ABSTRACT
OBJECTIVE Lapachol is a natural naphthoquinone compound that possesses extensive biological activities. The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo, as well as the potential mechanisms. METHODS The antitumor effect of lapachol was firstly evaluated in the C6 glioma model in Wistar rats. The effects of lapachol on C6 cell proliferation, apoptosis and DNA damage were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)/ phenazinemethosulfate (PMS) assay, hoechst 33358 staining, annexinⅤ-FITC/PI staining, and comet assay. Effects of lapachol on topoisomerase I (TOP I) and topoi?somerase Ⅱ (TOP Ⅱ) activities were detected by TOP Ⅰ and TOP Ⅱ mediated supercoiled pBR322 DNA relaxation assays and molecular docking. TOPⅠ and TOPⅡ expression levels in C6 cells were also determined. RESULTS High dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats (P<0.05). It was showed that lapachol could inhibit proliferation, induce apoptosis and DNA damage of C6 cells in dose dependent manners. Lapachol could inhibit the activities of both TOPⅠ and Ⅱ. Lapachol-TOPⅠ showed relatively stronger interaction than that of lapachol-TOPⅡ in molecular docking study. Also, lapachol could inhibit TOPⅡ expression levels, but not TOPⅠ expression levels. CONCLUSION These results showed that lapachol could significantly inhibit C6 glioma both in vivo and in vitro, which might be related with inhibiting TOPⅠ and TOPⅡ activities, as well as TOPⅡ expression.
ABSTRACT
Glutamine is an important conditionally necessary amino acid in human body. The effort is to establish a new and high efficient L-glutamine production system instead of traditional fermentaion. In this paper, high efficiency of L-glutamine production is obtained by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system. Glutamine Synthetase gene (glnA) was amplified from Bacillus subtilis genomic DNA with primers designed according to sequences reported in EMBL data bank, then it was inserted into expression vector PET28b, the sequence of glnA was proved to be the same as that reported in the data bank by DNA sequencing. After transformation of this recombinant plasmid PET28b-glnA into BL-21 (DE3) strain, Lactose and IPTG were used to induce GS expression at 37 degrees C separately. Both of them can induce GS expression efficiently. The induced protein is proved to be soluble and occupies about 80% of the total proteins by SDS-PAGE analysis. The soluble GS was purified by Ni2+ chelating sepharose colum. After purification, the purified enzyme was proved active. Results reveal that the optmum temperature of this enzyme is 60 degrees C and optmum pH is 6.5 in biosynthetic reaction by using glutamate, ammonium choloride and ATP as substrates. After induction, the enzyme activity in crude extract of BL-21/PET28b-glnA is 83 times higher than that of original BL-21 extract. Mn2+ can obviously increase the activity and stability of this enzyme. Experiments show that the transformation efficiency of glutamate to glutamine is more than 95%. Because of the high cost from ATP, a system coupling GS with yeast for ATP regenaration was established. In this system, GS utilizes ATP released by yeast fermentation to synthesize L-glutamine. Yeast was treated by 2% toluence to increase its permeability and a yeast named YC001 with high yield of glutamine by coupling with recombinant GS was obtained. The good efficiency was achieved with the presence of 250 mmol/L glucose and 200 mmol/L phosphate, the transformation efficiency of glutamate to glutamine in this system is more than 80%, the average yield of glutamine is about 22g/L. This provides the basis for future large scale production of L-glutamine.
