Pro-apoptotic effects of luteolin on hepatoma HepG2 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of luteolin on cell growth and apoptosis of HepG2 cells in vitro.</p><p><b>METHODS</b>Cultured HepG2,HL60,A549 and LO2 cells were treated with luteolin for different doses (0 μg/ml,2.5 μg/ml,10 μg/ml and 20 μg/ml) and varied times (0 h,24 h,48 h and 72 h). Cell viability was measured with MTT assay and IC50 was calculated. The reactive oxygen species (ROS) levels in HepG2 cells treated with luteolin for 6 h and 12 h were measured with flow cytometry. Cell apoptosis of HepG2 cells treated with luteolin for 24h was examined with flow cytometry and Annexin V-FITC/PI. Expression levels of apoptosis pathway proteins (p53,ASPP2 and iASPP) in HepG2 cells were detected with western blot and the dose and time-effect was analyzed.</p><p><b>RESULTS</b>Luteolin effectively inhibited tumor cell proliferation in a dose-and time-dependent manner,and the inhibition rates of 20 μg/ml Luteolin for 72 h were 39.34%,62.90%,57.57% and 62.90% to LO2,HepG2, HL60 and A549 cells,respectively. The intracellular ROS level was decreased in HepG2 cells by 13.88% and 41.11% after being treated with luteolin for 6 h and 12 h,respectively. The apoptosis rate of HepG2 cells treated with luteolin for 24 h was 14.43%,and western blot showed that luteolin reduced the expression level of iASPP by 77.07% and up-regulated the expression of p53 by 179.37% and ASPP2 by 725.02% in HepG2 cells treated with luteolin for 12 h.</p><p><b>CONCLUSION</b>Luteolin has ant-proliferative and pro-apoptotic activity on hepatoma HepG2 cells, which is associated with the altered expression of pro-apoptotic factors and decreased ROS level in HepG2 cells.</p>

Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present here the expression, purification, and bioactivity of a GST (glutathione S-transferase)-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl beta-D-thiogalactopyranoside (IPTG)-regulated expression, and the fused protein was purified using GSH (glutathione) affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems employed in earlier Src expression.

Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Total RNA was isolated from the hybridoma cell line (LC-1), which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FR1 (framework region 1) and FR4 conserved regions of LC-1 gene, the variable regions of heavy chain (Vh) and light chain (Vl) were amplified, and the Vh and modified Vl were connected to single chain Fv (ScFv) by SOE-PCR (splice overlap extension PCR). The modified ScFv was fused with green fluorescent protein (GFP) and introduced into E. coli JM109. The fusion protein induced by IPTG (Isopropylthiogalactoside) was about 57000 on a 10% SDS-PAGE gel (10% Sds Polyacrylamide Gel Electrophoresis), and primarily manifested as inclusion bodies. The renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-1 lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.