ABSTRACT
Global alterations in the metabolic network provide substances and energy to support tumor progression. To fuel these metabolic processes, extracellular matrix (ECM) plays a dominant role in supporting the mass transport and providing essential nutrients. Here, we report a fibrinogen and thrombin based coagulation system to construct an artificial ECM (aECM) for selectively cutting-off the tumor metabolic flux. Once a micro-wound is induced, a cascaded gelation of aECM can be triggered to besiege the tumor. Studies on cell behaviors and metabolomics reveal that aECM cuts off the mass transport and leads to a tumor specific starvation to inhibit tumor growth. In orthotopic and spontaneous murine tumor models, this physical barrier also hinders cancer cells from distant metastasis. The in vivo gelation provides an efficient approach to selectively alter the tumor mass transport. This strategy results in a 77% suppression of tumor growth. Most importantly, the gelation of aECM can be induced by clinical operations such as ultrasonic treatment, surgery or radiotherapy, implying this strategy is potential to be translated into a clinical combination regimen.
Subject(s)
Biomimetic Materials/administration & dosage , Extracellular Matrix/chemistry , Neoplasms/therapy , Animals , Biological Transport/drug effects , Biological Transport/radiation effects , Biomimetic Materials/chemistry , Biomimetic Materials/radiation effects , Cell Line, Tumor/transplantation , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Chemoradiotherapy/methods , Disease Models, Animal , Extracellular Matrix/metabolism , Extracellular Matrix/radiation effects , Female , Fibrinogen/administration & dosage , Fibrinogen/chemistry , Fibrinogen/radiation effects , Gels , Humans , Injections, Intravenous , Metabolomics , Mice , Neoplasms/metabolism , Thrombin/administration & dosage , Thrombin/chemistry , Thrombin/radiation effects , Ultrasonic Therapy/methods , Ultrasonic WavesABSTRACT
Using two DNA aptamers selectively recognizing anion-binding exosites 1 and 2 of thrombin as a model, it has been demonstrated that their conjugation by a poly-(dT)-linker (ranging from 5 to 65 nt in length) to produce aptamer heterodimeric constructs results into affinity enhancement. The apparent dissociation constant (Kd(app)) measured at the optical biosensor Biacore-3000 for complexes of thrombin with the heterodimeric constructs reached minimum values (Kd(app) = 0.2-0.4 nM) which were approximately 30-fold less than for the complexes with the primary aptamers. A photoaptamer heterodimeric construct was designed connecting photoaptamer and aptamer sequences with the poly-(dT)-linker of 35 nt long. The photoaptamer used could form photo-induced cross-links with the exosite 2 of thrombin and the aptamer used could bind to the exosite 1. The measured value of Kd(app) for the photoaptamer construct was approximately 40-fold less than that for the primary photoaptamer (5.3 and 190 nM, respectively). Upon exposure to the UV radiation at 308 nm of the equimolar mixtures of thrombin with the photoaptamer construct, the equal yield of the crosslinked complexes was observed at concentrations which were lower by two orders of magnitude than in the case of the primary photoaptamer. It was found that concurrently with crosslinking to thrombin a photo-induced inactivation of the photoaptamer occurs presumably due to formation of the intermolecular crosslinking.
Subject(s)
Aptamers, Nucleotide/radiation effects , Thrombin/radiation effects , Ultraviolet Rays , Aptamers, Nucleotide/chemistry , Biosensing Techniques , Dimerization , Humans , Lasers , Thrombin/antagonists & inhibitors , Thrombin/chemistryABSTRACT
To study the interactions between oligonucleotides and proteins, an original photoaffinity radiolabeling probe has been synthesized. Starting with a 5'-pyridyldithio-3'-amino-oligonucleotide, the photophore benzophenone was first coupled to the 3' end, through acylation by an activated ester of benzoylbenzoic acid. A fluorescein molecule was grafted by alkylation of the free 5'-SH. This compound was finally radiolabeled with 125I using IodoBeads. The selective photolabeling of thrombin in a complex protein mixture by the radioiodinated probe validates this strategy to identify oligonucleotide-binding proteins.
Subject(s)
Fluorescent Dyes/chemistry , Oligodeoxyribonucleotides/chemistry , Photoaffinity Labels/chemistry , Proteins/chemistry , Thrombin/chemistry , Animals , Benzophenones/chemistry , Cattle , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes/radiation effects , Humans , Iodine Radioisotopes , Microscopy, Confocal , Oligodeoxyribonucleotides/radiation effects , Photoaffinity Labels/radiation effects , Thrombin/radiation effects , Ultraviolet RaysABSTRACT
In the rabbit vessels of a. mesenterica, total lysis of fibrin occurred within 60-90 minutes whereas in the renovascular basin the process took 30 minutes longer. This seems to be due to synthesis of a plasminogen activator.
Subject(s)
Fibrinolysis/physiology , Mesenteric Arteries/physiology , Renal Artery/physiology , Animals , Female , Fibrinogen/pharmacokinetics , Fibrinogen/radiation effects , Fibrinolysis/radiation effects , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Male , Mesenteric Arteries/radiation effects , Rabbits , Renal Artery/radiation effects , Thrombin/pharmacokinetics , Thrombin/radiation effects , Time Factors , Ultraviolet RaysABSTRACT
The effect of gamma-irradiation on purified prothrombin and thrombin in aqueous solution has been assessed with reference to bifunctional activities, e.g., clotting and esterase functions, physico-chemical changes in structure, and kinetics. The inactivation curves indicated that the clotting activity was more susceptible to gamma-radiation than the esterolytic function in both the proteins. Prothrombin was comparatively more sensitive to radiation than thrombin. The irradiation of prothrombin (100 kR) caused modifications in the protein resulting in reduced formation of thrombin after activation by Factor Xa. The modifications caused by irradiation were assessed in these proteins by changes in spectral characteristics, levels of tryptophan and disulphides, electrophoretic mobility and amino acid composition. Radiation-induced changes in thrombin were reflected in its kinetic behaviour. The clotting activity of thrombin was almost completely lost at 100 kR, while esterolysis was relatively less affected. The modification of tyrosine and tryptophan residues in thrombin influenced the clotting activity, while these were not involved for esterolysis. Histidine had involvement in both these activities.