Guang-Hong Tan

The antitumour activities induced by pegylated liposomal cytochalasin D in murine models

Cytochalasin D targets actin and is ubiquitous in eukaryotic cells. When cytochalasin D is used as a cytotoxic agent in cancer therapy, it causes significant side effects. To prevent this, cytochalasin D can be encapsulated in polyethylene liposomes. In this study, high-performance liquid chromatography observation of the biodistribution of pegylated liposomal cytochalasin D in tumour-bearing mice showed that liposomal cytochalasin D could be conveniently dissolved in water for i.v. injection and that it specifically accumulated in tumour tissues, more than natural cytochalasin D did. The half-time of liposomal cytochalasin D in the plasma was also significantly longer than that of natural cytochalasin D (4h versus 10 min). MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that liposomal cytochalasin D treatment could cause significant inhibition of cell proliferation in vitro in a manner similar to that of natural cytochalasin D. The antitumour activities of liposomal cytochalasin D were investigated in B16 melanoma, CT26 colorectal carcinoma and H22 hepatoma models, and the results indicated that liposomal cytochalasin D could significantly inhibit tumour growth and prolong survival in a manner similar to that of cisplatin. TUNEL-based apoptosis assays showed that liposomal cytochalasin D induced significant tumour cell apoptosis. Significant inhibition of tumour angiogenesis was observed in mice treated with liposomal cytochalasin D. In addition, no significant side effects were observed in mice treated with liposomal cytochalasin D. Our results show that liposomal cytochalasin D increases solubility and bioavailability, a lower incidence of side effects and improves antitumour effects, indicating its potential as a chemical agent for cancer therapy.

A recombinant DNA plasmid encoding the human interleukin-5 breaks immunological tolerance and inhibits airway inflammation in a murine model of asthma.

Background: Eosinophils play a pivotal role in the generation of asthma inflammation. Interleukin (IL)-5 is the major activator of eosinophils. We hypothesize that modulating IL-5 activity could be an effective strategy for asthma therapy. In this study, we tested whether the plasmid encoding human IL-5 as a xenogeneic DNA vaccine could induce the production of autoantibodies, and be used for asthma treatment. Methods: A eukaryotic plasmid encoding the human IL-5 was constructed, and used as a DNA vaccine. A mouse model of asthma was established to observe its antiasthma activities. Eosinophils in tissue, blood and the bronchoalveolar lavage were stained and counted. Airway hyperresponsiveness (AHR) was determined by whole body plethysmography. Antibody characters and cytokines were detected with immunological methods. Results: Immunization with a plasmid encoding the human IL-5 as DNA vaccine reduced airway inflammation, reversed Th2 cytokines, and decreased AHR in mice. In addition, this immunization induced the production of polyclonal antibodies that were cross-reactive with native murine IL-5, and IgG1 and IgG2a were the major subclasses. Adoptive transfer of the purified antibodies from the sera of mice immunized with the plasmid encoding the human IL-5 resulted in similar antiasthma effects. Conclusions: Our results suggest that active vaccination against IL-5 may be a rational therapeutic approach for the treatment of asthma and potentially other eosinophilic disorders.

Cytochalasin D, a tropical fungal metabolite, inhibits CT26 tumor growth and angiogenesis

OBJECTIVE To investigate whether cytochalasin D can induce antitumor activities in a tumor model. METHODS Murine CT26 colorectal carcinoma cells were cultured in vitro and cytochalasin D was used as a cytotoxic agent to detect its capabilities of inhibiting CT26 cell proliferation and inducing cell apoptosis by MTT and a TUNEL-based apoptosis assay. Murine CT26 tumor model was established to observe the tumor growth and survival time. Tumor tissues were used to detect the microvessel density by immunohistochemistry. In addition, alginate encapsulated tumor cell assay was used to quantify the tumor angiogenesis in vivo. RESULTS Cytochalasin D inhibited CT26 tumor cell proliferation in time and dose dependent manner and induced significant CT26 cell apoptosis, which almost reached the level induced by the positive control nuclease. The optimum effective dose of cytochalasin D for in vivo therapy was about 50 mg/kg. Cytochalasin D in vivo treatment significantly inhibited tumor growth and prolonged the survival times in CT26 tumor-bearing mice. The results of immunohistochemistry analysis and alginate encapsulation assay indicated that the cytochalasin D could effectively inhibited tumor angiogenesis. CONCLUSIONS Cytochalasin D inhibits CT26 tumor growth potentially through inhibition of cell proliferation, induction of cell apoptosis and suppression of tumor angiogenesis.

Toxicarioside A Inhibits Tumor Growth and Angiogenesis: Involvement of TGF-β/Endoglin Signaling

Toxicarioside A is a cardenolide isolated mainly from plants and animals. Emerging evidence demonstrate that cardenolides not only have cardiac effects but also anticancer effects. In this study, we used in vivo models to investigate the antitumor activities of toxicarioside A and the potential mechanisms behind them. Murine colorectal carcinoma (CT26) and Lewis lung carcinoma (LL/2) models were established in syngeneic BALB/c and C57BL/6 mice, respectively. We found that the optimum effective dose of toxicarioside A treatment significantly suppressed tumor growth and angiogenesis in CT and LL/2 tumor models in vivo. Northern and Western blot analysis showed significant inhibition of endoglin expression in toxicarioside A-treated human umbilical vein endothelial cells (HUVECs) in vitro and tumor tissues in vivo. Toxicarioside A treatment significantly inhibited cell proliferation, migration and invasion, but did not cause significant cell apoptosis and affected other membrane protein (such as CD31 and MHC I) expression. In addition, TGF-β expression was also significantly inhibited in CT26 and LL/2 tumor cells treated with toxicarioside A. Western blot analysis indicated that Smad1 and phosphorylated Smad1 but not Smad2/3 and phosphorylated Smad2/3 were attenuated in HUVECs treated with toxicarioside A. Smad1 and Smad2/3 signaling remained unchanged in CT26 and LL/2 tumor cells treated with toxicarioside A. Endoglin knockout by small interfering RNA against endoglin induced alternations in Smad1 and Smad2/3 signaling in HUVECs. Our results indicate that toxicarioside A suppresses tumor growth through inhibition of endoglin-related tumor angiogenesis, which involves in the endoglin/TGF-β signal pathway.

Antigen 43/Fcε3 chimeric protein expressed by a novel bacterial surface expression system as an effective asthma vaccine.

The IgE Fcε3 domain is an active immunotherapeutic target for asthma and other allergic diseases. However, previous methods for preparing IgE fusion protein vaccines are complex. Antigen 43 (Ag43) is a surface protein found in Escherichia coli that contains α and β subunits (the α subunit contains multiple T epitopes). Here we constructed a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against IgE. The Ag43 system was constructed from the E. coli strain Tan109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43) expressing a partial Ag43 gene was introduced. The Fcε3 domain of the IgE gene was then subcloned into plasmid pETAg43, resulting in a recombinant plasmid pETAg43/Fcε3, which was used to transform Tan109 for Ag43/Fcε3 surface expression. Thereafter, Ag43/Fcε3 was investigated as an asthma vaccine in a mouse model. Ag43/Fcε3 was expressed on and could be separated from the bacterial surface by heating to 60° while retaining activity. Ag43/Fcε3, as a protein vaccine, produced neutralizing autoantibodies to murine IgE, induced significant anti‐asthma effects, and regulated IgE and T helper cytokines in a murine asthma model. Data show that Ag43/Fcε3 chimeric protein is a potential model vaccine for asthma treatment, and that the Ag43 system may be an effective tool for novel vaccine preparation to break immune tolerance to other self‐molecules.

Cytochalasin D promotes pulmonary metastasis of B16 melanoma through expression of tissue factor

Cytochalasin D (CytD) targets actin, a ubiquitous protein in eukaryotic cells. Previous studies have focused mainly on the antitumor effects of CytD. We previously found CytD to promote lung metastasis in B16 melanoma cells, which we had not anticipated, and, therefore, in the present study we investigated the possible underlying mechanisms. B16 melanoma cells were co-cultured with CytD and other agents and used to establish a lung metastatic model. In this B16 melanoma metastatic model, significantly increased lung metastasis and lung weight were found in CytD-treated mice, which was almost completely suppressed by tissue factor (TF) RNA interference expressed via lentivirus. The results of northern and western blot, and real-time RT-PCR analysis showed that the expression of TF was significantly upregulated in B16 cells treated with CytD but was significantly inhibited by TF RNA interference. In addition, upregulation and phosphorylation of mitogen-activated protein kinase p38 were also found in the metastatic lung tissues treated with CytD and in the B16 cells co-cultured with CytD and factor VIIa (FVIIa), but not in cells cultured with CytD, dimethyl sulfoxide or FVIIa alone. These results indicate that CytD stimulates the expression of TF in B16 melanoma cells, activating both coagulation-dependent and -independent pathways via binding to FVIIa, eventually promoting lung metastasis. TF interference is a potential approach to the prevention of B16 melanoma metastasis.

Bacterial surface display of endoglin by antigen 43 induces antitumor effectiveness via bypassing immunotolerance and inhibition of angiogenesis

Various angiogenesis‐related self‐molecules have been considered to be therapeutic targets. However, the direct use of self‐molecules as vaccines is not recommended because of the inherent ability of the host to develop immune tolerance. Antigen 43 (Ag43) is a surface protein found in E. coli and contains an α and a β subunits, which contains multiple T epitopes in α subunit. Here we construct a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against self‐molecules. The Ag43 system was constructed from an Escherichia coli strain Tan109, derived from JM109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43′) expressing a partial Ag43 gene was introduced. The extracellular domain of angiogenesis‐related endoglin gene was then subcloned into plasmid pETAg43′, resulting in a recombinant plasmid pETAg43′/ENDe which was then used to transform Tan109 for protein expression. We found that Ag43 and endoglin chimeric protein (Ag43′/ENDe) was expressed on the bacterial surface. The chimeric protein could be separated from the bacterial surface by heating to 60°C and yet retain activity. We used Ag43′/ENDe as a protein vaccine and found that it could disrupt immune tolerance against endoglin by inducing significant antitumor activities and inhibit angiogenesis in several tumor models without significant side effects. These data suggest that Ag43′/ENDe chimeric protein is a potential model vaccine for active tumor immunotherapy, and that Ag43 system could be an effective tool for novel vaccine preparation to break immune tolerance to other angiogenesis‐related self‐molecules for cancer therapy.

Toxicarioside A, isolated from tropical Antiaris toxicaria, blocks endoglin/TGF-β signaling in a bone marrow stromal cell line.

OBJECTIVE To investigate possible mechanism of toxicarioside A in HS-5 bone stromal cells. METHODS HS-5 bone stromal cells were cultured in media supplemented with various concentrations of toxicarioside A or control DMSO (not treatment). Endoglin and TGF-β were detected by Northern and Western blot analysis and quantified in a standard method. Downstream molecules of endoglin and TGF-β (Smad1, Smad2 and their active phosphorylated counterparts, pSmad1 and pSmad2) were also detected and quantified by Western blot analysis. In addition, cell proliferation assay and small interfering RNA (siRNA) against endoglin were used to certificate the function of endolgin in the HS-5 cells. RESULTS Compared with the not treated (0 μg/mL) or DMSO treated control HS-5 cells, HS-5 cells treated with toxicarioside A were found significant attenuation of endolgin and TGF-β expression. Significant inhibition of cell proliferation was also found in the HS-5 cells treated with toxicarioside A. ALK1-related Smad1 and ALK5-related Smad2 were decreased in HS-5 cells treated with toxicarioside A. In addition, phosphorylated Smad1 (pSmad1) and Smad2 (pSmad2) were also found attenuation in toxicarioside A-treated HS-5 cells. RNA interference showed that blockage of endoglin by siRNA also decreased Smad1 and Smad2 expression in HS-5 cells. CONCLUSIONS Our results indicate that toxicarioside A can influence bone marrow stromal HS-5s function and inhibit HS-5 cell proliferation by alteration of endoglin-related ALK1 (Smad1) and ALK5 (Smad2) signaling.

Isolation and identification of multidrug–resistant Staphylococcus haemolyticus from a laboratory–breeding mouse

OBJECTIVE To analysis and identify a bacterium strain isolated from laboratory breeding mouse far away from a hospital. METHODS Phenotype of the isolate was investigated by conventional microbiological methods, including Gram-staining, colony morphology, tests for haemolysis, catalase, coagulase, and antimicrobial susceptibility test. The mecA and 16S rRNA genes were amplified by the polymerase chain reaction (PCR) and sequenced. The base sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank database by phylogenetic analysis and multiple sequence alignment. RESULTS The isolate in this study was a gram positive, coagulase negative, and catalase positive coccus. The isolate was resistant to oxacillin, methicillin, penicillin, ampicillin, cefazolin, ciprofloxacin erythromycin, et al. PCR results indicated that the isolate was mecA gene positive and its 16S rRNA was 1 465 bp. Phylogenetic analysis of the resultant 16S rRNA indicated the isolate belonged to genus Saphylococcus, and multiple sequence alignment showed that the isolate was Saphylococcus haemolyticus with only one base difference from the corresponding 16S rRNA deposited in the GenBank. CONCLUSIONS 16S rRNA gene sequencing is a suitable technique for non-specialist researchers. Laboratory animals are possible sources of lethal pathogens, and researchers must adapt protective measures when they manipulate animals.

Effect of hepatocyte growth factor signaling pathway activation on Plasmodium berghei infection.

Abstract Objective To explore the effect of hepatocyte growth factor signaling pathway activation on Plasmodium berghei infection. Methods In this study, hepatocyte growth factor was detected by ELISA and Western blotting assay. Hepatocyte injury was detected by FITC-dextran absorption assay, and hepatocyte growth factor expression was shown to be expressed in the same injury cells by immunofluorescence against hepatocyte growth factor. In addition, Activation of hepatocyte growth factor and its receptor signaling pathway was detected with immunoprecipitation and detection of phosphorylation status. Results It was found that injury of hepatocytes by sporozoite migration induced the secretion of hepatocyte growth factor and it was hepatocyte growth factor that rendered hepatocytes susceptible to Plasmodium sporozoite infection. In addition, hepatocyte infections depended on activation of the hepatocyte growth factor and its receptor signaling pathway. Conclusions Our results indicate that hepatocyte growth factor and its receptor may possibly be potential targets for new approaches to malaria treatment.