RESUMEN
Cetuximab improves the survival of patients with metastatic colorectal cancer. The main limitation is primary and secondary resistance, the underlying mechanism of which requires extensive investigation. We proved that PRSS expression levels are significantly negatively associated with the sensitivity of cancer cells to cetuximab. Detailed mechanistic analysis indicated that PRSS can cleave cetuximab, leading to resistance. Cetuximab or bevacizumab combined with SPINK1, a PRSS inhibitor, inhibited cell growth more efficiently than cetuximab or bevacizumab alone in xenograft models. PRSS levels in the serum of 156 patients with mCRC were analyzed, and poor efficacy of cetuximab therapy was observed in patients with aberrant PRSS expression. PRSS expression in monoclonal antibody (mAb)-treated patients with cancer from The Cancer Genome Atlas database was also evaluated to determine whether patients with higher PRSS expression have significantly reduced progression-free survival. Our work provides a strong scientific rationale for targeting PRSS in combination with cetuximab therapy.
Asunto(s)
Cetuximab/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Inhibidor de Tripsina Pancreática de Kazal/genética , Tripsina/genética , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/efectos adversos , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Bevacizumab , Línea Celular Tumoral , Cetuximab/efectos adversos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Xenoinjertos , Humanos , Masculino , Ratones , Persona de Mediana Edad , Metástasis de la NeoplasiaRESUMEN
Directed differentiation of human pluripotent stem cells (hPSCs) into functional insulin-producing cells (IPCs) holds great promise for cell therapy for diabetic patients. Despite recent advances in developing beta cell differentiation protocols, it is becoming clear that the hPSC-derived beta-like cells are functionally immature, and the efficiencies of differentiation can be variable depending on the hPSC lines used. Therefore, advanced methodologies are highly desirable for the development and refinement of beta cell differentiation protocols from hPSCs. In this report, we first derived and validated a Pdx1-mRFP/insulin-hrGFP dual-reporter cell line from MRC5-iPSCs. Then, using this dual-reporter cell line, we developed and optimized an in vitro beta cell differentiation protocol through real-time monitoring expression of Pdx1 and insulin. We demonstrated that DNA demethylation could increase the efficiency of beta cell differentiation. Furthermore, three-dimensional induction not only significantly increased the efficiency of pancreatic progenitor specification and the yield of IPCs, but also produced more mature IPCs. The current study indicates that this dual-reporter cell line is of great value for developing and optimizing the beta cell differentiation protocols. It will facilitate the development of novel protocols for generating IPCs from hPSCs and the investigation of beta cell differentiation mechanisms.
RESUMEN
BACKGROUND: TEM8 is a cell membrane protein predominantly expressed in tumor endothelium, which serves as a receptor for the protective antigen (PA) of anthrax toxin. However, the physiological ligands for TEM8 remain unknown. RESULTS: Here we identified uPA as an interacting partner of TEM8. Binding of uPA stimulated the phosphorylation of TEM8 and augmented phosphorylation of EGFR and ERK1/2. Finally, TEM8-Fc, a recombinant fusion protein comprising the extracellular domain of human TEM8 linked to the Fc portion of human IgG1, efficiently abrogated the interaction between uPA and TEM8, blocked uPA-induced migration of HepG2 cells in vitro and inhibited the growth and metastasis of human MCF-7 xenografts in vivo. uPA, TEM8 and EGFR overexpression and ERK1/2 phosphorylation were found co-located on frozen cancer tissue sections. CONCLUSIONS: Taken together, our data provide evidence that TEM8 is a novel receptor for uPA, which may play a significant role in the regulation of tumor growth and metastasis.
Asunto(s)
Receptores ErbB/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proliferación Celular , Humanos , Cinética , Proteínas de Microfilamentos , Metástasis de la Neoplasia , Fosforilación , Dominios Proteicos , Receptores del Activador de Plasminógeno Tipo Uroquinasa/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/químicaRESUMEN
Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. VEGF receptor1 (VEGFR1) acts as a decoy VEGF receptor that enables the regulation of VEGF on the vascular endothelium. In the present study, the recombinant human VEGFR1D13/Fc (rhVEGFR1), which contains key domains for VEGF binding, was cloned and expressed in Chinese hamster ovary (CHO) cells. The rhVEGFR1 protein was purified using proteinA affinity chromatography. The molecular weight of rhVEGFR1 was found to be ~162 and 81 kD in nonreducing and reducing SDSPAGE, respectively. The majority of the final protein products were in the dimeric conformation. Western blot analysis revealed that rhVEGFR1 was only capable of binding to the full glycan form of rhVEGF165 and rhVEGF121. The dissociation constant for the binding of rhVEGFR1 to VEGF165, detected using Biacore, was 285 pM. In addition, rhVEGFR1 inhibited the proliferation and migration of human microvascular endothelial cells. In vivo experiments also demonstrated that rhVEGFR1 inhibited chicken chorioallantoic membrane neovascularization and angiogenesis in nude mice. In conclusion, an antiangiogenic recombinant soluble VEGFR was expressed (up to 5 mg/l) in CHO cells and was shown to be capable of inhibiting neovascularization in vivo and in vitro.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Recombinantes de Fusión , Receptor 1 de Factores de Crecimiento Endotelial Vascular/farmacología , Animales , Células CHO , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Clonación Molecular , Cricetulus , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Ratones , Ratones Desnudos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2) and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.
Asunto(s)
Antitoxinas , Simulación por Computador , Fragmentos Fc de Inmunoglobulinas , Inmunoglobulina G , Modelos Moleculares , Receptores de Péptidos , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antitoxinas/química , Antitoxinas/genética , Bacillus anthracis/genética , Bacillus anthracis/patogenicidad , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/química , Inmunoglobulina G/genética , Mutación Missense , Receptores de Péptidos/química , Receptores de Péptidos/genéticaRESUMEN
Human lysozyme (hLYZ) has important potential applications as antimicrobial medicine and food additive. To develop a robust expression vector that ensures expression of large amounts of hLYZ in milk, here a 26,267 bp chimeric mouse whey acidic protein (mWAP)::hLYZ cassette was constructed and used as a mammary gland-specific expression vector, in which a 3,010 bp genomic sequence in the 24,466 bp mWAP gene locus was substituted by a 4,811 bp genomic sequence of hLYZ, exactly from the start codon to the stop codon. Corresponding transgenic mice were generated, and enzymatically-active hLYZ was expressed at 18.4-35 g l(-1) in the milk of most transgenic mouse lines. Our transgenic mice carrying chimeric mWAP::hLYZ represent a model system for cost-effective production of hLYZ.
Asunto(s)
Proteínas de la Leche/biosíntesis , Leche Humana/química , Muramidasa/biosíntesis , Animales , Vectores Genéticos , Humanos , Ratones , Ratones Transgénicos , Proteínas de la Leche/genética , Muramidasa/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Urokinase (uPA) and its receptor (uPAR) play an important role in tumor growth and metastasis, and overexpression of these molecules is strongly correlated with poor prognosis in a variety of malignant tumors. In this study, ATF-Fc, an antibody-like molecule comprising the amino-terminal fragment of human uPA (ATF) linked to the Fc fragment of human IgG1 via a flexible linker was developed. Its antitumor activities were evaluated in vitro and in vivo. The results showed that ATF-Fc had obvious cytotoxic effect on several types of tumor cells, which is dependent on cellular expression of uPAR and its Fc fragment. Treatment with ATF-Fc caused a significant suppression on tumor growth and metastasis of xenograft human tumors (MCF-7 breast cancer and BGC-823 gastric cancer) in athymic nude mice. Furthermore, we demonstrated that ATF-Fc had an anti-angiogenesis activity both in vitro and in vivo. In conclusion, we provided a novel therapeutic antibody-like molecule in the management of a variety of solid tumors by disrupting the uPA/uPAR interaction.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Neoplasias/terapia , Receptores de Superficie Celular/química , Activador de Plasminógeno de Tipo Uroquinasa/química , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Humanos , Inmunoglobulina G/química , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Neovascularización Patológica , Receptores del Activador de Plasminógeno Tipo UroquinasaRESUMEN
Tumor endothelial marker 8 (TEM8) was discovered as a cell membrane protein that is predominantly expressed in tumor endothelium and identified as a receptor for anthrax toxin. We developed an antibody-like molecule that consists of the protective antigen (PA)-binding domain of human TEM8 linked to the Fc portion of human immunoglobulin G1 (TEM8-Fc). This engineered protein bound to PA in a divalent cation-dependent manner and efficiently protected J774A.1 macrophage-like cells against anthrax toxin challenge in a dose-dependent manner. TEM8-Fc suppressed the growth and metastasis of xenograft human tumors in athymic nude mice (control versus 10 mg/kg TEM8-Fc, mean tumor weight: LS-180, 1.72 versus 0.16 g, difference = 1.56 g, 95% confidence interval [CI] = 0.96 to 2.16 g; P<.001; MCF-7, 1.12 versus 0.08 g, difference = 1.04 g, 95% CI = 0.77 to 1.31 g; P<.001; HepG2, 1.28 versus 0.35 g, difference = 0.93 g, 95% CI = 0.60 to 1.25 g; P<.001). Furthermore, TEM8 interacted with the M2 isoenzyme of pyruvate kinase (M2-PK), which has an important role in tumor growth and metastasis. TEM8-Fc is a novel therapeutic antibody-like agent in the management of solid tumors that may act by trapping M2-PK.