Methotrexate (MTX) is a widely used anticancer and antirheumatic drug that has been postulated to protect against metabolic risk factors associated with type 2 diabetes, although the mechanism remains unknown. MTX inhibits 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) and thereby slows the metabolism of 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranosyl-5'-monophosphate (ZMP) and its precursor AICAR, which is a pharmacological AMPK activator. We explored whether MTX promotes AMPK activation in cultured myotubes and isolated skeletal muscle. We found MTX markedly reduced the threshold for AICAR-induced AMPK activation and potentiated glucose uptake and lipid oxidation. Gene silencing of the MTX target ATIC activated AMPK and stimulated lipid oxidation in cultured myotubes. Furthermore, MTX activated AMPK in wild-type HEK-293 cells. These effects were abolished in skeletal muscle lacking the muscle-specific, ZMP-sensitive AMPK-gamma3 subunit and in HEK-293 cells expressing a ZMP-insensitive mutant AMPK-gamma2 subunit. Collectively, our findings underscore a role for AMPK as a direct molecular link between MTX and energy metabolism in skeletal muscle. Cotherapy with AICAR and MTX could represent a novel strategy to treat metabolic disorders and overcome current limitations of AICAR monotherapy. In parallel AMPK is a potential target for cancer tehrapy due to modified metaoblic pathways of cancer cells. Thus developed protocols and can be also implemented for analysis of potential anticancer role of AMPK activators.
COBISS.SI-ID: 31657689
In collaboration with the Institute for pathophysiology (Faculty of medicine, University of Ljubljana) we developed the electroporation protocol and analyzed the mechanisms of introduction of short RNAs into cultured primary human myoblast cells. The protocols were optimized using siRNA against HIF-1α (Hypoxia Inducible factor 1α) mRNA. HIF is a transcription factor and key regulator of cellular oxygen homeostasis and has an important role in several pathologies, most importantly cancer. New approaches seek to achieve inhibition of HIF activation eithe genetically or phyrmacologically. We optimized the electroporation medium and the parameters of electric pulses, which enabled us to achieve 80% silencing of HIF-1α mRNA. In parallel, we performed viability studies for the same electroporation conditions. Suppression of HIF-1α expression was first estimated by measuring HIF-1α mRNA with qPCR. Depletion of HIF-1α protein was subsequently confirmed with Western Blot. Due to involvement of HIF-1α in several muscle pathologies, the effect of HIF-1α silencing was confirmed also on two HIF-1α’s downstream gene targets; PGK (phosphoglycerate kinase) and VEGF (vascular endothelial growth factor). Manuscript of this research for the publication in an international journal is in preparation. The developed protocol will not only serve as a mean to study the mechanisms of introduction of gene material, but also for therapeutic purposes. HIF, PDK and VEGF (involved in angiogenesis) are involved in cellular metabolism and were identified as targets for cancer treatment.
COBISS.SI-ID: 11125588
Our paper focuses on analysis of mechanisms of gene electrotransfer, which has in last years emerged as the most promising non-viral method for delivery of plasmid DNA, oligonucleotides and short RNA molecules. We present new experimental and theoretical results on different steps involved in gene electrotransfer of plated cells and cells in a suspension combined with theoretical analysis of the underlying biophysical phenomena. In our in vitro study we addressed opened questions of this multistep process: how electropermeabilization is related to electrotransfer efficiency; the role of DNA electrophoresis for contact and transfer across the membrane, visualization and theoretical analysis of DNA-membrane interaction and its relation to final transfection efficiency, and the differences between plated and suspended cells. Combinations of high-voltage and low-voltage pulses were used. We demonstrate that the crucial step is DNA insertion into the electropermeabilized membrane which is governed by electrophoretic force. The inserted DNA is then slowly transferred into the cytosol, where also nuclear entry is a limiting factor for optimal transfection. The quantification and theoretical analysis of the crucial parameters reveals that number of DNA molecules interacting with the electropermeabilized cell membrane increases with higher DNA concentration or addition of electrophoretic LV pulses to HV, while transfection reach saturation suggesting that there is a maximal number of DNA molecule which can be successfully transferred. We also explain the observed differences between transfection of cell suspensions and plated cells due to more homogeneous size, shape and movement of suspended cells. From the presented results we propose that DNA is most probably translocated through the stable electropores or alternatively through electro-stimulated endocytosis, possibly dependent on pulse parameters. Understanding the relation between permeabilization, electrophoresis, interaction, viability and transfection efficiency can aid to faster optimization of optimal electric protocol for specific application.
COBISS.SI-ID: 10952788