[Optimization of expression conditions of recombinant Fuantai-03 and detection of its biological activities].

Fuantai-03(FAT-03), isolated from the Dasyatis akajei, has a strong antiangiogenic activity. The recombinant Fuantai-03 (GST/rFAT-03) fusion protein can be obtained with the DNA recombination technology. In this study, expression conditions of GST/rFAT-03 were optimized by response surface experimental design method. The constructed engineering bacteria containing GST/rFAT-03 plasmid was induced by isopropy-beta-D-thiogalactosid (IPTG), the GST affinity column was used for isolation and purification, and then the effects of different culture time, IPTG concentration, induction temperature and induction time on the amount of soluble GST/rFAT-03 fusion protein were compared. The culture time for optimal expression was 6.13 h, IPTG concentration was 0.36 mmol/L, induction temperature was 19.71 degrees C, and induction time was 13.60 h. The amount of soluble GST/rFAT-03 fusion protein was 7.57 mg/L under above mentioned expression conditions. The results also showed that rFAT-03 significantly inhibited angiogenesis in chicken chorioallantoic membrane in a dose-dependent manner. Moreover, the soluble form of the target protein is useful for further work on purification and on studying its biological function.

Anti-microtubule activity of tubeimoside I and its colchicine binding site of tubulin.

BACKGROUND: Tubeimoside I (TBMS1) was isolated from the tubers of Bolbostemma paniculatum (Maxim.) Franquet. TBMS1 shows potent anti-tumor activity. The present study was conducted to investigate the anti-microtubule role of TBMS1 and its binding site of tubulin. METHODS: Cell growth inhibition was measured by MTT after treatment with TBMS1. Uptake kinetics of TBMS1 by human nasopharyngeal carcinoma CNE-2Z cell line (CNE-2Z) was assayed by HPLC. Microtubule protein (MTP) was prepared from porcine brain through two cycles of polymerization-depolymerization in a high molarity buffer. Inhibition of MTP polymerization induced by TBMS1 was determined by a turbidity measurement and a sedimentation assay; the interactions of TBMS1 with tubulin within CNE-2Z cells were investigated by immunofluorescence microscopy and immunoblotting. TBMS1 was tested for its ability to inhibit binding of known tubulin ligands through competitive binding assay. RESULTS: TBMS1 displayed growth inhibitory activity against CNE-2Z cells with IC(50) value of 16.7 microM for 72 h. HPLC analysis of TBMS1 uptake by CNE-2Z cells displayed the initial slow TBMS1 uptake and then gradually reaching an maximum uptake near 18 h. CNE-2Z cells treated with TBMS1 (25 microM, 3 h) were sufficient to cause the microtubular network disruption. Immunoblot analysis showed that the proportion of cytosolic tubulin of cells treated with TBMS1 increased in a time- and concentration-dependent manner. TBMS1 did not inhibit the binding of vinblastine to tubulin. Colchicine binding to tubulin was inhibited in the presence of TBMS1. CONCLUSIONS: TBMS1 is an anti-microtubule agent, and its binding site of tubulin is the colchicine binding site of tubulin.

[Cell cycle arrest and apoptosis induced by tubeimoside in HeLa cell].

BACKGROUND & OBJECTIVE: Tubeimoside, which is composed of tubeimoside I (79%) and II (21%), was isolated from the tubers of Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), a traditional Chinese medicine, "Tu-Bei-Mu". This study was designed to investigate the anti-tumor mechanism of tubeimoside. METHODS: Growth inhibition was measured by MTT assay. Induction of cell cycle arrest and apoptosis was determined by flow cytometry, fluorescence and electron microscopy, and gel electrophoresis of fragmented DNA. RESULTS: Tubeimoside display strong growth inhibitory effect in a dose- and time-dependant manner against HeLa cells with estimated IC50 values of 20.0, 18.8, and 8.8 mumol/L after 24, 48, and 72 h of treatment with tubeimoside. The flow cytometry profiles revealed that treatment with tubeimoside (5 h; 15, 30, 35 mumol/L) led to a dose-dependant shift from 9.80% up to 21.90%, and 27.00% in percentage of cells with a G2/M-like DNA content. On the other hand, treatment with tubeimoside (12 h, 15, 30, 35 mumol/L) led to a time-dependant shift from 8.20% up to 21.40%, 31.15%, and 34.55%, respectively. Exposure of HeLa cells to 40 mumol/L of tubeimoside induced nuclear shrinkage, chromation condensation and margination against nuclear envelope, subdiploid peak, and DNA fragmentation, characteristic as seen in apoptotic cells. CONCLUSION: Induction of cell cycle arrest and apoptosis may play an important role in the anti-tumor effect of tubeimoside.