and the damage of mitochondria occurred with DeltaPsim, cardiolipin decreased, and the activity of Caspase-9 and cytochrome c increased. The Caspase-9 activity at 24 hour was (48.12+/-2.21)micromol.L(-1).h(-1).mg(-1) protein. Mitochondrial damage induced by TRAIL could be inhibited by Caspase inhibitor Z-VAD. fmk.

CONCLUSIONMitochondrial pathways involved in the apoptosis of colon carcinoma cell induced by TRAIL. Cytochrome c was released and Caspase-9 was activated in the Caspase-dependent manner after the damage of mitochondrial.

Apoptosis ; Cardiolipins ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Humans ; Mitochondria ; metabolism ; Mitochondrial Membranes ; metabolism ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; metabolism

OBJECTIVETo explore the enhanced sensitivity of leukemia cell line KG-1a to activated immune cell-mediated cytolysis after treated with resveratrol.

METHODSThe value of 50% inhibition concentration (IC₅₀) for KG-1a by resveratrol was analyzed using trypan blue staining. Peripheral blood mononuclear cells were separated, and then activated by interleukin (IL)-2 and IL-15. The sensitivity of KG-1a treated with and without resveratrol to activated immune cell-mediated cytolysis was assayed by lactate dehydrogenase (LDH) -releasing assay. The expression of tumor necrosis factor related apoptosis inducing ligand (TRAIL) on the surface of activated immune cells and its receptors (DR4/5 and DcR1/2) on the surface of KG-1a were detected by flow cytometry.

RESULTSResveratrol could inhibit the proliferation of KG-1a and IC50 at 24 h was 25 mmol/L. At a ratio of 10:1 or 20:1 between effect and target, the cytolytic rates of treated KG-1a by activated immune cells were (55.80 ± 10.88)% and (72.31 ± 13.06)%, significantly higher than (24.96 ± 9.25)% and (37.93 ± 5.21)% of untreated KG-1a (P<0.05). The expression of DR5 on the surface of KG-1a treated with resveratrol was (9.05 ± 3.57)%, significantly higher than (3.11 ± 0.54)% of untreated KG-1a (P<0.05). Conversely, the expression of DcR1 on the surface of treated KG-1a was (13.23 ± 3.56)%, lower than (53.75 ± 10.51)% of KG-1a (P<0.05). When TRAIL pathway on the surface of activated immune cells was blocked, the cytolytic rates of treated KG-1a were (35.97 ± 6.36)% and (49.80 ± 10.68)%, significantly lower than (52.92 ± 6.98)% and (70.73 ± 9.79)% of untreated KG-1a (P<0.05) at the same ratio of effector and target.

CONCLUSIONResveratrol could enhance cytolytic sensitivity of KG-1a by activated immune cells through TRAIL pathway.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Leukemia ; metabolism ; pathology ; Leukocytes, Mononuclear ; drug effects ; immunology ; metabolism ; Male ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Receptors, Tumor Necrosis Factor, Member 10c ; metabolism ; Stilbenes ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; metabolism

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 investigate the effect of 2-deoxy-D-glucose (2-DG) in enhancing the sensitivity of oral cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis.

METHODSThe oral cancer cell line KB was incubated in the presence of different concentrations (0, 0.625, 1.25, 2.5, 5, and 10 mmol/L) of 2-DG with or without TRAIL (200 ng/ml). The cell viability was measured using MTT assay and cell apoptosis was detected using flow cytometry with propidium iodide (PI) staining. KB cells treated with 5 mmol/L 2-DG with or without TRAIL for 0, 6, 16, or 24 h were examined with Western blotting for protein expressions of death receptor 5 (DR5) and caspase-3.

RESULTSTreatment of the cells with 5 mmol/L 2-DG for 24, 48 and 72 h resulted in a cell viability of 25.25%, 69.06%, and 59.19%, respectively. Combined treatment with 5 mmol/L 2-DG with TRAIL for 24 significantly enhanced the cell apoptotic rate (72.5%) as compared to the rate induced by TRAIL alone (45.3%) and by 2-DG (15.9%) alone. 2-DG treatment markedly up-regulated DR5 and caspase-3 expression and enhanced the inhibitory effect of TRAIL on cell colony formation.

CONCLUSION2-DG sensitizes oral cancer cells to TRAIL- induced apoptosis by up-regulating DR5 and caspase-3 expressions.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Deoxyglucose ; pharmacology ; Drug Synergism ; Gene Expression Regulation, Neoplastic ; Humans ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology

To investigate the cell-killing effect and its possible mechanism of rClone30-hDR5 in combination with TRAIL on human hepatic carcinoma (HCC) cell line, first of all, recombinant plasmid pee12.4-hDR5 was introduced into HepG2 cells by liposome transfection. After five rounds of screening by flow cytometry, HepG2 cells expressing high levels of DR5 on cell surface were isolated. The cytotoxicity of TRAIL to selected cells was higher than that of TRAIL to HepG2 cells by MTT method (P < 0.01). The result suggested that the cloned hDR5 gene had biological activity. MTT assay showed that, rClone30- hDR5 in combination with TRAIL more efficiently inhibited the tumor growth of HepG2 cells compared to rClone30-hDR5 or TRAIL in vitro. The results of Annexin V-FITC/PI staining and Quantitative Real-time PCR indicated that rClone30-hDR5 in combination with TRAIL significantly increased the mRNA levels of caspase 3 and caspase 8, and induced the apoptosis of tumor cells. HepG2 cells were infected with rClone30-hDR5 or rClone30 at MOI of 1. The expression of hDR5 on tumor surface increased significantly by rClone30-hDR5 compared to that by rClone30, which contributed to the sensitivity to TRAIL. In conclusion, rClone30-hDR5 in combination with TRAIL has potential application value in cancer treatment.
Apoptosis ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Drug Synergism ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; Real-Time Polymerase Chain Reaction ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Transfection

OBJECTIVETo evaluate the effects of hypobaric hypoxia on the expressions of death receptor 5 (DR5) and cellular FLICE-like inhibitory protein (c-FLIP) and the distribution of c-FLIP in the rat testis.

METHODSForty adult male SD rats were randomly divided into four groups of equal number: normoxia control, 3 d hypoxia, 15 d hypoxia and 30 d hypoxia. The control rats were raised at 300 m above the sea level, while the latter three groups of rats in a hypobaric chamber at a simulated altitude of 4000 m for 5, 15 and 30 days, respectively. Then the expressions of DR5 and c-FLIP were detected by immunoblotting and the distribution of c-FLIP in the testis observed by immunofluorescence.

RESULTSThe expressions of DR5 were 2.04 +/- 0.11, 1.97 +/- 0.12 and 2.34 +/- 0.11 in the 3 d, 15 d and 30 d hypoxia groups, respectively, significantly higher than 1.78 +/- 0.09 in the normoxia group (P < 0.05). The expressions of c-FLIP were 0.87 +/- 0.03 and 0.74 +/- 0.07 in the 15 d and 30 d hypoxia groups, respectively, significantly lower than 1.03 +/- 0.02 in the normoxia group (P < 0.05).

CONCLUSIONSimulated hypobaric hypoxia at 4000 m above the sea level increased the expression of DR5 and inhibited that of c-FLIP in the rat testis.

Animals ; CASP8 and FADD-Like Apoptosis Regulating Protein ; metabolism ; Hypoxia ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Testis ; metabolism