Breast cancer is the most prevalent cancer in the female population. The prolonged action of estrogens may affect tumor proliferation. Additionally, a fat-rich diet may show various effects on cancer proliferation, depending on the type of fat. Vitamin D, similarly to estrogens, is a fat-soluble cholesterol derivative. The deficiency of vitamin D correlates with increased proliferation of breast cancer cells. In turn, doxorubicin is commonly used cytostatic in chemotherapy. The study aimed to assess whether vitamin D enhances the anti-cancer effect of doxorubicin (DOX) in the MCF-7 cell line.

The cells were divided into four groups: untreated control, DOX- and vitamin D-treated cells, and cells treated with the combination of compounds in a 1:1 ratio. We applied MTT colorimetric assay (cell viability analysis), Annexin V/PI assay (cell death analysis), flow cytometry (cell cycle distribution), and fluorescence staining of cytoskeletal proteins (F-actin and vimentin). The type of DOX and vitamin D interaction was estimated based on the Chou-Talalay method.

Our results showed that vitamin D and doxorubicin in a 1:1 ratio act synergistically. We observed a decrease in the survival of MCF7 cells. The combination of DOX and vitamin D enhanced changes in morphology and organization in F-actin and vimentin network compared to the treatment with the substances separately.

In summary, we suggest that natural compounds such as vitamin D may be useful in anticancer treatment in the context of enhancing the cytostatic effects of drugs.

The past few decades have seen an increasing number of both space travels and studies aimed at investigating the effects induced by the space flights and environment on humans. One of the main feature of these conditions, is the presence of an altered gravity mostly represented by microgravity experienced by astronauts. Microgravity is well known to induce deleterious effects at cellular, organ and systemic levels, including alterations in the male and female reproductive systems.

In the present study, we investigated the effect of simulated microgravity on the metabolic activity of male germ cells using TCam-2 line as cell model. These cells were cultured in the Random Positioning Machine that simulated microgravity conditions and were grown as 2D monolayers or 3D spheroids to assay the effects on single cells or on organ-like structure. After a 24 hour-exposure to simulated microgravity, TCam-2 monolayers showed: 1) a decreased proliferation rate and a delay in cell cycle progression; 2) increased anaerobic metabolism; 3) increased levels of reactive oxygen species and superoxide anion; 4) modifications in mitochondrial morphology. After the same 24 hour-exposure, TCam-2 spheroids showed: 1) an increased anaerobic and aerobic activity in 40% and 26% of samples, respectively; 2) alterations in the redox balance with a decrease in catalase activity in about 65% of cell samples, therefore a deficit in the cellular antioxidant capacity; 3) increases in oxidative damage to proteins and lipids in more than 50% of cell samples.

In conclusion, these data demonstrated a clear inference of simulated microgravity on the metabolic activity of TCam-2 cells, which is expressed through the activation of an oxidative stress state, that, if not compensated for, could result deleterious over time.

Metabolic dysregulation harms brain health. Early-life (pre- and perinatal) metabolic stress has been demonstrated to affect central nervous system (CNS), multigenerationally affecting brain plasticity and cognitive functions in adult offsprings. . In our previous work, we reported that maternal high fat diet (HFD) impairmed synaptic plasticity, learning and memory of descendants until the third generation. Neural stem and progenitor cells (NSPCs) represent the cellular source of newborn neurons in the subgranular zone of the hippocampus, and their fate is finely modulated by metabolic signals. Epigenetic mechanisms are key factors controlling the neural fate of NSPCs and they dynamically regulate CNS development and adult neurogenesis. Here, we demonstrate that progenitor HFD altered both the proliferation of NSPCs and the hippocampal adult neurogenesis on second and third generations of progeny (F2HFD and F3HFD), leading to the depletion of neurogenic niche in the descendants. Moreover, NSPCs derived from HFD descendants showed altered expression of several genes involved in the regulation of stem cell proliferation and neurodifferentiation (i.e., Hes1, NeuroD1, Bdnf). Furthermore, maternal HFD-related metabolic stress induced a rearrangement of STAT3/5 transcription factors occurring on the regulatory sequences of NeuroD1 and Gfap genes, causing the epigenetic repression of pro-neurogenic and the activation of pro-glial differentiation genes. Collectively, our data indicate that maternal HFD multigenerationally affects hippocampal adult neurogenesis via an epigenetic inhibition of pro-neurogenic gene expression in NSPCs.

Hsa-miR-1249-3p is dysregulated in several neoplasms, including hepatocellular and breast carcinomas. However, its function in human epithelial cells is unknown. Herein, we functionally investigated the effect of hsa-miR-1249-3p on the proliferation, migration, clonogenicity and apoptosis of human epithelial cells and explored the underlying mechanism.

Droplet digital PCR was used to evaluate the hsa-miR-1249-3p expression in human keratinocyte cell lines HaCaT and NCTC and in control human uterine cervical carcinoma cell lines SiHa, CaSki and HeLa. Then, the hsa-miR-1249-3p mimic, inhibitor and negative/positive controls were transfected onto HaCaT cells. Upon transfections, cell proliferation, clonogenicity, migration and apoptosis were assessed by WST, clonogenic, wound healing and western blot assays, respectively.

Results indicate that hsa-miR-1249-3p is overexpressed in HaCaT and NCTC cell lines, respectively, compared to pooled human cervical carcinoma cell lines. Upon transfections, hsa-miR-1249-3p resulted as undetectable in miR-inhibitor HaCaT condition, while being strongly overexpressed miR-mimic HaCaT, compared to untreated cells. Hsa-miR-1249-3p inhibition modestly favored cell proliferation and migration potential in HaCaT cells, without perturbing apoptosis. Contrariwise, a strong clonogenic effect was detected in hsa-miR-1249-3p-inhibited HaCaT cells. Furthermore, in silico analyses conducted with TargetScan tool identified the oncogene Homeobox A13 (HOXA13) as a hsa-miR-1249-3p downstream target. Mechanistically, hsa-miR-1249-3p inhibition prompted the up-regulation of HOXA13 transcript in HaCaT cells in vitro.

Our data indicate that hsa-miR-1249-3p can target HOXA13 to regulate the clonogenic potential of HaCaT cells. These data will allow the set-up of further studies aimed in investigating the role of has-miR-1249-3p/HOXA13 axis in epithelial cell clonogenicity, such as evaluating the relationship between this miRNA/target gene axis and its downstream genes implicated in cell-cell adhesion pathways, i.e., β-catenin, c-Met and c-Jun.

After recalling the slow emergence of the cell theory, stating that cells are the fundamental units of structures and functions in all living organisms, the widespread observations of extracellular vesicles (EVs) will be somemore detailed. The current EV classification, and their main characteristics will be recalled, together with their promising assets for the theranostics of many human diseases, including cancers. However, many challenges remain to be solved before achieving this goal.

There is a missing step between the accumulated biological knowledge about EVs during two decades and the many recent preclinical searches for efficient EVs applications in oncology. The main reason is the current complete lack of knowledge about how EVs are completing the current cell theory centered on cells acting as powerful "biological factories" highly protected from their environment by their plasma membrane. In a great breakthrough, EVs are now known to mediate important cell's interconnections, which were completely ignored before, and which are resting on many mechanisms, which need to be urgently deciphered. In preclinal studies dealing with a few human patients compared to controls, the huge amount of different cells-derived EVs generates an inextricable complexity.

To evidence unknown EV-mediated mechanisms, a simple cell model would be much more convenient. The microorganism Dictyostelium discoideum (Dd) is ideal to achieve this goal as a wonderful eukaryotic in vitro and in vivo cell model. In 1998, we have discovered Dd EVs as mediating a new multidrug resistance mechanism, and then the normal and physiological Dd cells-release of different EVs during both the well-separated growth and starvation-induced differentiation. Moreover, Dd cells are devoted with many other assets, and axenic Dd cells are very well suited for conditioned-medium experiments to study the influence of specifically generated Dd EVs upon naive Dd cells, as will be shown in this presentation.

Ageing is associated with an exponential increase in mortality, but paradoxically, in many organisms mortality rates decline late in life, a phenomenon known as late-life mortality deceleration. How late-life differs to ageing physiologically, and if mortality deceleration implies that ageing stops or reverses at a specific point of an organism’s life remains unknown. Therefore, to examine the cellular and metabolic basis for mortality deceleration, we used a novel model of ageing – that of the African killifish, an extremely short-lived vertebrate that displays mortality deceleration. Using skeletal muscle, where the stereotypic hallmarks of ageing are well characterized, we highlight that ageing and late-life phases are physiologically distinct. Using a systems metabolomics approach, we demonstrate that during ageing there is a striking depletion of triglycerides, mimicking a state of calorie restriction, which triggers mitohormesis, a reactive oxygen species mediated stress resistance mechanism. This improves lipid and mitochondrial metabolism, subsequently maintaining nutrient homeostasis during late-life and driving mortality deceleration. Our results not only provide evidence of mitohormesis in regulating lifespan in vertebrates that naturally live-longer, but they also collectively show that the metabolic hallmarks of ageing are reversible.

Succinic semialdehyde dehydrogenase deficiency (SSADH-D) is a rare monogenic disorder of the γ-amino butyric acid (GABA) metabolism. Various pathogenic mutations in aldehyde dehydrogenase 5 family member A1 (ALDH5A1) gene are responsible for the enzymatic dysfunction of the succinic semialdehyde dehydrogenase (SSADH), an enzyme that plays a key role in the breakdown of GABA. As a consequence, GABA and its potentially toxic metabolite γ–hydroxybutyrate (GHB) accumulate in the brain and physiological fluids. The aim of this study was to produce and test different recombinant SSADH proteins for an enzyme-replacement therapy for SSADH-D. The intracellular delivery of large bioactive molecules, such as enzymes, requires that these molecules traverse not only the plasma membrane, but also further intracellular membranes. Thus, a cell-penetrating peptide (Trans-activator of Transcription; Tat) was fused to the N-terminal part of SSADH. This sequence was followed by mitochondrial targeting sequence (MTS), as SSADH is a mitochondrial enzyme (rHis-Tat-MTS-SSADH). The sequence of human SSADH as well as MTS and Tat were optimized for efficient bacterial overexpression. As a control, optimized sequences lacking MTS and Tat were produced either with (rHis-SSADH) or without His-tag (rSSADH). In-vitro, purified rHis-SSADH and rSSADH, but not in rHis-Tat-MTS-SSADH, exhibited SSADH activity. Interestingly, all produced recombinant enzymes displayed a highly efficient cellular and mitochondrial uptake in SSADH-D patient fibroblasts. However, only rHis-SSADH and rSSADH were able to fully reconstitute the missing SSADH activity. These effects were His-independent. Although rHis-Tat-MTS-SSADH reached the mitochondrial compartment, it was not processed in the mature form and thus showed no SSADH activity. These results indicate that rHis-SSADH and rSSADH are suitable candidates for further testing in an animal model for SSADH-D.

P16INK4A is a tumor suppressor and cell cycle regulator that has been linked to aging and senescence. In development, a potential role of p21 and of p19ARF has been postulated, but little is known about p16. Our previous results revealed a highly dynamic expression pattern of p16 in development and in different organs and cell types assessed by qRT-PCR and immunohistochemistry (IHC). In addition, we also noticed through IHC observation that p16 expression in old liver is mainly in the endothelial cells (EC) compared to parenchymal cells. Therefore, we aimed at better understanding the role of p16 in biological processes of liver ECs such as proliferation, migration, apoptosis, and tube formation. We also performed RNA sequencing to identify genes differentially expressed between young and old ECs. We used small hairpin (shRNA) constructs and a p16 cDNA- GFP vector to knockdown and overexpress p16 in-vitro, in two types of liver ECs, CD31+ vascular ECs and CD146+ sinusoidal endothelial cells. Afterwards, we assessed p16 down and up regulation effect on ECs function. Brdu incorporation assays showed that p16 upregulation was associated with slower proliferation compared to control cells whereas its down-regulation induced higher proliferation compared to control cells. Scratch assay and trans-well migration assays showed attenuated migration in p16 overexpressed cells compared to baseline expression, while only transwell assays showed ameliorated migration of p16 knockdown cells compared to controls. similar migration between p16 knockdown and control was observed in scratch assays. We also observed in β-gal staining, a marker of senescence, a higher number of stained cells in p16 overexpression conditions compared to controls while less cells were stained in case of knockdown. Further experiments that aim to further decipher p16 effect in EC’s tube formation, apoptosis, and telomeres shortening are ongoing which might contribute to the invention of more specialized anti-aging therapies.

Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive cancer, with over half of patients presenting with metastatic PDAC at diagnosis. Most patients receive conventional chemotherapy which invariably faces resistance, and a key facilitator in this is the PDAC stroma which acts as a functional mediator of disease progression through bilateral crosstalk between stromal cells and cancer cells. ‘Migrastatics’ are a new drug class which target cell migration pathway effector proteins to attenuate cancer cell invasion. Improvement in PDAC treatment strategy is well-overdue and migrastatics as adjuvant therapy is one avenue gaining traction. The p21-activated kinase (PAK) family is frequently overexpressed and/or amplified in PDAC where it regulates cytoskeletal actin contractility as well as transcription. Pre-clinical PAK inhibitors have shown reduced 3D PDAC cell invasion in vitro, yet it is unknown how the PDAC stroma would respond to a PAK inhibitor and how this could affect PDAC invasion. My PhD project investigates the stellate cells response to PAK inhibition.

In diabetic retinopathy (DR), Müller cell gliosis contributes to retinal degeneration and inflammation. In this context, we highlight annexin A1 (AnxA1), an anti-inflammatory protein able to regulate neurodegeneration and angiogenesis, however, its mechanisms of action were poorly explored in DR. This study evaluates the function of AnxA1 in streptozotocin (STZ)-induced DR in wild-type (WT) and knockout (AnxA1^-/-) mice after 12 weeks. In addition, in silico analysis was performed with GSE111465 (whole retinas from 6-weeks-old STZ-diabetic or control animals) and GSE160306 (human retina with different stages of DR). Retina from 6-weeks-old STZ-diabetic mice showed raised transcripts of AnxA1 and GFAP compared to controls. After 12 weeks, RD was associated with increased levels of AnxA1, formyl peptide receptor 2 (Fpr2) in the WT retina, as well as cleaved caspase 3 and vascular endothelial growth factor (VEGF) compared to control samples. Lack of AnxA1 caused increased glutamine synthetase expression (Müller cell marker) in retinas from RD animals compared to WT RD group. On the other hand, no alterations in the levels of caspase 3 and VEGF expression were showed in AnxA1^-/- groups. Despite both genotypes presented gliosis in peripheral retina, as shown by glial fibrillary acid protein (GFAP) immunostaining, AnxA1^-/- RD group exhibited decreased levels of GFAP compared to RD WT group. In silico study with human retinas, the severity of DR is associated with higher levels of AnxA1 mRNA expression. Additionally, a positive correlation between AnxA1 and GFAP mRNA levels was detected. These results allow us to conclude that AnxA1 participates in the progression of RD and that this protein can regulate the expression of GFAP.