Pathologically altered energy metabolism in cancer cells offers the possibility of targeted approach. Some approaches test inhbitors of glycolisis such as 2-deoxy-D-glucose in order to reverse Warburg effect as an anticaner strategy or in combination with standard chemotherapy, while other approaches test alredy approved drugs such as anti-diabetic drug metformin that is already in over 100 clinical trials. We focused on the process of anchorage independent growth. Triple negative breast cancer has an increased rate of distant metastasis and consequently poor prognosis. To metastasize cancer cells must detach from the main tumour and resist anoikis, a programmed cell death induced by lack of cell-extracellular matrix communication. Although cancer cells must detach to metastasize in vivo, the viability of floating cancer cells in vitro is rarely investigated. Here we show that co-treatment of anoikis-resistant MDA-MB-231 cells with metformin and 2-deoxy-D-glucose (2-DG) increased the percentage of floating cells, of which, strikingly, about 95% were viable. Floating cells resumed their proliferation once they were reseeded in the pharmacological compound-free medium. Similar effects on detachment were observed on anoikis-prone MCF-7 cells. Co-treatment of MDA-MB-231 cells with metformin and 2-DG induced a strong activation of AMP-activated protein kinase (AMPK). However, direct AMPK activators A-769662 and AICAR did not have any major effect on the percentage of floating MDA-MB-231 cells. We show that AMPK activation is necessary but not sufficient for triggering detachment of cancer cells. Our results also demonstrate that separate analysis of floating and attached cancer cells might be important for evaluation of anti-cancer agents. In general, we have described a novel phenomena of detachment of live cancer cells that my be important for understanding of the metastatic processes.
COBISS.SI-ID: 11739732
Epidemiological studies indicate that metformin, reduce cancer risk and mortality in patients with type 2 diabetes for some type of cancers. Different studies show that metformin might protect against breast cancer indirectly by ameliorating systemic glucose homeostasis and/or targeting breast cancer cells directly as inhibitor of complex I and AMPK activator. However, experiments using MDA-MB-231 cells , a standard in vitro breast cancer model, produced inconsistent results. We therefore analysed how glucose level in the medium and medium renewal protocol alter sensitivity of MDA-MB-231 cells. We found that metformin suppressed the viability and proliferation only if medium was not renewed due to reduced glucose concentration. We also show synergistic effect of 2-deoxy-D-glucose (2-DG) and metformin on reduced viability and proliferation of MDA-MB-231 cells with or without medium renewal, however at physiological cencetrations the proliferation was blocked only for low glucose levels. In summury, we have show that medium renewal blocks anti-proliferative effects of metformin during prolonged treatments in low-glucose medium.
COBISS.SI-ID: 11347540
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. Objaveljno v reviji Scientific Reports NPG: A'', IF=5.08 http://www.nature.com/scientificreports
COBISS.SI-ID: 10952788
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.
COBISS.SI-ID: 31657689
Background: At first glance, there appears to be a high degree of similarity between the metabolism of yeast (the Crabtree effect) and human cancer cells (the Warburg effect). At the root of both effects is accelerated metabolic flow through glycolysis which leads to overflows of ethanol and lactic acid, respectively. It has been proposed that enhanced glycolytic flow in cancer cells is triggered by the altered kinetic characteristics of the key glycolytic regulatory enzyme 6-phosphofructo-1-kinase (Pfk). Through a posttranslational modification, highly active shorter Pfk-M fragments, which are resistant to feedback inhibition, are formed after the proteolytic cleavage of the C-terminus of the native human Pfk-M. Alternatively, enhanced glycolysis is triggered by optimal growth conditions in the yeast Saccharomyces cerevisiae. Results: To assess the deregulation of glycolysis in yeast cells, the sfPFKM gene encoding highly active human shorter Pfk-M fragments was introduced into pfk-null S. cerevisiae. No growth of the transformants with the sfPFKM gene was observed on glucose and fructose. Glucose even induced rapid deactivation of Pfk1 activities in such transformants. However, Pfk1 activities of the sfPFKM transformants were detected in maltose medium, but the growth in maltose was possible only after the addition of 10 mM of ethanol to the medium. Ethanol seemed to alleviate the severely unbalanced NADH/NADPH ratio in the sfPFKM cells. However, the transformants carrying modified Pfk-M enzymes grew faster than the transformants with the human native human Pfk-M enzyme in a narrow ecological niche with a low maltose concentration medium that was further improved by additional modifications. Interestingly, periodic extracellular accumulation of phenylacetaldehyde was detected during the growth of the strain with modified Pfk-M but not with the strain encoding the human native enzyme. Conclusions: Highly active cancer-specific shorter Pfk-M fragments appear to trigger several controlling mechanisms in the primary metabolism of yeast S. cerevisiae cells. These results suggest more complex metabolic regulation is present in S. cerevisiae as free living unicellular eukaryotic organisms in comparison to metazoan human cells. However, increased productivity under broader growth conditions may be achieved if more gene engineering is performed to reduce or omit several controlling mechanisms.
COBISS.SI-ID: 6147098