Synthesis and anticancer properties of celastrol derivatives involved in the inhibition of VEGF.

In this study, fourteen celastrol derivatives (1-14) were synthesised by esterification of celastrol at the 29th position. The in vitro anticancer activity of them was determined by the MTT assay. All the synthetic compounds showed significant antiproliferative activity against six cancer cells, with IC50 of the submicron molar level. The most promising compound (2) blocked the cell cycle in the G2 phase and inhibited the expression of VEGF and MMP-9 in gastric cancer cell line MGC-803. In flow cytometry analysis, compound 2 induced cancer cell apoptosis in a dose-dependent manner. In the mouse tumour xenograft model, compound 2 showed significant anti-tumour activity in vivo at the dosage of 2.5 mg/kg and 1 mg/kg, with a higher inhibition rate than 5-FU (10 mg/kg). What's more, the anticancer mechanism involved in the inhibition of VEGF and the toxicity evaluation of compound 2 were also investigated.

Antiseizure Properties of Histamine H3 Receptor Antagonists Belonging 3,4-Dihydroquinolin-2(1H)-Ones.

H3R is becoming an attractive and promising target for epilepsy treatment as well as the discovery of antiepileptics. In this work, a series of 6-aminoalkoxy-3,4-dihydroquinolin-2(1H)-ones was prepared to screen their H3R antagonistic activities and antiseizure effects. The majority of the target compounds displayed a potent H3R antagonistic activity. Among them, compounds 2a, 2c, 2h, and 4a showed submicromolar H3R antagonistic activity with an IC50 of 0.52, 0.47, 0.12, and 0.37 µM, respectively. The maximal electroshock seizure (MES) model screened out three compounds (2h, 4a, and 4b) with antiseizure activity. Meanwhile, the pentylenetetrazole (PTZ)-induced seizure test gave a result that no compound can resist the seizures induced by PTZ. Additionally, the anti-MES action of compound 4a fully vanished when it was administrated combined with an H3R agonist (RAMH). These results showed that the antiseizure role of compound 4a might be achieved by antagonizing the H3R receptor. The molecular docking of 2h, 4a, and PIT with the H3R protein predicted their possible binding patterns and gave a presentation that 2h, 4a, and PIT had a similar binding model with H3R.

Knockdown of METTL14 suppresses the malignant progression of non-small cell lung cancer by reducing Twist expression.

Non-small cell lung cancer (NSCLC) is one of the most malignant cancer types. N6-methyladenosine (m6A), an abundant eukaryotic mRNA modification, has been observed in multiple diseases, particularly cancer. Methyltransferase-like 14 (METTL14) is a central component of the m6A methyltransferase complex and has been reported to promote tumor development in several cancer types. The present study aimed to investigate the role of METTL14 in NSCLC. Relevant clinical and mRNA sequencing data for m6A-related genes were downloaded from The Cancer Genome Atlas database. R software was used to evaluate the expression of m6A regulators in NSCLC. The biological functions of METTL14 were evaluated using Cell Counting Kit-8, colony formation, Transwell migration and western blot analyses. The results demonstrated that METTL14 expression was upregulated in NSCLC tissues and cell lines, and its expression was high in cancer tissues from patients with NSCLC with all four stages (I, II, III and IV) of disease. METTL14 downregulation inhibited cell proliferation and migration in A549 and SK-MES-1 lung cancer cell lines. Knockdown of METTL14 in lung cancer cell lines increased E-cadherin expression and suppressed N-cadherin expression. Furthermore, METTL14 downregulation reduced the expression levels of the transcription factor Twist and the p-AKT/AKT ratio. In conclusion, the present findings revealed that silencing of METTL14 suppressed NSCLC malignancy by inhibiting Twist-mediated activation of AKT signaling. These data suggest that METTL14 may be a potential therapeutic target for NSCLC.

Fibrinogen/AKT/Microfilament Axis Promotes Colitis by Enhancing Vascular Permeability.

BACKGROUND & AIMS: Increased vascular permeability (VP) has been indicated to play an important role in the pathogenesis of inflammatory bowel disease (IBD). However, the pathological causes of increased intestinal VP in IBD remain largely unknown. METHOD: Fibrinogen level was measured in dextran sulphate sodium (DSS)-induced colitis and patients with ulcerative colitis. Gly-Pro-Arg-Pro acetate (GPRP), an Fg inhibitor, was used to detect the effect of Fg inhibition on the pathogenesis of DSS-induced colitis, as indicated by tissue damage, cytokine release and inflammatory cell infiltration. Miles assay was used to detect vascular permeability. RESULTS: Through tandem mass tag-based quantitative proteomics, fibrinogen (Fg) was found to be upregulated in the colon of DSS-treated mice, which was consistent with increased Fg level in colon sample of patients with ulcerative colitis. Gly-Pro-Arg-Pro acetate (GPRP), an Fg inhibitor, significantly alleviated DSS-induced colitis as indicated by improvement of body weight loss and mortality. GPRP decreased colonic inflammation and VP in DSS-treated mice. In vivo, Fg enhanced VP as indicated by Miles assay, which was significantly inhibited by GRPR, AKT (serine/threonine kinase 1) inhibitors and low doses of Jasplakinolide which induced actin polymerization, while was dramatically enhanced by Cytochalasin D (an actin polymerization inhibitor). Moreover, activation of AKT was found in vessels of DSS-treated mice. In vitro, Fg induced activation of AKT and depolymerization of microfilament and promoted cell-to-cell disaggregation. Furthermore, inhibition of AKT decreased Fg-induced microfilament depolymerization. CONCLUSIONS: Our findings highlight the importance of Fg in regulating colitis by modulation of VP via activating AKT and subsequent depolymerization of microfilament and suggest Fg as an attractive target for anti-colitis treatment.

Reelin Promotes Cisplatin Resistance by Induction of Epithelial-Mesenchymal Transition via p38/GSK3ß/Snail Signaling in Non-Small Cell Lung Cancer.

BACKGROUND Emerging evidence suggests the involvement of Reelin in chemoresistance in various cancers. However, its function in cisplatin (DDP) sensitivity of non-small cell lung cancer (NSCLC) needs to be investigated. MATERIAL AND METHODS Reelin expression in cisplatin-sensitive A549 cells and cisplatin-resistant NSCLC (A549/DDP) cells was analyzed by western blot analysis. qRT-PCR, western blotting, immunofluorescence, CCK-8 assays, Annexin V/propidium iodide apoptosis assay, and Transwell migration assays were carried out to determine the function of Reelin on DDP resistance. RESULTS Reelin was markedly increased in A549/DDP cells relative to A549 cells. Knockdown of Reelin enhanced DDP chemosensitivity of A549/DDP cells, whereas overexpression of Reelin enhanced DDP resistance of A549, H1299, and H460 cells. Reelin induced DDP resistance in NSCLC cells via facilitating epithelial-mesenchymal transition (EMT). Furthermore, Reelin modulated p38/GSK3ß signal transduction and promoted Snail (EMT-associated transcription factor) expression. Suppression of p38/Snail reversed Reelin-induced EMT and resistance of NSCLC cells to DDP. CONCLUSIONS These data indicated that Reelin induces DDP resistance of NSCLC by regulation of the p38/GSK3ß/Snail/EMT signaling pathway and provide evidence that Reelin suppression can be an effective strategy to suppress DDP resistance in NSCLC.

Dehydrogenase/reductase SDR family member 2 silencing sensitizes an oxaliplatin­resistant cell line to oxaliplatin by inhibiting excision repair cross­complementing group 1 protein expression.

Oxaliplatin (Oxa)­based chemotherapy is widely used as the first­line treatment for colorectal cancer (CRC). However, Oxa­resistance is common for many postoperative CRC patients. To explore drug resistance in CRC, an Oxa­resistant cell line, HCT116/Oxa, was established from parental HCT116 cells. These Oxa­resistant cells exhibited characteristics of epithelial­mesenchymal transition (EMT) and a higher migratory capacity than parental cells. Protein profiles of HCT116/Oxa and HCT116 cells were compared using a tandem mass tag­based quantitative proteomics technique. The protein dehydrogenase/reductase SDR family member 2 (DHRS2) was revealed to be highly expressed in HCT116/Oxa cells. Silencing of DHRS2 in HCT116/Oxa cells effectively restored Oxa­sensitivity by suppressing the expression of excision repair cross­complementing group 1 protein via a p53­dependent pathway, and reversed the EMT phenotype. Overall, the suppression of DHRS2 expression may be a promising strategy for the prevention of Oxa­resistance in CRC.

Blockade of the NLRP3/Caspase-1 Axis Ameliorates Airway Neutrophilic Inflammation in a Toluene Diisocyanate-Induced Murine Asthma Model.

Multiple studies have addressed the vital role of Nod-like receptor protein 3(NLRP3)/caspase-1/IL-1ß signaling in asthma. Yet, the role of NLRP3/caspase-1 in toluene diisocyanate (TDI)-induced asthma is still obscure. The aim of this study is to investigate the role of the NLRP3/caspase-1 axis in TDI-induced asthma. Using an established murine model of TDI-induced asthma as described previously, we gave the asthmatic mice a highly selective NLRP3 inhibitor, MCC950, as well as the specific caspase-1 inhibitors VX-765 and Ac-YVAD-CHO for therapeutic purposes. Airway resistance was measured and bronchoalveolar lavage fluid was analyzed. Lungs were examined by histology, immunohistochemistry, Western blotting, and flow cytometry. TDI exposure elevated the expression of NLRP3 and caspase-1 that was coupled with increased airway hyperresponsiveness (AHR), neutrophil-dominated cell infiltration, pronounced goblet cell metaplasia, extensive collagen deposition, and increased TH2/TH17 responses. Both VX-765 and Ac-YVAD-CHO effectively inhibited the activation of caspase-1 in TDI-asthmatic mice that was accompanied by dramatic attenuation of AHR, airway inflammation, and airway remodeling, in addition to a decreased TH2 response and lower levels of IL-18 and IL-1ß. MCC950 blocked the activation of NLRP3 and downregulated protein expression of caspase-1, IL-1ß, and IL-18 in TDI-exposed mice. Furthermore, MCC950 remarkably alleviated AHR, airway inflammation, airway remodeling, and significantly suppressed TH2/TH17 responses. These findings suggested that blockade of the NLRP3/caspase-1 axis effectively prevents the progression of TDI-induced asthma and could be used as therapeutic targets for asthmatics.

Inhibition of HtrA2 alleviated dextran sulfate sodium (DSS)-induced colitis by preventing necroptosis of intestinal epithelial cells.

Necroptosis of intestinal epithelial cells has been indicated to play an important role in the pathogenesis of inflammatory bowel disease (IBD). The identification of dysregulated proteins that can regulate necroptosis in dextran sulfate sodium (DSS)-induced colitis is the key to the rational design of therapeutic strategies for colitis. Through tandem mass tag (TMT)-based quantitative proteomics, HtrA2 was found to be downregulated in the colon of DSS-treated mice. UCF-101, a specific serine protease inhibitor of HtrA2, significantly alleviated DSS-induced colitis as indicated by prevention of body weight loss and decreased mortality. UCF-101 decreased DSS-induced colonic inflammation, prevented intestinal barrier function loss and inhibited necroptosis of intestinal epithelial cells. In vitro, UCF-101 or silencing of HtrA2 decreased necroptosis of HT-29 and L929 cells. UCF-101 decreased phosphorylation of RIPK1 and subsequent phosphorylation of RIPK3 and MLKL during necroptosis. Upon necroptotic stimulation, HtrA2 translocated from mitochondria to cytosol. HtrA2 directly interacted with RIPK1 and promoted its degradation during a specific time phase of necroptosis. Our findings highlight the importance of HtrA2 in regulating colitis by modulation of necroptosis and suggest HtrA2 as an attractive target for anti-colitis treatment.

A pan-RAF inhibitor LY3009120 inhibits necroptosis by preventing phosphorylation of RIPK1 and alleviates dextran sulfate sodium-induced colitis.

A dramatic increase in the incidence of inflammatory bowel disease (IBD) has been observed in the past two decades, mainly in developed countries and also in developing regions. Necroptosis has been found to play an important role in the pathogenesis of IBD, suggesting its inhibitors are promising in clinic. However, clinical drugs targeting necroptosis are seriously lacking. Through screening a clinical compound library that contains 611 inhibitors, a pan-RAF inhibitor LY3009120 was found to be promising as a necroptosis inhibitor. LY3009120 inhibited necroptosis in vitro, and its inhibition against necroptosis was independent of its well-known activity to inhibit RAF. Surprisingly, LY3009120 prevented phosphorylation of receptor interacting serine/threonine kinase 1 (RIPK1) and subsequently phosphorylation of receptor interacting serine/threonine kinase 3 (RIPK3) and mixed lineage kinase domain like pseudokinase (MLKL) which happened during necroptosis. In vivo, LY3009120 significantly alleviated dextran sulfate sodium (DSS)-induced colitis as indicated by prevention of body weight loss, colon shortening, and decreased mortality. Furthermore, LY3009120 inhibited necroptosis of intestinal epithelial cells (IECs) and prevented intestinal barrier function loss. Consistently, LY3009120 decreased DSS-induced colonic inflammation, as indicated by decreased infiltration of macrophages and neutrophils, and decreased colonic TNF-α, IL-6, and IL-1ß level in DSS treated mice. These results indicate that an anti-cancer pan-RAF inhibitor LY3009120 is a necroptosis inhibitor and may serve as a potential therapeutic drug for colitis.

High Platelet Levels Attenuate the Efficacy of Platinum-Based Treatment in Non-Small Cell Lung Cancer.

BACKGROUND/AIMS: The correlation between platelet levels and clinical outcomes has received increasing attention, but it is not yet clear whether and how platelet levels affect the therapeutic response in non-small cell lung cancer (NSCLC). In the current study, we aimed to explore the role of platelet levels in responsive to platinum-based chemotherapy and investigated the underlying mechanism. METHODS: We evaluated the possibility of platelet level as a biomarker for response to platinum-based therapy in NSCLC by retrospective analysis of NSCLC patients. Cell proliferation was evaluated using cell counter and flow cytometry. Cell capillary-like structures of HPMEC were estimated with ECMatrix. The effect of platelets on A549, H1299, and HPMEC apoptosis was measured by flow cytometry. A A549-bearing NOD/ SCID mice model was employed to determine whether platelets could counteract cisplatin-induced apoptosis in vivo. In vivo cell proliferation and apoptosis were evaluated with Ki-67 antibody and TUNEL staining respectively. The angiogenesis of tumor was estimated by CD31 microvessel density. The protein levels of Akt, Bad and Bcl-2 were assessed by western blot. To further examine platelet-driven effects of the chemotherapeutic response, we used platelet depletion and platelet transfusion in A549-bearing NOD/SCID mice. RESULTS: Thrombocytosis at NSCLC diagnosis was associated with lower progression-free survival and median overall survival. Platelet levels before chemotherapy in the no response group were markedly higher than in the responsive group. Platelets rescued the inhibition of cell proliferation and angiogenesis and protected against cell apoptosis induced by cisplatin, platelets rescued cisplatin-induced apoptosis via the Akt/Bad/Bcl-2 signaling pathway under endoplasmic reticulum stress. Platelet transfusion decreased the therapeutic effect of cisplatin, while it was increased by platelet depletion. CONCLUSION: We confirmed an important anti-apoptosis mechanism mediated by platelets and found that platelets could counteract cisplatin-induced apoptosis. Reducing platelet levels or blocking platelet-based cytoprotection may represent new methods for improving the chemotherapeutic effect.

Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR.

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.1 Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compound 21 is currently being evaluated in phase-I clinical trials of mutant EGFR driven NSCLC.

Net platelet angiogenic activity (NPAA) correlates with progression and prognosis of non-small cell lung cancer.

Circulating platelets are abundant sources of angiogensis molecules for the tumor vasculature affecting tumor growth and metastasis. The relationship between non-small cell lung cancer (NSCLC) and intra-platelet levels of VEGF, TSP-1 and net platelet angiogenic activity (NPAA) is unclear. The aim of this study was to better understand the role of these factors in the progression of NSCLC cancer and to assess its clinical significance. Platelet VEGF and TSP-1 and NPAA were measured preoperatively in 68 patients with NSCLC by ELISA or Capillary tube formation assay. VEGF, TSP-1 and NPAA distributions in cancer patients and healthy volunteers were compared using the Mann-Whitney U test. The Kaplan-Meier method, univariate and multivariate regression analysis was used to analyze the correlation between these factors and clinicopathological features, overall survival and disease-free survival. Mean intra-platelet TSP-1 level was slightly higher in patients than in healthy subjects (p = 0.092). Intra-platelet TSP-1 levels were significantly higher in patients with involvement greater than T2 or stage III, compared to other patients. Mean intra-platelet VEGF level was 40.8 pg/106 in patients compared to 21.9 ng/106 in healthy subjects (p = 0.041). Median value of NPAA in patients was significantly higher than that in healthy controls (p<0.001). Patients with high NPAA are more likely to exhibit aggressive clinical pathological features. NPAA greater than the median are associated with poor prognosis. The elevated NPAA have better correlation with tumor microvessel density (MVD) than platelet-derived VEGF. The areas under receiver operating curve (AUROC) of NPAA were higher than that of platelet derived VEGF in different groups. A multivariate analysis showed that NPAA are independent prognostic factors. These results indicated that NPAA may be a clinically useful indicator for diagnostic and prognostic evaluation in NSCLC patients.

Overexpression of tumstatin in genetically modified megakaryocytes changes the proangiogenic effect of platelets.

BACKGROUND: Thrombocytopenia is a common side effect of tumor chemotherapy, the main management approach to which is based on platelet (PLT) transfusion. However, PLTs, containing angiogenesis regulators, play a major role in boosting tumor growth and metastasis. The purpose of the study was to determine whether PLTs have the capacity to overexpress tumstatin by modified megakaryocyte (MK) and PLT precursors using lentivirus-mediated gene transfer, which might lead to alteration in proangiogenic effect of PLTs. STUDY DESIGN AND METHODS: CD34+ hematopoietic stem cells (HSCs) were transduced with recombinant lentivirus carrying tumstatin and induced to produce MKs and PLTs in the culture medium containing a cytokine cocktail. Flow cytometry and aggregation test were used to detect the generation and function of MKs and PLTs. Western blot analysis and confocal microscopy were applied to examine the expression and distribution of tumstatin in transgenic MKs and PLTs. Capillary tube formation of human umbilical vein endothelial cells (HUVECs) was used to evaluate the inhibitory effect of transgenic PLTs. RESULTS: CD34+ HSCs can be efficiently transduced with lentivirus vectors and successfully differentiated into MKs and PLTs. Large amounts of functional MKs and PLTs could be generated and had correct biologic characteristics. The tests demonstrated the feasibility of tumstatin expression in MKs and PLTs under control of the cytomegalovirus promoter, that thus tumstatin was stored in the α-granules of PLTs, and that the releasate of thrombin or A543 cell-stimulated transgenic PLTs obviously inhibited the growth of capillary tube network structures of HUVECs. CONCLUSION: Gene-modified CD34+ HSCs not only successfully differentiated into MKs and PLTs but also expressed tumstatin protein. Release of tumstatin in transgenic PLT granules led to antiangiogenic effect of PLTs.

Decreased tumstatin-mRNA is associated with poor outcome in patients with NSCLC.

Tumstatin is a candidate tumor suppressor that plays an important role in tumor growth and angiogenesis. The purpose of this study was to evaluate the correlation between tumstatin-mRNA expression and the clinicopathologic characteristics, tumor angiogenesis, outcome of patients with non-small cell lung cancer (NSCLC). Specimens from 68 patients with NSCLC were recruited in this study. Tumstatin-mRNA expression and protein level in tumor tissues were quantified respectively by quantitative RT-PCR and ELISA. Microvessel density (MVD) was determined by CD34 immunostaining. The correlation of tumstatin-mRNA expression levels with clinicopathologic variables, tumor angiogenesis, and prognosis was analyzed. Tumstatin-mRNA expression levels were decreased in tumor. Tumstatin-mRNA expression level was significantly correlated with its protein level in tumor (r = 0.562; P = 0.001). Tumstatin-mRNA expression levels in poorly differentiated tumor tissues were significantly lower than in well-differentiated tumor tissues (P < 0.004). Furthermore, tumor tumstatin-mRNA expression were also significantly related to tumor pathologic stage (P = 0.032) and MVD (r = -0.77, P < 0.0001). Overall survival (OS) and disease-free survival (DFS) analysis indicated that NSCLC patients with low tumstatin-mRNA expression had poorer OS and DFS than those with high expression (P = 0.015 and 0.037; respectively). Multivariable Cox regression analysis revealed that the tumstatin-mRNA expression could be an independent prognostic indicators in both DFS and OS. Tumstatin-mRNA expression levels are down-regulated in NSCLC tissues. Tumstatin-mRNA expression level correlates with prognosis, which suggests that tumstatin-mRNA is a new potential independent marker of favorable prognosis in NSCLC.

Development of an ELISA for quantification of tumstatin in serum samples and tissue extracts of patients with lung carcinoma.

BACKGROUND: Tumstatin, an angiogenesis inhibitor with anti-tumor activity in mice, is the bioactive NC1 domain of Col IVa3, the potential significance of tumstatin as an endogenous angiogenesis inhibitor in human is not yet completely understood. This study aimed to develop an enzyme-linked immunoassay (ELISA) for tumstatin to accurately measure its concentrations in biological samples. METHODS: Recombinant tumstatin as an immunogen was expressed by E.coli. The purified tumstatin was injected into mice for antibody generation. An ELISA was developed with the monoclonal antibodies. RESULTS: The detection limit of the ELISA was 1.4ng/ml, and the intra- and inter- assay CVs were within 10%. Recovery of tumstatin added to sera was 92.7-115%. Patients with metastases had serum tumstatin levels 50% lower than patients without metastases (P=0.039). Tumstatin levels in poorly differentiated tumor tissues were significantly lower than in nontumorous tissues and well-differentiated tumor tissues (P<0.001). CONCLUSIONS: The development of a highly sensitive and reliable ELISA method capable of quantifying tumstatin in human serum samples and tissue extracts is reported. This assay for tumstatin in biological samples may be helpful in evaluating the therapeutic and/or prognostic value of tumstatin in cancer patients.

Expression of soluble, biologically active recombinant human tumstatin in Escherichia coli.

Tumstatin, a 28-kDa C-terminal fragment of collagen IV, is a potent anti-angiogenic protein and inhibitor of tumour growth. Recombinant tumstatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human tumstatin in E. coli by the coding region of tumstatin being linked to the 3'-end of the maltose-binding protein (MBP) gene. The fusion protein was expressed as the soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tumstatin was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 20% of the total soluble protein in E. coli, allowing approximately 8.6 mg of highly purified protein to be obtained per litre of bacterial culture. The purified tumstatin specifically inhibited the proliferation of endothelial cells in a dose-dependent manner. Annexin V-FITC apoptotic assay showed that recombinant tumstatin induced significant increase of apoptotic endothelial cells after 20 h of exposure to 20 microg/ml tumstatin, and when tumstatin was incubated on the chicken embryo, chorioallantoic membrane at doses of 1-15 microg, there was a dramatic decrease in the microvasculature allantoids of chicken embryos neovascular vessel test in vivo demonstrated that tumstatin treatment at doses of 1-15 microg gives rise to dramatically decrease the number of neovascular vessel. Our study provides a feasible and convenient approach to produce soluble and biologically active tumstatin.

Construction, expression, and characterization of a new targeted bifunctional fusion protein: tumstatin45-132-TNF.

The anti-angiogenic activity of tumstatin45-132 is mediated by binding to alphaVbeta3 on endothelial cells and tumor vascular endothelium showing increased expression of alphaVbeta3. Tumor necrosis factor alpha (TNF-alpha) is known to not only possess direct cytotoxicity against tumor cells, but also induces tumor vessel disruption, however, clinical use of TNF-alpha as an anticancer drug is hampered by severe systemic toxicity. In this study, we explore the possibility of fusing tumstatin45-132 with human TNF-alpha in the hope of generating a targeting, bi-functional protein in tumor treatment. Tumstatin45-132-TNF was constructed and expressed in E. coli. The recombinant fusion protein was shown to be insoluble and in an inclusion body form. An effective strategy for refolding and purification of tumstatin45-132-TNF resulted in final purified yields of 3 mg purified fusion protein recovered from 1 liter of E. coli culture. The refolded tumstatin45-132-TNF with a purity of 98% assessed by denaturing SDS - PAGE showed a single band on gels. Endothelial cell proliferation assay and standard cytolytic assays against L929 indicated that the fusion protein maintains tumstatin45-132 and TNF-alpha activity. More importantly, tumstatin45-132-TNF inhibits endothelial cell proliferation more than tumstatin45-132 alone. Cell adhesion assays and competitive binding experiments with anti-integrin antibodies showed that the tumstatin45-132 moiety specifically interacts with alphaVbeta3 integrin. These results lay the solid foundation for further investigation of antitumor activity of tumstatin45-132-TNF in vivo.

[The cloning and expression of angiogenic inhibitor tumstatin(45-132) in E. coli].

AIM: To clone tumstatin(45-132) gene and to express recombinant human tumstatin(45-132) in E. coli BL21. METHODS: Tumstatin gene was cloned by RT-PCR and then tumstatin(45-132) gene amplified from tumstatin gene was cloned into pBV220. The recombinant plasmid pBV220-tumstatin(45-132) was sequenced and transformed into E.coli BL21. E. coli BL21 transformed with the recombinant plasmid pBV220-tumstatin(45-132) was induced at 42 degrees Celsius. After the recombinant tumstatin(45-132) was purified, its bioactivity was detected by endothelia cell proliferation test. RESULTS: 264 bp tumstatin(45-132) fragment was cloned and its sequence was identical with that in GenBank. The tumstatin(45-132) was expressed in E. coli BL21. Expressed product accounted for about 10% of total bacterial proteins and its relative molecular mass (M(r)) was 9,600. The purified protein showed inhibitory effect on proliferation of endothelia cells ECV304 in vitro. CONCLUSION: tumstatin(45-132) gene has been cloned and expressed successfully in E.coli BL21. Expressed tumstatin(45-132) can inhibit the proliferation of endothelial cell ECV304.

WITHDRAWN: Combining tumstatin45-132 with tumor necrosis factor-α improves its antineoplastic activity.

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