Search Results (348)

Pancreatic cancer (PC) is an aggressive malignancy with a poor prognosis, requiring innovative approaches to evaluate new therapies. Considering the high activity of unsymmetrical bisacridines (UAs) in PC monolayer cultures, we employed multicellular tumor spheroids (MCTS) to assess whether UAs retain pro-apoptotic activity under more physiologically relevant conditions. Ultra-low attachment plates were used to form spheroids from three PC cell lines (Panc-1, MIA PaCa-2, and AsPC-1) with different genotypes and phenotypes. The effects of UA derivatives (C-2028, C-2045, and C-2053) were evaluated using microscopy and flow cytometry (7-AAD for viability and annexin V-FITC/PI for membrane integrity). UAs altered the morphology of the spheroids and reduced their growth. Notably, Panc-1 spheroids exhibited compromised integrity. The increase in 7-AAD⁺ cells confirmed diminished cell viability, and annexin V-FITC assays showed apoptosis as the dominant death pathway. Interestingly, the exact derivative was most active against a given cell line regardless of culture conditions. These results confirm that UAs maintain anticancer activity in 3D cultures and induce apoptosis, with varying efficacy across different cell lines. This underscores the value of diverse cellular models in compound evaluation and supports UAs as promising candidates for pancreatic cancer therapy. Full article

Background/Objectives: Pancreatic cancer (PC) is the seventh leading cause of cancer-related deaths worldwide, with its incidence rising each year. Despite its relatively low incidence, the aggressiveness of pancreatic cancer results in high mortality, with only 12% of patients surviving five years post-diagnosis. Surgical resection remains the only potentially curative treatment, but the tumor is often diagnosed at an advanced stage. The goal of this work is to identify vulnerabilities that can affect the efficacy of treatments and improve the efficacy of therapy. Methods: MAP17 overexpression in pancreatic cancer cell lines, RT-qPCR analysis, xenografts, in vitro and in vivo treatments, analysis of data from pancreatic tumors in transcriptomic patient databases. Results: We studied the prognostic and predictive value of MAP17 (PDZK1IP1) expression in pancreatic cancer, and we found that high MAP17 mRNA expression was associated with poor prognosis. In addition, single-cell analysis revealed that high MAP17 expression was present only in tumor cells. We investigated whether the response to various antitumor agents depended on MAP17 expression. In 2D culture, MAP17-expressing pancreatic cancer cells responded better to gemcitabine and 5-fluorouracil. However, in vivo xenograft tumors with MAP17 expression showed resistance to all treatments. Additionally, MAP17-expressing cells had a high NAD pool, which seems to be effectively depleted in vivo by NAMPT inhibitors, the primary enzyme for NAD biosynthesis. Conclusions: Our findings suggest that MAP17 expression could enhance the prognostic stratification of pancreatic cancer patients. Moreover, the coadministration of NAMPT inhibitors with current treatments may sensitize tumors with high MAP17 expression to chemotherapy and improve the efficacy of chemotherapy. Full article

Background and Objectives: The efficacy of newly synthesized water-soluble octa-mercaptopyridine-substituted oxotitanium (IV) phthalocyanine (oxo-TiPc) and copper (II) phthalocyanine (CuPc) compounds in photodynamic therapy (PDT) was investigated using human tongue squamous cell cancer cell line (SCC-9, ATCC) cultures. Materials and Methods: A laser light source with a wavelength of 635 nm was used for this study. The cytotoxic values of the cancerous (SCC-9) and healthy (L-929) cell samples to which different Pc concentrations were applied under laser light were evaluated spectroscopically with the XTT method. Results: The oxo-TiPc compound exhibited a significantly lower IC50 value (46.8 µM) for SCC-9 cells compared to the CuPc compound (286.2 µM), indicating higher anticancer activity. This cytotoxicity may be due to decreased aggregation and enhanced reactive oxygen species (ROS) formation. Double-staining tests confirmed that oxo-TiPc-induced cell death included both apoptosis and necrosis. Conclusions: The findings show that the oxo-TiPc compound, unlike the CuPc compound, exhibited more selective toxicity to the SCC-9 cell line and has a higher phototoxic effect. Full article

The cellular prion protein (PrP^C) is studied in prion diseases, where its misfolded isoform (PrP^Sc) leads to neurodegeneration. PrP^C has also been implicated in several physiological functions. The protein is abundant in the nervous system, and it is critical for cell signaling in cellular communication, where it acts as a scaffold for various signaling molecules. The Reelin signaling pathway, implicated both in Alzheimer’s and prion diseases, engages Dab1, an adaptor protein influencing APP processing and amyloid beta deposition. Here, we show, using Prnp knockout models (Prnp^0/0), that PrP^C modulates Reelin signaling, affecting Dab1 activation and downstream phosphorylation in both neuronal cultures and mouse brains. Notably, Prnp^0/0 mice showed reduced responsiveness to Reelin, associated with altered Dab1 phosphorylation and Fyn kinase activity. Even though no direct interaction between PrP^C and Reelin/ApoER2 was found, Prnp^0/0 neurons showed lower NCAM levels, a well-established PrP^C interactor. Prion infection further disrupted the Reelin signaling pathway, thus downregulating Dab1 and Reelin receptors and altering Reelin processing, like Alzheimer’s disease pathology. These findings emphasize PrP^C indirect role in Dab1 signaling via the NCAM and Fyn pathways, which influence synaptic function and neurodegeneration in prion diseases. Full article

The brain actively obtains nutrients through various transporters on brain microvessel endothelial cells (BMECs). Major facilitator superfamily domain–containing protein 2a (MFSD2A) serves as a key transporter of docosahexaenoic acid (DHA) at the blood–brain barrier (BBB) and is exclusively expressed in BMECs. Although brain pericytes (PCs) regulate MFSD2A expression in BMECs, the underlying mechanism remains unclear. To determine whether PDGF-BB/PDGFRβ signaling between endothelial cells (ECs) and PCs affects MFSD2A protein expression and plasma membrane localization in ECs, we examined the impact of AG1296 (a PDGF receptor inhibitor) and Pdgfrb-knockdown PCs on a non-contact coculture BBB model comprising the primary cultures of rat brain ECs and PCs. The effects of PCs on MFSD2A expression, localization, and brain endothelial DHA uptake was assessed using Western blot, immunofluorescence staining, and [¹⁴C]DHA uptake by ECs, respectively. In ECs cocultured with PCs, MFSD2A expression and plasma membrane localization were significantly higher than in EC monolayers. Moreover, conditioned medium derived from PCs failed to enhance MFSD2A expression. The increased expression and membrane localization of MFSD2A were inhibited by AG1296 and Pdgfrb-knockdown PCs. Furthermore, PCs significantly increased [¹⁴C]DHA uptake by ECs. These findings suggest that PCs enhance MFSD2A expression and plasma membrane localization in ECs through PDGF-BB/PDGFRβ signaling. Full article

A killed, whole-cell vaccine was produced to induce immunity in dogs against leptospirosis. The vaccine, containing serovar Copenhageni, was produced and administered to 12 beagle dogs at both 8 and 12 weeks of age. Ten unvaccinated dogs of the same age group served as the control group. A live, virulent inoculum of Leptospira (1.52 × 10⁹–4.40 × 10⁹ leptospires per dog) was used to challenge the dogs at 2 weeks (Study 1) and 14 months (Study 2) post-booster vaccination. At regular intervals, pre- and post-challenge (PC), the microscopic agglutination test (MAT) was performed to measure antibody titers. Leptospiremia and leptospiruria were determined via culture, and the cytokine, biochemical, and pathological profiles of vaccinates and controls were also assessed. A high antibody response was measurable after booster administration. In Study 1 (onset of immunity), acute leptospirosis was observed in five (100%) out of five unvaccinated dogs. In contrast, no acute clinical leptospirosis developed in vaccinated dogs, except in one (20%) dog with mild clinical signs. In Study 2 (duration of immunity), mild clinical signs were observed in two (40%) of the control dogs, while all vaccinated dogs remained clinically normal. The incidence of leptospiruria and leptospiremia PC was lower in the vaccinated dogs compared to the unvaccinated group. Severe thrombocytopenia occurred in 100% (5/5) of the unvaccinated dogs in Study 1 that exhibited acute severe leptospirosis, whereas 80% (4/5) of the unvaccinated dogs in Study 2 showed mild to moderate thrombocytopenia 3 days after challenge. Four out of five unvaccinated dogs (80%) in Study 1 exhibited icteric tissues and hemorrhages in the lungs and mucosal surfaces of the stomach and intestines. A high IL-10 to TNF-α ratio, observed in the control group of both studies, and severe thrombocytopenia observed in the control group of Study 1, indicative of acute leptospiral disease, were detected. The vaccine prevented acute clinical leptospirosis and reduced the renal carrier state in beagle dogs, and further investigation is required using a larger sample size. Full article

Background/Objectives: Pancreatic cancer (PC) is among the most aggressive malignancies, often diagnosed at late stages. MicroRNAs (miRNAs) and proteins released from the tumor microenvironment into body fluids represent promising non-invasive biomarkers for early cancer detection. In this study, we took advantage of an innovative ultrasound (US)-based instrument (SonoWell^®, Inno-Sol srl, Rome, Italy) to treat PC cells in order to promote and amplify the release of molecules, with the aim of identifying novel putative diagnostic PC biomarkers. Methods: Three human pancreatic adenocarcinoma cell lines (T3M-4, Panc02.03, and PaCa-44) and a non-cancerous pancreatic epithelial line (HPanEPic) were subjected to US using the SonoWell instrument. MiRNAs released in the supernatants were profiled by TaqMan-based qRT-PCR microfluidic cards, while proteins were analyzed by antibody arrays. Publicly available datasets of circulating miRNAs in PC patients were also reviewed. Results: Expression levels of 22 miRNAs in T3M-4 cells, 11 in Panc02.03, and 22 in PaCa-44, none of which were identified in the non-cancerous cell line profiling, were increased in the supernatant of US-treated as opposed to control cells. Among the statistically significant miRNAs or miRNAs common to at least two tumor cell lines, the expression levels of miR-155-5p, miR-320a, miR-32-5p, and miR-93-5p were also found to be significantly upregulated in sera from PC patients compared to the results for healthy controls. With regard to proteins released after sonication, several molecules were identified as candidate biomarkers in cancer US supernatants (Beta-2 microglobulin, CA125, CA19-9, CEA, CRP, Galectin-3, TIMP-1, uPA, and VEGF-A). Conclusions: We demonstrated that US-mediated sonoporation can promote and amplify the release of small molecules, miRNAs, and proteins into cell culture supernatants for consideration as putative biomarkers, thus encouraging further studies aimed at directly validating their expression levels in sera/plasma from PC patients and at deepening their role in the treatment of PC. Full article

The Rotimer (rotifer-specific biopolymer) like SCO-spondin (R-SSPO/1), predicted as the main component of this biopolymer, is an adequate base for the design of functional small peptides. This macromolecule is interactive and protective against neurotoxic human-type beta-amyloid 1-42 aggregates (agg-Aβ). The current work presents biological investigations and predictable molecular interaction analysis of DSSNDL and PNCRDGSDE peptides that were synthesized based on the sequences of R-SSPO/1. Viability assays (NADH-dependent cellular reduction capacity, intracellular esterase activity, and motility) were performed on differentiated neuro-type cell cultures (SH-SY5Y and PC12) and on Rotimer-depleted rotifers (Euchlanis dilatata and Lecane bulla). A control peptide (STTRPTGTT), not found in Rotimer, was also included in the study. All three peptides are present in both rotifer and human proteomes. Among these small molecules, DSSNDL showed a significant protective effect against the toxicity of agg-Aβ both in vitro and in vivo and presumably interacted with its aggregates. The stagogram analysis of amyloid–peptide complexes and the possible bonding competition of these small molecules against aggregation-specific dyes on agg-Aβ surface suggest that DSSNDL affects the properties of these neurotoxic macromolecules. This effective hexapeptide can serve as a promising candidate for further investigations into the inactivation of beta-amyloid toxicity. Full article

Pleural carcinomatosis (PC) and malignant pleural effusion (MPE) affect up to 20% of patients with non-small cell lung cancer (NSCLC) and are usually synonymous with poor prognosis. Pressurized Intra-Thoracic Aerosol Chemotherapy (PITAC) is a novel and promising technique to control MPE in PC-NSCLC. This pilot study aimed to assess the feasibility, safety, and efficacy of PITAC in terms of palliative pleurodesis and evaluate the local antineoplastic control by analyzing patient-derived primary cell cultures. From January to December 2023, seven patients underwent PITAC with tailored doses of cisplatin and doxorubicin. There were four males and three females, with a median age of 65 (IQR:19) years. No operating room contamination by aerosolized chemotherapeutics was observed. No intraoperative complications occurred, and 30-day mortality was nil. One patient developed a postoperative prolonged air leak. The median chest tube stay was 2 (IQR:2) days, and the median hospital stay was 4 (IQR:2) days. No systemic toxicity nor hypersensitivity to chemotherapeutics were observed. All patients developed effective pleurodesis in 30 days. Cell cultures obtained from biopsy of PC-NSCLC sampled before PITAC formed confluent and monolayer sheets of attached tumor cells, while after 30 min from PITAC, cultures exhibited a significant reduction in the cancer cells’ growth. Effective pleurodesis was observed three and six months after surgery in all patients. PITAC is a safe and effective technique to control MPE recurrence and might revolutionize loco-regional therapy for PC-NSCLC. Further research should assess its oncological role. Full article

Tumor-Treating Fields (TTFields), a novel therapeutic avenue, is approved for therapy in Glioblastoma multiforme, malignant pleural mesothelioma, and metastatic non-small cell lung cancer (NSCLC). In pancreatic ductal adenocarcinoma (PDAC), several clinical trials are underway to improve outcomes, yet a significant knowledge gap prevails involving the cell-extracellular matrix (ECM) crosstalk. Herein, we hypothesized that treatment with TTFields influence this crosstalk, which is reflected by the dynamic alteration in nanomechanical properties (NMPs) of cells and the ECM in a co-culture system. We employed an ECM gel comprising collagen, fibronectin, and laminin mixed in 100:1:1 stoichiometry to co-culture of Panc1 and AsPC1 individually. This ECM mixture mimics the in vivo tumor microenvironment closely when compared to the individual ECM components studied before. A comprehensive frequency-dependent study revealed the optimal TTFields frequency to be 150 kHz. We also observed that irrespective of the ECM’s presence, TTFields increase cell membrane stiffness and decrease deformation several-folds in both Panc1 and AsPC1 cells at both 48 h and 72 h. Although adhesion for AsPC1 decreased at 48 h, at 72 h it was observed to increase irrespective of ECM’s presence. Moreover, it significantly alters the NMPs of ECM gels when co-cultured with PDAC cell lines. However, AsPC1 cells were observed to be more detrimental to these changes. Lastly, we attribute the stiffness changes in Panc1 cells to the membrane F-actin reorganization in the presence of TTFields. This study paves a path to study complex PDAC TME as well as the effect of various chemotherapeutic agents on such TME with TTFields in the future. Full article

Background:Mesenchymal stem/stromal cells (MSCs) are non-hematopoietic, plastic-adherent, and self-renewing cells capable of in vitro trilineage differentiation into fat, bone, and cartilage tissue. Suggestively, MSCs have additional plasticity, as demonstrated by their ability to differentiate in vitro into myocytes, neuron-like cells, and hepatocytes. MSCs are ideal for therapeutic application owing to their numerous advantages; they exhibit limited growth and differentiation abilities, leading to heterogeneous cell populations with inconsistent functions. However, highly purified MSCs, namely, rapidly expanding clones (RECs) that are isolated by single-cell sorting, display uniform functionality. RECs have the potential to offer many benefits, such as transplantable cells for treating several disorders of bone, heart, peripheral nerves, brain, and other organs. This study aimed to assess the effects of RECs on the pheochromocytoma (PC12) cell line, a well-known neuronal cell model.Methods: PC12 cells were cultured under the following conditions: co-culture with RECs, treatment with REC-derived conditioned medium (CM), or co-culture with RECs using Transwell inserts for 7 days. The cells were stained with anti-βIII-tubulin antibody; the lengths of neurites were measured by image analysis. Results: Regarding the co-culture with RECs, PC12’s outgrowth was significantly increased. The RECs expressed nerve growth factor (NGF), a neurotrophic factor that could act on PC12 cells to trigger cellular differentiation.Conclusions: Our findings suggest that RECs via direct culture, intercellular communication in Transwell culture, and RECs CM promoted PC12 cell survival and outgrowth via NGF signaling. Full article

Background/Objectives: Metastatic prostate cancer (PCa) remains a major clinical challenge with limited therapeutic options. The long non-coding RNA H19 has been implicated in regulating cell adhesion molecules and collective migration, key features of metastatic dissemination. This study investigates the role of the Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 in the H19-dependent transcriptional regulation of cell adhesion molecules. Currently, the major effects of BET inhibitors require androgen receptor (AR) expression. Methods: H19 was stably silenced in PC-3 (AR-null) and 22Rv1 (AR-positive) castration-resistant PCa cells. The cells were treated with the pan-BET inhibitors JQ1 and OTX015 or the BET degrader dBET6. In vivo, the effects of JQ1 were evaluated in xenograft mouse models. Chromatin immunoprecipitation (ChIP) and RNA-ChIP were used to assess BET protein recruitment and interaction with cell adhesion gene loci and H19. Organotypic slice cultures (OSCs) from fresh PCa surgical specimens were used as ex vivo models to validate transcriptional changes and BRD4 recruitment. Results: BET inhibition significantly reduced the expression of β4 integrin and E-cadherin and cell proliferation in both basal conditions, and following H19 knockdown in PC-3 and 22Rv1 cells. These effects were mirrored in JQ1-treated tumor xenografts, which showed marker downregulation and tumor regression. ChIP assays revealed that BRD4, more than BRD2/3, was enriched on β4 integrin and E-cadherin promoters, especially in regions marked by H3K27ac. H19 silencing markedly enhanced BRD4 promoter occupancy. RNA-ChIP confirmed a specific interaction between BRD4 and H19. These findings were validated in OSCs, reinforcing their clinical relevance. Conclusions: Our study demonstrates that BRD4 epigenetically regulates the H19-mediated transcriptional control of adhesion molecules involved in collective migration and metastatic dissemination. Importantly, these effects are independent of AR status, suggesting that targeting the H19/BRD4 axis may represent a promising therapeutic avenue for advanced PCa. Full article

The success of assisted reproductive technology is contingent upon the growth potential of embryos post-vitrification process. When compared to in vivo embryos, it has been found that the high intracellular lipid accumulation inside the in vitro-derived embryos results in poor survival during vitrification. Based on this finding, the present study assessed the impact of incorporating forskolin and linoleic acid (FL) entering in vitro culture (IVC) on the embryos’ cryo-survival, lipid content, and viability throughout vitrification. Lipid metabolomics and single-cell RNA sequencing (scRNA-seq) techniques were used to determine the underlying mechanism that the therapies were mimicking. It was observed that out of 726 identified lipids, 26 were expressed differentially between the control and FL groups, with 12 lipids upregulated and 14 lipids downregulated. These lipids were classified as Triacylglycerol (TG), Diacylglycerol (DG), Phosphatidylcholine (PC), and so on. A total of 1079 DEGs were detected between the FL and control groups, consisting of 644 upregulated genes and 435 downregulated genes. These DEGs were significantly enhanced in the arachidonic acid metabolism, lipolysis, fatty acid metabolism, cAMP signaling pathway, and other critical developmental pathways. Based on the observation, it was concluded that forskolin and linoleic acid decreased the droplet content of embryos by modulating lipid metabolism, thus enhancing the vitrified bovine embryos’ cryo-survival. Full article

Gut-derived uremic toxins (UTs) contribute to cardiovascular disorders like atherosclerosis and cardiomyopathy in patients with chronic kidney disease (CKD), causing increased cardiovascular morbidity and mortality. The intermediate steps between higher concentrations of gut-derived UTs and organ damage caused by UTs are still insufficiently understood. Glycosaminoglycans (GAGs) as components of the extracellular matrix are known to interact with various ligands such as growth factors or receptors, thereby influencing (patho)physiological processes. We previously found that the UT inorganic phosphate (Pi) induces the synthesis and sulphation of the GAGs heparan sulphate and chondroitin sulphate in the rat vascular smooth muscle cell (VSMC) line A7r5 and in the human endothelial cell (EC) line EA.Hy926. The aim of this study was to investigate if other organic UTs modulate GAGs in vascular cells as well. We treated ex vivo cultures of rat aortic rings as well as primary rat VSMCs and human ECs with the UTs Pi, indoxylsulphate (IS), p-cresylsulphate (pCS), trimethylamine N-oxide (TMAO), and urea, and analyzed the samples by histological staining, qPCR, western blot, HPLC, and colorimetric assays. The UT treatment of aortic rings and cells increased contents of sulphated GAGs and hyaluronic acid. UT-treated cells contained higher amounts of 4S- and 6S-sulphated GAGs compared to controls. This was accompanied by altered expressions of genes and proteins relevant for GAG metabolism. Mechanistically, the effects of the UTs on GAGs involve the activation of the PI3K/Akt pathway and of the transcription factor NF-κB. In conclusion, the UT-induced remodeling of the cardiovascular matrix by upregulation of sulphated GAGs and hyaluronic acid in aortic tissue and vascular cells might be a missing link between gut-derived UT and pathophysiological alterations in the cardiovascular system in the sense of a gut–matrix axis. Full article

Applications of lanthanide chemistry have been successful in metallics and the petroleum industry. In the medical realm, lanthanides have shown utility in radiotherapy agents, photodynamic therapy agents, and magnetic resonance imaging (MRI) contrast agents. The lanthanide group elements have a few known biological roles, notably among some bacteria and the yeast Saccharomyces cerevisiae, which have been used as models for changes in gene expression. However, the systematic effects of lanthanide nitrates on eukaryotic cell model systems have not yet been reported. This study presents the first documented effects on cell viability, after acute incubations of various lanthanide nitrate salts, using axenic C6 glial or PC12 neuronal cells in vitro. Cultures were exposed to a 1 mM concentration of lanthanide nitrate salts for 24 h. In comparison to the saline control, several cultures demonstrated significantly lower cell viability, as measured by the MTT viability assay. Data were analyzed as an average absorbance of n = 4 replicate samples, corrected for the average absorbance of cell-free blanks. The reported results were normalized to the average of the saline control cells. Among the 13 lanthanides tested, Praseodymium, Holmium, Erbium, Thulium, and Ytterbium nitrates exhibited the most pronounced inhibitory effects, resulting in over 40% reduction in cell viability at 1 mM for either or both cell types. Recovery after lanthanide exposure also was cell-type-dependent as well as lanthanide-type-dependent, with Lutetium having the greatest effect on both cell types. PC12 cells displayed greater sensitivity for inhibition than the C6 cells with some of the lanthanides but not all. Furthermore, the controls of sodium nitrate and calcium nitrate showed only modest discernible impacts on cell viability for PC12 and C6 cells, highlighting the role of the lanthanides in influencing cell viability. Full article