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*

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy. The identification of small molecules that can establish the sensitivity of prostate cancer (PCa) cells to TRAIL-induced apoptosis is crucial for the targeted treatment of PCa. PC3, DU145, JAC-1, TsuPr1, and LNCaP cells were treated with Andrographolide (Andro) and TRAIL, and the apoptosis was measured using the Annexin V/PI double staining method. Real time-polymerase chain reaction (PCR) and Western blot analysis were performed to measure the expression levels of target molecules. RNA interference technique was used to down-regulate the expression of the target protein. We established a nude mouse xenograft model of PCa, which was used to measure the caspase-3 activity in the tumor cells using flow cytometry. In this research study, our results demonstrated that Andro preferentially increased the sensitivity of PCa cells to TRAIL-induced apoptosis at subtoxic concentrations, and the regulation mechanism was related to the up-regulation of DR4. In addition, it also increased the p53 expression and led to the generation of reactive oxygen species (ROS) in the cells. Further research revealed that the DR4 inhibition, p53 expression, and ROS generation can significantly reduce the apoptosis induced by the combination of TRAIL and Andro in PCa cells. In conclusion, Andro increases the sensitivity of PCa cells to TRAIL-induced apoptosis through the generation of ROS and up-regulation of p53 and then promotes PCa cell apoptosis associated with the activation of DR4.
Animals ; Antineoplastic Agents/pharmacology* ; Apoptosis/drug effects* ; Cell Line, Tumor ; Diterpenes/pharmacology* ; Drug Synergism ; Humans ; Male ; Mice ; Mice, Nude ; Neoplasm Transplantation ; PC-3 Cells ; Prostatic Neoplasms/metabolism* ; Reactive Oxygen Species/metabolism* ; Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism* ; TNF-Related Apoptosis-Inducing Ligand/pharmacology* ; Tumor Suppressor Protein p53/metabolism* ; Xenograft Model Antitumor Assays

OBJECTIVETo study the molecular mechanism of cisplatin to enhance the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in reversing multidrug resistance in vincristine-resistant human gastric cancer SGC7901/VCR cells.

METHODSMTT assay was used to measure the 50% inhibiting concentration (IC₅₀) and cell survival in SGC7901 and SGC7901/VCR cells after different treatments. SGC7901/VCR cells were treated with different concentrations of DDP, different concentrations of TRAIL alone or in combination, and then the mRNA and protein levels of several genes were determined by RT-PCR, RT-qPCR and Western-blot analysis. After targeted silencing with specific siRNA and transfection of recombinant plasmid c-myc into the SGC7901/VCR cells, the mRNA and protein levels of DR4, DR5 and c-myc were determined by RT-PCR and Western-blot analysis.

RESULTSAfter combined treatment with TRAIL and DDP of the SGC7901/VCR cells, the IC₅₀ of VCR, DDP, ADM, and 5-Fu treatment was significantly decreased compared with the control group or TRAIL-treated group (P < 0.05). After treatment with 0, 10, 50 ng/ml TRAIL in combination with 0.4 µg/ml DDP, the SGC7901/VCR cells showed significantly higher activation of caspase 3, down-regulation of DNA-PKcs/Akt/GSK-3β signaling pathway, and higher inhibition of MDR1(P-gp) and MRP1 than those treated with TRAIL alone (P < 0.01 for all). The mRNA and protein levels of DR4, DR5, c-myc were significantly decreased after silencing c-myc with specific siRNA in the SGC7901/VCR cells (P < 0.01 for all), and were significantly increased after transfection of recombinant plasmid c-myc into the SGC7901/VCR cells (P < 0.01 foe all). After the treatment with 10 ng/ml TRAIL, 0.25 µg/ml DDP + 10 ng/ml TRAIL and 0.5 µg/ml DDP + 10 ng/ml TRAIL, the relative expression level of c-myc protein in the SGC7901/VCR cells was 0.314 ± 0.012, 0.735 ± 0.026, and 0.876 ± 0.028, respectively, and the relative expression of cytochrome C was 0.339 ± 0.036, 0.593 ± 0.020 and 0.735 ± 0.031, respectively, and the relative expression levels of DR4, DR5, active-caspase 3 and active-caspase 9 in the SGC7901/VCR cells were also increased along with increasing DDP concentrations.

CONCLUSIONSThe activation of DNA-PKcs/Akt/GSK-3β signaling pathway and high expression of MDR1 and MRP1 play an important role in the multi-drug resistance properties of SGC7901/VCR cells. After combining with TRAIL, DDP can enhance the expression of DR4 and DR5 through up-regulating c-myc and enhancing the activation of caspase 3 and caspase 9 by facilitating mitochondrial release of cytochrome C. It may be an important molecular mechanism of DDP-induced sensitization of TRAIL to reverse the multidrug resistancein SGC7901/VCR cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; administration & dosage ; pharmacology ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cisplatin ; administration & dosage ; pharmacology ; Down-Regulation ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Fluorouracil ; administration & dosage ; pharmacology ; Formazans ; Genes, myc ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; Inhibitory Concentration 50 ; Multidrug Resistance-Associated Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Plasmids ; Proto-Oncogene Proteins c-myc ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Stomach Neoplasms ; drug therapy ; pathology ; TNF-Related Apoptosis-Inducing Ligand ; administration & dosage ; pharmacology ; Tetrazolium Salts ; Transfection ; methods

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 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 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

OBJECTIVETo investigate the effect of doxorubicin on TRAIL resistance and TRAIL receptor expression in lymphoma cell line SNK-6 cells.

METHODSSNK-6 cells treated with doxorubicin at different concentrations alone or in combination with tumor necrosis factor related apoptosis inducing ligand (TRAIL). Cell proliferation was evaluated by MTT assay. Apoptosis and the expression of TRAIL receptors were determined by flow cytometry.

RESULTSMTT assay showed that treatment with 100 and 1000 ng/ml doxorubicin for 24 h, the survival rates of SNK-6 cells were (80.9 ± 7.2)% and (53.7 ± 2.8)%, significantly higher than that by treatment combined with 500 ng/ml TRAIL (64.9 ± 1.1)% and (34.0 ± 3.9)%, respectively (P < 0.05). Flow cytometry showed that after treatment with 100 and 1000 ng/ml doxorubicin for 48 h, the survival rates of SNK-6 cells were (69.9 ± 6.1)% and (31.1 ± 1.9)%, while treated in combination with 500 ng/ml TRAIL, the cell survival rates were (37.5 ± 6.4)% and (15.0 ± 1.8)%, respectively. The early apoptosis rate was (14.8 ± 0.6)% and (30.8 ± 1.5)%, significantly lower than that [(28.7 ± 0.6)% and (46.6 ± 2.8)%] after treatment in combination with TRAIL (P < 0.05). The expressions of TRAIL receptors and decoy receptors were increased when SNK-6 cells were treated with 100 ng/ml doxorubicin for 24 hours.

CONCLUSIONSDoxorubicin can overcome to a certain extent the TRAIL resistance of SNK-6 cells and induce upregulation of TRAIL death receptors and decoy receptors on the surface of SNK-6 cells. However, a higher dose is needed.

Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Doxorubicin ; administration & dosage ; pharmacology ; Drug Resistance, Neoplasm ; Drug Synergism ; Humans ; Lymphoma, Extranodal NK-T-Cell ; metabolism ; pathology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Tumor Necrosis Factor Decoy Receptors ; metabolism

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

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

Induction of apoptosis in target cells is a key mechanism by which chemotherapy promotes cell killing. The purpose of this study was to determine whether Indole-3-Carbinol (I3C) and Genistein in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induce apoptosis in endometrial cancer cell (Ishikawa) and to assess apoptotic mechanism. The MTT assay and flow cytometry were performed to determine cell viability and cell cycle. The induction of apoptosis was measured by caspase-3 activity test, DNA fragmentation assay, annexin V binding assay and western blot analysis. There was no effect in cell growth inhibition and cell cycle progression alone or in two-combination. However, the treatment of I3C and Genistein followed by TRAIL showed significant cell death and marked increase in sub-G1 arrest. Three-combination treatment revealed elevated expression of DR4, DR5 and cleaved forms of caspase-3, caspase-8, PARP. The Flip was found down regulated. Moreover, increase in caspase-3 activity and DNA fragmentation indicated the induction of apoptosis. The results indicate that I3C and Genistein with TRAIL synergistically induced apoptosis via death receptor dependent pathway. Our findings might provide a new insight into the development of novel combination therapies against endometrial cancer.
Anticarcinogenic Agents/*pharmacology ; Apoptosis/*drug effects ; Caspase 3/metabolism ; Caspase 8/metabolism ; Cell Line, Tumor ; Drug Synergism ; Endometrial Neoplasms/metabolism/pathology ; Female ; G1 Phase Cell Cycle Checkpoints/drug effects ; Genistein/*pharmacology ; Humans ; Indoles/*pharmacology ; Poly(ADP-ribose) Polymerases/metabolism ; Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism ; TNF-Related Apoptosis-Inducing Ligand/*pharmacology