Song-lin Zhou

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.

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.

Strophalloside induces apoptosis of SGC-7901 cells through the mitochondrion-dependent caspase-3 pathway.

Cardenolides with special chemical structures have been considered as effective anti-cancer drugs in clinic trials. Strophalloside is a cardenolide we recently isolated from Antiaris toxicaria obtained from Hainan, China. The aim of this study was to investigate the possible anticancer effects induced by strophalloside and the underlying molecular mechanism. Gastric carcinoma SGC-7901 cells were treated with strophalloside at various concentrations for different times, and resulting cell viability was determined by the MTT assay, and the motility and invasion of tumor cells were assessed by the Transwell chamber assay. Apoptosis were measured by Annexin V-FITC/PI and Hoechst staining. The changes of mitochondrial transmembrane potential were examined by a JC-1 kit. The expressions of pro-apoptotic protein cytochrome c, caspase-3 and caspase-9 were detected by western blotting analysis. The results showed that strophalloside was capable of reducing cell viability, inhibiting cell growth, and suppressing cell migration and invasion in a time- and dose-dependent manner. Mitochondrial membrane potential declined and the concentration of cytochrome c increased in cytoplasm and caspase-3 and caspase-9 were cleaved into activated states, suggesting that cytochrome c was released from the mitochondrion to cytoplasm and finally activated the caspase-dependent apoptosis pathway. Our results indicate that strophalloside is a potential anticancer drug.

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.

Sanpao herbs inhibit development of atopic dermatitis in Balb/c mice.

OBJECTIVE To explore the effects of SANPAOCAO (SPC), a compound traditional Chinese folk medicine, on chronic dermatitis/eczema in mice induced by 2, 4-dinitrochlorobenzene (DNCB). METHODS Thirty-three Balb/c mice were randomly divided into a negative control group, a positive control group, a prednisolone treatment group, an SPC ethanol extract treatment group, a Cardiospermum halicacabum ethanol extract treatment group, a Physalis minima ethanol extract treatment group, and a Jussiaea repens ethanol extract treatment group. Mice in the six treatment groups had twenty-five microliters of 0.1% DNCB in acetone/olive oil (3: 1) applied to each side of their right ears and dorsal skin three times a week, over a 5 week period. They were treated with prednisolone or the various kinds of ethanol extract after each challenge. The weight difference between the two ears, pathological changes in the right ears, dermal inflammatory cell numbers, and total serum Ig E levels were used to assess the effects of the drugs. RESULTS after the 5 weeks of challenges, the weight differences of the ears in the SPC group and the prednisolone group were significantly less than those in the other groups. There was evidence of significant suppression of the development of dermatitis, as determined by a histological examination and the serum Ig E levels. CONCLUSION SPC has beneficial effects when used in the treatment of chronic dermatitis-eczema in mice.

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.

RNA interference against interleukin-5 attenuates airway inflammation and hyperresponsiveness in an asthma model *

Interleukin-5 (IL-5) accompanies the development of airway inflammation and hyperresponsiveness through the activation of eosinophils. Therefore, interference of IL-5 expression in lung tissue seems to be an accepted approach in asthma therapy. In this study, we designed a small interfering RNA (siRNA) to inhibit the expression of IL-5. The siRNAs against IL-5 were constructed in a lentivirus expressing system, and 1.5×106 IFU (inclusion-forming unit) lentiviruses were administered intratracheally to ovalbumin (OVA)-sensitized murine asthmatic models. Our results show that lentivirus-delivered siRNA against IL-5 efficiently inhibited the IL-5 messenger ribonucleic acid (mRNA) expression and significantly attenuated the inflammation in lung tissue. Significant decrease of eosinophils and inflammatory cells were found in peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue. In addition, significant inhibition of airway hyperresponsiveness (AHR) was found in the mice treated with siRNA against IL-5. These observations demonstrate that siRNA delivered by means of the lentivirus system is possibly an efficacious therapeutic approach for asthma.

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.