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

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

To study the effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on PC-3M cell line, PC-3M cell line was incubated with gradient concentrations of TRAIL for 4--24 h. Annixin-V fluorescence staining and TUNEL method were employed to detect the apoptosis of PC-3M cells. The morphology of apoptotic PC-3M cells was observed by electron microscopy. The relationship between TRAIL concentrations and the percentage of apoptotic cells was evaluated by flow cytometry. The proliferation inhibitory ratio was calculated by using MTT colorimetry. Our results showed that apoptosis of PC-3M cells could be induced by treatment with TRAIL for at most 4 h. The results of flow cytometry and MTT colorimetry demonstrated a time- and concentration-dependent relationship between cell apoptosis rate and TRAIL concentration. It is concluded that apoptosis of PC-3M cells can be induced by TRAIL. Because of the selective killing effect of TRAIL on tumor cells, it may become a potential alternative for the treatment of advanced prostate cancer.
Apoptosis/*drug effects ; Cell Line, Tumor ; Prostatic Neoplasms/metabolism ; Prostatic Neoplasms/*pathology ; 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 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 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 study the effect of recombinant tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) on the expression of apoptosis and inflammatory related genes in human colon cancer cell line HCT-116.

METHODSAfter 24-hour treatment with recombinant human TRAIL protein, the expressions of apoptosis-related genes(Bcl-2, Bad, caspase-3, and caspase-8) and inflammation-related genes(TNF-α, IL-1β, and COX-2) were measured by real-time PCR and appropriate kits in HCT-116.

RESULTSAfter treatments of 10 μg/L and 100 μg/L recombinant human TRAIL proteins, the apoptotic rates of HCT-116 cells were 27.4% and 45.9%, respectively. Expressions of anti-apoptosis gene Bcl-2, pro-apoptosis gene Bad and apoptotic markers caspase-3 and caspase-8 were significantly up-regulated, which was more significant in the group of 100 μg/L treatment(P<0.05). Moreover, after TRAIL treatments, expressions of inflammation-related genes TNF-α, IL-1β, COX-2 were also dramatically increased, and 100 μg/L treatment group showed higher up-regulation(P<0.05).

CONCLUSIONSRecombinant TRAIL protein induces both apoptosis and inflammation of human colon cancer cells.

Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Colonic Neoplasms ; metabolism ; pathology ; HCT116 Cells ; Humans ; Inflammation ; Recombinant Proteins ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology

This study was aimed to explore whether bortezomib can sensitize HL-60 cells to TNF-related apoptosis-inducing ligand (TRAIL) and to investigate its possible mechanism. The HL-60 cells were treated by different concentrations of TRAIL combined with subtoxic concentration of bortezomib. The proliferative inhibition of treated HL-60 cell was analysed by MTT assay. The cell apoptosis was determined by flow cytometry with Annexin V/PI double staining and the expression of caspase-8 was detected by Western blot. The results showed that the subtoxic concentration of bortezomib combined with 10 ng/ml of TRAIL enhanced apoptosis of HL-60 cells, as compared with TRAIL used alone; the expression of caspase-8 increased correspondingly. It is concluded that subtoxic concentration of bortezomib can sensitize HL-60 cells to TRAIL and its mechanism may be related to upregulation of caspase-8 expression.
Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Caspase 8 ; metabolism ; HL-60 Cells ; Humans ; Pyrazines ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology

OBJECTIVETo determine the sensitivity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis in Hep-2 cells by means of systematically evaluating the cytotoxicity of TRAIL alone and TRAIL in combination with chemotherapeutic agents (cisplatin, paclitaxel) or radiation in Hep-2 cells in vitro, and whether the synergistic killing effects correlated with the expression level of TRAIL receptors and the activity of caspase-8 or caspase-9.

METHODSThe cytotoxicities of TRAIL, cisplatin and paclitaxel were investigated by cell counting kit-8 (CCK-8) assay. The expression levels of four TRAIL receptors in Hep-2 cells after treated by TRAIL, chemotherapeutic agents or radiation alone and by combined treatments were measured by flow cytometry and Western blotting respectively. The growth inhibition effects of caspase-8 or caspase-9 inhibitor on Hep-2 cells were determined by CCK-8 assay, and the activities of caspase-8, caspase-9 and caspase-3 were measured by Western blotting.

RESULTSThe half maximal inhibitory concentration (IC50) of TRAIL to Hep-2 cells on 24 h was 596.92 microg/L. Cisplatin, paclitaxel and radiation had synergistic inhibitory effects with TRAIL on the growth of Hep-2 cell line. After the activity of caspase-9 was inhibited by Z-LETD-FMK, the inhibition effects of TRAIL, cisplatin and paclitaxel on Hep-2 cells decreased significantly (all P<0.05). The expressions of caspase-8, caspase-9 and the death receptors (DR4 and DR5) increased significantly (all P<0.05) after combined administration.

CONCLUSIONSHep-2 cells are resistant to the apoptosis induced by TRAIL and TRAIL can induce the apoptosis of Hep-2 cells through the endogenous apoptotic pathway. The increase of death receptors expression by chemotherapeutic agents or radiation could enhance the sensitivity of Hep-2 cells to TRAIL and the synergistic killing effects.

Antineoplastic Agents ; pharmacology ; Apoptosis ; Carcinoma, Squamous Cell ; metabolism ; pathology ; radiotherapy ; therapy ; Cell Line, Tumor ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; radiotherapy ; therapy ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology