Search Results (1,001)

Background: several experimental findings and epidemiological observations indicated that aspirin/acetylsalicylic acid (ASA) may be endowed with anticancer effects against a variety of human malignancies, including thyroid carcinomas. Among these, undifferentiated/anaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal human cancers, refractory to all currently available therapies. Methods: we here evaluated in a preclinical setting the effects of ASA on a panel of three ATC-derived cell lines: the CAL-62, the 8305C, and the 8505C. Results: the data obtained demonstrated the ability of ASA to inhibit, in a dose- and time-dependent manner, the proliferation of all ATC cell lines investigated, with IC₅₀ values comprised between 2.0 and 4.3 mM. Cell growth was restrained with the same efficacy when the ASA treatment was applied to three-dimensional soft-agar cultures. In addition, ASA significantly reduced migration and invasion in two of the three ATC cell lines. We finally investigated the effects of ASA on the MAPK and PI3K/Akt signaling pathways, which are often altered in ATC. The results showed that the phosphorylation status of the Akt1/2/3 kinases was significantly reduced following ASA treatment, while ERK1/2 phosphorylation was either unaffected or slightly upregulated. Conclusions: our findings support epidemiological evidence on the anticancer potential of ASA. On this basis, further investigations should be carried out to assess the usefulness of ASA as adjuvant therapy in patients affected by ATC. Full article

Gliomas are a group of primary brain tumors characterized by their aggressive nature and resistance to treatment. Infiltration of surrounding normal tissues limits surgical approaches, wide inter- and intratumor heterogeneity hinders the development of universal therapeutics, and the presence of the blood–brain barrier reduces the efficiency of their delivery. As a result, patients diagnosed with gliomas often face a poor prognosis and low survival rates. The spectrum of anti-glioma drugs used in clinical practice is quite narrow. Alkylating agents are often used as first-line therapy, but their effectiveness varies depending on the molecular subtypes of gliomas. This highlights the need for new, more effective therapeutic approaches. Standard drug-screening methods involve the use of two-dimensional cell cultures. However, these models cannot fully replicate the conditions present in real tumors, making it difficult to extrapolate the results to humans. We describe the advantages and disadvantages of existing glioma cell-based models designed to improve the situation and build future prospects to make drug discovery comprehensive and more effective for each patient according to personalized therapy paradigms. Full article

Mucopolysaccharidosis (MPS) comprises a group of inherited metabolic diseases. Each MPS type is caused by a deficiency in the activity of one kind of enzymes involved in glycosaminoglycan (GAG) degradation, resulting from the presence of pathogenic variant(s) of the corresponding gene. All types/subtypes of MPS, which are classified on the basis of all kinds of defective enzymes and accumulated GAG(s), are severe diseases. However, neuronopathy only occurs in some MPS types/subtypes (specifically severe forms of MPS I and MPS II, all subtypes of MPS III, and MPS VII), while in others, the symptoms related to central nervous system dysfunctions are either mild or absent. The early diagnosis of neuronopathy is important for the proper treatment and/or management of the disease; however, there are no specific markers that could be easily used for this in a clinical practice. Therefore, in this work, a comparative analysis of shared and specific gene expression alterations in neuronopathic and non-neuronopathic MPS types was performed using cultures of cells derived from patients. Using transcriptomic analyses (based on the RNA-seq method, confirmed by measuring the levels of a selected gene product), we identified genes (including PFN1, ADAMTSL1, and ABHD5) with dysregulated expression that are common for all, or almost all, types of MPS, suggesting their roles in MPS pathogenesis. Moreover, a distinct set of genes (including ARL6IP6 and PDIA3) exhibited expression changes only in neuronopathic MPS types/subtypes, but not in non-neuronopathic ones, suggesting their possible applications as biomarkers for neurodegeneration in MPS. These findings provide new insights into both the molecular mechanisms of MPS pathogenesis and the development of differentiation method(s) between neuronopathic and non-neuronopathic courses of the disease. Full article

Background: Glioblastoma is an aggressive and incurable type of brain cancer. Little progress has been made in the development of effective new therapies in the past decades. The blood–brain barrier (BBB) and drug efflux pumps, which together hamper drug delivery to these tumors, play a pivotal role in the gap between promising preclinical findings and failure in clinical trials. Therefore, selecting drugs that can reach the tumor region in pharmacologically effective concentrations is of major importance. Methods: In the current study, we utilized a drug selection platform to identify candidate drugs by combining in vitro oncological drug screening data and pharmacokinetic (PK) profiles for central nervous system (CNS) penetration using the multiparameter optimization (MPO) score. Furthermore, we developed intracranial patient-derived xenograft (PDX) models that recapitulated the in situ characteristics of glioblastoma and characterized them in terms of vascular integrity, BBB permeability and expression of ATP-binding cassette (ABC) transporters. Omacetaxine mepesuccinate (OMA) was selected as a proof-of-concept drug candidate to validate our drug selection pipeline. Results: We assessed OMA’s PK profile in three different orthotopic mouse PDX models and found that OMA reaches the brain tumor tissue at concentrations ranging from 2- to 11-fold higher than in vitro IC₅₀ values on patient-derived glioblastoma cell cultures. Conclusions: This study demonstrates that OMA, a drug selected for its in vitro anti-glioma activity and CNS- MPO score, achieves brain tumor tissue concentrations exceeding its in vitro IC₅₀ values in patient-derived glioblastoma cell cultures, as shown in three orthotopic mouse PDX models. We emphasize the importance of such approaches at the preclinical level, highlighting both their significance and limitations in identifying compounds with potential clinical implementation in glioblastoma. Full article

Antigen presentation plays a critical role in the pathogenesis of immune-mediated disorders. This study aimed to investigate the effects of IFN-γ and a cytokine mix (5MIX: IL-1α, IL-17A, IL-22, OsM, and TNF-α) on the antigen-presenting capabilities of keratinocytes, with a specific focus on immune-mediated dermatological conditions such as psoriasis (Ps). To achieve this, keratinocytes were treated with IFN-γ and 5MIX, and their impact on the expression of key antigen-presentation molecules, HLA-DRα and CD74, was assessed. Transcriptomic analysis revealed that IFN-γ alone altered the expression of 254 genes, highlighting its central role in modulating immune responses, including the recruitment of immune cells and regulation of inflammation. Temporal experiments further demonstrated that IFN-γ and 5MIX enhanced early endocytic activity and lysosomal degradation pathways, both essential for effective antigen presentation and T-cell activation. To extend these findings to a clinical context, a co-culture model using keratinocytes derived from psoriatic patients was established. This model revealed increased cytokine production following antigen stimulation, indicating robust and consistent CD4+ and naïve T-cell responses. These results elucidate the complex dynamics of cytokine signaling and antigen presentation in keratinocytes, providing insights into potential therapeutic strategies for immune-mediated skin disorders like Ps. Full article

Interleukin-6 (IL-6) expression in mesenchymal stem cells (MSCs) has been shown to play a pivotal role in modulating cartilage regeneration and immune responses, particularly in the context of diseases that involve both degenerative processes and inflammation, such as osteoarthritis (OA). However, the precise mechanism through which IL-6 and other immune-regulatory factors influence the therapeutic efficacy of autologous adipose-derived stem cells (ASCs) transplantation in OA treatment remains to be fully elucidated. This study aims to investigate the relationship between IL-6 expression in autologous ASCs isolated from OA patients and their impact on immune modulation, particularly focusing on the regulation of Receptor Activator of Nuclear factor Kappa-Β Ligand (RANKL), a key mediator of immune-driven cartilage degradation in OA. Autologous ASCs were isolated from the stromal vascular fraction (SVF) of adipose tissue obtained from 22 OA patients. The isolated ASCs were cultured and characterized using reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and flow cytometry to the phenotype and immune regulatory factors of MSCs. Based on IL-6 expression levels, ASCs were divided into high and low IL-6 expression groups. These groups were then co-cultured with activated peripheral blood mononuclear cells (PBMCs) to evaluate their immune-modulatory capacity, including the induction of regulatory T cells, inhibition of immune cell proliferation, and regulation of key cytokines, such as interferon-gamma (IFN-γ). Additionally, RANKL expression, a critical factor in osteoclastogenesis and cartilage degradation, was assessed in both ASC groups. High IL-6-expressing ASCs demonstrated a significantly greater capacity to inhibit immune cell proliferation and IFN-γ production compared to their low IL-6-expressing counterparts under co-culture conditions. Moreover, the group of ASCs with high IL-6 expression showed a marked reduction in RANKL expression, suggesting enhanced potential to control osteoclast activity and subsequent cartilage defect in OA. Conclusion: Autologous ASCs with elevated IL-6 expression exhibit enhanced immunomodulatory properties, particularly in regulating over-activated immune response and reducing osteoclastogenesis through RANKL suppression. These findings indicate that selecting ASCs based on IL-6 expression could enhance the therapeutic efficacy of ASC-based treatments for OA by mitigating immune-driven joint inflammation and cartilage degradation, potentially slowing disease progression. Full article

Background: Obesity, characterized by the secretion of several pro-inflammatory cytokines and hormones, significantly increases the risk of developing breast cancer and is associated with poorer outcomes. Mitochondrial and antioxidant status are crucial in both tumor progression and treatment response. Methods: This study investigates the impact of an ELIT cocktail (17β-estradiol, leptin, IL-6, and TNFα), which simulates the obesity-related inflammation condition in postmenopausal women, using a 3D culture model. We examined the effects of ELIT exposure on mammosphere formation, oxidative stress and mitochondrial markers, and treatment sensitivity in luminal (T47D, MCF7) and triple-negative (MDA-MB-231) breast cancer cell lines. After that, 3D-derived cells were re-cultured under adherent conditions focusing on the mechanisms leading to dissemination and drug sensitivity. Results: Our results indicated that ELIT condition significantly increased mammosphere formation in luminal breast cancer cell lines (from 3.26% to 6.38% in T47D cell line and 0.68% to 2.32% in MCF7 cell line) but not in the triple-negative MDA-MB-231 cell line. Further analyses revealed a significant decrease in mitochondrial and antioxidant-related markers, particularly in the T47D cell line, where higher levels of ESR2, three-fold increased by ELIT exposure, may play a critical role. Importantly, 3D-derived T47D cells exposed to ELIT showed reduced sensitivity to tamoxifen and paclitaxel, avoiding a 34.2% and 75.1% reduction in viability, respectively. Finally, through in silico studies, we identified specific biomarkers, including TOMM20, NFE2L2, CAT, and ESR2, correlated with poor prognosis in luminal breast cancer. Conclusions: Taken together, our findings suggest that antioxidant and mitochondrial markers are key factors that reduce treatment sensitivity in obesity-related luminal breast cancer. The identified biomarkers may serve as valuable tools for the prognosis and development of more effective therapies in these patients. Full article

Background: Insulin resistance (IR) is central to the progression of non-alcoholic fatty liver disease (MAFLD). While aerobic exercise reduces hepatic fat and enhances insulin sensitivity, the specific mechanisms—particularly those involving exosomal pathways—are not fully elucidated. Method: Exosomes were isolated from 15 MAFLD patients’ plasma following the final session of a 12-week aerobic exercise intervention. Liver fat content was measured using MRI-PDFF, and metabolic parameters were assessed via OGTT, HOMA-IR, QUICKI, and VO₂ max. Co-culture experiments evaluated the effects of exercise-derived exosomes on IR signaling pathways. miRNA microarray analysis identified miR-324, which was quantified in high-fat diet (HFD) mice with and without exercise and compared between athletes and sedentary controls. Functional assays assessed miR-324’s role in glucose and lipid metabolism, while luciferase reporter and Western blot assays confirmed ROCK1 as its direct target. Result: Aerobic exercise significantly reduced liver fat and improved insulin sensitivity in both MAFLD patients and HFD mice. Notably, exosomal miR-324 levels were lower in athletes than sedentary controls, indicating an inverse association with insulin sensitivity. Post-exercise, precursor and mature miR-324 increased in adipose tissue and decreased in muscle, suggesting its adipose origin and inverse regulation. Functional assays demonstrated that miR-324 modulates insulin resistance by targeting ROCK1. Conclusion: Exercise-induced exosomal miR-324 from adipose tissue targets ROCK1, revealing a novel mechanism by which aerobic exercise confers hepatoprotection against insulin resistance in MAFLD. These findings enhance our understanding of how exercise influences metabolic health and may inform future therapeutic strategies for managing MAFLD and related conditions. Full article

Background/Objectives: Indolo[2,3-a]pyrrolo[3,4-c]carbazole scaffold is successfully used as an efficient structural motif for the design and development of different antitumor agents. In this study, we investigated the anti-glioblastoma therapeutic potential of glycosylated indolocarbazole analog LCS1269 utilizing in vitro, in vivo, and in silico approaches. Methods: Cell viability was estimated by an MTT assay. The distribution of cell cycle phases was monitored using flow cytometry. Mitotic figures were visualized by fluorescence microscopy. Quantitative RT-PCR was used to evaluate the gene expression. The protein expression was assessed by Western blotting. Molecular docking and computational ADMET were approved for the probable protein target simulations and predicted pharmacological assessments, respectively. Results: Our findings clearly suggest that LCS1269 displayed a significant cytotoxic effect against diverse glioblastoma cell lines and patient-derived glioblastoma cultures as well as strongly suppressed xenograft growth in nude mice. LCS1269 exhibited more potent anti-proliferative activity toward glioblastoma cell lines and patient-derived glioblastoma cultures compared to conventional drug temozolomide. We further demonstrated that LCS1269 treatment caused the severe G2 phase arrest of cell cycle in a dose-dependent manner. Mechanistically, we proposed that LCS1269 could affect the CDK1 activity both by targeting active site of this enzyme and indirectly, in particular through the modulation of the Wee1/Myt1 and FOXM1/Plk1 signaling pathways, and via p21 up-regulation. LCS1269 also showed favorable pharmacological characteristics in in silico ADME prediction in comparison with staurosporine, rebeccamycin, and becatecarin as reference drugs. Conclusions: Further investigations of LCS1269 as an anti-glioblastoma medicinal agent could be very promising. Full article

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, due in part to early invasion and metastasis, which in turn involves epithelial–mesenchymal transition (EMT) of the cancer cells. Prompted by the discovery that two PDAC cell lines of the quasi-mesenchymal subtype (PANC-1, MIA PaCa-2) exhibit neuroendocrine differentiation (NED), we asked whether NED is associated with EMT. Using real-time PCR and immunoblotting, we initially verified endogenous expressions of various NED markers, i.e., chromogranin A (CHGA), synaptophysin (SYP), somatostatin receptor 2 (SSTR2), and SSTR5 in PANC-1 and MIA PaCa-2 cells. By means of immunohistochemistry, the expressions of CHGA, SYP, SSTR2, and the EMT markers cytokeratin 7 (CK7) and vimentin could be allocated to the neoplastic ductal epithelial cells of pancreatic ducts in surgically resected tissues from patients with PDAC. In HPDE6c7 normal pancreatic duct epithelial cells and in epithelial subtype BxPC-3 PDAC cells, the expression of CHGA, SYP, and neuron-specific enolase 2 (NSE) was either undetectable or much lower than in PANC-1 and MIA PaCa-2 cells. Parental cultures of PANC-1 cells exhibit EM plasticity (EMP) and harbor clonal subpopulations with both M- and E-phenotypes. Of note, M-type clones were found to display more pronounced NED than E-type clones. Inducing EMT in parental cultures of PANC-1 cells by treatment with transforming growth factor-β1 (TGF-β1) repressed epithelial genes and co-induced mesenchymal and NED genes, except for SSTR5. Surprisingly, treatment with bone morphogenetic protein (BMP)-7 differentially affected gene expressions in PANC-1, MIA PaCa-2, BxPC-3, and HPDE cells. It synergized with TGF-β1 in the induction of vimentin, SNAIL, SSTR2, and NSE but antagonized it in the regulation of CHGA and SSTR5. Phospho-immunoblotting in M- and E-type PANC-1 clones revealed that both TGF-β1 and, surprisingly, also BMP-7 activated SMAD2 and SMAD3 and that in M- but not E-type clones BMP-7 was able to dramatically enhance the activation of SMAD3. From these data, we conclude that in EMT of PDAC cells mesenchymal and NED markers are co-regulated, and that mesenchymal–epithelial transition (MET) is associated with a loss of both the mesenchymal and NED phenotypes. Analyzing NED in another tumor type, small cell carcinoma of the ovary hypercalcemic type (SCCOHT), revealed that two model cell lines of this disease (SCCOHT-1, BIN-67) do express CDH1, SNAI1, VIM, CHGA, SYP, ENO2, and SSTR2, but that in contrast to BMP-7, none of these genes was transcriptionally regulated by TGF-β1. Likewise, in BIN-67 cells, BMP-7 was able to reduce proliferation, while in SCCOHT-1 cells this occurred only upon combined treatment with TGF-β and BMP-7. We conclude that in PDAC-derived tumor cells, NED is closely linked to EMT and TGF-β signaling, which may have implications for the therapeutic use of TGF-β inhibitors in PDAC management. Full article

The gold standard assay for radiation response is the clonogenic assay, a normalized colony formation assay (CFA) that can capture a broad range of radiation-induced cell death mechanisms. Traditionally, this assay relies on two-dimensional (2D) cell culture conditions with colonies counted by fixing and staining protocols. While some groups have converted these to three-dimensional (3D) conditions, these models still utilize 2D-like media compositions containing serum that are incompatible with stem-like cell models such as brain tumor initiating cells (BTICs) that form self-aggregating spheroids in neural stem cell media. BTICs are the preferred patient-derived model system for studying glioblastoma (GBM) as they tend to better retain molecular and phenotypic characteristics of the original tumor tissue. As such, it is important that preclinical radiation studies should be adapted to BTIC conditions. In this study, we describe a series of experimental approaches for performing CFA experiments with BTIC cultures. Our results indicate that serum-free clonogenic assays are feasible for combination drug and radiation testing and may better facilitate translatability of preclinical findings. Full article

Background/Objectives: A specialized microenvironment in the bone marrow, composed of stromal cells including mesenchymal stem cells (MSCs), supports hematopoietic stem cell (HSC) self-renewal, and differentiation bands play an important role in leukemia development and progression. The reciprocal direct interaction between MSCs and CD34⁺ HSCs under physiological and pathological conditions is yet to be fully characterized. Methods: Here, we established a direct co-culture model between MSCs and CD34⁺ HSCs or MSCs and acute myeloid leukemia cells (THP-1, Molm-13, and primary cells from patients) to study heterocellular communication. Results: Following MSCs-CD34⁺ HSCs co-culture, the expression of adhesion markers N-Cadherin and connexin 43 increased in both cell types, forming gap junction channels. Moreover, the clonogenic potential of CD34⁺ HSCs was increased. However, direct contact of acute myeloid leukemia cells with MSCs reduced the expression levels of connexin 43 and N-Cadherin in MSCs. The impairment in gap junction formation may potentially be due to a defect in the acute myeloid leukemia-derived MSCs. Interestingly, CD34⁺ HSCs and acute myeloid leukemia cell lines attenuated MSC osteoblastic differentiation upon prolonged direct cell–cell contact. Conclusions: In conclusion, under physiological conditions, connexin 43 and N-Cadherin interaction preserves stemness of both CD34⁺ HSCs and MSCs, a process that is compromised in acute myeloid leukemia, pointing to the possible role of gap junctions in modulating stemness. Full article

Medulloblastoma (MB) is a common primary brain cancer in children. The sonic hedgehog (SHH) pathway is indispensable for the normal development of the cerebellum, and MB is often caused by persistent SHH activation owing to mutations in pathway components. Patched1 (PTCH1) is the primary receptor for the SHH ligand and a negative regulator of the SHH signal transduction pathway. Mice heterozygous for the Ptch1 gene (Ptch1^+/−) are predisposed to MB development. Irradiation of newborn Ptch1^+/− mice dramatically increases MB occurrence. A genetic background carrying the Ptch1 mutation significantly influences the risk of developing MB. This study aims to investigate the genetic background-related mechanisms that regulate radiation-induced cellular response and oncogenesis in the cerebellum. We employed multiple approaches, including: (a) analysis of cellular radiosensitivity in granule cell precursors (GCPs), the MB cells of origin, derived from Ptch1 mice with a genetic background that is sensitive (CD1) or resistant (C57Bl/6) to the induction of radiogenic MB; (b) identification of genes differentially expressed in spontaneous and radiation-induced MBs from these two mouse strains; (c) bioinformatic analysis to correlate the expression of radiation-induced genes with survival in MB patients; and (d) examining the expression of these genes in ex vivo MBs induced by single or repeated radiation doses. We have identified a potential gene expression signature—Trp53bp1, Bax, Cyclin D1, p21, and Nanog—that influences tumor response. In ex vivo cultured spontaneous MBs, the expression levels of these genes increase after irradiation in CD1 mice, but not in mice with a C57Bl/6 genetic background, suggesting that this signature could predict tumor response to radiation therapy and help develop strategies for targeting DNA damage repair in tumors. A detailed understanding of the mechanisms behind genetic background-related susceptibility to radiation-induced oncogenic responses is crucial for translational research. Full article

Osteoporosis is a progressive metabolic bone disease characterized by decreased bone density and microarchitectural deterioration, leading to an increased risk of fracture, particularly in postmenopausal women and the elderly. Increasing evidence suggests that inflammatory processes play a key role in the pathogenesis of osteoporosis and are strongly associated with the activation of osteoclasts, the cells responsible for bone resorption. In the present study, we investigated, for the first time, the influence of proinflammatory cytokines on the osteogenic differentiation, proliferation, and metabolic activity of primary human osteoblast-like cells (OBs) derived from the femoral heads of elderly patients. We found that all the proinflammatory cytokines, IL-1β, TNF-α, IL-6, and IL-8, enhanced the extracellular matrix mineralization of OBs under differentiation-induced cell culture conditions. In the cases of IL-1β and TNF-α, increased mineralization was correlated with increased osteoblast proliferation. Additionally, IL-1β- and TNF-α-increased osteogenesis was accompanied by a rise in energy metabolism due to improved glycolysis or mitochondrial respiration. In conclusion, we show here, for the first time, that, in contrast to findings obtained with cell lines, mesenchymal stem cells, or animal models, human OBs obtained from patients exhibited significantly enhanced osteogenesis upon exposure to proinflammatory cytokines, probably in part via a mechanism involving enhanced cellular energy metabolism. This study significantly contributes to the field of osteoimmunology by examining a clinically relevant cell model that can help to develop treatments for inflammation-related metabolic bone diseases. Full article

The incidence of arbovirus infections has increased in recent decades. Other than dengue, chikungunya, and West Nile viruses, the data on arbovirus in solid organ transplant (SOT) are limited to case reports, and infections in renal transplant recipients account for most of the reported cases. Dengue and West Nile infections seem to be more severe with higher mortality in SOT patients than in the general population. Acute kidney injury is more frequent in patients with dengue and chikungunya although persistent arthralgia with the latter is less frequent. There is no clear relationship between arboviral infection and acute cellular rejection. Pre-transplant screening of donors should be implemented during increased arboviral activity but, despite donor screening and negative donor nucleic acid amplification test (NAT), donor derived infection can occur. NAT may be transiently positive. IgM tests lack specificity, and neutralizing antibody assays are more specific but not readily available. Other tests, such as immunohistochemistry, antigen tests, PCR, metagenomic assays, and viral culture, can also be performed. There are a few vaccines available against some arboviruses, but live vaccines should be avoided. Treatment is largely supportive. More data on arboviral infection in SOT are needed to understand its epidemiology and clinical course. Full article