RESUMO
Targeting fibroblast growth factor receptor 1 (FGFR1) is a promising therapeutic strategy for various cancers associated with alterations in the FGFR1 gene. In this study, we developed a highly cytotoxic bioconjugate based on fibroblast growth factor 2 (FGF2), which is a natural ligand of this receptor, and two potent cytotoxic drugs-α-amanitin and monomethyl auristatin E-with completely independent mechanistic modes of action. Utilizing recombinant DNA technology, we produced an FGF2 N- to C-end dimer that exhibited superior internalization capacity in FGFR1-positive cells. The drugs were site-specifically attached to the targeting protein using SnoopLigase- and evolved sortase A-mediated ligations. The resulting dimeric dual-warhead conjugate selectively binds to the FGFR1 and utilizes receptor-mediated endocytosis to enter the cells. Moreover, our results demonstrate that the developed conjugate exhibits about 10-fold higher cytotoxic potency against FGFR1-positive cell lines than an equimolar mixture of single-warhead conjugates. The diversified mode of action of the dual-warhead conjugate may help to overcome the potential acquired resistance of FGFR1-overproducing cancer cells to single cytotoxic drugs.
Assuntos
Antineoplásicos , Neoplasias , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Alfa-Amanitina , Oligopeptídeos/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias/tratamento farmacológicoRESUMO
Fibroblast growth factor 2 (FGF2) is a heparin-binding growth factor with broad mitogenic and cell survival activities. Its effector functions are induced upon the formation of 2:2 FGF2:FGFR1 tetrameric complex. To facilitate receptor activation, and therefore, to improve the FGF2 biological properties, we preorganized dimeric ligand by a covalent linkage of two FGF2 molecules. Mutations of the FGF2 WT protein were designed to obtain variants with a single surface-exposed reactive cysteine for the chemical conjugation via maleimide-thiol reaction with bis-functionalized linear PEG linkers. We developed eight FGF2 dimers of defined topology, differing in mutual orientation of individual FGF2 molecules. The engineered proteins remained functional in terms of FGFR downstream signaling activation and were characterized by the increased stability, mitogenic potential and anti-apoptotic activity, as well as induced greater migration responses in normal fibroblasts, as compared to FGF2 monomer. Importantly, biological activity of the dimers was much less dependent on the external heparin administration. Moreover, some dimeric FGF2 variants internalized more efficiently into FGFR overexpressing cancer cells. In summary, in the current work, we showed that preorganization of dimeric FGF2 ligand increased the stability of the growth factor, and therefore, enhanced its biological activity.
Assuntos
Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/farmacologia , Mitógenos/farmacologia , Mitose/efeitos dos fármacos , Engenharia de Proteínas/métodos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cisteína/química , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparina/farmacologia , Humanos , Maleimidas/química , Camundongos , Mitógenos/química , Células NIH 3T3 , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Polietilenoglicóis/química , Multimerização Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Equine Metabolic Syndrome (EMS) is a steadily growing life-threatening endocrine disorder linked to insulin resistance, oxidative stress, and systemic inflammation. Inflammatory microenvironment of adipose tissue constitutes the direct tissue milieu for various cell populations, including adipose-derived mesenchymal stromal cells (ASCs), widely considered as a potential therapeutic cell source in the course of the treatment of metabolic disorders. Moreover, elevated oxidative stress induces inflammation in intestinal epithelial cells (IECs)-the first-line cells exposed to dietary compounds. In the conducted research, we showed that in vitro application of Spirulina platensis contributes to the restoration of ASCs' and IECs' morphology and function through the reduction of cellular oxidative stress and inflammation. Enhanced viability, suppressed senescence, and improved proliferation of ASCs and IECs isolated from metabolic syndrome-affected individuals were evident following exposition to Spirulina. A protective effect of the investigated extract against mitochondrial dysfunction and degeneration was also observed. Moreover, our data demonstrate that Spirulina extract effectively suppressed LPS-induced inflammatory responses in macrophages. In vivo studies showed that horses fed with a diet based on Spirulina platensis supplementation lost weight and their insulin sensitivity improved. Thus, our results indicate the engagement of Spirulina platensis nourishing as an interesting alternative approach for supporting the conventional treatment of equine metabolic syndrome.
Assuntos
Síndrome Metabólica/metabolismo , Mitocôndrias/efeitos dos fármacos , Spirulina/metabolismo , Tecido Adiposo/metabolismo , Animais , Células Epiteliais/metabolismo , Cavalos , Resistência à Insulina , Mucosa Intestinal/metabolismo , Intestinos/citologia , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Obesidade/metabolismo , Estresse Oxidativo/efeitos dos fármacosRESUMO
Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials. One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Maleimidas/química , Compostos de Sulfidrila/química , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/química , Neoplasias/tratamento farmacológico , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Domínios Proteicos , Engenharia de ProteínasRESUMO
Type 2 diabetes (T2D) is a chronic metabolic disorder affecting increasing number of people in developed countries. Therefore new strategies for treatment of T2D and its complications are of special interest. Nowadays, cellular therapies involving mesenchymal stromal cells that reside in adipose tissue (ASCs) constitute a promising approach; however, there are still many obstacles concerning safety and effectiveness that need to be overcome before ASCs could be engaged for the treatment of diabetes mellitus. One of the challenges is preventing ASCs from deterioration caused by elevated oxidative stress present in diabetes milieu. In the current study we investigated the effect of basic fibroblast growth factor (bFGF) treatment on ASCs isolated from patients with diagnosed T2D. We demonstrate here that cell exposition to bFGF in 5 and 10 ng/mL dosages results in improved morphology, increased proliferative activity, reduced cellular senescence and apoptosis, and decreased oxidative stress, indicating recovery of ASCs' function impaired by T2D. Therefore our results provide a support for bFGF as a potential therapeutic agent for improving stem cell-based approaches for the treatment of diabetes mellitus and its complications.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Idoso , Apoptose , Proliferação de Células , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-IdadeRESUMO
[This corrects the article DOI: 10.1155/2017/3027109.].