[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.

Repair of glutamate-induced excitotoxic neuronal damage mediated by intracerebroventricular transplantation of neural stem cells in adult mice.

OBJECTIVE: To investigate a possibility of repairing damaged brain by intracerebroventricular transplantation of neural stem cells (NSCs) in the adult mice subjected to glutamate-induced excitotoxic injury. METHODS: Mouse NSCs were isolated from the brains of embryos at 15-day postcoitum (dpc). The expression of nestin, a special antigen for NSC, was detected by immunocytochemistry. Immunofluorescence staining was carried out to observe the survival and location of transplanted NSCs. The animals in the MSG + NSCs group received intracerebroventricular transplantation of NSCs (approximately 1.0 x 10(5) cells) separately on day 1 and day 10 after 10-d MSG exposure (4.0 g/kg per day). The mice in control and MSG groups received intracerebroventricular injection of Dulbecco's minimum essential medium (DMEM) instead of NSCs. On day 11 after the last NSC transplantation, the test of Y-maze discrimination learning was performed, and then the histopathology of the animal brains was studied to analyze the MSG-induced functional and morphological changes of brain and the effects of intracerebroventricular transplantation of NSCs on the brain repair. RESULTS: The isolated cells were Nestin-positive. The grafted NSCs in the host brain were region-specifically survived at 10-d post-transplantation. Intracerebroventricular transplantation of NSCs obviously facilitated the brain recovery from glutamate-induced behavioral disturbances and histopathological impairs in adult mice. CONCLUSION: Intracerebroventricular transplantation of NSCs may be feasible in repairing diseased or damaged brain tissue.

[Tubeimoside I-induced ultrastructural changes of human cervical carcinoma HeLa cell line and the protective effect of cyclosporine A].

OBJECTIVE: To observe the ultrastructural changes of HeLa cells in response to tubeimoside I (TBMS1) treatment and the protective effect of cyclosporine A (CsA), and explore the role of intrinsic apoptosis pathway in TBMS1-induced HeLa cell apoptosis. METHODS: HeLa cells were treated with TBMS1 (10-50 micromo/L) alone or in combination with 2 micromol/L CsA for 12 and 24 h and observed with transmission electron microscope (TEM) for the ultrastructural changes of the cells. RESULTS: TBMS1 induced apoptosis of HeLa cells in a concentration- and time-dependant manner. Under TEM, the treated cells progressively shrunk and the intercellular space widened with loss of microvillus, mitochondrial swelling, rough endoplasmic reticulum enlargement, chromatin condensation, nuclear shrinkage and nuclear pyknosis as TBMS1 concentration increased. At low concentrations, CsA offered partial protection of the mitochondria from TBMS1-induced damage whereas high-concentration CsA did not. CONCLUSION: TBMS1 induces ultrastructural changes typical for apoptosis of the HeLa cells, which provides morphological evidence for the role of intrinsic apoptosis pathway in TBMS1-induced apoptosis.

Role of mitochondria and mitochondrial cytochrome c in tubeimoside I-mediated apoptosis of human cervical carcinoma HeLa cell line.

BACKGROUND: Tubeimoside I (TBMS1), a triterpenoid saponin, isolated from the tubers of Bolbostemma paniculatum, showed potent antitumor and antitumor-promoting effects. The objective of this study is to investigate the role of mitochondria and mitochondria cytochrome c in TBMS1-mediated apoptosis of human cervical carcinoma HeLa cell line. METHODS: Viability of HeLa cells was measured by MTT assay. Apoptotic induction by TBMS1 was determined by fluorescence microscopy, flow cytometry and gel electrophoresis of fragmented DNA. Mitochondrial transmembrane potential (Deltapsim) was assayed by flow cytometry. Cytochrome c (Cyt c) was detected by Western blotting. RESULTS: The results showed that Cyclosporin A (CsA) partly protected HeLa cells from growth inhibitory effect of TBMS1, and partly countered the ability of TBMS1 to rapidly induce apoptosis in HeLa cells, and that TBMS1 decreased Deltapsim and induced Cyt c release by a mechanism inhibited by CsA, and that TBMS1 induced apoptosis of HeLa cells dose-dependently in accordance with increase of cytosolic Cyt c. CONCLUSIONS: TBMS1 opens the permeability transition (PT) pore, thereby decreasing Deltapsim, releasing Cyt c from mitochondria, and further causing a series of events consistent with established mechanistic models of apoptosis.

Potent protection of ferulic acid against excitotoxic effects of maternal intragastric administration of monosodium glutamate at a late stage of pregnancy on developing mouse fetal brain.

The present study was conducted to investigate a possible protection of ferulic acid against excitotoxic effects of maternal intragastric (ig) administration of monosodium glutamate (MSG) at a late stage of pregnancy on developing mouse fetal brain. [(3)H]-labeled glutamate was used as radiotracer to study the effect of ferulic acid on distribution of MSG in mouse fetal brain. MSG dissolved in distilled water (2.0 g/kg body weight, 640 kBq of [(3)H]glutamate/mouse, ig) or/and sodium ferulate (SF) (20, 40, 80 mg/kg body weight, ip), was given to pregnant mice at 17-19 days; the distribution of [(3)H] glutamate in the mouse fetal brains was measured at 30, 60, 90, 120 min after administration of MSG or/and SF. Maternal mice were given MSG (1.0, 2.0, 4.0 g/kg body weight, ig) or/and SF (20, 40, 80 mg/kg body weight, ip) simultaneously at 17-19 days of pregnancy, and then behavioral tests and histopathological observations were used to analyze glutamate-induced functional and morphological changes of the brains of their offspring, and Western blot analysis was performed for examining expressions of bcl-2 and caspase-3. The results showed that SF obviously inhibited the uptake of labeled glutamate in fetal brain. In addition, SF countered the effects of MSG on behavior, histopathology, genetic toxicity, and expression of apoptosis-related gene. The results suggest that ferulic acid is a novel competitive N-methyl-D-aspartate (NMDA) receptor antagonist and neuroprotector. In conclusion, maternal administration of ferulic acid has potent protective effects against glutamate-induced neurotoxicity in their filial mice.

[Role of mitochondria in Tubeimoside I-mediated apoptosis in human cervical carcinoma HeLa cell line].

OBJECTIVE: To investigate the role of mitochondria in TBMS1-medited apoptosis of human cervical carcinoma HeLa cell line. METHOD: Mitochondrial transmembrane potentia (deltapsim) was assayed by flow cytometry. Apoptotic induction by TBMS1 was determined by gel electrophoresis of fragmented DNA. Cytochrome c (Cyt c) was detected by Western blotting. RESULT: The results showed that TBMS1 induced apoptosis in HeLa cells, that TBMS1 decreased deltapsim and facilitated Cyt c release, and that TBMS1 induced apoptosis of HeLa cells dose-and time-dependently in accordance with increase of cytosolic Cyt c. TBMS1 had direct effect on isolated rat mitochondria which induced a time- and dose-dependent release of Cyt c from mitochondria. The results also showed that CsA protected partly HeLa cells from the effect of TBMS1. CONCLUSION: The mitochondrial apoptosis pathway has effects on TBMS1 induced human cervical carcinoma HeLa cell line apoptosis.

[Apoptosis of human nasopharyngeal carcinoma CNE-2Z cells induced by tubeimoside I].

BACKGROUND & OBJECTIVE: Tubeimu, Bolbostemma pani- culatum(Maxim.) Franquet (Cucurbitaceae), is a kind of traditional Chinese medicinal herb. Our previous studies revealed that tubeimoside I, a triterpenoid saponin isolated from the tubers of Bolbostemma paniculatum, showed potent antitumor effect. This study was designed to investigate the effect of tubeimoside I on the apoptosis of human nasopharyngeal carcinoma cell line CNE-2Z. METHODS: Cell growth inhibition mediated by tubeimoside I was measured by MTT assay after treatment with tubeimoside I. The effect of tubeimoside I on apoptotic induction in CNE-2Z cells was determined using the fluorescent microscopy, electronic microscopy,DNA agarose gel electrophoresis, and flow cytometry,respectively. Western blot analysis was performed to detect the changes of apoptosis-related genes bcl-2 and bax protein expression. RESULTS: Tubeimoside I displayed growth inhibitory activity against CNE-2Z cells with IC(50) values of 32.5, 20.7, and 16.7 micromol/L for 24, 48, and 72 hours, and CNE-2Z cells showed typical apoptotic morphological features observed by fluorescent microscopy and electronic microscopy. In CNE-2Z cells occurred typical "Ladder"bands after being exposed to tubeimoside I (10 micromol/L, 24, 48, 72 hours; 30, 40, 50, 60 micromol/L, 12 hours). Sub-G1 peak was found using flow cytometry. When CNE-2Z cells were exposed to tubeimoside I (50 micromol/L, 12 hours), the apoptosis index was 72.8%. The down regulation and phosphorylation of bcl-2 (an inhibitor of apoptosis) were detected at 1 hour after the addition of tubeimoside I. In contrast, the levels of bax appeared to be significantly upregulated at 1, 3, 5, and 24 hours after the addition of tubeimoside I. CONCLUSION: Tubeimoside I can induce the apoptosis of CNE-2Z cells, and the induction of apoptosis by tubeimoside I is closely associated with downregulation and phosphorylation of bcl-2 and bax activation.

[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.