Maria Koffa

Chromosomal instability (CIN) arising from mitotic errors is a hallmark of cancer progression, yet how specific spindle assembly factors are co-opted to support aggressive tumor phenotypes remains incompletely understood. Hepatoma Upregulated Protein (HURP/DLGAP5), a Ran-regulated microtubule-associated protein essential for kinetochore fiber stabilization and chromosome congression, is frequently overexpressed in aggressive cancers. Here, we investigated HURP’s role across a breast cancer metastatic gradient—immortalized MCF10A, the low-metastatic luminal T47D, and the highly metastatic triple-negative MDA-MB-231 cell lines—integrating quantitative spindle analysis, kinetochore tension measurements, spindle checkpoint profiling, migration dynamics, and three-dimensional spheroid modeling. We show that total HURP protein levels increase with metastatic potential, yet spindle-bound HURP is paradoxically reduced in MDA-MB-231 cells, indicating cytoplasmic mislocalization despite increased total protein levels. HURP silencing induced cell-line-specific defects: moderate disorganization and misorientation in MCF10A and T47D cells, but catastrophic spindle collapse, apoptosis, and G2/M arrest in MDA-MB-231 cells. Mechanistically, HURP depletion disrupted the spindle-associated levels and distributions of TPX2, Aurora-A, and NuMA in a subtype-dependent manner, implicating HURP as a context-dependent stabilizer of this mitotic regulatory axis. HURP loss reduced interkinetochore tension in all cell lines, but only MCF10A and T47D cells mounted a proportional BubR1-dependent checkpoint response; MDA-MB-231 cells showed reduced checkpoint signaling, consistent with constitutive spindle assembly checkpoint (SAC) attenuation in triple-negative breast cancer. Beyond mitosis, HURP depletion impaired collective migration and converted MDA-MB-231 cells from super-diffusive, amoeboid-like motility to sub-diffusive behavior, while minimally affecting the less aggressive cell lines. HURP-depleted MDA-MB-231 spheroids were significantly larger, less compact, and less spherical than controls, linking spindle regulation to tissue-level architectural coherence. These findings establish HURP as a multifunctional regulator coordinating mitotic fidelity, migration plasticity, and tumor architecture in breast cancer, with a selective dependency in highly metastatic cells, positioning it as a promising therapeutic target for aggressive breast cancers.

Thirty 2-methyl-quinazolinone fussed hydroxamic acids (3-OH) and their 3-OEt and 3-OBn derivatives were evaluated for their affinity towards calf-thymus (CT) DNA using UV-vis absorption, viscosity and fluorescence spectroscopy. DNA photocleavage activity was assessed by incubating the compounds with plasmid DNA followed by UV-A and visible light irradiation, which enabled identification of the most potent derivatives active at concentrations of 100 nΜ and 10 μΜ, respectively. Mechanistic studies on the most active compounds indicated the formation of oxygen radical species and a decrease in efficiency under argon. Measurements of singlet oxygen release verified these findings. Molecular docking studies provided further insight into the interactions between the compounds and DNA. UV-A irradiation of the most potent DNA photocleavers in three cell lines, two malignant melanoma lines (A375 and COLO-800) and the immortalized keratinocyte line HaCaT, identified three derivatives that, at a concentration up to 10 μΜ, reduced cell viability by approximately 50%. Taken together, these results indicate that these 2-methylquinazolinone-based hydroxamic acid derivatives are promising candidates for the development of photodynamic therapy agents.

Biofilm-forming pathogens are a major cause of persistent infections, showing limited response to antibiotic treatment. The search for alternative strategies has therefore driven extensive research into the antimicrobial potential of beneficial microorganisms. In the present study, the antibacterial and antibiofilm activity of the commercial probiotic strain Lacticaseibacillus casei ATCC 393 (Lc393) was examined against clinical isolates of Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Enteritidis and Escherichia coli. Lc393 reduced pathogen viability and attachment to the colon adenocarcinoma cell line HT-29, with maximal effects recorded against S. aureus. Confocal microscopy visualization of the lactobacilli-pathogens-host interface revealed that Lc393 binds loosely to both host cells and pathogens. The Lc393 cell-free culture supernatant (CFCS) significantly reduced planktonic growth, biofilm mass, and viability of cells in biofilm (>2 logCFU reduction, p < 0.05) and downregulated genes involved in the early stages of biofilm formation in S. aureus (i.e., icaA, fnbpA, eno). In silico analysis of the Lc393 genome identified two bacteriocin clusters, along with genes related to ethanol and organic acid production. Based on in silico predictions and a bacteriocin zymogram, the strain cannot produce functional antimicrobial peptides. Untargeted metabolomics based on UPLC/MS further revealed the presence of putative antimicrobial metabolites. Collectively, our findings highlight the antimicrobial potential of Lc. casei ATCC 393 and support its further investigation for combating clinically relevant human pathogens.

Legionella pneumophila (L. pneumophila) infection is characterized by a wide spectrum of manifestations, from influenza-like illness to life-threatening atypical pneumonia with multiorgan failure. The aim of our study was the assessment of in vitro and ex vivo neutrophil activation in L. pneumophila infections, as well as the role of neutrophils’ peptides such as LL-37 in infection. The ability of neutrophils to form ex vivo extracellular traps (NETs) in response to bacterial infection was examined by immunofluorescence. In parallel, patients’ sera, as well as opsonized standard L. pneumophila strains, were used for in vitro activation of neutrophils from healthy individuals. The serum levels of interleukins were assessed using the LEGENDplexTM Multi-Analyte Flow Assay Kit. Furthermore, citrullinated cf-DNA as a marker of neutrophil extracellular traps (NETs) was detected in the serum of patients with acute infection. It was demonstrated that neutrophils released NETs in vitro and ex vivo upon L. pneumophila (interaction in an autophagy-independent manner. Notably, IL-1b was detected on NETs, but an antimicrobial peptide LL-37 was absent. The lack of antimicrobial activity failed to inhibit bacterial proliferation. In addition, in vitro and ex vivo NETs formation was observed during the Clarithromycin treatment. Those NETs were decorated with bioactive antimicrobial peptide LL-37, which inhibits bacterial proliferation. The findings provide evidence that neutrophils release NETs in vitro and ex vivo by expressing the IL1β protein in them. The lack of expression of the antimicrobial peptide LL-37 on the NETs demonstrates the inability of the cells to inhibit proliferation, and consequently the elimination of L. pneumophila. Clarithromycin plays a dual role in the elimination.

Chromosomal instability (CIN) arising from mitotic errors is a hallmark of cancer progression. Hepatoma Upregulated Protein (HURP), a microtubule-associated protein essential for spindle assembly, is frequently overexpressed in aggressive cancers. Here, we investigated HURP's role in breast cancer progression using a cell line model of increasing metastatic potential: immortalized MCF10A, low-metastatic T47D, and highly metastatic MDA-MB-231 cells. Elevated HURP expression was observed in cancer cells, with MDA-MB-231 showing the highest levels. Paradoxically, spindle-bound HURP was reduced in MDA-MB-231 cells despite increased total protein, suggesting cytoplasmic mislocalization. HURP silencing induced cell line-specific effects: moderate spindle defects in MCF10A and T47D cells, but severe abnormalities, apoptosis, and G2/M arrest in MDA-MB-231 cells. Mechanistically, HURP depletion perturbed spindle-bound NuMA, TPX2, and Aurora-A levels, implicating HURP in mitotic regulation and metastatic behavior. HURP silencing also impaired collective migration and converted MDA-MB-231 cells from super-diffusive to sub-diffusive motility, while having minimal effects on less aggressive cell lines. These findings establish HURP as a multifunctional regulator coordinating mitotic fidelity, migration plasticity, and tissue architecture in breast cancer, with pronounced effects in highly metastatic cells, positioning it as a promising therapeutic target for aggressive breast cancers.

Diacylhydrazine bridged anthranilic acids with aryl and heteroaryl domains have been synthesized as the open flexible scaffold of arylamide quinazolinones in order to investigate flexibility versus rigidity towards DNA photocleavage and sensitivity. Most of the compounds have been synthesized via the in situ formation of their anthraniloyl chloride and subsequent reaction with the desired hydrazide and were obtained as precipitates, in moderate yields. All compounds showed high UV-A light absorption and are eligible for DNA photocleavage studies under this “harmless” irradiation. Despite their reduced UV-B light absorption, a first screening indicated the necessity of a halogen at the p-position in relation to the amine group and the lack of an electron-withdrawing group on the aryl group. These characteristics, in general, remained under UV-A light, rendering these compounds as a novel class of UV-A-triggered DNA photocleavers. The best photocleaver, the compound 9, was active at concentrations as low as 2 μΜ. The 5-Nitro-anthranilic derivatives were inactive, giving the opposite results to their related rigid quinazolinones. Molecular docking studies with DNA showed possible interaction sites, whereas cytotoxicity experiments indicated the iodo derivative 17 as a potent cytotoxic agent and the compound 9 as a slight phototoxic compound.

Faithful chromosome segregation during cell division requires accurate mitotic spindle formation. As mitosis occurs rapidly within the cell cycle, the proteins involved in mitotic spindle assembly undergo rapid changes, including their interactions with other proteins. The proper localization of the HURP protein on the kinetochore fibers, in close proximity to chromosomes, is crucial for ensuring accurate congression and segregation of chromosomes. In this study, we employ photoactivation and FRAP experiments to investigate the impact of alterations in microtubule flux and phosphorylation of HURP at the Ser627 residue on its dynamics. Furthermore, through immunoprecipitations assays, we demonstrate the interactions of HURP with various proteins, such as TPX2, Aurora A, Eg5, Dynein, Kif5B, and Importin β, in mammalian cells during mitosis. We also find that phosphorylation of HURP at Ser627 regulates its interaction with these partners during mitosis. Our findings suggest that HURP participates in at least two distinct complexes during metaphase to ensure its proper localization in close proximity to chromosomes, thereby promoting the bundling and stabilization of kinetochore fibers.

Biomedical research requires both in vitro and in vivo studies in order to explore disease processes or drug interactions. Foundational investigations have been performed at the cellular level using two-dimensional cultures as the gold-standard method since the early 20th century. However, three-dimensional (3D) cultures have emerged as a new tool for tissue modeling over the last few years, bridging the gap between in vitro and animal model studies. Cancer has been a worldwide challenge for the biomedical community due to its high morbidity and mortality rates. Various methods have been developed to produce multicellular tumor spheroids (MCTSs), including scaffold-free and scaffold-based structures, which usually depend on the demands of the cells used and the related biological question. MCTSs are increasingly utilized in studies involving cancer cell metabolism and cell cycle defects. These studies produce massive amounts of data, which demand elaborate and complex tools for thorough analysis. In this review, we discuss the advantages and disadvantages of several up-to-date methods used to construct MCTSs. In addition, we also present advanced methods for analyzing MCTS features. As MCTSs more closely mimic the in vivo tumor environment, compared to 2D monolayers, they can evolve to be an appealing model for in vitro tumor biology studies.

Aldehyde dehydrogenase 3A1 (ALDH3A1) oxidizes medium-chain aldehydes to their corresponding carboxylic acids. It is expressed at high rates in the human cornea, where it has been characterized as a multi-functional protein displaying various cytoprotective modes of action. Previous studies identified its association with the DNA damage response (DDR) pathway. Here, we utilized a stable transfected HCE-2 (human corneal epithelium) cell line expressing ALDH3A1, to investigate the molecular mechanisms underlying the cytoprotective role(s) of ALDH3A1. Our data revealed morphological differences among the ALDH3A1-expressing and the mock-transfected HCE-2 cells accompanied by differential expression of E-cadherin. Similarly, the ALDH3A1/HCE-2 cells demonstrated higher mobility, reduced proliferation, upregulation of ZEB1, and downregulation of CDK3, and p57. The expression of ALDH3A1 also affected cell cycle progression by inducing the sequestration of HCE-2 cells at the G2/M phase. Following 16 h cell treatments with either H₂O₂ or etoposide, a significantly lower percentage of ALDH3A1/HCE-2 cells were apoptotic compared to the respective treated mock/HCE-2 cells. Interestingly, the protective effect of ALDH3A1 expression under these oxidative and genotoxic conditions was accompanied by a reduced formation of γ-H2AX foci and higher levels of total and phospho (Ser15) p53. Finally, ALDH3A1 was found to be localized both in the cytoplasm and the nucleus of transfected HCE-2 cells. Its cellular compartmentalization was not affected by oxidant treatment, while the mechanism by which ALDH3A1 translocates to the nucleus remains unknown. In conclusion, ALDH3A1 protects cells from both apoptosis and DNA damage by interacting with key homeostatic mechanisms associated with cellular morphology, cell cycle, and DDR.