Echinacoside, an active constituent of Herba Cistanche, suppresses epileptiform activity in hippocampal CA3 pyramidal neurons

Echinacoside, an active compound in the herb Herba Cistanche, has been reported to inhibit glutamate release. In this study, we investigated the effects of echinacoside on spontaneous excitatory synaptic transmission changes induced by 4-aminopyridine (4-AP), by using the in vitro rat hippocampal slice technique and whole-cell patch clamp recordings from CA3 pyramidal neurons. Perfusion with echinacoside significantly suppressed the 4-AP-induced epileptiform activity in a concentration-dependent manner. Echinacoside reduced 4-AP-induced increase in frequency of spontaneous excitatory postsynaptic currents (sEPSCs) but it did not affect the amplitude of sEPSCs or glutamate-activated currents, implicating a presynaptic mechanism of action. Echinacoside also potently blocked sustained repetitive firing, which is a basic mechanism of antiepileptic drugs. These results suggest that echinacoside exerts an antiepileptic effect on hippocampal CA3 pyramidal neurons by simultaneously decreasing glutamate release and blocking abnormal firing synchronization. Accordingly, our study provides experimental evidence that echinacoside may represent an effective pharmacological agent for treating epilepsy.

Tracking of CFSE-labeled endothelial progenitor cells in laser-injured mouse retina / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Endothelial progenitor cells (EPCs) transplantation is a promising therapeutic strategy for ischemic retinopathy. The current study aimed to establish a simple, reliable and fluorescent labeling method for tracking EPCs with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) in laser-injured mouse retina.</p><p><b>METHODS</b>EPCs were isolated from human umbilical cord blood mononuclear cells, cultivated, and labeled with various concentrations of CFSE. Based on fluorescence intensity and cell morphology, a 15 minutes incubation with 5 µmol/L CFSE at 37°C was selected as the optimal labeling condition. The survival capability and the apoptosis rate of CFSE-labeled EPCs were measured by Trypan blue staining and Annexin V/PI staining assay respectively. Fluorescence microscopy was used to observe the label stability during the extended culture period. Labeled EPCs were transplanted into the vitreous cavity of pigmented mice injured by retinal laser photocoagulation. Evans Blue angiography and flat mounted retinas were examined to track the labeled cells.</p><p><b>RESULTS</b>EPCs labeled with 5 µmol/L CFSE presented an intense green fluorescence and maintained normal morphology, with no significant changes in the survival capability or apoptosis rate after being labeled for 2 days, 1 and 4 weeks. The fluorescence intensity gradually decreased in the cells at the end of 4 weeks. Evans Blue angiography of the retina displayed the retinal capillarity network clearly and fluorescence leakage was observed around photocoagulated spots in the laser-injured mouse model. One week after transplantation of labeled EPCs, the fluorescent cells were identified around the photocoagulated lesions. Four weeks after transplantation, fluorescent tube-like structures were observed in the retinal vascular networks.</p><p><b>CONCLUSION</b>EPCs could be labeled by CFSE in vitro and monitored in vivo for at least 4 weeks, and participate in the repair of injured retinal vessels.</p>

Ultrasound-mediated microbubble destruction enhances LMP-1 gene transfection into dendritic cells in vivo / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the transfection efficiency and the optimal conditions of delivering latent membrane protein-1 (LMP-1) gene to dendritic cells (DCs) by ultrasound exposure combined with contrast agent.</p><p><b>METHODS</b>Human DCs were cultured in vivo and transfected with the recombinant plasmid pEGFP-C3-LMP1 under varying conditions including ultrasound intensities, exposure time and microbubble contrast agent concentration. The transfection efficiency was assessed by fluorescent microscopy and flow cytometry, and the cell viability by trypan blue exclusion test.</p><p><b>RESULTS</b>An exposure time of 60 s at MI 1.0 with a microbubble contrast agent concentration of 20% resulted in the optimal effect of delivering the recombinant plasmid pEGFP-C3-LMP1 into the DCs, with a transfection efficiency of (14.37∓2.12)%. Over 90% of the transfected cells were viable after the transfection.</p><p><b>CONCLUSION</b>Microbubble contrast agent combined with ultrasound exposure can enhance the delivery of recombinant plasmid pEGFP-C3-LMP1 into the DCs.</p>

In vitro cytotoxicity of 131I-Rituximab against B-cell lymphoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the specific cytotoxicity of (131)I-Rituximab against CD20-positive B-cell lymphoma cells.</p><p><b>METHODS</b>Rituximab was labeled with (131)I using IODO-GEN method, and the dose-effects of various concentrations of (131)I-Rituximab, (131)I alone and Rituximab in Raji cells were evaluated by MTT assay to determine the optimal dose according to the dose-effect curves. The cytotoxicity of (131)I-Rituximab, (131)I and Rituximab was assessed in CD20-positive Ramos (RA-1) cells, Raji cells and CD20-negative Molt-4 cells according to the changes of the survival rates. Giemsa staining was used to evaluate the antitumor effect of (131)I-Rituximab in Raji cells by measuring the mitosis index (MI).</p><p><b>RESULTS</b>(131)I-Rituximab presented with a dose-dependent cytotoxicity against Raji cells. At the specific activity of 60 microCi/ml, (131)I-Rituximab resulted in significantly higher growth inhibition rate of the cells than Rituximab (P<0.05). The inhibition rate of Raji cells treated with (131)I-Rituximab, (131)I, or Rituximab for 96 h were comparable with the rates in Ramos RA-1 cells, but significantly higher than the rates in Molt-4 cells. The MI values in (131)I-Rituximab group were significantly lower than those in the other groups (P<0.001).</p><p><b>CONCLUSION</b>(131)I-Rituximab can induce specific cytotoxicity against CD20-positive tumor cells, and may potentially serve as an agent for targeted radioimmunotherapy for CD20-positive B-cell lymphoma.</p>

Sorafenib reverses multidrug resistance of hepatoma cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of sorafenib in reversing multidrug resistance (MDR) in hepatoma BEL-7402/FU cells and its possible mechanisms.</p><p><b>METHODS</b>MTT colorimetric assay was used to obtain the dose-response curve of sorafenib in BEL-7402/FU cells, and flow cytometry performed to assess the effect of sorafenib on Rho123 concentration in the cells. The optimal dose of sorafenib for cell treatment was determined according to the results of MTT assay and flow cytometry. MTT assay was employed to evaluate the effect of sorfenib on the cytotoxicity of the antitumor drugs, flow cytometry performed to determine the expression of cell membrane transport protein (P-gp), and RT-PCR used to detect mdr1 gene expression in the cells treated with sorafenib at the optimal dose.</p><p><b>RESULTS</b>Sorafenib at the concentration of 4 micromol/L, efficiently reversed the MDR of the cells with minimal side effects. At the concentration of 4 micromol/L, sorafenib partially reversed the drug resistance of BEL-7402/FU cells to ADM, 5-FU, GEM and DDP, with reversal indexes of 2.98, 7.16, 1.99 and 10.08, respectively. Treatment of the cells with 4 micromol/L, sorafenib also partially down-regulated P-gp expression in BEL-7402/FU cells, and caused a reduction of mdr1 gene expression by 27.3% in comparison with the control cells.</p><p><b>CONCLUSION</b>Sorafenib can reverse MDR in human hepatoma cells probably in association with down-regulation of mdr1 gene expression and increased accumulation of the chemotherapeutic agents in the cells.</p>

Clinical significance of VEGF levels in serums of colorectal cancer patients at stage IV / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the clinical significance of vascular endothelial growth factor (VEGF) levels in serums of colorectal cancer patients at stage IV.</p><p><b>METHODS</b>Using enzyme linked immunosorbent assay (ELISA) to detect the VEGF levels in serums of 45 colorectal cancer patients at stage IV, and 20 healthy served as normal control.</p><p><b>RESULTS</b>The mean concentration of VEGF in 45 colorectal cancer patients at the 7 day after operation were significantly lower than that before operation (P<0.01). The mean concentration of VEGF in the patients who benefit from bevacizumab showed no statistical difference from the levels of who did not benefit (P=0.554).</p><p><b>CONCLUSION</b>The VEGF levels in colorectal patients at stage IV are lowed as the load of tumor decrease. The circulating levels of VEGF seem not predict the response to bevacizumab in colorectal cancer patients at stage IV.</p>

Effects of bevacizumab and cisplatin on human lung adenocarcinoma A549/DDP xenografts in nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effects of bevacizumab with or without cisplatin (DDP) on the growth of lung adenocarcinoma A549/DDP cell xenografts in mice.</p><p><b>METHODS</b>Human lung cancer A549/DDP cells was subcutaneously transplanted in to 25 nude mice, which were randomly divided into control group (group A), bevacizumab group (group B), DDP group (group C), combined treatment group (group D) and half-dose combined treatment group (group E). After corresponding treatments for 4 consecutive weeks, the tumor inhibition rate was evaluated, tumor microvessel density (MVD) measured with immunohistochemistry, and the mRNA expression of apoptosis-associated gene (bcl-2) and multidrug resistance genes (LRP and GST-pi) assessed by RT-PCR.</p><p><b>RESULTS</b>The tumor growth inhibition rates in groups B, D, and E with bevacizumab treatment were 20.96%, 51.67% and 50.95%, respectively, and the two combined treatment groups showed better effects. MVD in these 3 groups were 18.6-/+1.14, 13.6-/+1.14, and 14.4-/+0.55, respectively, and no significant difference was found in MVD between DDP group and the control group. Compared with the control group, the 3 bevacizumab-treated groups showed decreased expression of bcl-2 genes in A549/DDP tumors at a comparable amplitude, and LRP and GST-pi mRNA expression showed no significant differences between the 5 groups.</p><p><b>CONCLUSION</b>Bevacizumab has synergetic inhibitory effect with conventional chemotherapy against lung adenocarcinoma A549/DDP cell xenografts in mice by inhibiting angiogenesis of the tumor, and may enhance the sensitivity of A549/DDP cells to DDP by inducing cell apoptosis.</p>

Main factors affecting the effect of photodynamic therapy against human esophageal cancer cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the tumor cell-killing effect of photodynamic therapy against human esophageal cancer cells in vitro and identify the main factors affecting the effect.</p><p><b>METHODS</b>Human esophageal cancer Eca-109 cells were incubated for 24 h in vitro with hematoporphyrin derivative (HpD) and Photofrin at different concentrations prior to exposure to a light energy density of 15 J/cm(2) delivered from a DIOMED 630 PDT system. The cell killing effect was also evaluated for different HpD concentrations combined with 3 light energy densities (10, 30, and 50 J/cm(2)), respectively. The cell survival rate was measured using MTT assay, and fluorescence spectrometry was used to detect the intracellular photosensitizer fluorescence of the tumor cells after incubation with HpD for 4 h.</p><p><b>RESULTS</b>The cell survival rate after incubation with the two photosensitizers at different concentrations were significantly different, and under the 3 different light energy densities, incubation of the cells with different HpD concentrations also resulted in significantly different cell survival rates (P<0.05). At the 4 low photosensitizer concentrations and with different light energy densities, the cell survival rates were similar (P>0.05), but the 4 higher photosensitizer concentrations resulted in significant difference in the cells survival (P<0.05). Correlation analysis showed that the intracellular photosensitizer concentration was positively correlated to the photosensitizer concentrations in cell incubation (r=0.997).</p><p><b>CONCLUSION</b>When the light source remains constant, the light energy density, the kinds of photosensitizers and their concentrations are the main factors affecting the Eca-109 cell-killing effect of PDT.</p>

Hematoporphyrin derivative-mediated photodynamic therapy for human nasopharyngeal carcinoma: a comparative study with CNE2 and C666-1 cell lines in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate biological effect of hematoporphyrin derivative (HpD) photodynamic therapy (PDT) on in vitro cultured nasopharyngeal carcinoma (NPC) cell lines CNE2 and C666-1.</p><p><b>METHODS</b>CNE2 and C666-1 cells cultured in vitro were incubated in a medium containing HpD at different concentrations (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 microg/ml) for 4 h followed by exposure to different light doses (2, 5, 10, and 20 J/cm2) using a diode laser at 630 nm with power density of 20 mW/cm2. After 24 h of incubation with HpD-PDT, the survival rate of CNE2 and C666-1 cells were analyzed by MTT assay.</p><p><b>RESULTS</b>HpD-PDT produced effective killing of CNE2 and C666-1 cells cultured in vitro, and the killing effects were positively correlated with HpD concentration and the irradiation dose. Exposure of CNE2 and C666-1 cells to irradiation dose of 20 J/cm2 resulted in the IC50 of 0.7 and 1.2 microg/ml, respectively (P<0.01). With the same HpD concentration and irradiation dose, the survival rate of C666-1 cells, however, was significantly higher than that of CNE2 cells (P<0.05).</p><p><b>CONCLUSION</b>HpD-PDT may result in effective killing of CNE2 and C666-1 cells cultured in vitro, although C666-1 cells are less sensitive to HpD-PDT than CNE2 cells.</p>

Killing effect of sequential Herceptin and adriamycin treatment on breast cancer cell line in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the killing effect of Herceptin and adriamycin sequentially applied on breast cancer cell line in vitro.</p><p><b>METHODS</b>BT-474 human breast cancer cells in exponential growth phase were treated with Herceptin alone, adriamycin alone and their sequential administration (Herceptin before adriamycin and vice versa), respectively. Under optical microscope, the morphological changes of the cells were observed before and after drug administration. The expression rate and mean fluorescence intensity (MFI) of HER-2/neu and cell death rate were detected by flow cytometry.</p><p><b>RESULTS</b>Microscopically, the cells treated with different protocols all exhibited such changes as darkening and increase of cellular debris with irregular cell morphology. Flow cytometry revealed no significant difference in the expression rate of HER-2/neu in each group before and after treatment, but the MFI of HER-2/neu and death rate of the treated cells were significant different from those of the control group (P<0.05). The cell death rate of Herceptin-pretreated cells was significantly higher than that of adriamycin-pretreated ones (P<0.05).</p><p><b>CONCLUSION</b>Herceptin pretreatment enhances the killing effect of adriamycin on breast cancer cell line BT-474, which provides experimental evidence for designing clinical sequential biochemotherapy of breast cancer.</p>