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 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

The objective of this study was to investigate the synergistic effect of soluble human recombinant tumor necrosis factor related apoptosis inducing ligand (TRAIL) protein combined with anti-vascular endothelial growth factor (anti-VEGF) antibody on inducing apoptosis of leukemia K562 cells. The inhibitory rates and apoptotic rates of K562 cells treated with TRAIL and anti-VEGF antibody alone and their combination for 48 hours were examined by CCK-8 assay and flow cytometry respectively. The results indicated that the apoptotic rates of K562 cells induced with 75, 100 and 150 ng/ml TRAIL after culture for 48 hours were (4.26±0.67)%, (8.91±0.55)% and (11.71±0.78)% respectively. The apoptotic rates of K562 cells induced with 2.5, 5 and 7.5 µg/ml anti-VEGF antibody after culture for 48 hours were (3.95±0.69)%, (7.98±0.74)% and (10.26±0.83)% respectively. The apoptotic rates of K562 cells treated with combination use of 2.5 µg/ml anti-VEGF antibody and 75 ng/ml TRAIL, 5 µg/ml anti-VEGF antibody and 100 ng/ml TRAIL, and 7.5 µg/ml anti-VEGF antibody and 150 ng/ml TRAIL for 48 hours were (22.16±0.93)%, (36.32±1.31)% and (49.19±0.71)% respectively. The combined use of above mentioned agents induced significantly higher apoptosis and cytotoxicity than that of TRAIL or anti-VEGF antibody alone (p<0.05). It is concluded that the combination use of TRAIL and anti-VEGF antibody can significantly increase the sensitivity of K562 cells to apoptosis.
Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Humans ; K562 Cells ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Vascular Endothelial Growth Factor A ; immunology

The aim of study was to investigate the combined effect of recombinant mutant human TRAIL (rmhTRAIL) with daunorubicin (DNR) or alone on K562 and U937 leukemia cell lines and its mechanism. The fibroblasts (MRC-5) of normal-human embryonic lung were used as control cells. After being treated with rmhTRAIL and DNR or only with rmTRAIL, the cytotoxic effect and the apoptosis rate in K562, U937 cells were measured by MTT assay. The expression levels of TRAIL death receptor and TRAIL decoy receptor mRNA in these three cell lines were assayed by semiquantitive RT-PCR before and after treatment with DNR. The results indicated that K562 and U937 were sensitive to rmhTRIAL. DNR had synergistic inhibitory effect with rmhTRAIL on the growth of K562 and U937 cell lines (P < 0.05). The expression level of DR4 and DR5 mRNA was significantly higher in K562 and U937 with combined treatment of rmhTRAIL and DNR than that in those alone, while the expressions of DcR1 and DcR2 mRNA were not influenced. It is concluded that in vitro, rmhTRAIL alone or in combination with DNR can obviously inhibit the growth of leukemia cell lines and induce cell apoptosis, DNR and rmhTRAIL have a synergistic inhibitory effect on growth of K562 and U937. The mechanism may correlate with the up-regulation of DR4 and DR5 of K562 and U937.
Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Daunorubicin ; pharmacology ; Drug Synergism ; Humans ; K562 Cells ; Mutation ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics ; Recombinant Proteins ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; U937 Cells ; Up-Regulation

OBJECTIVE: To study the apoptosis inducing effect of tumor necrosis factor related apoptosis-ligand (TRAIL) on human laryngeal squamous carcinoma Hep-2 cells and its effect mechanism. METHOD: The human laryngeal squamous carcinoma Hep-2 cell line was treated with different concentration of TRAIL in vitro. The inhibition ratio of tumor cells was determined by MTT colorimetric assay, the incidence of cell apoptosis was determined by flow cytometry method. The morphologic changes of laryngeal squamous carcinoma Hep-2 cell were observed with transmission electron microscope. RESULT: In vitro, all the different concentrations of TRAIL inhibited laryngeal squamous carcinoma cell's growth. The inhibited growth ratio showed significant concentration-dependence. The concentrations for inducing apoptosis-ratio(TRAIL 1, 10, 100 microg/L) determined by flow cytometry was (11.49 +/- 0.36)%, (22.31 +/- 0. 82)%, (59.64 +/- 1.10)% respectively in the study group, and (3.13 +/- 0.12)% in the control group, which was significantly different between these two groups (P < 0. 01). CONCLUSION In vitro, TRAIL inhibited the growth of human laryngeal squamous carcinoma Hep-2 cells. The induced apoptosis of TRAIL shows significant concentration- independence. TRAIL inhibits the growth of human laryngeal squamous carcinoma Hep-2 cells trough inducing apoptosis.
Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; pathology ; Cell Line, Tumor ; Humans ; Laryngeal Neoplasms ; pathology ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology

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

OBJECTIVETo investigate synergistic killing effect of anti-human DR5 (death receptor 5 of TRAIL) monoclonal antibody (mDRA-6) and adriamycin(Adr) on HL-60 cells.

METHODSmDRA-6 was prepared by immunizing BALB/c mice with DR5 protein. DR5 expression on Adr-treated HL-60 cells was detected by flow cytometry. Morphologic changes of HL-60 cells were observed under fluorescence microscope. Cytotoxic and apoptotic effects of mDRA-6 and Adr on HL-60 cells were measured by MTT analysis. DNA fragmentation was detected by agarose gel electrophoresis.

RESULTSAdr induce DR5 expression on HL-60 cells. Cell budding, chromatin condensation and apoptotic body formation were observed in HL-60 cells treated by mDRA-6 and Adr. Death and apoptosis of these cells and DNA ladder were exhibited on agarose gel electrophoresis.

CONCLUSIONmDRA-6 and Adr have synergistic killing effect on HL-60 cells.

Animals ; Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Dose-Response Relationship, Drug ; Doxorubicin ; pharmacology ; HL-60 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; immunology ; TNF-Related Apoptosis-Inducing Ligand ; immunology

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 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