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 p21^waf1 and p27^kip. 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.

Angiotensin II (Ang II) has various cardiac effects and causes vasoconstriction. Ang II activates the type-1 angiotensin receptor—G_q/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.

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-β2 (TGFβ2), 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 TGFβ2-induced human TM cells regarding cell proliferation; ECM-related functions, including F-actin reorganization; and the accumulation of ECM elements. TGFβ2 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 TGFβ2, suggesting that S1R agonists could be potential candidates for preserving TM function and thus maintaining normal IOP.

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.

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.

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.