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

Escherichia coli was genetically engineered to produce recombinant tumor necrosis factor-related apoptosis inducing ligand (Apo2L/TRAIL) using a temperature-inducible expression system. To create a fed-batch culture condition that allows efficient production of TRAIL, different feeding strategy including discontinuous, DO-stat and pH-stat feeding strategies were compared. Then, a special 2-stage feeding strategy was developed. High concentration of biomass (300g wet cell weight per liter of culture broth) and active soluble TRAIL protein (1.1g/L) was obtained by applying a high-cell-density cultivation procedure with the 2-stage feeding strategy. Cultivation of recombinant E. coli was started as a batch process at 30 degrees C and then followed by fed-batch culture when the dissolved oxygen concentration presented a steep increase resulted from the exhaustion of glucose in the medium. At the first phase of fermentation (batch phase), agitation rate was enhanced to control dissolved oxygen at 30 percent. When glucose in the medium was used up, indicated by a sudden rise in pH value and dissolved oxygen, the second phase (fed-batch phase) was started with glucose and nitrogen resource being supplied automatically. At the beginning of fed-batch operation, stirrer rate was cascaded with dissolved oxygen signals to keep it at 20 percent (DO-stat). During the fed-batch phase, glucose was limited to control the specific growth rate under the critical value microcrit, to avoid acetic acid excretion. When the stirrer speed arrived at its up-limit, the flow rate of feed was kept constant. In the inducing phase(42 degrees C for 4h) glucose was fed as a pH regulating agent (pH-stat) and the specific growth rate and dissolved oxygen decreased sharply. Aqueous ammonia was used for maintaining pH value at 7.0 throughout the first two phases. In the whole fermentation, acetic acid concentration didn't exceed 2.9 g/L. At the end of the high-cell-density cultivation process, no acetic acid could be detected in the medium. These results indicated that our fed-batch strategy was able to prevent acetate accumulation significantly. Although high cell density has been achieved, the induction process was not optimized satisfactorily and much work should be done further. Furthermore, since no special ways, like pure oxygen, pressure, has been used in our experiments, this efficient approaches would be useful not only in a pilot scale but also in an industry scale. Finally, simple purification procedure based on immobilized metal affinity column (IMAC) and CM-Sepharose column was implemented to isolate the TRAIL. Yields of more than 800mg TRAIL per liter of culture broth were obtained, the final purity reaching more than 95%. The purified TRAIL showed strong cytotoxity activity against human pancreatic 1990 tumor cells, with ED50 about 1.6 microg/mL.
Escherichia coli ; genetics ; growth & development ; metabolism ; Fermentation ; Genetic Engineering ; methods ; Humans ; Recombinant Proteins ; biosynthesis ; chemistry ; isolation & purification ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics

To construct soluble TNF related apoptosis inducing ligand (TRAIL) expression system and investigate the effect of the expression product on tumor cell. It may provide valuable information for research into the immune system of the finless porpoise. The full-length cDNA of TRAIL (designated fTRAIL) was cloned from the total RNA of the finless porpoises blood using RT-PCR techniques and then the extracellular soluble fragments of fTRAIL (designated fsTRAIL) was ligated into pET43.1a. Recombinant soluble fTRAIL (pET43.1a-fsTRAIL) fused with Nus-his tag was efficiently expressed in Escherichia coli BL21 (DE3) and the Nus-His-fsTRAIL protein was purified. The expression of Nus-His-fsTRAIL was verified by Western blotting. In vitro, the effects of the purified Nus-His-fsTRAIL protein on Jurkat and HeLa cells were etected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) assay, TrypanBlue and Flow Cytometry analysis. The expression system pET43.1a-fsTRAIL was constructed and Nus-His-fsTRAIL protein was expressed successfully. In vitro, the Nus-His-fsTRAIL protein was able to inhibit the proliferation and induce apoptosis of Jurkat and HeLa cells in a dose-dependent manner. The Nus-His-fsTRAIL protein has anti-tumor activity against Jurkat and HeLa cells in vitro.
Animals ; Apoptosis ; Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; HeLa Cells ; Humans ; Jurkat Cells ; Porpoises ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis

OBJECTIVETo investigate the antitumor efficacy of death receptor 5, its ligand (TRAIL) and DR5mAb in human hepatocellular carcinoma.

METHODSExpression of DR5 in the HCC cell lines HepG2, SMMC 7721 and normal human liver cell line LO2 was measured at mRNA and protein level by semi-quantitative RT-PCR and Western blot, respectively. MTT method was used to measure the cell viability and flow cytometry assay was used to detect apoptosis so as to observe the inhibitory effect of TRAIL and DR5mAb on HCC cells.

RESULTSDeath receptor 5 was highly expressed in the HCC cell lines, but rarely expressed in normal human liver cell line (P < 0.01). With the increase of TRAIL concentration, the cell viability of HCC cells decreased gradually. However, when the concentration of TRAIL was above 1000 ng/ml, HCC cells were resistant to TRAIL, but still sensitive to DR5mAb. After incubation with DR5mAb (1000 ng/ml) for 24 h, the rate of apoptosis in HCC cells reached to 52.45% +/- 0.57%, which was higher than that incubated with TRAIL under the same condition (14.74% +/- 0.48%) (P < 0.05). The cell viability of normal human liver cell line treated with TRAIL tended to decline with the increase of the concentration, which was significantly different from that of matched control group. But DR5mAb had little effect on normal human liver cell line.

CONCLUSIONDeath receptor 5 as a target plays an important role in the course of HCC apoptosis induction. Agonistic monoclonal antibody specific for human DR5 can selectively and effectively kill hepatocellular carcinoma cells in vitro, while is not harmful to normal human hepatocytes. It reveals that DR5mAb might provide a new direction in hepatocellular carcinoma treatment research.

Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line ; Cell Line, Tumor ; Cell Survival ; drug effects ; Humans ; Liver Neoplasms ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics ; immunology ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis

This study was aimed to detect the expression of TNF related apoptosis-inducing ligand (TRAIL) and its receptors on acute myeloid leukemic (AML) cells, and explore its possible role in leukemia therapy. RT-PCR and flow cytometry were used to detect the expression of TRAIL and its receptors on AML cells of 39 cases (patient group), AML cells of 18 cases with complete remission (CR group) and BMMNC or PBMNC of 21 normal persons (control group). The results showed that (1) TRAIL, DR4 and DR5 were highly expressed in both patient group and CR group, while the DcR1 and DcR2 were poorly expressed. (2) The level of DR5 expression in CR group was higher than that in patient group. (3) The level of DR5 was higher than DR4 in both patient group and CR group. (4) TRAIL and its receptors were expressed similarly in different subtypes of AML. In conclusion, there are differences between the expressions of TRAIL and its receptors in AML cells. DR5 may play an important role in TRAIL-inducing apoptosis of AML cells.
Adult ; Female ; Flow Cytometry ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics

OBJECTIVETo clone and identify novel proteins binding to the death domain of the death receptor 4 (DR4).

METHODSThe yeast two-hybrid system was used for this study. Automatic sequencing was carried out for DNA sequencing. The sequence homology and the functional domains were analyzed by BLAST and the ScanProsite Tool softwares, respectively. Co-immunoprecipitate method was used to confirm human formyl peptide receptor-like 1 (FPRL1) binding specifically with DR4CD (the cytoplasmic domain of DR4) in HEK293T cells.

RESULTSTwo positive clones, named as pADB1 and pADB2, were obtained. BLAST searching showed that the homology of the insert sequence of pADB1 with the mRNA of FPRL1 was 97%. The insert of pADB2 shared no homology with any known peptides in GeneBank. Co-immunoprecipitate analysis further confirmed that FPRL1 could bind to DR4CD in vivo specifically.

CONCLUSIONSFPRL1 may associate with DR4CD in vivo specifically. The functional studies of FPRL1 in signaling pathway mediated by TNF-related apoptosis inducing ligand (TRAIL) are in active progress in our laboratory.

Amino Acid Sequence ; Apoptosis ; Apoptosis Regulatory Proteins ; Base Sequence ; Carrier Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Humans ; Membrane Glycoproteins ; metabolism ; Molecular Sequence Data ; Protein Structure, Tertiary ; Receptors, Formyl Peptide ; metabolism ; Receptors, Lipoxin ; metabolism ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; Receptors, Tumor Necrosis Factor ; genetics ; metabolism ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha ; metabolism

To compare the activity of RGD-TRAIL in different expression systems, RGD-TRAIL in both Escherichia coli (E.coli) and Pichia pastoris was constructed and expressed. In vitro activity of RGD-TRAIL from Pichia pastoris expression system was also analyzed. Genetic engineering techniques were used to construct recombinant plasmid pET30-rgd-trail and pHBM-rgd-trail. The recombinant protein RGD-TRAIL was purified with Ni ion affinity chromatography after induction. MTT assay, ELISA, scratch wound healing, transwell migration assay and Hoechst 33342 staining were performed to detect the effects of RGD-TRAIL on proliferation, binding activity, migration and apoptosis. The expression of apoptosis-associated proteins was detected by Western blotting. Recombinant protein RGD-TRAIL was successfully expressed in a form of inclusion body in E.coli, while expressed secretorily in Pichia pastoris. It possessed more potent cytotoxicity than RGD-TRAIL in E.coli by MTT assay. The RGD-TRAIL expressed by Pichia pastoris showed powerful binding affinity with cancer cells expressing α(v), DR4, DR5 and highly potent cytotoxicity through inducing apoptosis of cancer cells. Nuclear fragmentation was examined by Hoechst 33342 staining. Cleaved PARP and caspase-3 were also detected after incubation with RGD-TRAIL. Additionally, RGD-TRAIL inhibited migration significantly in A549 and HT1080 cells. The results demonstrate that Pichia pastoris expression system is more suitable for the recombinant protein RGD-TRAIL. Its binding affinity and antitumor activity might make RGD-TRAIL a promising candidate for cancer therapy.
Antineoplastic Agents ; pharmacology ; Apoptosis ; Blotting, Western ; Cell Line, Tumor ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Humans ; Oligopeptides ; biosynthesis ; pharmacology ; Pichia ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; pharmacology

OBJECTIVETo evaluate the expression and activity of TNF-related apoptosis-inducing ligand (TRAIL) gene expressed from the hTERT promoter on colon cancer cell line HT-29.

METHODSGFP/TRAIL gene expressed from the hTERT promoter was transfected into HT-29 with adenoviral vectors system, expression and apoptosis inducing ability of GFP/TRAIL protein were determined with fluorescence-activated cell sorting (FACS) method.

RESULTSThe expression of GFP gene was 31.4 % and 67.0 % with either hTERT promoter or CMV promoter in DLD1 cells; GFP/TRAIL gene was able to inhibit cell growth (74.2%) and induce apoptosis (25.8%) of HT-29 cells. There was significant difference between Ad/hTERT-gTRAIL and the other two control groups (PBS and Ad/CMV-GFP, P<0.05).

CONCLUSIONThe GFP/TRAIL gene with hTERT promoter transfected by adenoviral vector was successfully expressed in HT-29 cell, which can both inhibit cell growth and induce apoptosis of colon cancer cell line HT-29.

Adenoviridae ; genetics ; Apoptosis ; physiology ; Colonic Neoplasms ; enzymology ; pathology ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; HT29 Cells ; Humans ; Promoter Regions, Genetic ; genetics ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics ; Telomerase ; genetics ; Tumor Cells, Cultured

TNF-related apoptosis-inducing ligand (TRAIL) is a member of factor TNF family, which could be potentially developed as novel antitumor agent due to its selective and efficient induction of apoptosis in tumor cells. Gene recombinant expression is an important tool for production of pharmaceutical protein. In this paper, the gene encoding human soluble TRAIL (114-281aa fragment) was cloned by PCR and then inserted into the Pichia Pastoris expression vector pPIC9K. The transformants were double-screened on plates containing neomycin G418 and many clones with high levels of G418-resistance were selected for further studies on protein expression. The recombinant human soluble TRAIL was secreted into the BMMY media under the condition of 3% methanol. And the recombinant protein was purified to homogeneity (-80% purity) by using Ni-agarose affinity chromatography. The yield of this protein is about 1-2 mg per liter culture. Cell viability assays demonstrated that human soluble TRAIL was cytotoxic in both leukemia cells Jurkat and lung cancer cells A549. After treatment with 0.05 microg/ml TRAIL, the survival rate of Jurkat cells was about 10%. The expressed TRAIL showed dose-dependent cytotoxicity in A549 cells within the range of 0.1-1 microg/ml. When the protein concentration reached 1 microg/ml, the survival rates of A549 cells were about 30%. However, the recombinant human soluble TRAIL did not show obvious cytotoxicity in human skin fibroblast cells (HSF) at concentrations tested. There results demonstrate that human soluble TRAIL is selectively cytotoxic in tumor cells. The expression system constructed in this experiment might contribute to further production of soluble TRAIL and TRAIL-based novel fusion proteins in large quantities.
Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cloning, Molecular ; Cytotoxicity Tests, Immunologic ; methods ; Genetic Vectors ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics

The brain is particularly vulnerable to oxygen free radicals, and these radicals have been implicated in the pathology of several neurological disorders. In this study, the modulation of TNF-related apoptosis-inducing ligand (TRAIL) expression by oxidative stress was shown in LN215 cells, an astroglioma cell line. Hydrogen peroxide (H2O2) treatment increased TRAIL expression in LN215 cells and H2O2-induced TRAIL augmented apoptosis in Peer cells, a cell line sensitive to TRAIL- mediated cell death. Our findings suggest that the upregulation of TRAIL in astroglial cells may abrogate immune cell effector functions.
*Up-Regulation ; TNF-Related Apoptosis-Inducing Ligand/*biosynthesis ; T-Lymphocytes/*metabolism ; Ribonucleases/metabolism ; Oxidative Stress ; Immunosuppressive Agents/pharmacology ; Hydrogen Peroxide/*pharmacology ; Humans ; *Gene Expression Regulation, Neoplastic ; Cyclosporine/pharmacology ; Cell Line, Tumor ; Astrocytes/*metabolism ; *Apoptosis ; Anoxia ; Allergy and Immunology