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.

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.

Potent anti-cancer activity of Alnus nitida against lung cancer cells; in vitro and in vivo studies.

Alnus nitida is used for multiple disorders in norther areas of Pakistan. In this study we have evaluated methanol extract of leaf (ANL) and stem bark (ANB) of A. nitida against two lung cancer cell lines; A-549 and H460 (Human non-small lung cancer cell lines) during in vitro assays for growth inhibition. Treatment with ANL and ANB markedly inhibited the growth of both cancer cell lines. Exposure of A-549 and H460 cancer cell lines to ANL and ANB inhibited cell survival, colony growth and migration of cells. Further, treatment of A-549 and H460 with ANL and ANB indicated alteration in actin fibers after staining with rhodamine-phalloidin. Both extracts cause shrinkage and cell cycle arrest during G1 phase. Treatment of A-549 and H460 cancer cells with ANL and ANB repressed the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL along with downregulation of NFκB, cyclin D1 and PI3-K protein. In addition, intraperitoneal injection of ANL and ANB (10 mg/kg bw and 20 mg/kg bw) to C57BL/6 J mice implanted with B16F10 (Mouse melanoma cancer cell line) cells significantly (p < 0.01) decreased the number of nodules per lung and the level of various proteins reciprocating the in vitro studies. These results suggest that ANL and ANB be explored further for therapeutic use in lung cancer.

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.

3ß-Hydroxy-urs-12-en-28-oic Acid Modulates Dietary Restriction Mediated Longevity and Ameliorates Toxic Protein Aggregation in C. elegans.

Species from lower invertebrates to a spectrum of mammals show antiaging health benefits of phytochemical(s). Here, we explored the pro-longevity effects of a natural triterpenoid, ursolic acid (3ß-hydroxy-urs-12-en-28-oic acid; UA) in Caenorhabditis elegans with maximal life span being evident at 25 µM UA. Similar to eat-2 mutants, UA uptake by worm results in reduced fat storage and attenuation of reactive oxygen species (ROS), independent of superoxide dismutase(s) activation. The genetic requirements for UA-mediated longevity are quite similar to dietary restriction (DR) achieved through SKN-1/NRF-2 exhibiting upregulation of downstream target genes gcs-1 and daf-9. Longevity mechanism was independent of PHA-4/FOXA and attributed to partial dependence on sir-2.1. Altogether, our study suggests differential use of UA-elicited signaling cascades in nutrient sensing for longevity. Both the redox state and the proteostasis of an organism play critical role in aging and disease resistance. Interestingly, we observed a reduction of toxic protein aggregation in transgenic polyglutamine (polyQ) C. elegans model and UA-mediated JNK-1 (c-Jun-NH2-terminal kinase) activation in wild-type animals. Thus, our study demonstrates a small extent of prevention against proteotoxic stress by UA coupled with positive aspects of DR-mediated longevity.

3ß-Hydroxy-urs-12-en-28-oic acid confers protection against ZnONPs induced adversity in Caenorhabditis elegans.

Despite their well reported potent risk towards human health and environment Zinc oxide nanoparticles (ZnONPs) find an extensive commercial usage due to their antimicrobial properties. Here, we evaluated the efficacy of a natural triterpene ursolic acid (3ß-hydroxy-urs-12-en-28-oic acid; UA) for overcoming the cytotoxic challenges of ZnONPs employing Ceanorhabditis elegans. The 24h LC50 of Zn-ONPs (<50nm TEM) was deduced as 4.75mgL-1. UA (25µM) was observed to defend against this toxicity by attenuating Reactive Oxygen Species (ROS) resulting into better survival at 2mgL-1 in a time dependent behavior. However, reproductive health remains compromised. Our study identifies UA as a natural inducer of metallothionein proteins along with antioxidant potential. We demonstrate that UA induces upregulation of predominantly antioxidant genes, including the superoxide dismutases (sod-1, sod-5 and sod-3), glutathione S-transferase 7 (gst-7), heat shock protein (hsp-16.2) along with the metal exposure responsive metallothionein (mtl-1 and mtl-2). Moreover, UA also restores elevated transcript levels of gst-4 during ZnONPs stress conditions to normal by directly scavenging ROS owing to its own antioxidant potential. Altogether, the toxic aspects of NPs that can be avoided compensated or postponed by supplementation of phytochemical(s) in biological system underscore their potential implications in near future.

3ß-Hydroxy-urs-12-en-28-oic acid prolongs lifespan in C. elegans by modulating JNK-1.

Remarkably the c-Jun-NH2-terminal kinase (JNK) pathway is all evolutionarily conserved across species. In view of the hypothesis that increased stress resistance subdue aging, we investigated the role of ursolic acid (3ß-Hydroxy-urs-12-en-28-oic acid; UA) in the pioneering aging model Caenorhabditis elegans with an increase in mean and maximum lifespan by up to 30%. Our genetic study unravelled the underlying pathway where JNK-1 is acting independently of insulin-IGF-1 signalling (IIS) pathway to modulate longevity. In support of in vivo results in silico docking study of UA with C. elegans JNK-1 ATP-binding site suggested promising binding affinity exhibiting binding energy of -8.11 kcalmol-1. UA induced JNK-1 activation in wild-type animals underlie the importance of pharmacological interventions in the delineation of molecular targets for aging and associated pathologies.

Binding of anterior gradient 2 and estrogen receptor-α: Dual critical roles in enhancing fulvestrant resistance and IGF-1-induced tumorigenesis of breast cancer.

Anterior gradient 2 (AGR2), an essential cancer biomarker, has been widely reported to be associated with estrogen receptor (ER) positive breast cancer development. Here, we uncovered the role of cytoplasmic and exogenous AGR2, through interaction with ER-α, in enhancing fulvestrant resistance and IGF-1-induced carcinogenesis respectively. Our present study revealed that the endogenous AGR2 level positively correlates with fulvestrant resistance in MCF-7 and T47D cells. AGR2-knockdown in MCF-7 cells strongly enhances the fulvestrant-induced G1 phase arrest and accelerates the fulvestrant-induced ER-α degradation. Furthermore, intracellular AGR2 exhibits a functional interaction with ER-α. On the other hand, extracellular AGR2 remarkably promotes the IGF-1-induced cell proliferation, migration, cell cycle progression and epithelial-mesenchymal transition. Extracellular AGR2 also enhances IGF-1 downstream signaling. We also showed that ER-α specifically interacts with both extracellular AGR2 and IGF-1 receptor as a potential intermediator. Finally, we revealed that the adjuvant therapy of AGR2 monoclonal antibody enhances the inhibitory effects of fulvestrant and linsitinib toward breast cancer development. Our findings, for the first time, point out the different functions of intra- and extra-cellular AGR2, providing new insights into the development of anti-tumor therapies targeting AGR2.

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.

Induction of anterior gradient 2 (AGR2) plays a key role in insulin-like growth factor-1 (IGF-1)-induced breast cancer cell proliferation and migration.

Anterior gradient 2 (AGR2) is a promising anti-tumor target associated with estrogen receptor expression and metastatic progression of breast cancer. Insulin-like growth factor-1 (IGF-1) is another potent factor that stimulates breast cancer progression and mediates anti-estrogen drug resistance. However, the precise mechanism and connections between these two factors in breast cancer drug resistance have not been fully elucidated. Here, for the first time, we decipher that IGF-1 remarkably induces AGR2 in the MCF7 cell line, through an estrogen response element (ERE) between -802 and -808 bp and a leucine zipper transcription factor-binding site located between -972 and -982 bp on the AGR2 promoter. We also found that the ERK1/2 and AKT pathways mediate estrogen receptor-α at the upstream of ERE and that the JNK pathway activates the leucine zipper site through the c-Jun/c-Fos complex. Additionally, our data suggest that knockdown of AGR2 reduces IGF-1-induced cell proliferation, migration and cell cycle progression. Therefore, we report that AGR2 is a key modulator involved in IGF-1-induced breast cancer development. We propose that the identification of the mechanism linking the IGF-1/insulin signal and AGR2 promoter activation is important, because it provides insights into the development of anti-breast cancer drugs.

Sal-like protein 2 upregulates p16 expression through a proximal promoter element.

Sal-like protein 2 (Sall2), a homeotic transcription factor, is a putative tumor suppressor. We have previously shown that Sall2 activates the transcription of tumor suppressor gene p21 and suppresses tumorigenesis through cell cycle inhibition and induction of apoptosis. To investigate additional Sall2-regulated downstream genes, we analyzed the differences in mRNA expression profiles with and without exogenously expressed Sall2. We identified 1616 Sall2-responsive genes through gene expression arrays. Promoter-reporter assays of p16(INK4A) and several other tumor-related genes indicated that the Sall2 regulation of these promoters was not significantly different between the two major forms of Sall2 with alternative exon 1 or exon 1A. Additional analysis showed that Sall2-induced p16 promoter activation was Sall2 dose-dependent. Deletion and site-directed mutagenesis of the p16 promoter identified a consensus Sall2 binding site (GGGTGGG) proximal to the p16 transcription start site and was critical for p16 promoter activation. Finally, to confirm the significance of Sall2-activated p16 expression in cell cycle regulation, we co-transfected the SKOV3 cells with a Sall2 expression construct and a p16 minigene and also co-transfected the ES-2 cells with a Sall2 expression construct and the siRNA against p16 for flow cytometry analysis. Our results showed that Sall2 enhanced the p16 minigene blocking of cell cycle progression and p16 knockdown with siRNA abolished most of the Sall2 inhibition of cell cycle progression. These findings indicate that Sall2 targets multiple cell cycle regulators, including p16, through their promoters, adding knowledge to the understanding of Sall2 and p16 gene regulation, and how Sall2 deregulation may promote cancer formation.