Serum starvation: caveat emptor Pirkmajer, Sergej; Chibalin, Alexander V
American Journal of Physiology: Cell Physiology,
08/2011, Letnik:
301, Številka:
2
Journal Article
Recenzirano
Serum starvation is one of the most frequently performed procedures in molecular biology and there are literally thousands of research papers reporting its use. In fact, this method has become so ...ingrained in certain areas of research that reports often simply state that cells were serum starved without providing any factual details as to how the procedure was carried out. Even so, we quite obviously lack unequivocal terminology, standard protocols, and perhaps most surprisingly, a common conceptual basis when performing serum starvation. Such inconsistencies not only hinder interstudy comparability but can lead to opposing and inconsistent experimental results. Although it is frequently assumed that serum starvation reduces basal activity of cells, available experimental data do not entirely support this notion. To address this important issue, we studied primary human myotubes, rat L6 myotubes and human embryonic kidney (HEK)293 cells under different serum starvation conditions and followed time-dependent changes in important signaling pathways such as the extracellular signal-regulated kinase 1/2, the AMP-activated protein kinase, and the mammalian target of rapamycin. Serum starvation induced a swift and dynamic response, which displayed obvious qualitative and quantitative differences across different cell types and experimental conditions despite certain unifying features. There was no uniform reduction in basal signaling activity. Serum starvation clearly represents a major event that triggers a plethora of divergent responses and has therefore great potential to interfere with the experimental results and affect subsequent conclusions.
Modulating body temperature, mostly through the use of antipyretics, is a commonly employed therapeutic intervention in medical practice. However, emerging evidence suggests that hyperthermia could ...serve as an adjuvant therapy for patients with infection. We performed a narrative review to explore the application of therapeutic hyperthermia in the treatment of infection. A number of studies have been performed in the pre-antibiotic era, enrolling patients with neurosyphilis and gonococcal infections, with reported cure rates at around 60%-80%. We have outlined the potential molecular and immunological mechanisms explaining the possible beneficial effects of therapeutic hyperthermia. For some pathogens increased temperature exerts a direct negative effect on virulence; however, it is presumed that temperature driven activation of the immune system is probably the most important factor affecting microbial viability. Lastly, we performed a review of modern-era studies where modulation of body temperature has been used as a treatment strategy. In trials of therapeutic hypothermia in patients with infection worse outcomes have been observed in the hypothermia group. Use of antipyretics has not been associated with any improvement in clinical outcomes. In modern-era therapeutic hyperthermia achieved by physical warming has been studied in one pilot trial, and better survival was observed in the hyperthermia group. To conclude, currently there is not enough data to support the use of therapeutic hyperthermia outside clinical trials; however, available studies are in favor of at least a temperature tolerance strategy for non-neurocritical patients.
AMP-activated protein kinase (AMPK) is a cellular energy gauge and a major regulator of cellular energy homeostasis. Once activated, AMPK stimulates nutrient uptake and the ATP-producing catabolic ...pathways, while it suppresses the ATP-consuming anabolic pathways, thus helping to maintain the cellular energy balance under energy-deprived conditions. As much as ~ 20–25% of the whole-body ATP consumption occurs due to a reaction catalysed by Na
+
,K
+
-ATPase (NKA). Being the single most important sink of energy, NKA might seem to be an essential target of the AMPK-mediated energy saving measures, yet NKA is vital for maintenance of transmembrane Na
+
and K
+
gradients, water homeostasis, cellular excitability, and the Na
+
-coupled transport of nutrients and ions. Consistent with the model that AMPK regulates ATP consumption by NKA, activation of AMPK in the lung alveolar cells stimulates endocytosis of NKA, thus suppressing the transepithelial ion transport and the absorption of the alveolar fluid. In skeletal muscles, contractions activate NKA, which opposes a rundown of transmembrane ion gradients, as well as AMPK, which plays an important role in adaptations to exercise. Inhibition of NKA in contracting skeletal muscle accentuates perturbations in ion concentrations and accelerates development of fatigue. However, different models suggest that AMPK does not inhibit or even stimulates NKA in skeletal muscle, which appears to contradict the idea that AMPK maintains the cellular energy balance by always suppressing ATP-consuming processes. In this short review, we examine the role of AMPK in regulation of NKA in skeletal muscle and discuss the apparent paradox of AMPK-stimulated ATP consumption.
Triple naegative breast cancer has an increased rate of distant metastasis and consequently poor prognosis. To metastasize, breast cancer cells must detach from the main tumour mass 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 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), which was reduced by AMPK inhibitor compound C that prevented detachment of MDA-MB-231 cells. However, direct AMPK activators A-769662 and AICAR did not have any major effect on the percentage of floating MDA-MB-231 cells, indicating that AMPK activation is necessary but not sufficient for triggering detachment of cancer cells. Our results demonstrate that separate analysis of floating and attached cancer cells might be important for evaluation of anti-cancer agents.
Oximes, investigated as antidotes against organophosphates (OP) poisoning, are known to display toxic effects on a cellular level, which could be explained beyond action on acetylcholinesterase as ...their main target. To investigate this further, we performed an in vitro cell-based evaluation of effects of two structurally diverse oxime groups at concentrations of up to 800 μM, on several cell models: skeletal muscle, kidney, liver, and neural cells. As indicated by our results, compounds with an imidazolium core induced necrosis, unregulated cell death characterized by a cell burst, increased formation of reactive oxygen species, and activation of antioxidant scavenging. On the other hand, oximes with a pyridinium core activated apoptosis through specific caspases 3, 8, and/or 9. Interestingly, some of the compounds exhibited a synergistic effect. Moreover, we generated a pharmacophore model for each oxime series and identified ligands from public databases that map to generated pharmacophores. Several interesting hits were obtained including chemotherapeutics and specific inhibitors. We were able to define the possible structural features of tested oximes triggering toxic effects: chlorine atoms in combination with but-2(E)-en-1,4-diyl linker and adding a second benzene ring with substituents such as chlorine and/or methyl on the imidazolium core. Such oximes could not be used in further OP antidote development research, but could be introduced in other research studies on new specific targets. This could undoubtedly result in an overall improved wider use of unexplored oxime database created so far in OP antidotes field of research in a completely new perspective.
The cardiotonic steroids (CTS), such as ouabain and marinobufagenin, are thought to be adrenocortical hormones secreted during exercise and the stress response. The catalytic α-subunit of Na,K-ATPase ...(NKA) is a CTS receptor, whose largest pool is located in skeletal muscles, indicating that muscles are a major target for CTS. Skeletal muscles contribute to adaptations to exercise by secreting interleukin-6 (IL-6) and plethora of other cytokines, which exert paracrine and endocrine effects in muscles and non-muscle tissues. Here, we determined whether ouabain, a prototypical CTS, modulates IL-6 signaling and secretion in the cultured human skeletal muscle cells. Ouabain (2.5–50 nM) suppressed the abundance of STAT3, a key transcription factor downstream of the IL-6 receptor, as well as its basal and IL-6-stimulated phosphorylation. Conversely, ouabain (50 nM) increased the phosphorylation of ERK1/2, Akt, p70S6K, and S6 ribosomal protein, indicating activation of the ERK1/2 and the Akt-mTOR pathways. Proteasome inhibitor MG-132 blocked the ouabain-induced suppression of the total STAT3, but did not prevent the dephosphorylation of STAT3. Ouabain (50 nM) suppressed hypoxia-inducible factor-1α (HIF-1α), a modulator of STAT3 signaling, but gene silencing of HIF-1α and/or its partner protein HIF-1β did not mimic effects of ouabain on the phosphorylation of STAT3. Ouabain (50 nM) failed to suppress the phosphorylation of STAT3 and HIF-1α in rat L6 skeletal muscle cells, which express the ouabain-resistant α1-subunit of NKA. We also found that ouabain (100 nM) promoted the secretion of IL-6, IL-8, GM-CSF, and TNF-α from the skeletal muscle cells of healthy subjects, and the secretion of GM-CSF from cells of subjects with the type 2 diabetes. Marinobufagenin (10 nM), another important CTS, did not alter the secretion of these cytokines. In conclusion, our study shows that ouabain suppresses the IL-6 signaling via STAT3, but promotes the secretion of IL-6 and other cytokines, which might represent a negative feedback in the IL-6/STAT3 pathway. Collectively, our results implicate a role for CTS and NKA in regulation of the IL-6 signaling and secretion in skeletal muscle.
Electrotransfection is based on application of high-voltage pulses that transiently increase membrane permeability, which enables delivery of DNA and RNA in vitro and in vivo. Its advantage in ...applications such as gene therapy and vaccination is that it does not use viral vectors. Skeletal muscles are among the most commonly used target tissues. While siRNA delivery into undifferentiated myoblasts is very efficient, electrotransfection of siRNA into differentiated myotubes presents a challenge. Our aim was to develop efficient protocol for electroporation-based siRNA delivery in cultured primary human myotubes and to identify crucial mechanisms and parameters that would enable faster optimization of electrotransfection in various cell lines.
We established optimal electroporation parameters for efficient siRNA delivery in cultured myotubes and achieved efficient knock-down of HIF-1α while preserving cells viability. The results show that electropermeabilization is a crucial step for siRNA electrotransfection in myotubes. Decrease in viability was observed for higher electric energy of the pulses, conversely lower pulse energy enabled higher electrotransfection silencing yield. Experimental data together with the theoretical analysis demonstrate that siRNA electrotransfer is a complex process where electropermeabilization, electrophoresis, siRNA translocation, and viability are all functions of pulsing parameters. However, despite this complexity, we demonstrated that pulse parameters for efficient delivery of small molecule such as PI, can be used as a starting point for optimization of electroporation parameters for siRNA delivery into cells in vitro if viability is preserved.
The optimized experimental protocol provides the basis for application of electrotransfer for silencing of various target genes in cultured human myotubes and more broadly for electrotransfection of various primary cell and cell lines. Together with the theoretical analysis our data offer new insights into mechanisms that underlie electroporation-based delivery of short RNA molecules, which can aid to faster optimisation of the pulse parameters in vitro and in vivo.
Gene immunotherapy has become an important approach in the treatment of cancer. One example is the introduction of genes encoding immunostimulatory cytokines, such as interleukin 2 and interleukin ...12, which stimulate immune cells in tumours. The aim of our study was to determine the effects of gene electrotransfer of plasmids encoding interleukin 2 and interleukin 12 individually and in combination in the CT26 murine colon carcinoma cell line in mice. In the in vitro experiment, the pulse protocol that resulted in the highest expression of IL-2 and IL-12 mRNA and proteins was used for the in vivo part. In vivo, tumour growth delay and also complete response were observed in the group treated with the plasmid combination. Compared to the control group, the highest levels of various immunostimulatory cytokines and increased immune infiltration were observed in the combination group. Long-term anti-tumour immunity was observed in the combination group after tumour re-challenge. In conclusion, our combination therapy efficiently eradicated CT26 colon carcinoma in mice and also generated strong anti-tumour immune memory.
Na
+
,K
+
-ATPase (NKA) is essential for maintenance of cellular and whole-body water and ion homeostasis. In the kidney, a major site of ion transport, NKA consumes ~ 50% of ATP, indicating a tight ...coordination of NKA and energy metabolism. AMP-activated protein kinase (AMPK), a cellular energy sensor, regulates NKA by modulating serine phosphorylation of the α1-subunit, but whether it modulates other important regulatory phosphosites, such as Tyr10, is unknown. Using human kidney (HK-2) cells, we determined that the phosphorylation of Tyr10 was stimulated by the epidermal growth factor (EGF), which was opposed by inhibitors of Src kinases (PP2), tyrosine kinases (genistein), and EGF receptor (EGFR, gefitinib). AMPK activators AICAR and A-769662 suppressed the EGF-stimulated phosphorylation of EGFR (Tyr1173) and NKAα1 at Tyr10. The phosphorylation of Src (Tyr416) was unaltered by AICAR and increased by A-769662. Conversely, ouabain (100 nM), a pharmacological NKA inhibitor and a putative adrenocortical hormone, enhanced the EGF-stimulated Tyr10 phosphorylation without altering the phosphorylation of EGFR (Tyr1173) or Src (Tyr416). Ouabain (100–1000 nM) increased the ADP:ATP ratio, while it suppressed the lactate production and the oxygen consumption rate in a dose-dependent manner. Treatment with ouabain or gene silencing of NKAα1 or NKAα3 subunit did not activate AMPK. In summary, AMPK activators and ouabain had antagonistic effects on the phosphorylation of NKAα1 at Tyr10 in cultured HK-2 cells, which implicates a role for Tyr10 in coordinated regulation of NKA-mediated ion transport and energy metabolism.
Graphical Abstract
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-β-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-γ3 subunit and in HEK-293 cells expressing a ZMP-insensitive mutant AMPK-γ2 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.
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