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

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

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

Interferon beta (IFN-beta) and TNF-related apoptosis-inducing ligand (TRAIL) are effective anticancer agents. Adeno-associated virus (AAV) is one of the current most promising gene delivery vectors. Previously, we constructed tumor-targeting AAV-hTERT-IFN-beta and AAV-hTERT-TRAIL by inserting IFN-beta or TRAIL gene into AAV controlled by hTERT promoter. The studies showed that either single IFN-beta or TRAIL gene therapy exhibited a certain extent anticancer effect. Here, we report their inhibitory effects on A549 lung cancer cell growth in vitro and in vivo by combined AAV-hTERT-IFN-beta and AAV-hTERT-TRAIL. Expression of secreted IFN-beta in lung cancer A549 cells infected by AAV-hTERT-IFN-beta was detected by enzyme-linked immunosorbent assay (ELISA). The growth-suppressing effect of AAV-hTERT-IFN-beta in combination with AAV-hTERT-TRAIL on several cancer cell lines was assessed by MTT assay. Apoptosis of A549 cancer cells infected by AAV-hTERT-IFN-beta alone, AAV-hTERT-TRAIL alone, and their combination was evaluated by apoptotic cell staining and flow cytometry (FCM), respectively. The antitumor effect of the combination of AAV-hTERT-IFN-beta with AAV-hTERT-TRAIL in vivo was further evaluated through A549 lung cancer xenograft in nude mice. The results showed that the combinational treatment was superior to any alone and presented intensified tumor cytotoxic and apoptotic effect on A549 cancer cells. Most importantly, the combination of AAV-hTERT-IFN-beta with AAV-hTERT-TRAIL exhibited significant antitumor effect and eliminated all tumor masses in nude mice, which lay a foundation for exploring the molecular mechanisms of combined IFN-beta and TRAIL anti-tumor activity.
Animals ; Antineoplastic Agents ; metabolism ; pharmacology ; Cell Line, Tumor ; Dependovirus ; genetics ; metabolism ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Interferon-beta ; genetics ; pharmacology ; Lung Neoplasms ; pathology ; therapy ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; pharmacology

In order to explore the expression of TRAIL in primary acute leukemic cells and the effect of chemotherapeutic drug on TRAIL expression in acute leukemic cells, the expression of TRAIL was assessed by flow cytometry on day 0, day 1, day 3 and day 5 in 16 patients with acute leukemia received chemotherapy. Meanwhile, the bone marrow mononuclear cells of acute leukemia patients were cultured in vitro with VP-16 and INFalpha-2a. Expression of TRAIL was analyszed by flow cytometry at 24, 48 and 72 hours after treatment. The results showed that the expression of TRAIL in the peripheral blood mononuclear cells was upregulated significantly from day 1 after chemotherapy (P < 0.05). In in vitro culture test, VP-16 upregulated the expression of TRAIL on acute leukemia bone marrow mononuclear cells (P < 0.05). Compared with VP-16 alone, the combination of VP-16 with IFNalpha-2a showed no synergic effects on the expression of TRAIL. It is concluded that the expression of TRAIL increases after chemotherapy in vivo and after treatment with VP-16 and IFN in vitro, which suggests that the apoptosis induced by TRAIL may play an important role in chemotherapy of leukemia.
Acute Disease ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Bone Marrow Cells ; metabolism ; Etoposide ; pharmacology ; Humans ; Interferon-alpha ; pharmacology ; Leukemia ; drug therapy ; metabolism ; pathology ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics ; Tumor Cells, Cultured

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

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

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

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

OBJECTIVETo study the differential expression of four TRAIL receptors on bone marrow mononuclear cells (BMMNC) from multiple myeloma (MM) patients and myeloma cell line KM3 cells, to compare their altered expressions after chemotherapy and to explore the mechanisms by which TRAIL selectively kills tumor cells.

METHODSSemi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry were used to investigate the expression of four TRAIL receptors on BMMNCs in 23 MM patients, KM3 cells and 15 controls, and the changes of their expression pattern after chemotherapy and after incubation of KM3 cells with sub-clinical concentration of doxorubicin.

RESULTSDR4 and DR5 were highly expressed on KM3 cells with no expression of DcR1 and DcR2. Expressions of DR4 and DR5 on BMMNCs from MM patients were higher and expression of DcR1 and DcR2 were lower than that of controls (P < 0.05). The expression of DR5 on MM and KM3 cells was up-regulated after chemotherapy and exposure to doxorubicin (P < 0. 05).

CONCLUSIONSThe expressions of four TRAIL receptors on myeloma cells and normal controls were different, which might account for the selective killing effect of TRAIL on MM cells. Up-regulated DR5 on KM3 cells after incubating with doxorubicin and after chemotherapy suggests the cytotoxic agents might enhance the apoptosis of MM cells.

Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cells, Cultured ; Doxorubicin ; pharmacology ; Female ; Flow Cytometry ; Gene Expression ; drug effects ; Humans ; Leukocytes, Mononuclear ; cytology ; drug effects ; metabolism ; Male ; Middle Aged ; Multiple Myeloma ; drug therapy ; genetics ; pathology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction