Subcellular Localization of Thioredoxin/Thioredoxin Reductase System-A Missing Link in Endoplasmic Reticulum Redox Balance.

The lumen of the endoplasmic reticulum (ER) is usually considered an oxidative environment; however, oxidized thiol-disulfides and reduced pyridine nucleotides occur there parallelly, indicating that the ER lumen lacks components which connect the two systems. Here, we investigated the luminal presence of the thioredoxin (Trx)/thioredoxin reductase (TrxR) proteins, capable of linking the protein thiol and pyridine nucleotide pools in different compartments. It was shown that specific activity of TrxR in the ER is undetectable, whereas higher activities were measured in the cytoplasm and mitochondria. None of the Trx/TrxR isoforms were expressed in the ER by Western blot analysis. Co-localization studies of various isoforms of Trx and TrxR with ER marker Grp94 by immunofluorescent analysis further confirmed their absence from the lumen. The probability of luminal localization of each isoform was also predicted to be very low by several in silico analysis tools. ER-targeted transient transfection of HeLa cells with Trx1 and TrxR1 significantly decreased cell viability and induced apoptotic cell death. In conclusion, the absence of this electron transfer chain may explain the uncoupling of the redox systems in the ER lumen, allowing parallel presence of a reduced pyridine nucleotide and a probably oxidized protein pool necessary for cellular viability.

LPA-Induced Thromboxane A2-Mediated Vasoconstriction Is Limited to Poly-Unsaturated Molecular Species in Mouse Aortas.

We have previously reported that, in aortic rings, 18:1 lysophosphatidic acid (LPA) can induce both vasodilation and vasoconstriction depending on the integrity of the endothelium. The predominant molecular species generated in blood serum are poly-unsaturated LPA species, yet the vascular effects of these species are largely unexplored. We aimed to compare the vasoactive effects of seven naturally occurring LPA species in order to elucidate their potential pathophysiological role in vasculopathies. Vascular tone was measured using myography, and thromboxane A2 (TXA2) release was detected by ELISA in C57Bl/6 mouse aortas. The Ca2+-responses to LPA-stimulated primary isolated endothelial cells were measured by Fluo-4 AM imaging. Our results indicate that saturated molecular species of LPA elicit no significant effect on the vascular tone of the aorta. In contrast, all 18 unsaturated carbon-containing (C18) LPAs (18:1, 18:2, 18:3) were effective, with 18:1 LPA being the most potent. However, following inhibition of cyclooxygenase (COX), these LPAs induced similar vasorelaxation, primarily indicating that the vasoconstrictor potency differed among these species. Indeed, C18 LPA evoked a similar Ca2+-signal in endothelial cells, whereas in endothelium-denuded aortas, the constrictor activity increased with the level of unsaturation, correlating with TXA2 release in intact aortas. COX inhibition abolished TXA2 release, and the C18 LPA induced vasoconstriction. In conclusion, polyunsaturated LPA have markedly increased TXA2-releasing and vasoconstrictor capacity, implying potential pathophysiological consequences in vasculopathies.

Sigma-1 Receptor Agonist Fluvoxamine Ameliorates Fibrotic Response of Trabecular Meshwork Cells.

Primary open-angle glaucoma remains a global issue, lacking a definitive treatment. Increased intraocular pressure (IOP) is considered the primary risk factor of the disease and it can be caused by fibrotic-like changes in the trabecular meshwork (TM) such as increased tissue stiffness and outflow resistance. Previously, we demonstrated that the sigma-1 receptor (S1R) agonist fluvoxamine (FLU) has anti-fibrotic properties in the kidney and lung. In this study, the localization of the S1R in TM cells was determined, and the anti-fibrotic efficacy of FLU was examined in both mouse and human TM cells. Treatment with FLU reduced the F-actin rearrangement, inhibited cell proliferation and migration induced by the platelet-derived growth factor and decreased the levels of fibrotic proteins. The protective role of the S1R in fibrosis was confirmed by a more pronounced increase in alpha smooth muscle actin and F-actin bundle and clump formation in primary mouse S1R knockout TM cells. Furthermore, FLU demonstrated its protective effects by increasing the production of nitric oxide and facilitating the degradation of the extracellular matrix through the elevation of cathepsin K. These findings suggest that the S1R could be a novel target for the development of anti-fibrotic drugs and offer a new therapeutic approach for glaucoma.

Reduced O-GlcNAcylation and tubular hypoxia contribute to the antifibrotic effect of SGLT2 inhibitor dapagliflozin in the diabetic kidney.

Diabetic kidney disease is a worldwide epidemic, and therapies are incomplete. Clinical data suggest that improved renal outcomes by Na+-glucose cotransporter 2 inhibitor (SGLT2i) are partly beyond their antihyperglycemic effects; however, the mechanisms are still elusive. Here, we investigated the effect of the SGLT2i dapagliflozin (DAPA) in the prevention of elevated O-GlcNAcylation and tubular hypoxia as contributors of renal fibrosis. Type 1 diabetes was induced by streptozotocin in adult male Wistar rats. After the onset of diabetes, rats were treated for 6 wk with DAPA or DAPA combined with losartan (LOS). The effect of hyperglycemia was tested in HK-2 cells kept under normal or high glucose conditions. To test the effect of hypoxia, cells were kept in 1% O2 for 2 h. Cells were treated with DAPA or DAPA combined with LOS. DAPA slowed the loss of renal function, mitigated renal tubular injury markers (kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin), and reduced tubulointerstitial fibrosis. DAPA diminished high glucose-induced protein O-GlcNAcylation and moderated the tubular response to hypoxia through the hypoxia-inducible factor pathway. DAPA alone was as effective as combined treatment with LOS in all outcome parameters. These data highlight the role of ameliorated O-GlcNAcylation and diminished tubular hypoxia as important benefits of SGLT2i treatment. Our results support the link between glucose toxicity, tubular hypoxia, and fibrosis, a vicious trio that could be targeted by SGLT2i in kidney diseases of other origins as well.

Stress-Induced, p53-Mediated Tumor Growth Inhibition of Melanoma by Modulated Electrohyperthermia in Mouse Models without Major Immunogenic Effects.

Modulated electrohyperthermia (mEHT), an innovative complementary technique of radio-, chemo-, and targeted oncotherapy modalities, can induce tumor apoptosis and contribute to a secondary immune-mediated cancer death. Here, we tested the efficiency of high-fever range (~42 °C) mEHT on B16F10 melanoma both in cell culture and allograft models. In vivo, mEHT treatment resulted in significant tumor size reduction when repeated three times, and induced major stress response as indicated by upregulated cytoplasmic and cell membrane hsp70 levels. Despite the increased PUMA and apoptosis-inducing factor 1, and moderate rise in activated-caspase-3, apoptosis was not significant. However, phospho-H2AX indicated DNA double-strand breaks, which upregulated p53 protein and its downstream cyclin-dependent kinase inhibitors p21waf1 and p27kip. Combined in vitro treatment with mEHT and the p53 activator nutlin-3a additively reduced cell viability compared to monotherapies. Though mEHT promoted the release of damage-associated molecular pattern (DAMP) damage signaling molecules hsp70, HMGB1 and ATP to potentiate the tumor immunogenicity of melanoma allografts, it reduced MHC-I and melan-A levels in tumor cells. This might explain why the number of cytotoxic T cells was moderately reduced, while the amount of natural killer (NK) cells was mainly unchanged and only macrophages increased significantly. Our results suggest that mEHT-treatment-related tumor growth control was primarily mediated by cell-stress-induced p53, which upregulated cyclin-dependent kinase inhibitors. The downregulated tumor antigen-presenting machinery may explain the reduced cytotoxic T-cell response despite increased DAMP signaling. Decreased tumor antigen and MHC-I levels suggest that natural killer (NK) cells and macrophages were the major contributors to tumor eradication.

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control.

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.

Digoxin-Mediated Inhibition of Potential Hypoxia-Related Angiogenic Repair in Modulated Electro-Hyperthermia (mEHT)-Treated Murine Triple-Negative Breast Cancer Model.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer type with no targeted therapy and hence limited treatment options. Modulated electrohyperthermia (mEHT) is a novel complementary therapy where a 13.56 MHz radiofrequency current targets cancer cells selectively, inducing tumor damage by thermal and electromagnetic effects. We observed severe vascular damage in mEHT-treated tumors and investigated the potential synergism between mEHT and inhibition of tumor vasculature recovery in our TNBC mouse model. 4T1/4T07 isografts were orthotopically inoculated and treated three to five times with mEHT. mEHT induced vascular damage 4-12 h after treatment, leading to tissue hypoxia detected at 24 h. Hypoxia in treated tumors induced an angiogenic recovery 24 h after the last treatment. Administration of the cardiac glycoside digoxin with the potential hypoxia-inducible factor 1-α (HIF1-α) and angiogenesis inhibitory effects could synergistically augment mEHT-mediated tumor damage and reduce tissue hypoxia signaling and consequent vascular recovery in mEHT-treated TNBC tumors. Conclusively, repeated mEHT induced vascular damage and hypoxic stress in TNBC that promoted vascular recovery. Inhibiting this hypoxic stress signaling enhanced the effectiveness of mEHT and may potentially enhance other forms of cancer treatment.

Correction to "Digoxin-Mediated Inhibition of Potential Hypoxia-Related Angiogenic Repair in Modulated Electro-Hyperthermia (mEHT)-Treated Murine Triple-Negative Breast Cancer Model".

[This corrects the article DOI: 10.1021/acsptsci.3c00296.].

Heat shock factor 1 inhibition enhances the effects of modulated electro hyperthermia in a triple negative breast cancer mouse model.

Female breast cancer is the most diagnosed cancer worldwide. Triple negative breast cancer (TNBC) is the most aggressive type and there is no existing endocrine or targeted therapy. Modulated electro-hyperthermia (mEHT) is a non-invasive complementary cancer therapy using an electromagnetic field generated by amplitude modulated 13.56 MHz frequency that induces tumor cell destruction. However, we have demonstrated a strong induction of the heat shock response (HSR) by mEHT, which can result in thermotolerance. We hypothesized that inhibition of the heat shock factor 1 (HSF1) can synergize with mEHT and enhance tumor cell-killing. Thus, we either knocked down the HSF1 gene with a CRISPR/Cas9 lentiviral construct or inhibited HSF1 with a specific small molecule inhibitor: KRIBB11 in vivo. Wild type or HSF1-knockdown 4T1 TNBC cells were inoculated into the mammary gland's fat pad of BALB/c mice. Four mEHT treatments were performed every second day and the tumor growth was followed by ultrasound and caliper. KRIBB11 was administrated intraperitoneally at 50 mg/kg daily for 8 days. HSF1 and Hsp70 expression were assessed. HSF1 knockdown sensitized transduced cancer cells to mEHT and reduced tumor growth. HSF1 mRNA expression was significantly reduced in the KO group when compared to the empty vector group, and consequently mEHT-induced Hsp70 mRNA upregulation diminished in the KO group. Immunohistochemistry (IHC) confirmed the inhibition of Hsp70 upregulation in mEHT HSF1-KO group. Demonstrating the translational potential of HSF1 inhibition, combined therapy of mEHT with KRIBB11 significantly reduced tumor mass compared to either monotherapy. Inhibition of Hsp70 upregulation by mEHT was also supported by qPCR and IHC. In conclusion, we suggest that mEHT-therapy combined with HSF1 inhibition can be a possible new strategy of TNBC treatment with great translational potential.

Sigma-1 Receptor Activation Is Protective against TGFß2-Induced Extracellular Matrix Changes in Human Trabecular Meshwork Cells.

The trabecular meshwork (TM) route is the principal outflow egress of the aqueous humor. Actin cytoskeletal remodeling in the TM and extracellular matrix (ECM) deposition increase TM stiffness, outflow resistance, and elevate intraocular pressure (IOP). These alterations are strongly linked to transforming growth factor-ß2 (TGFß2), a known profibrotic cytokine that is markedly elevated in the aqueous humor of glaucomatous eyes. Sigma-1 receptor (S1R) has been shown to have neuroprotective effects in the retina, but data are lacking about its role in the TM. In this study, we identified the presence of S1R in mouse TM tissue and investigated the effect of an S1R agonist fluvoxamine (FLU) on TGFß2-induced human TM cells regarding cell proliferation; ECM-related functions, including F-actin reorganization; and the accumulation of ECM elements. TGFß2 increased the proliferation, cytoskeletal remodeling, and protein levels of fibronectin, collagen type IV, and connective tissue growth factor, and decreased the level of matrix metalloproteinase-2. Most importantly, FLU reversed all these effects of TGFß2, suggesting that S1R agonists could be potential candidates for preserving TM function and thus maintaining normal IOP.

Angiotensin II-Induced Cardiac Effects Are Modulated by Endocannabinoid-Mediated CB1 Receptor Activation.

Angiotensin II (Ang II) has various cardiac effects and causes vasoconstriction. Ang II activates the type-1 angiotensin receptor-Gq/11 signaling pathway resulting in the release of 2-arachidonoylglycerol (2-AG). We aimed to investigate whether cardiac Ang II effects are modulated by 2-AG-release and to identify the role of type-1 cannabinoid receptors (CB1R) in these effects. Expression of CB1R in rat cardiac tissue was confirmed by immunohistochemistry. To characterize short-term Ang II effects, increasing concentrations of Ang II (10-9-10-7 M); whereas to assess tachyphylaxis, repeated infusions of Ang II (10-7 M) were administered to isolated Langendorff-perfused rat hearts. Ang II infusions caused a decrease in coronary flow and ventricular inotropy, which was more pronounced during the first administration. CB agonist 2-AG and WIN55,212-2 administration to the perfusate enhanced coronary flow. The flow-reducing effect of Ang II was moderated in the presence of CB1R blocker O2050 and diacylglycerol-lipase inhibitor Orlistat. Our findings indicate that Ang II-induced cardiac effects are modulated by simultaneous CB1R-activation, most likely due to 2-AG-release during Ang II signalling. In this combined effect, the response to 2-AG via cardiac CB1R may counteract the positive inotropic effect of Ang II, which may decrease metabolic demand and augment Ang II-induced coronary vasoconstriction.

Crosslinked Hyaluronic Acid Gels with Blood-Derived Protein Components for Soft Tissue Regeneration.

Hyaluronic acid (HA) is an ideal initial material for preparing hydrogels, which may be used as scaffolds in soft tissue engineering based on their advantageous physical and biological properties. In this study, two crosslinking agents, divinyl sulfone (DVS) and butanediol diglycidyl ether, were used to investigate their effect on the properties of HA hydrogels. As HA hydrogels alone do not promote cell adhesion on the scaffold, fibrin and serum from platelet-rich fibrin (SPRF) were combined with the scaffold; the aim was to create a material intended to be used as soft tissue implant that facilitates new tissue formation, and degrades over time. The chemical changes were characterized and cell attachment capacity of the protein-containing gels was examined using human mesenchymal stem cells, and viability was assessed using live-dead staining. Fourier-transform infrared measurements revealed that linking fibrin into the gel was more effective than linking SPRF. The scaffolds were found to be able to support cell adherence onto the hydrogels, and the best result was achieved when HA was crosslinked with DVS and contained fibrin. The most promising derivative, 5% DVS-crosslinked fibrin-containing hydrogel, was injected subcutaneously into C57BL/6 mice for 12 weeks. The scaffold was proven to be biocompatible, remodeling, and vascularization occurred, while shape and integrity were maintained. Impact statement Fibrin was combined with crosslinked hyaluronic acid (HA) for regenerative application, the structure of the combination of crosslinked HA with blood-derived protein was analyzed and effective coating was proven. It was observed that the fibrin content led to better mesenchymal stem cell attachment in vitro. The compositions showed biocompatibility, connective tissue and vascularization took place when implanted in vivo. Thus, a biocompatible, injectable gel was produced, which is a potential candidate for soft tissue implantation.

[Preclinical and clinical investigation and development of electromagnetic oncological device - experience with solid tumors]. / Elektromágneses daganatterápiás készülék preklinikai és klinikai vizsgálatai, valamint mûszaki továbbfejlesztése: tapasztalatok szolid tumorokkal.

Our objective was to develop an electromagnetic tumor therapy device in a consortial cooperation between Semmelweis University and Oncotherm Ltd., to provide data and contribute to the development of the next generation of devices through preclinical, clinical and developmental modules via in vivo, in vitro studies, and patient treatments. Our numerous preclinical studies support the efficacy of mEHT. Clinical treatments were performed in 181 patients with inoperable and/or oligometastatic solid tumors. The protocols were developed, an international guideline was completed, and the planned steps of device development were realized. By optimizing previous selective RF techniques based on recent research findings, we can provide the most modern evidence-based treatment in the future.

The SH3 domain of Caskin1 binds to lysophosphatidic acid suggesting a direct role for the lipid in intracellular signaling.

Src homology 3 or SH3 domains constitute one of the most common protein domains in signal transduction, generally characterized by their binding to proline-rich sequences on interacting signaling proteins. Caskin1, a scaffold protein regulating cortical actin filaments, enriched in neural synapses in mammals, has an atypical SH3 domain. Key aromatic residues necessary for ligand binding that are present in canonical SH3 domains are missing from Caskin1 SH3. In concordance, proline-rich interacting partner could not be identified yet. Based on previous reports that several SH3 domains are able to bind phospholipids, we sought for lipid interacting partners of the SH3 domain of human Caskin1. We investigated the signaling-born lysophospholipid mediators, such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) as potential binding partners for this SH3 domain. These lipid mediators as first messengers activate G protein-coupled receptors. They also exert several G protein-coupled receptor-independent functions but their intracellular target proteins are mostly unknown. Here we provide evidence that the SH3 domain of human Caskin1 selectively binds to LPA in vitro. The binding strength and stoichiometry depend on the association-state of the lipid, with nanomolar affinity to LPA-containing membraneous surfaces. The amino acids involved in the interaction are located in a ß-strand structure and are distinct from those corresponding to the canonical proline-rich ligand-binding groove in the SH3 domain of Src kinase. Our results suggest that the SH3 domain of human Caskin1 is a lipid-binding domain rather than a proline-rich motif interacting domain.