RESUMO
The natural product family of macrocyclic lipodepsipeptides containing the 4-amido-2,4-pentadienoate functionality possesses intriguing cytotoxic selectivity toward hypoxic cancer cells. These subpopulations of cancer cells display increased metastatic potential and resistance to chemo- and radiotherapy. In this paper, we present studies on the mechanism of action of these natural products in hypoxic cancer cells and show that this involves rapid and hypoxia-selective collapse of mitochondrial integrity and function. These events drive a regulated cell death process that potentially could function as a powerful tool in the fight against chemo- and radiotherapy-resistant cancer cells. Toward that end, we demonstrate activity in two different mouse tumor models.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Mitocôndrias/efeitos dos fármacos , Hipóxia Tumoral/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Depsipeptídeos/uso terapêutico , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
The α-oxoaldehyde methylglyoxal is a ubiquitous and highly reactive metabolite known to be involved in aging- and diabetes-related diseases. If not detoxified by the endogenous glyoxalase system, it exerts its detrimental effects primarily by reacting with biopolymers such as DNA and proteins. We now demonstrate that during ketosis, another metabolic route is operative via direct non-enzymatic aldol reaction between methylglyoxal and the ketone body acetoacetate, leading to 3-hydroxyhexane-2,5-dione. This novel metabolite is present at a concentration of 10%-20% of the methylglyoxal level in the blood of insulin-starved patients. By employing a metabolite-alkyne-tagging strategy it is clarified that 3-hydroxyhexane-2,5-dione is further metabolized to non-glycating species in human blood. The discovery represents a new direction within non-enzymatic metabolism and within the use of alkyne-tagging for metabolism studies and it revitalizes acetoacetate as a competent endogenous carbon nucleophile.
Assuntos
Acetoacetatos/química , Corpos Cetônicos/química , Aldeído Pirúvico/sangue , Acetoacetatos/metabolismo , Alcinos/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Hexanonas/análise , Hexanonas/sangue , Hexanonas/metabolismo , Humanos , Corpos Cetônicos/metabolismo , Espectrometria de Massas , Aldeído Pirúvico/análise , Aldeído Pirúvico/metabolismo , Albumina Sérica/química , Albumina Sérica/metabolismoRESUMO
We here present a conceptually novel reaction-based ELISA principle (ReactELISA) for quantitation of the carbon nucleophilic lipid metabolite acetoacetate. Key to the assay is the utilization of a highly chemoselective Friedländer reaction that captures and simultaneously stabilizes the nucleophilic metabolite directly in the biological matrix. By developing a bifunctional biotinylated capture probe, the Friedländer-acetoacetate adduct can be trapped and purified directly in streptavidin coated wells. Finally, we outline the selection and refinement of a highly selective recombinant antibody for specific adduct quantitation. The setup is very robust and, as we demonstrate via miniaturization for microplate format, amenable for screening of compounds or interventions that alter lipid metabolism in liver cell cultures. The assay-principle should be extendable to quantitation of other nucleophilic or electrophilic and perhaps even more reactive metabolites provided suitable capture probes and antibodies.