ABSTRACT
<p><b>OBJECTIVE</b>To explore the effects of Osthole on apoptosis of HL-60 cells induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its possible mechanism.</p><p><b>METHODS</b>The proliferative inhibition of HL-60 cells treated with different concentrations of Osthole, TRAIL alone and Osthole combined with TRAIL was measured by MTT assay. The HL-60 cells were treated with Osthole, TRAIL alone and Osthole combined with TRAIL at the concentration<ICvalue, i.e. 100µmol/L for Osthole and 40 ng/ml for TRAIL. Apoptosis and mitochondrial membrane potential (MMP) of HL-60 cells were detected by flow cytometry; the mRNA expression of BCL-2, BAX and DR5 was determined by RT-PCR; and the levels of Caspase-3,-8,-9 activity were detected by spectrophotometry.</p><p><b>RESULTS</b>The combined treatment (100µmol/L Osthole + 40 ng/ml TRAIL) of HL-60 cells for 48 h induced an apoptotic rate of (33.9±2.7) %, which was significantly higher than that of cells treated with Osthole or TRAIL alone (P<0.05); at the same time, the combined treatment promoted the decrease of MMP and the expression rate of BCL-2/BAX, and potentiated the expression of DR5 and Caspase-3,-8,-9 activity.</p><p><b>CONCLUSION</b>Osthole can sensitize HL-60 cells to TRAIL-induced apoptosis, which may be related with the activation of mitochondrial pathways and up-regulation of DR5.</p>
Subject(s)
Humans , Apoptosis , Coumarins , HL-60 Cells , TNF-Related Apoptosis-Inducing LigandABSTRACT
To investigate the effects of gambognic acid (GA) on TRAIL-induced apoptosis of cancer cells, human colon HT-29 cancer cells were treated with GA to promote apoptosis. Inhibition of the cell proliferation was measured with MTT assay and cell apoptosis was detected with formation of DNA ladders in agarose gel electrophoresis, and activation of caspase activity. The content of cytosolic reactive oxygen species (ROS) was measured with flow cytometry. The activities of Caspase-3, -8, -9 were detected using spectrophotometric assay. The levels of c-FLIP, CHOP, DR4 and DR5 in cells were tested by Western blot. Combination of GA (1 µg · mL(-1)) and TRAIL (40 ng · mL(-1)) significantly reduced proliferation and increased apoptosis of HT-29 cells over those induced by each agent alone. Percentage of apoptotic cells was increased to 45.5%. GA markedly enhanced the intracellular ROS generation. Expression of CHOP, DR4 and DR5 was up-regulated to 7.38, 5.41, and 4.85 times of the control group, respectively. GA promoted activation of Caspase-3, -8, and -9 by TRAIL (P<0.05). Furthermore, the expression of anti-apoptotic protein c-FLIP was down-regulated to 0.22 ± 0.08 times of the control group. In conclusion, GA sensitizes HT-29 cells to TRAIL-induced apoptosis by promoting ROS-activated ERS pathways, up-regulating of DR4 and DR5, and inhibiting c-FLIP expression.
Subject(s)
Humans , Apoptosis , Apoptosis Regulatory Proteins , Metabolism , Caspases , Metabolism , Cell Line, Tumor , Cell Proliferation , Colonic Neoplasms , Metabolism , Down-Regulation , HT29 Cells , Reactive Oxygen Species , Metabolism , TNF-Related Apoptosis-Inducing Ligand , Pharmacology , Up-Regulation , Xanthones , PharmacologyABSTRACT
BCMA is one of the transmembrane receptors belonging to BAFF and APRIL. In order to identify the feasibility of sBCMA as decoy receptor and obtain active sBCMA for its structural and functional research, full length of hBCMA was amplified with total RNA from Raji cell line by RT-PCR, and the cDNA encoding the extracelluar soluble domain of hBCMA was inserted into pET43.1a(+) vector. The recombinant vector pET43.1a(+)-sBCMA was transformed into E. coli Origami B(DE3) pLyS which is helpful for disulfide bond construction of expression proteins. After IPTG induction, the recombinant protein was expressed as soluble fusion protein, sBCMA-NusA-His6, and identified by western blotting. Then the target protein was purified by Ni(+)-chelating Sepharose Fast Flow. The binding activity between recombinant sBCMA and BAFF was detected by ELISA. Also, Recombinant sBCMA inhibited proliferation of mouse B cell stimulating by rhsBAFF. It was proved that recombinant sBCMA has good bioactivity and the method to express those proteins rich in disulfide bond is feasible and effectual.