Design, synthesis and biological evaluation of novel diarylacylhydrazones derivatives for the efficient treatment of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized with high mortality, unknown etiology, and lack of effective treatment. Many evidences validate that inhibiting the activation of STAT3 is an attractive therapeutic strategy for IPF. Herein, based on our previous findings that nifuroxazide (NIF) could effectively attenuate pulmonary fibrosis by inhibiting STAT3 activation, a series of diarylacylhydrazones derivatives have been designed and synthesized. Among them, compounds 44 and 52 could inhibit TGF-ß1-induced abnormal activation of NIH-3T3 and A549 cells, as well as migration and EMT of A549 cells. In a bleomycin-induced mouse pulmonary fibrosis model, the oral administration of 44 and 52 (bioavailability F = 31.75% and 42.08%) improved mouse lung function and slowed the progression of IPF. Moreover, 52 could reverse the pulmonary fibrosis in treatment model. Collectively, this work shows 44 and 52 could be a potential lead compound for the treatment of IPF, and it is worthy of further study.

Niclosamide-loaded nanoparticles (Ncl-NPs) reverse pulmonary fibrosis in vivo and in vitro.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF), a life-threatening interstitial lung disease, is characterized by excessive activation and proliferation of fibroblasts and epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AEC) accompanied by a large amount of extracellular matrix aggregation. There are no therapies to reverse pulmonary fibrosis, and nintedanib and pirfenidone could only slow down the decline of lung function of IPF patients and delay their survival time. Niclosamide (Ncl) is an antihelminthic drug approved by FDA, which has been reported to have pleiotropic pharmacological activities in recent years, but it's almost complete insolubility in water limits its clinical application. OBJECTIVES: To improve the water solubility of Ncl, explore its ability to reverse BLM-induced pulmonary fibrosis and its specific mechanism of action. METHODS: The Niclosamide-loaded nanoparticles (Ncl-NPs) were formed by emulsification solvent evaporation method. A mouse model induced by bleomycin (BLM) was established to evaluate its effects and mechanisms of inhibiting and reversing fibrosis in vivo. The cell models treated by transforming growth factor-ß1 (TGF-ß1) were used to examine the mechanism of Ncl-NPs inhibiting fibrosis in vitro. Flow cytometry, IHC, IL-4-induced macrophage model and co-culture system were used to assess the effect of Ncl-NPs on M2 polarization of macrophages. RESULTS: The Ncl-NPs improved the poor water solubility of Ncl. The lower dose of Ncl-NPs (2.5 mg/kg) showed the same effect of reversing established pulmonary fibrosis as free Ncl (5 mg/kg). Mechanistic studies revealed that Ncl-NPs blocked TGF-ß/Smad and signaling transducer and activator of transcription 3 (Stat3) signaling pathways and inhibited the M2 polarization of macrophages. Additionally, H&E staining of the tissues initially showed the safety of Ncl-NPs. CONCLUSION: These results indicate Ncl-NPs may serve as a new idea for the treatment of pulmonary fibrosis.

An oral phenylacrylic acid derivative suppressed hepatic stellate cell activation and ameliorated liver fibrosis by blocking TGF-ß1 signalling.

BACKGROUND AND AIMS: Liver fibrosis is an excessive wound-healing response governed by activated hepatic stellate cells (HSCs). To date, there is no drug available for liver fibrosis. Although ferulic acid (FA) has multiple pharmacological functions, its anti-hepatic fibrosis activity is weak. Based on the activity modification of the FA structure, we synthesized a series of phenylacrylic derivatives and found a superior compound, FA11. In this study, we investigated its antifibrotic effect and mechanism. METHODS: Activated HSC and CCl4 -induced mouse liver fibrosis were established and followed by FA11 treatment. Cell viability was measured by CCK-8 assay. Apoptosis and cell cycle analysis were conducted by flow cytometry. Western blot and Real-time qPCR were used to examine the expression of fibrotic and M1/M2-type macrophages markers. Degree of liver fibrosis was shown by histological staining. RESULTS: In vitro, FA11 inhibited TGF-ß1-induced LX-2 proliferation and led to apoptosis and cycle arrest. Furthermore, elevation of fibrotic markers in TGF-ß1-induced LX-2 and primary activated HSC was reversed by FA11. In vivo, FA11 administration alleviated collagen deposition and blocked HSC activation and epithelial-mesenchymal transition (EMT). Additionally, FA11 reduced macrophage infiltration in fibrotic liver and prevented macrophage polarization to a profibrotic phenotype. Meanwhile, the systemic toxicity of CCl4 was also ameliorated by FA11. Mechanistically, FA11 reversed the phosphorylation of canonical and noncanonical TGF-ß1 signalling, as well as FGFR1 signalling. CONCLUSIONS: We reported an oral phenylacrylic acid derivative, FA11, which showed excellent antifibrotic activity and was expected to be an anti-hepatic fibrosis candidate.

Discovery and evaluation of phenacrylanilide derivatives as novel potential anti-liver fibrosis agents.

Liver fibrosis is characterized by the excessive deposition of extracellular matrix components and results from chronic liver injury. At present, there is no approved drug for the treatment of liver fibrosis by the Food and Drug Administration. Here, we have reported a series of novel compounds with phenacrylanilide scaffolds that potently inhibit the transfer growth factor ß1 (TGF-ß1)-induced activation of LX-2, a hepatic stellate cell (HSC) line. Among them, compound 42 suppressed TGF-ß1-induced upregulation of fibrotic markers (α-SMA and fibronectin) and showed excellent safety in vitro. Furthermore, in a carbon tetrachloride (CCl4) -induced liver fibrosis model, 42 at a dose of 30 mg/kg/day through oral administration for 3 weeks effectively improved liver function, restored damaged liver structures, and reduced collagen deposition, with a greater effect than Tranilast. In addition, epithelial-mesenchymal transition (EMT) is inhibited by compound 42 in the process of fibrosis. Meanwhile, the imbalanced immune microenvironment could also be effectively reversed. More interestingly, compound 42 prolongs the survival of CCl4 mice and ameliorates CCl4-induced injury to spleen, kidney, lung and heart. Altogether, these results suggest that 42 could be a potential drug candidate for the treatment of liver fibrosis.

Nifuroxazide ameliorates pulmonary fibrosis by blocking myofibroblast genesis: a drug repurposing study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a serious interstitial lung disease with a complex pathogenesis and high mortality. The development of new drugs is time-consuming and laborious; therefore, research on the new use of old drugs can save time and clinical costs and even avoid serious side effects. Nifuroxazide (NIF) was originally used to treat diarrhoea, but more recently, it has been found to have additional pharmacological effects, such as anti-tumour effects and inhibition of inflammatory diseases related to diabetic nephropathy. However, there are no reports regarding its role in pulmonary fibrosis. METHODS: The therapeutic effect of NIF on pulmonary fibrosis in vivo was measured by ELISA, hydroxyproline content, H&E and Masson staining, immunohistochemistry (IHC) and western blot. Immune cell content in lung tissue was also analysed by flow cytometry. NIF cytotoxicity was evaluated in NIH/3T3 cells, human pulmonary fibroblasts (HPFs), A549 cells and rat primary lung fibroblasts (RPLFs) using the MTT assay. Finally, an in vitro cell model created by transforming growth factor-ß1 (TGF-ß1) stimulation was assessed using different experiments (immunofluorescence, western blot and wound migration assay) to evaluate the effects of NIF on the activation of NIH/3T3 and HPF cells and the epithelial-mesenchymal transition (EMT) and migration of A549 cells. RESULTS: In vivo, intraperitoneal injection of NIF relieved and reversed pulmonary fibrosis caused by bleomycin (BLM) bronchial instillation. In addition, NIF inhibited the expression of a variety of cellular inflammatory factors and immune cells. Furthermore, NIF suppressed the activation of fibroblasts and EMT of epithelial cells induced by TGF-ß1. Most importantly, we used an analytical docking experiment and thermal shift assay to further verify that NIF functions in conjunction with signal transducer and activator of transcription 3 (Stat3). Moreover, NIF inhibited the TGF-ß/Smad pathway in vitro and decreased the expression of phosphorylated Stat3 in vitro and in vivo. CONCLUSION: Taken together, we conclude that NIF inhibits and reverses pulmonary fibrosis, and these results support NIF as a viable therapeutic option for IPF treatment.

Liver Fibrosis: Therapeutic Targets and Advances in Drug Therapy.

Liver fibrosis is an abnormal wound repair response caused by a variety of chronic liver injuries, which is characterized by over-deposition of diffuse extracellular matrix (ECM) and anomalous hyperplasia of connective tissue, and it may further develop into liver cirrhosis, liver failure or liver cancer. To date, chronic liver diseases accompanied with liver fibrosis have caused significant morbidity and mortality in the world with increasing tendency. Although early liver fibrosis has been reported to be reversible, the detailed mechanism of reversing liver fibrosis is still unclear and there is lack of an effective treatment for liver fibrosis. Thus, it is still a top priority for the research and development of anti-fibrosis drugs. In recent years, many strategies have emerged as crucial means to inhibit the occurrence and development of liver fibrosis including anti-inflammation and liver protection, inhibition of hepatic stellate cells (HSCs) activation and proliferation, reduction of ECM overproduction and acceleration of ECM degradation. Moreover, gene therapy has been proved to be a promising anti-fibrosis method. Here, we provide an overview of the relevant targets and drugs under development. We aim to classify and summarize their potential roles in treatment of liver fibrosis, and discuss the challenges and development of anti-fibrosis drugs.

Epigenetic regulation in fibrosis progress.

Fibrosis, a common process of chronic inflammatory diseases, is defined as a repair response disorder when organs undergo continuous damage, ultimately leading to scar formation and functional failure. Around the world, fibrotic diseases cause high mortality, unfortunately, with limited treatment means in clinical practice. With the development and application of deep sequencing technology, comprehensively exploring the epigenetic mechanism in fibrosis has been allowed. Extensive remodeling of epigenetics controlling various cells phenotype and molecular mechanisms involved in fibrogenesis was subsequently verified. In this review, we summarize the regulatory mechanisms of DNA methylation, histone modification, noncoding RNAs (ncRNAs) and N6-methyladenosine (m6A) modification in organ fibrosis, focusing on heart, liver, lung and kidney. Additionally, we emphasize the diversity of epigenetics in the cellular and molecular mechanisms related to fibrosis. Finally, the potential and prospect of targeted therapy for fibrosis based on epigenetic is discussed.

A novel multikinase inhibitor SKLB-YTH-60 ameliorates inflammation and fibrosis in bleomycin-induced lung fibrosis mouse models.

OBJECTIVES: Idiopathic pulmonary fibrosis (IPF) is marked by the excessive accumulation of extracellular matrix, which participates in a variety of chronic diseases or injuries and seriously threatens human health. Due to the side effects of clinical drugs, there is still a need to develop novel and less toxic drugs to treat pulmonary fibrosis. MATERIALS AND METHODS: SKLB-YTH-60 was developed through computer-aided drug design, de novo synthesis and high-throughput screening. We employed the bleomycin (BLM)-induced lung fibrosis animal models and used TGF-ß1 to induce the epithelial-mesenchymal transition (EMT) of A549 cells in vitro. Meanwhile, the protein expression of collagen I and the α-smooth muscle actin (α-SMA), E-cadherin, p-FGFR1, p-PLCγ, p-Smad2/3 and p-Erk1/2 was detected by western blot. RESULTS: YTH-60 has obvious anti-proliferative activity on fibroblasts and A549 cells. Moreover, YTH-60 could impair the EMT of A549 cells and suppressed fibrosis by inhibiting FGFR and TGF-ß/Smad-dependent pathways. Intraperitoneal administration of preventive YTH-60 could significantly reduce the degree of fibrosis in mice and regulate the imbalance of the immune microenvironment. In addition, we observed that therapeutic YTH-60 treatment attenuated fibrotic changes in mice during the period of fibrosis. Importantly, YTH-60 has shown an acceptable oral bioavailability (F = 17.86%) and appropriate eliminated half-life time (T1/2  = 8.03 hours). CONCLUSIONS: Taken together, these preclinical evaluations suggested that YTH-60 could be a promising drug candidate for treating IPF.

Hemangiopericytoma: Incidence, Treatment, and Prognosis Analysis Based on SEER Database.

BACKGROUND: Hemangiopericytomas are rare tumors derived from pericytes surrounding the blood vessels. The clinicopathological characteristics and prognosis of hemangiopericytoma patients remain mostly unknown. In this retrospective cohort study, we assessed the clinicopathological characteristics of hemangiopericytoma patients, as well as the clinical usefulness of different treatment modalities. Material and Methods. We collected the clinicopathological data (between 1975 and 2016) of hemangiopericytoma and hemangioendothelioma patients from the Surveillance, Epidemiology, and End Results (SEER) database. Incidence, treatment, and patient prognosis were assessed. RESULTS: Data from 1474 patients were analyzed in our study cohort (hemangiopericytoma: n = 1243; hemangioendothelioma: n = 231). The incidence of hemangiopericytoma in 2016 was 0.060 per 100,000 individuals. The overall survival (OS) and cancer-specific survival (CSS) did not differ between patients with hemangioendothelioma and those with hemangiopericytoma (P = 0.721, P = 0.544). The tumor grade had no effect on the OS of hemangiopericytoma patients. Multivariate analysis revealed the clinical usefulness of surgery in hemangiopericytoma patients (HR = 0.15, 95% confidence interval: 0.05-0.41, P < 0.001). In contrast, radiotherapy did not improve OS (P = 0.497) or CSS (P = 0.584), and chemotherapy worsened patient survival (P < 0.001). Additionally, the combination of surgery and radiotherapy had a similar effect with surgery alone on hemangiopericytoma patient survival (OS: P = 0.900; CSS: P = 0.156). Surgery plus chemotherapy provided a worse clinical benefit than surgery alone (P < 0.001). CONCLUSIONS: Our findings suggested that hemangiopericytoma had a similar prognosis with hemangioendothelioma. Surgery was the only effective treatment that provided survival benefits in hemangiopericytoma patients, while the clinical usefulness of adjuvant chemotherapy or radiotherapy was limited.

Cryptotanshinone reverses the epithelial-mesenchymal transformation process and attenuates bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic interstitial pneumonia that causes pulmonary tissue damage and functional impairment. To investigate the effects of cryptotanshinone on pulmonary fibrosis, the expression of NIH/3T3, HPF, and rat primary pulmonary fibroblasts was measured and found to be inhibited by CPT in a time- and concentration-dependent manner, and the upregulation of α-SMA expression in NIH/3T3 and HPF cells, which had been stimulated by TGFß-1, was decreased after CPT administration. We observed that CPT could reverse the increase in α-SMA expression and vimentin and the decrease in E-cad expression in A549 cells, which had been induced by 5 ng/mL TGFß-1, indicating that CPT has inhibitory effects in the EMT process. A BLM-induced pulmonary fibrosis model was established in C57BL/6 mice. The lung coefficient and hydroxyproline content increased significantly in the BLM-induced group and were decreased in the CPT-treated group. The expression levels of collagen-I and α-SMA and the phosphorylation level of Stat3 were significantly increased, and CPT treatment decreased these levels. Furthermore, the results from the flow cytometry analysis indicated that, in lung tissues, the frequencies of MDSCs, macrophages, DCs and T cells were considerably increased in the BLM-induced group, while CPT treatment reduced these immunocyte populations.

Therapeutic effects of Erbin inhibitor on spinal cord contusion in mice.

Erbin has been shown to maintain the integrity of cell structure, regulate the proliferation and differentiation of cell and transconduct signals in the pathways. This study was conducted to assess the therapeutic effects of an Erbin inhibitor on spinal cord contusion in mice. Spinal contusion models of mouse were constructed and treated with an Erbin inhibitor. The experimental animals were divided into control (normal animal without any treatment), models with spinal cord injury (SIM), and models receiving Erbin inhibitor (Inhibitor). The contents of 5-hydroxytryptamine (5-HT) and reactive oxygen species (ROS) in the brain and spinal cord tissues were measured using ELISA. The expression of ERK1/2, MAPK, NF-kB and NRG1 was quantified using qRT-PCR, Western blot analysis and immunohistochemistry. Flow cytometry was used to determine the formation of macrophages. Erbin interference vector was constructed and its interference effect on the expression of these genes was characterized in cultured bone marrow cells. Spinal contusion models were successfully constructed. Administering Erbin inhibitor inhibited the expression of ERK1/2, MAPK and NF-kB and up-regulated the expression of NRG1. Flow cytometry showed that Erbin inhibitor induced the formation of a large number of macrophages, which are beneficial to the recovery of spinal cord injury. Experiments with Erbin interference vector showed similar impacts on the expression of genes at cellular level as the inhibitor did. Our work has demonstrated that the Erbin inhibitor is very effective to treat spinal cord contusion in mice. The possible mechanism of therapeutic effect is that the inhibitor suppresses the ERK1/2/MAPK and/or NF-kB/MAPK signal pathways and enhances the NRG1-ErbB signaling pathway by reducing the expression of Erbin, leading to the inhibition of apoptosis, promotion of proliferation and differentiation, and subsequent repair of the damaged spinal cord.

Netrin-1 with stem cells promote angiogenesis in limb ischemic rats.

BACKGROUND: Recent findings have elucidated that netrin-1 has ability of promoting angiogenesis besides the functions in nervous system. Autologous mesenchymal stem cells (MSCs) transplantation is now proved to be an effective method to treat peripheral arterial disease. However there are still many patients who cannot complete full treatments. Therefore it is necessary to improve the effectiveness. This study estimated the curative effects in chronic limb ischemia when MSCs allied with netrin-1. MATERIALS AND METHODS: Thirty-six rats were made into chronic limb ischemia models. They were randomly assigned to four groups, netrin-1 + MSCs group (treated with netrin-1 and MSCs derived from peripheral blood), MSCs group (treated with MSCs individually), netrin-1 group (treated with netrin-1 individually), and control group (treated with saline). Measurements of murine behaviors, vascular endothelial growth factor expression, and capillary density in ischemia limb were performed on days 7, 14, and 28 after treatments; measurements of contraction force in ischemia limb was performed on day 28 after treatments to compare differences among the groups. RESULTS: Netrin-1 allied with MSCs significantly increased Tarlov score, vascular endothelial growth factor expression, capillary density, and muscular strength in ischemia limb. CONCLUSIONS: Netrin-1 allied with MSCs derived from peripheral blood significantly promoted angiogenesis in aged rats with chronic limb ischemia. It may be a promising method of treating peripheral arterial disease in the future.

Construction and expression of D-dimer and GPIIb/IIIa single-chain bispecific antibody.

The aim of this study was to construct a plasmid expressing glycoprotein IIb-IIIa (GPIIb/IIIa) and D-dimer single-chain bispecific antibody for the targeted therapy of thrombosis. The phosphorylated gene encoding the anti-GPIIb/IIIa single-chain variable fragment (scFv) and the gene encoding the anti-D-dimer scFv were amplified by PCR and linked in tandem by blunt-end ligation. The recombinant plasmid was transfected into the competent cell line HB2151 and identified by PCR and DNA sequencing. Then, the soluble recombinant antibody in bacterial lysates was purified by an NTA column and molecular sieve chromatography in turn. Finally, the binding specificity of the purified antibody was tested by enzyme-linked immunosorbent assay (ELISA). Results demonstrated that the construction of the expression plasmid was successful and the purified recombinant protein, which had a molecular weight of ∼56 kDa, was specific to GPIIb/IIIa and D-dimer. In conclusion, a plasmid expressing a bispecific antibody was constructed by a new method of blunt-end ligation. The soluble recombinant protein is a promising platform for target-oriented thrombolytic therapy.

Erbin is required for myelination in regenerated axons after injury.

Neuregulin 1 (NRG1) is an axon-derived factor that is critical for Schwann cell (SC) development and myelinogenesis in a manner dependent on transmembrane tyrosine kinases ErbB2 and ErbB3. Recent studies suggest that NRG1 signaling plays a role in remyelination of regenerated nerves after injury. In this study, we investigated the role of Erbin, a protein that interacts with ErbB2 in remyelination of injured nerves. We show that Erbin expression increased dramatically in injured nerves. Myelinated axons were fewer, and g-ratios of those that were myelinated were increased in erbin(-/-) mice, which were impaired in functional recovery from nerve injury. These results indicate a necessary role of Erbin in remyelination of regenerating axons. Erbin ablation had little effect on numbers of BrdU-labeled and TUNEL-labeled SCs, suggesting mechanisms independent of altered proliferation or apoptosis. We demonstrated that Erbin mutant mice were impaired in raising or maintaining the levels of ErbB2 and in producing NRG1 in axons. Together, these observations demonstrate that Erbin is required for remyelination of regenerated axons after injury, probably by regulating ErbB2 and NRG1 levels, identifying a novel player in regulating remyelination.

KLF6 inhibits estrogen receptor-mediated cell growth in breast cancer via a c-Src-mediated pathway.

Estrogen receptors play a key role in breast cancer development and progression. Kruppel-like factor 6 (KLF6) is a tumour-suppressing protein. The aim of this study was to identify the role of KLF6 inhibition in estrogen receptor(alpha) (ERalpha)-elicited breast cancer development. Protein expression levels were examined by western blot analysis and immunoprecipitation was used to analyse interactions between proteins. An MTT assay was used to study cell proliferation. We found that KLF6 mediates cell growth in ERalpha-positive breast cancer cells through interaction with the c-Src protein. This interaction causes inactivation of the Erk and Akt proteins. These pathways are critical for the proliferation and survival of breast cancer cells. We also established that KLF6 could not mediate cell growth in ERalpha-negative cells. We conclude that KLF6 can modulate ERalpha-mediated cell growth in breast cancer cells. The unique role of KLF6 in mediating cell growth in breast cancer cells opens up the possibility of a new therapeutic strategy for treating breast cancer.

Construction and expression of anti-CD62P/anti-GPIIb-IIIa diabody.

OBJECTIVE: To construct 3 expression plasmids for the targeted therapy of thrombosis: single chain variable fragment (scFv) of monoclonal antibody (mAb) 7E3 that can identify and bind platelet glycoprotein GPIIb-IIIa complex, scFv of mAb WAPS12.2 that can identify and bind P selectin (CD62P), a diabody that can identify and bind GPIIb-IIIa and CD62P simultaneously, and to investigate whether the vectors can express correctly. METHODS: This study was carried out at the Laboratory of Hunan Yuantai Biological Technology Co. Ltd, Hubei, China from September 2007 to May 2008. Total RNA of mAb 7E3 cells and WAPS12.2 cells were obtained. Reverse transcriptase polymerase chain reaction (PCR) was carried out to obtain the genes of variable regions of light and heavy chains of 7E3 and WAPS12.2. Target genes were named 7E3VL, 7E3VH, CD62PVL, and CD62PVH. The 7E3VL-7E3VH and CD62PVL-CD62PVH were obtained by PCR and connected with pET-22b(+). Products were named pET-scFv7E3 and pET-scFvCD62P. The 7E3VL-CD62PVH and CD62PVL-7E3VH were obtained by using overlap PCR and were ligated to pET-22b(+). The products were named pET-ED1 and pET-ED2. The PCR was performed by taking pET-ED2 as a template to obtain the complete operon gene and was ligated to pET-ED1. The product was named pET-7ECD. RESULTS: The identification by restriction endonuclease cleavage and DNA sequencing confirmed that the construction of these expression plasmids was successful. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot confirmed that these plasmids expressed correctly. CONCLUSION: The expression plasmids pET-scFv7E3, pET-scFvCD62P, and pET-7ECD were constructed and expressed successfully, and laid a good foundation for further research on target-oriented thrombolytic agents.

Construction of humanized carcinoembryonic antigen specific single chain variable fragment and mitomycin conjugate.

AIM: To construct a new target-oriented conjugate of humanized carcinoembryonic antigen (CEA) specific single chain variable fragment (scFv) and mitomycin (MMC) against colorectal cancer, and to investigate its influence on the growth and apoptosis of colorectal cancer cells. METHODS: The primer was designed according to the gene sequence described in reference 16, which respectively contains restriction enzyme cleavage sites BamHI and EcoRI in its upstream and downstream. PCR was performed with the plasmid as template containing genes of humanized anti-CEA scFv. The product was digested by BamHI and EcoRI, and connected to an expression vector which also has the restriction enzyme cleavage sites BamHI and EcoR. Expression of the reaction was induced by isopropy-beta-D-thiogalactoside (IPTG). Then the expression product was covalently coupled with MMC by dextran T-40. The immunoreactivity of the conjugate against colorectal cancer cells as well as CEA was measured by enzyme linked immunosorbent assay (ELISA). The inhibiting ratio of conjugate on the growth of colorectal cancer cells was also measured by ELISA. The effect of conjugate on the apoptosis of colorectal cancer cells was determined by flow cytometry (FCM). RESULTS: Restriction endonuclease cleavage and gene sequencing confirmed that the expression vector was successfully constructed. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) confirmed that this vector correctly expressed the fusion protein. ELISA confirmed that the conjugate had quite a strong immunoreactivity against colorectal cancer cells and CEA. The conjugate had inhibitory effects on colorectal cancer cells in a concentration-dependent manner and could induce apoptosis of colorectal cancer cells in a concentration-dependent manner. CONCLUSION: The CEA-scFv-MMC conjugate can be successfully constructed and is able to inhibit the growth and induce apoptosis of colorectal cancer cells.

Combination of VEGF(165)/Angiopoietin-1 gene and endothelial progenitor cells for therapeutic neovascularization.

Previous studies have established that vascular endothelial growth factor (VEGF), Angiopoietin-1 (Ang1) and endothelial progenitor cells (EPCs) play important roles in neovascularization, suggesting that combination of them would be a promising therapy for ischemic diseases. So we constructed the adeno-associated virus-2 (AAV-2) vectors simultaneously encoding human VEGF(165) and Ang1 (AAV-Ang1/VEGF), and investigated the combination therapeutic effect of AAV-Ang1/VEGF with EPCs in a rabbit ischemic hindlimb model. In the present study we found that AAV-Ang1/VEGF could successfully and efficiently transfer VEGF(165) and Ang1 gene into bone marrow derived EPCs for gene therapy. Combined administration of AAV-Ang1/VEGF with EPCs had higher blood flow recovery, cellularity, capillary density and smooth muscle alpha-actin positive vessel density than administration of either of them alone. Furthermore, the strategy of pre-intramuscular injection of AAV-Ang1/VEGF followed by EPCs transplantation had a higher therapeutic effect than the strategy of transplantation of AAV-Ang1/VEGF transduced EPCs. It seemed that the former strategy may be a promising therapy for ischemic diseases.

Adeno-associated virus vectors simultaneously encoding VEGF and angiopoietin-1 enhances neovascularization in ischemic rabbit hind-limbs.

AIM: Angiopoietin-1 (Ang1) and vascular endothelial growth factor A (VEGF) play important roles in vascular formation and maturation, suggesting a combination of these 2 would be a promising therapy for ischemic diseases. So we constructed an adeno-associated virus (AAV) vector, simultaneously encoding human VEGF(165) and Ang1 (AAV-VEGF/Ang1), and investigated its therapeutic effect in a rabbit ischemic hind-limb model. METHODS: Four experimental groups were used to prepare the rabbit ischemic hind-limb model following AAV vectors intramuscular administration as follows: PBS (phosphate buffered solution), AAV-VEGF, AAV-Ang1, AAV-VEGF/Ang1. RESULTS: Eight weeks after administration, human VEGF(165) and Ang1 were detected by RT-PCR, Western blotting and histochemical staining methods in AAV-VEGF/Ang1 transduced muscles. Group AAV-VEGF/Ang1 showed a significantly increased blood-flow recovery in ischemic hind-limbs compared with the other groups. Histological staining for alkaline phosphatase showed that capillary density of group AAV-VEGF/Ang1 or AAV-VEGF was significantly higher than that of group PBS or AAV-Ang1. Histological immunostaining for smooth muscle alpha-actin (alpha-SMA) revealed that group AAV-VEGF/Ang1 had the highest density of alpha-SMA-positive vessels compared with the other groups. Vascular leakage, one of the major adverse effects induced by VEGF, was very severe in group AAV-VEGF, but the permeability was obviously reduced when VEGF was co-expressed with Ang1 in group AAV-VEGF/Ang1. CONCLUSION: AAV vectors can simultaneously encode 2 proteins which can be efficiently and stably co-expressed in transduced tissues. AAV-mediated VEGF and Ang1 gene transfer enhances neovascularization, prevents capillary leakage, and improves blood flow in a rabbit hind-limb ischemic model. These findings suggest that intramuscular administration of AAV-VEGF/Ang1 may be useful in the treatment of ischemic diseases.

Expression of Survivin in pancreatic cancer and its correlation to expression of Bcl-2.

AIM: To investigate the expression of Survivin in pancreatic cancer and its correlation to the expression of Bcl-2. METHODS: Survivin and Bcl-2 expressions were examined by immunohistochemistry in 42 tissue samples from pancreatic cancer and 10 from normal pancrease. RESULTS: No survivin expression was detected in the tissue samples from normal pancrease, while it was detected in 34 of 42 tissue samples from pancreatic cancer (81.95%). There was a correlation between survivin expression and differentiation and stages of pancreatic cancer. Survivin positive cases were strongly correlated to Bcl-2 expression (28/30 vs 6/12, P<0.05). CONCLUSION: Overexpression of survivin plays an important role in the development and progression of pancreatic cancer, and correlates to the expression of Bcl-2. Survivin expression can be used as a prognostic factor.