Inhibition of pyruvate dehydrogenase kinase (PDK) emerged as a potential strategy for treatment of cancer and metabolic disorders. Dichloroacetate (DCA), a prototypical PDK inhibitor, reduces the ...abundance of some PDK isoenzymes. However, the underlying mechanisms are not fully characterized and may differ across cell types. We determined that DCA reduced the abundance of PDK1 in breast (MDA-MB-231) and prostate (PC-3) cancer cells, while it suppressed both PDK1 and PDK2 in skeletal muscle cells (L6 myotubes). The DCA-induced PDK1 suppression was partially dependent on hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of PDK1, in cancer cells but not in L6 myotubes. However, the DCA-induced alterations in the mRNA and the protein levels of PDK1 and/or PDK2 did not always occur in parallel, implicating a role for post-transcriptional mechanisms. DCA did not inhibit the mTOR signaling, while inhibitors of the proteasome or gene silencing of mitochondrial proteases CLPP and AFG3L2 did not prevent the DCA-induced reduction of the PDK1 protein levels. Collectively, our results suggest that DCA reduces the abundance of PDK in an isoform-dependent manner via transcriptional and post-transcriptional mechanisms. Differential response of PDK isoenzymes to DCA might be important for its pharmacological effects in different types of cells.
Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific ...kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts.
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.
Genska terapija postaja čedalje bolj zanimiva tudi v onkologiji. Med aplikacijami je morda najzanimivejša imunostimulacija. Pripravimo lahko plazmidno DNA, ki nosi zapis za različne imunostimulatorne ...molekule, ki jih vnesemo v celice tumorjev ali normalnih tkiv. Ta tkiva postanejo proizvajalci teh molekul, ki lahko delujejo lokalno ali pa se izločajo tudi sistemsko v krvni obtok. Ker plazmidna DNA ne prehaja celične membrane, so potrebni dostavni sistemi, virusni ali nevirusni. V naših študijah uporabljamo predvsem nevirusni dostavni sistem – elektroporacijo. Interlevkin 12 (IL-12) je eden od zanimivih citokinov, za katerega je znano protitumorsko delovanje s spodbujanjem imunskega odziva in antiangiogenim delovanjem. Namen projekta SmartGene.si je bil pripraviti plazmid z zapisom za interlevkin 12 (plazmid phIL12) in pripraviti vse potrebno za njegovo klinično testiranje za zdravljenje kožnih tumorjev. V konzorciju smo združili moči s partnerji z akademskega in industrijskega področja. Treba je bilo pripraviti plazmid za uporabo v humani onkologiji po zahtevah Evropske agencije za zdravila (EMA). Za prijavo klinične študije na Javno agencijo za zdravila in medicinske pripomočke (JAZMP) smo morali izvesti tudi vse neklinične raziskave o varnosti in učinkovitosti zdravila. Nato je bilo treba razviti postopek priprave zdravila, zagotoviti primerne prostore za pripravo in izvedbo postopka priprave zdravila. V treh letih smo dosegli vse te zastavljene cilje in dobili dovoljenje za izvajanje klinične študije na kožnih tumorjih, ki ga je izdala JAZMP na osnovi pozitivnega mnenja Komisije Republike Slovenije za medicinsko etiko. Zdaj poteka klinična študija faze I preizkušanja plazmida phIL12 na kožnih tumorjih glave in vratu z namenom preveriti varnost in sprejemljivost genskega elektroprenosa plazmida v tumorje. Cilj študije je prav tako določiti primeren odmerek zdravila, ki bi ga v nadaljnji klinični študiji uporabili kot adjuvantno zdravljenje k ablativnim terapijam, kot sta radioterapija ali elektrokemoterapija.
Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To fulfill its clinical potential, the development is focused on localized delivery systems, such as gene ...electrotransfer, which can provide localized delivery of IL-12 to the tumor microenvironment. Gene electrotransfer of the plasmid encoding human IL-12 is already in clinical trials in USA, demonstrating positive results in the treatment of melanoma patients. To comply with EU regulatory requirements for clinical application, which recommend the use of antibiotic resistance gene-free plasmids, we constructed and developed the production process for the clinical grade quality antibiotic resistance gene-free plasmid encoding human IL-12 (p21-hIL-12-ORT) and its ortholog encoding murine IL-12 (p21-mIL-12-ORT). To demonstrate the suitability of the p21-hIL-12-ORT or p21-mIL-12-ORT plasmid for the first-in-human clinical trial, the biological activity of the expressed transgene, its level of expression and plasmid copy number were determined in vitro in the human squamous cell carcinoma cell line FaDu and the murine colon carcinoma cell line CT26. The results of the non-clinical evaluation in vitro set the basis for further in vivo testing and evaluation of antitumor activity of therapeutic molecules in murine models as well as provide crucial data for further clinical trials of the constructed antibiotic resistance gene-free plasmid in humans.
Circulating tumor cells (CTCs) have become an important biomarker in breast cancer. Different isolation tech-niques based on their biological or physical features were established. Currently, the ...most widely used methods for visualization after their separation are based on immunofluorescent staining, which does not provide the information on the morphology.
The aim of this study was to evaluate how two different separation techniques affect cell morphology and to analyse cell morphology with techniques used in routine cytopathological laboratory. A direct side-by-side comparison of physical (Parsortix®) and biological (MACS®) separation technique was performed.
In the preclinical setting, both isolation techniques retained the viability and antigenic characteristics of MCF7 breast cancer cells. Some signs of degeneration such as cell swelling, cytoplasmic blebs, villous projections and vacuolization were observed. In metastatic breast cancer patient cohort, morphological features of isolated CTCs were dependent on the separation technique. After physical separation, CTCs with preserved cell morphology were detected. After biological separation the majority of the isolated CTCs were so degenerated that their identity was difficult to confirm.
Taken together, physical separation is a suitable technique for detection of CTCs with preserved cell morphology for the use in a routine cytopathological laboratory.
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•Electrochemotherapy affects the early C2C12 myogenesis.•Electrochemotherapy has little effect on C2C12 myotubes.•Electrochemotherapy influenced differentiation but not proliferation ...of myoblasts.•Electrochemotherapy prevents formation of nAChR clusters.
Electrochemotherapy (ECT) is a local ablative therapy for the treatment of different skin and subcutaneous tumors and certain tumors in internal organs. Skeletal muscle represents a major tumor- surrounding tissue, exposed to side effects of ECT. At the cellular level, side-effects of ECT on skeletal muscle and underlying mechanisms have not been examined yet. Thus, we aimed to determine the effect of ECT in the mouse muscle cell line C2C12 during in vitro myogenesis. We evaluated the electroporation efficiency and viability of C2C12 myotubes at increasing voltages (200–1300 V/cm) using propidium iodide (PI). Permeabilization of PI into the cells was voltage-dependent accounting up to 97 % efficiency at the highest voltage. High cell viability and myotube integrity were maintained until 4 days after electroporation. ECT with the cytostatic drugs bleomycin and cisplatin decreased the viability of C2C12 myoblasts and myotubes in a dose-dependent manner. However, myoblasts were more sensitive to ECT than myotubes. Increased secretion of IL-6, observed 3 days after ECT, confirming its effects on early myogenesis. Only minor effects of ECT were observed in treated myotubes. These results contribute to the safety profile of ECT in tumor treatment.
The best established role of acetylcholinesterase (EC 3.1.1.7, AChE) is termination of neurotransmission at cholinergic synapses. However, AChE is also located at sites, where no other cholinergic ...components are present and there is accumulating evidence for non-cholinergic functions of this protein. In the process of skeletal muscle formation, AChE is expressed already at the stage of mononuclear myoblast, which is long before other cholinergic components can be demonstrated in this tissue. Myoblast proliferation is an essential step in muscle regeneration and is always accompanied by apoptosis. Since there are several reports demonstrating AChE participation in apoptosis one can hypothesize that early AChE expression in myoblasts reflects the development of the apoptotic apparatus in these cells. Here we tested this hypothesis by following the effect of siRNA AChE silencing on apoptotic markers and by determination of AChE level after staurosporine-induced apoptosis in cultured human myoblasts. Decreased apoptosis in siRNA AChE silenced myoblasts and increased AChE expression in staurosporine-treated myoblasts confirmed AChE involvement in apoptosis. The three AChE splice variants were differently affected by staurosporine-induced apoptosis. The hydrophobic (H) variant appeared unaffected, a tendency towards increase of tailed (T) variant was detected, however the highest, 8-fold increase was observed for readthrough (R) variant. In the light of these findings AChE appears to be a potential therapeutic target at muscle injuries including organophosphate myopathy.
► Expression of agrin, ColQ and AChE was compared in the developing rat spinal cord. ► Expression patterns support common targeting of AChE and agrin to the NMJ. ► AChE and agrin play more than one ...role in the development of the skeletal muscle.
Acetylcholinesterase (AChE) and agrin play unique functional roles in the neuromuscular junction (NMJ). AChE is a cholinergic and agrin a synaptogenetic component. In spite of their different functions, they share several common features: their targeting is determined by alternative splicing; unlike most other NMJ components they are expressed in both, muscle and motor neuron and both reside on the synaptic basal lamina of the NMJ. Also, both were reported to play various nonjunctional roles. However, while the origin of basal lamina bound agrin is undoubtedly neural, the neural origin of AChE, which is anchored to the basal lamina with collagenic tail ColQ, is elusive. Hypothesizing that motor neuron proteins targeted to the NMJ basal lamina share common temporal pattern of expression, which is coordinated with the formation of basal lamina, we compared expression of agrin isoforms with the expression of AChE-T and ColQ in the developing rat spinal cord at the stages before and after the formation of NMJ basal lamina. Cellular origin of AChE-T and agrin was determined by in situ hybridization and their quantitative levels by RT PCR. We found parallel increase in expression of the synaptogenetic (agrin 8) isoform of agrin and ColQ after the formation of basal lamina supporting the view that ColQ bound AChE and agrin 8 isoform are destined to the basal lamina. Catalytic AChE-T subunit and agrin isoforms 19 and 0 followed different expression patterns. In accordance with the reports of other authors, our investigations also revealed various alternative functions for AChE and agrin. We have already demonstrated participation of AChE in myoblast apoptosis; here we present the evidence that agrin promotes the maturation of heavy myosin chains and the excitation–contraction coupling. These results show that common features of AChE and agrin extend to their capacity to play multiple roles in muscle development.
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