A high efficient FVIII variant corrects bleeding in hemophilia A mouse model.

Hemophilia A is a bleeding disorder caused by quantitative or qualitative deficiencies in coagulation factor VIII (FVIII). Low FVIII expression due to its unstable mRNA and binding with immunoglobulin-binding protein (BiP) compromises gene therapy endeavors in hemophilia A. Site-directed mutagenesis have demonstrated an improvement in the expression of FVIII proteins. In this study, a targeted point mutation of Pro at position 290 to Thr (P290T) enhances the in vitro specific activity of B-domain-deleted factor VIII (BDD-FVIII). Hydrodynamic gene delivery of P290T cDNA into FVIII-deficient (FVIII-/-) mice corrected bleeding symptoms. P290T variant resulted in high plasma FVIII coagulant activity 24 h post-gene delivery. Furthermore, bleeding time and average blood loss was significantly reduced in FVIII-/- mice injected with P290T variant, whereas BDD-FVIII and PBS-injected mice experienced prolonged bleeding and excessive blood loss. Histological analysis of the liver biopsies revealed no apparent signs of liver damage. No signs of potential toxicity were seen in mice following mice bodyweights assessment. Altogether, our results demonstrate that the introduction of P290T mutation increases both in vitro and in vivo FVIII coagulant activity, supporting ongoing efforts to develop more effective replacement therapy for hemophilia A.

Extracellular AGR2 activates neighboring fibroblasts through endocytosis and direct binding to ß-catenin that requires AGR2 dimerization and adhesion domains.

Anterior gradient 2 (AGR2) is often overexpressed in several types of cancer. AGR2 is cytoplasmic or secreted as an extracellular signal. Intracellular AGR2 properties and role in cancer have been well studied, but its extracellular function is largely unclear. It has been shown that extracellular AGR2 activates endothelial cells and fibroblasts in culture, but the mechanism of AGR2 signaling is not well elucidated. Here, we report that tumor secreted or externally added AGR2 translocates into cytoplasm by endocytosis, binds to ß-catenin and further co-translocates to the nucleus in NIH3T3 fibroblasts. Externally added AGR2 also increased ß-catenin expression, stability, and accumulation in the nucleus in both fibroblasts and cancer cells. External AGR2 rescued the expression of ß-catenin, which was suppressed by EGFR inhibitor AG1478 indicating an alternative pathway to regulate ß-catenin independent of EGFR signal. These effects were abolished when a monoclonal antibody against AGR2 was added to the experiments, confirming the effects are caused by AGR2 only. Putting together, our results show that extracellular AGR2 signaling pathway involves endocytosis mediated cellular translocation, direct binding and regulating ß-catenin nuclear accumulation. It is also a target against tumor initiated AGR2 signaling to form and maintain tumor microenvironment.

A novel target anti-interleukin-13 receptor subunit alpha-2 monoclonal antibody inhibits tumor growth and metastasis in lung cancer.

IL13Rα2 shows high expression in different types of tumors and can be a target for cancer therapy in humans due to its poor prognosis. The aim of our study is to characterize and investigate the effect of interleukin-13 receptor subunit alpha-2monoclonal antibody mAb15D8 on lung cancer cells in vitro and in vivo by blocking its specific epitope in IL13Rα2 antigen. The mAb15D8 blocking epitope was analyzed through the mutagenesis of IL13Rα2 and confirmed with western blot. We found that the IL13Rα2 epitope recognized by mAb15D8 antibody is a new binding site localized in the fibronectin-III domain-1 of IL13Rα2 antigen. Moreover, the mAb15D8 obviously reduced cell proliferation, migration of H460, A549, SKOV3, and B16F10 cells. Treatment with mAb15D8 significantly reduced the H460 xenograft tumor formation and growth in nude mice and inhibited B16F10 tumor metastasis and increased survival in C57BL/6 mice. Pharmacokinetic and toxicological analysis demonstrated the safety of mAb15D8 as a potential therapeutic agent. We developed a novel mouse monoclonal antibody against IL13Rα2 which binds to specific epitope on IL13Rα2 antigen. In vivo treatment with the antibody significantly reduced tumor growth and lung metastasis and prolonged survival. These results suggest mAb15D8 antibody as a potential therapeutic agent for cancer therapy.

Withdrawal Notice: Emerging Novel Coronavirus is a Global Threat: Insight in the Biology of COVID-19 and its Hijacking Process of Hosts' Cell

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Paracrine signalling of AGR2 stimulates RhoA function in fibroblasts and modulates cell elongation and migration.

The most prominent cancer-associated fibroblasts (CAFs) in tumor stroma is known to form a protective structure to support tumor growth. Anterior gradient-2 (AGR2), a tumor secretory protein is believed to play a pivotal role during tumor microenvironment (TME) development. Here, we report that extracellular AGR2 enhances fibroblasts elongation and migration significantly. The early stimulation of RhoA showed the association of AGR2 by upregulation of G1-S phase-regulatory protein cyclin D1 and FAK phosphorylation through fibroblasts growth factor receptor (FGFR) and vascular endothelial growth factor receptor (VEGFR). Our finding indicates that secretory AGR2 alters fibroblasts elongation, migration, and organization suggesting the secretory AGR2 as a potential molecular target that might be responsible to alter fibroblasts infiltration to support tumor growth.

Anterior Gradient-2 monoclonal antibody inhibits lung cancer growth and metastasis by upregulating p53 pathway and without exerting any toxicological effects: A preclinical study.

Increased drug resistance and acute side effects on normal organs are the major disadvantages of traditional cancer chemotherapy and radiotherapy. This has increased the focus on targeted therapeutic strategies such as monoclonal antibody-based cancer therapies. The major advantage of antibody-based therapies is the specific inhibition of cancer-related targets, with reduced off-target side effects. Anterior gradient-2 (AGR2) is a prometastatic and proangiogenic tumor marker that is overexpressed in multiple cancers. Therefore, anti-AGR2 antibodies may be potential therapeutic agents for treating different cancers. In the present study, we examined a novel anti-AGR2 monoclonal antibody mAb18A4 and found that this antibody inhibited lung cancer progression and metastasis without exerting any adverse side effects on the major organs and blood in mice. Moreover, we found that mAb18A4 activated p53 pathway and attenuated ERK1/2-MAPK pathway. Furthermore, mAb18A4-treated cancer cell lines showed attenuated proliferation and colony formation, enhanced apoptosis, increased p53 expression, and reduced phosphorylated ERK1/2 expression. Treatment with mAb18A4 significantly reduced tumor size and suppressed tumor metastasis in and increased the survival of different xenograft tumor models. In addition, mAb18A4 potently suppressed AGR2-induced angiogenesis. Results of pharmacokinetic and toxicological analyses confirmed the safety of mAb18A4 as an antitumor treatment.

Anterior gradient 2 is induced in cutaneous wound and promotes wound healing through its adhesion domain.

Anterior gradient 2 (AGR2), a member of protein disulfide isomerase (PDI) family, is both located in cytoplasm and secreted into extracellular matrix. The orthologs of AGR2 have been linked to limb regeneration in newt and wound healing in zebrafish. In mammals, AGR2 influences multiple cell signaling pathways in tumor formation and in normal cell functions related to new tissue formation like angiogenesis. However, the function of AGR2 in mammalian wound healing remains unknown. This study aimed to investigate AGR2 expression and its function during skin wound healing and the possible application of external AGR2 in cutaneous wound to accelerate the healing process. Our results showed that AGR2 expression was induced in the migrating epidermal tongue and hyperplastic epidermis after skin excision. Topical application of recombinant AGR2 significantly accelerated wound-healing process by increasing the migration of keratinocytes (Kera.) and the recruitment of fibroblasts (Fibro.) near the wounded area. External AGR2 also promoted the migration of Kera. and Fibro. in vitro in a dose-dependent manner. The adhesion domain of AGR2 was required for the formation of focal adhesions in migrating Fibro., leading to the directional migration along AGR2 gradient. These results indicate that recombinant AGR2 accelerates skin wound healing through regulation of Kera. and Fibro. migration, thus demonstrating its potential utility as an alternative strategy of the therapeutics to accelerate the healing of acute or chronic skin wounds.

A human leucyl-tRNA synthetase as an anticancer target.

Several aminoacyl-tRNA synthetases have been reported to be overexpressed for charging essential aminoacyl-tRNAs in many cancer types. In this study, we aimed to explore the potential role of leucyl-tRNA synthetase (LARS) as an anticancer target. MTT assay was performed to screen inhibitors to human LARS (hsLARS) from compounds AN2690 and its derivatives, compounds 1-6, in U2OS and SKOV3 cells. The compound with the strongest inhibitory ability was further investigated for its inhibitory effect in cancer cell lines and in an animal tumor model. Additionally, a LARS-rescue experiment was performed to explore the potential target in U2OS using Western blot and flow cytometry. Luciferase reporter assay was designed to analyze the effect of of hsLARS inhibitor on p21 activation. We identified an hsLARS inhibitor (compound 2) that suppressed the proliferation of U2OS and SKOV3 cells in vitro. A LARS-rescue experiment demonstrated that the proliferation inhibition was induced by targeting intracellular LARS. In addition, the hsLARS inhibition was shown to activate the p21 early transcription and promote cell apoptosis, as well as reduce implanted EMT6 tumor progression in mice. Our results suggest that LARS might serve as a potential anticancer target through the p21 signaling pathway and that the nutritional signaling pathway may provide a valuable anticancer strategy for further investigation.

A new mouse model for wound healing in hemophilia A.

PURPOSE: To establish a new mouse model for wound healing studies on hemophilia A. METHODS: Total 54 male mice with different genotypes including wild-type nude mice, heterozygous mice (FVIII-/-/Nu) and FVIII deficient mice (FVIII-/-) were generated and verified by PCR. Mice were subjected to wound healing research by making a 5 mm-thickness wound on mice skin and applying recombinant human epidermal growth factor (EGF, 10 µg/g) ointment, FVIII ointment (30 IU) or the ointment base to heal the wounds. Furthermore, keratinocytes were isolated from these newborn mice and subjected to migration assay by stimulation of EGF (ng/ml), insulin (10 µM) or vehicle. RESULTS: A new hemophilic mouse model (FVIII-/-/Nu) was constructed successfully after genotyping verified by PCR. Compared to FVIII-/- mice, FVIII-/-/Nu and Nu mice showed greater degree of wound contraction and loss of the crust. Topical treatment with EGF exhibited faster wound healing than FVIII and ointment base. Insulin treatment showed more increased migration distance than treated with EGF or vehicle. FVIII-/-/Nu mice showed greater migration than FVIII-/- and Nu mice. CONCLUSIONS: A new mouse model (FVIII-/-/Nu) for wound healing in hemophilia A was constructed, and topical treatment of insulin may be a better therapy than EGF for healing wounds in hemophilia A.