each poster 3' presentation + 2' discussion
SFRR-E PP 2.1:
Role of Platanus derived compounds in stress induced premature senescence of human Mesenchymal Stem Cells
Dr. Varvara Trachana | Laboratory of Biology, Faculty of Medicine, School of Health Sciences, University of Thessaly | Greece
Cellular senescence -a process characterized by the irreversible cell cycle arrest- affects the function of eukaryotic cells, including stem cells, leading to tissue regeneration impairment, and mediating organismal aging and the development of age-related pathologies. Senescence can occur due to telomere shortening (replicative senescence), but can also be the result of damage, induced by internal or external stressors, such as oxidative stress (OS), leading to stress induced premature senescence (SIPS). Several plants and plant derived compounds could serve as potential anti-SIPS agents, possibly due to their antioxidant abilities. Here, we aimed at investigating the effects of Platanus derived compounds on Mesenchymal Stem Cells from the Wharton’s Jelly (WJ-MSCs) in the context of cellular senescence. Early passage WJ-MSCs treated or not with H2O2 in order to induce premature senescence were exposed to 3 different concentrations (0,1%, 1%, 10%) of compounds derived from various Platanus parts, extracted with different organic solvents and water. The activity of the senescence associated marker β-galactosidase (SA-β-gal) was then assessed. Our preliminary results showed that while some compounds, at higher concentrations exhibit a pro-senescence or even a cytotoxic function, at lower concentrations have the ability to protect from the establishment of cellular senescence. Compounds derived from Platanus have previously been reported to possess an antioxidative ability and given the well established relationship between oxidative stress and cellular senescence’s onset, it would be of utmost importance to analyze the underlying mechanisms of the observed outcome. We have previously reported that caveolin-1 -a component of the caveolae and recently recognized as major regulator of cellular senescence- plays an important role in the cells’ ability to repair OS induced DNA damage, and consequently to the development of WJ-MSCs premature senescence. Therefore, the involvement of caveolin-1-depended signaling in these compounds’ protective function against SIPS is currently extensively studied.
SFRR-E PP 2.2:
Fluorescent nanodiamonds as a biosensor for detecting free radicals during stress condition in Saccharomyces cerevisiae
Dr. Aryan Morita | Gadjah Mada University | Indonesia
Background. Free radicals play a significant role in physiological processes and pathological conditions such as degenerative diseases and the aging process. We have developed a new technology which allows nanoscale MRI measurements for quantifying these with subcellular resolution. We make use of diamond nanoparticles, which change their optical properties based on magnetic surrounding. While the method have already been proven in physics we demonstrate measurements in life cells for the first time. In this study we could measure radical production after provoking Saccharomyces cerevisiae . We were also able to clearly differentiate production rates between young and old as well as wild type and knock out strains. Additionally, we are able to follow how an antioxidant protects the cells from radical damage.
Methods. Saccharomyces cerevisiae BY4741 were grown in synthetic defined (SD) complete medium supplemented with 2% glucose and treated with zymolyase for creating spheroplasts which facilitated FNDs uptake. The cells were placed in 96-well plates and 4 compartments cell culture dishes then triggered with 1, 3, and 10% of hydrogen peroxide. They were analyzed by using MTT assay for evaluating metabolic activity and H2DCFDA for measuring ROS production. 0,1 M of L-ascorbic acid had been used for antioxidant during the measurements. Free radicals level during stress condition has been monitored by using diamond magnetometry technique with 50 µwatt laser power. All the measurements were performed triplicates.
Results. With our newly developed technique we are able to measure free radical generation with nanoscale spatial resolution.
SFRR-E PP 2.3:
Increased ROS can improve liver cell survival: stress response PACOS
Prof. Irina Milisav | University of Ljubljana, Faculty of Medicine and Faculty of Health Sciences | Slovenia
Excessive or too small amounts of reactive oxygen species (ROS) lead to oxidative imbalance, named oxidative stress or antioxidative/reductive stress, respectively, while moderate concentrations of ROS are required for normal cell function. H2O2 is the main redox signalling and redox regulation molecule. Increased levels of H2O2 are a part of a normal response to moderate stress in primary liver cells (hepatocytes) and trigger a stress response that prevents apoptosis triggering through caspase-9, called preapoptotic cell stress response (PACOS).
An increased amount of ROS production and lower apoptosis triggering are reversible in primary hepatocytes and their function is preserved at all times, in both, stress adapted and normal cells. Antioxidants, like N-acetylcysteine (NAC), annul the PACOS stress response and its protective role against apoptosis. A moderate increase of H2O2 can induce a stress response that prevents the cells from unnecessary apoptosis.
SFRR-E PP 2.4:
Diamond magnetometry to understand male reproductive decay during aging
Arturo Elias | UMC Groningen/University of Chile | Netherlands
Delaying fatherhood is a global trend. Aging affects the male germline decreasing the reproductive fitness and increasing the frequency of congenital anomalies in the progeny. Oxidative stress (OS) may play a leading role in the mechanisms behind age-related male reproductive problems. Understanding how OS affects the precursor cells of male gametes is critical to connect aging with inheritable genomic alterations that may affect the next generation. Nevertheless, most available methods to measure OS show several drawbacks that hinder the completion of such an ambitious goal. Diamond magnetometry (DM) is a promising technique that allows the measurement of free radical levels in real-time with subcellular resolution. DM uses fluorescent nanodiamonds (FNDs) with NV centers to translate the magnetic noise generated by free radicals into easily detectable fluorescent signals. Performing DM requires cell immobilization on a suitable coating and the optimization of FNDs concentration and incubation times to achieve the cellular internalization of the particles. Using a mouse model of aging we studied representative stages of spermatogenesis. We tested different coating conditions (0.4% gelatin, 0.01% poly-L-lysine, and 1 mg/ml hyaluronic acid) and concentrations of FNDs (2, 4, and 8 µg/ml) at three incubation times (2h, 4h, and 6h). Finally, we performed diamond magnetometry for the first time in male germ cells. Our results show that 0.4% gelatin, 4 µg/ml FNDs, and 6 hours of incubation are the optimal conditions to achieve more than 80% of attachment and at least 50% of cells with 3-5 internalized FNDs for all populations. Free radicals measurements allowed us to differentiate between germ cells from aged and control animals and to compare between different populations from the same group. In conclusion, we optimized the utilization of a novel tool to study OS in the male germline during aging, opening a myriad of potential applications in Reproductive Medicine.
SFRR-E PP 2.5 [YIA]:
Can physical activity counteract changes in redox-status biomarkers during chemotheraphy of breast cancer patients?
Chantalle Moulton | Unit of Biology and Genetics of Movement, Department of Movement, Human and Health Sciences, University of Rome Foro Italico, Rome, Italy | Italy
Breast cancer (BC) is one of the most commonly diagnosed types of cancer in women. Oxidative
stress may contribute to cancer aetiology through several mechanisms involving damage to DNA,
proteins and lipids leading to genetic mutations and genomic instability. The literature indicates that
physical activity (PA) has positive effects on every aspect of breast cancer evolution, including
negative effects from treatment. Specifically, how the beneficial association
between PA and BC survival are partially related to its influence on antioxidant status of the body.
Fifteen newly diagnosed BC patients (40-60 years), who underwent the same surgery and before
beginning cancer-related treatments, were recruited and divided randomly into a control group
(CG,n=5) undergoing usual care, and an exercise group (EG,n=10), which additionally participated in
a PA program. With the aim to verify the ability of PA to counteract the negative effects on systemic
redox-homeostasis induced by the BC treatment, we examined the impact of a 4-month exercise
program on the modulation of plasma markers of oxidative stress, inflammation and the stress
response, such as superoxide dismutase (SOD) and catalase (CAT) activity, total-glutathione (tGSH),
lipid-oxidation (TBARs), total-antioxidant-capacity (TAC) and total-free-thiols (tFTH), as well as
interleukin-6 (IL6), interleukin-8 (IL8), interleukin-10 (IL10), and tumor-necrosis-factor-alpha (TNFα).
Even in the absence of significant changes in CAT activity, TAC, tFTH and TBARs levels (p > 0.05),
exercise maintained SOD activity and tGSH levels in EG whereas in CG they were significantly
decreased (p < 0.05). Moreover, we found a significant decrease of IL8 in both groups, whereas only in
EG we observed a significant reduction in the pro-inflammatory IL6 and an increase of antiinflammatory IL10 (p < 0.05).
These results highlighted the importance of PA as a potential coadjuvant therapy, alongside usual
care of BC, able to counteract the chemotherapy-induced negative effects on an already compromised
SFRR-E PP 2.6:
Redox mosaic in breast cancer: At the intersection of cancer and adipose tissue
Andjelika Kalezic | Institute for Biological Research "Sinisa Stankovic"-National Institute of Republic of Serbia, University of Belgrade, Belgrade, Serbia | Serbia
Altered redox homeostasis is recognized as a hallmark of neoplastic transformation. However, data from various in vitro and in vivo studies often show increased or decreased transcriptional and translational levels of antioxidant defense (AD) enzymes. One of the underlying causes for such conflicting reports is cell heterogeneity within the complex tumor microenvironment, especially in breast cancer. To overcome barriers associated with bulk tissue gene and protein expression analysis, we choose an immunohistochemical approach. We cross-examined serial tissue sections of tumor and adipose tissue from premenopausal women with malignant invasive ductal carcinoma and benign fibroadenoma to gain a comprehensive overview of cell-specific AD enzymes expression and localization patterns. At the level of overall tissue architecture, malignant tumor tissue shows significantly higher immunopositivity for copper, zinc- and manganese- superoxide dismutase, catalase, and glutathione peroxidase compared to benign tumor tissue. Generally, AD enzymes are specifically localized in the cytoplasm (copper, zinc superoxide dismutase, catalase, glutathione peroxidase) and mitochondria (manganese superoxide dismutase, glutathione peroxidase) of cancer cells and cancer-associated adipocytes. Detailed analysis of different regions of the tumor tissue revealed significant heterogeneity in the degree of immunopositivity along the axis of tumor center–invasive front–adipose tissue. Clusters of cancer cells at the invasive front of the tumor often show a higher degree of immunopositivity for AD enzymes compared to cancer cells in the center of the tumor mass. Similarly, cancer-associated adipocytes that are in close proximity to cancer cells at the invasive front of the tumor show a higher degree of immunopositivity for AD enzymes compared to adipocytes from distant peritumoral adipose tissue. In conclusion, immunohistochemical approach confirms high AD enzymes expression in breast cancer and further reveals distinct regional mosaicism consistent with cell heterogeneity within the tumor microenvironment.
This research was supported by the Science Fund of the Republic of Serbia, #7750238-REFRAME.
SFRR-E PP 2.7 [YIA]:
Interaction between Glutathione S-transferase P and Nrf2 transcription factor in mouse hepatocarcinoma: a molecular and physical characterization
Dr. Desirée Bartolini | UNIVERSITA' DEGLI STUDI DI PERUGIA | Italy
Non-Alcoholic Fatty Liver Disease (NAFLD) is a common chronic liver disease (CLD) highly prevalent in obese patients. This is now becoming the first cause of liver transplantation, and its evolution to steatohepatitis increases the risk of cirrhosis and hepatocellular carcinoma (HCC). The latter is the second cause of cancer deaths worldwide, and the first one in CLD patients.
Drug-resistance (DR) limits the efficacy of chemotherapy in HCC. The oncogene Glutathione S-transferase P (GSTP) is involved in DR. Its expression increases in association with cancerogenic transformation of the liver tissue. In human HCC cell lines, we recently observed a possible functional interaction between GSTP and Nrf2, a transcription factor involved in the stress response to cellular electrophiles and in the carcinogenesis process.
Such interaction was investigated in the present study using the mouse model of N-nitrosodiethylamine (DEN)-induced HCC. In this model, the liver expression of GSTP and Nrf2 increased during tumor development in association with progressive desensitization to apoptotic stimuli and DR gene induction. Co-immunoprecipitation experiments showed a physical interaction of these proteins, that in HCC specifically involves the dimeric form of GSTP. Also, a nuclear translocation of GSTP was observed during tumor development in association with β−TrCP protease expression, suggesting a dynamic distribution of GSTP during the feedback response to Nrf2 nuclear translocation.
In conclusion, a physical (protein-protein) interaction of the chaperone GSTP with the transcription factors Nrf2 is confirmed in the DEN model of HCC. Its role in carcinogenesis and DR is worth investigating.
SFRR-E PP 2.8:
Crude extracts from pepper fruits show anti-proliferative activity against tumor cells altering their catalase and glucose-6-phosphate dehydrogenase profile
Prof. Dr. José M. Palma | Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas | Spain
Pepper (Capsicum annuum L.) fruit is characterized by its high amounts of antioxidants (vitamins C and A), polyphenols and capsaicinoids. It has been reported that capsaicin, which is exclusive from pepper fruits, shows anti-inflammatory, antiproliferative and analgesic activities. Recently, by the use of untargeted metabolomic approaches, it has been found that pepper (Capsicum annuum) fruits contain a series of compounds with potential therapeutic properties due to the presence, among others, of quercetin and its derivatives, with their content being modulated by nitric oxide (NO) (Guevara et al., 2021, IJMS 22, 4476). In this work, the anti-proliferative activity of crude extracts from four pepper fruits varieties (Melchor from type California, Padrón, Piquillo and Alegría riojana) against HEPG2 (hepatoma) and MIA Paca-2 (pancreas) cells was investigated. Interestingly, it was was demonstrated that pepper fruit tissues from the four varieties which contained the lower capsaicin content displayed the higher anti-proliferative activity in both cell lines. The antioxidant profile of the two tumor cell lines incubated with pepper fruit extracts from Alegría riojana was then investigated, and the activity profile of catalase, superoxide dismutase, glutathione peroxidase and several NADPH-generating enzyme systems was followed. As the most highlighting results, it has to be remarked that the anti-proliferative pattern of the pepper fruit extracts was linked to an altered profile of the (iso)enzymatic activity of catalase and the glucose-6-phosphate dehydrogenase. This research opens new windows on the pepper fruit’s bioactive compounds with nutraceutical and biomedical potentiality, as well as on their posible targets in the tumor cells.
[Supported by a European Regional Development Fund cofinanced grants from Junta de Andalucía (P18-FR-1359) and the Ministry of Science and Innovation (PID2019-103924GB-I00), Spain]
SFRR-E PP 2.9:
Compounds from sweet pepper (Capsicum annuum l.) fruits with potential therapeutic uses are boosted by nitric nitric oxide (NO)
Prof. Francisco J. Corpas | Estación Experimental del Zaidín (Spanish National Research Council, CSIC) | Spain
The treatment of sweet pepper (Capsicum annuum) fruits with nitric oxide (NO) provokes ripening delay and increases in the ascorbate content, linked to a moderate lipid peroxidation and protein nitration, as nitro-oxidative stress markers. This situation was considered as some sort of “phystress” (physiological stress). It has been also demonstrated that the NO treatment triggered changes at both transcriptional and proteomic levels in pepper fruits. Based on these data, a metabolomic study was conducted to investigate the metabolomic profiles in pepper fruits at different ripening stages (ripe red versus green immature) and as a consequence of the NO treatment, with the aim of searching for compounds which may have therapeutic potential, that boosting the nutraceutical value of this vegetable. Thus, high performance liquid chromatography (HPLC) coupled to metabolite identification by high resolution mass spectrometry (HRMS) was achieved, and different platforms and databases were used to characterize the metabolites. Twelve differential bioactive compounds were identified in sweet pepper fruits, including quercetin and its derivatives, L-tryptophan, phytosphingosine, FAD, gingerglycolipid A, tetrahydropentoxylin, blumenol C glucoside, colnelenic acid and capsoside A. The abundance of these metabolites varied depending on the ripening stage and on the presence of NO. Besides, the potential anti-proliferative activity of crude extracts from pericarp and placenta of pepper fruits with different pungency levels (capsaicin content) was investigated against seven tumor cell lines. Results proved that tissues with the highest capsaicin levels did not correlate with the greatest anti-proliferative capacity. Altogether, these data open new research perspectives on the pepper fruit’s bioactive compounds with therapeutic potentiality, where biotechnological strategies can be applied for optimizing the level of these beneficial molecules.
[Supported by a European Regional Development Fund cofinanced grants from Junta de Andalucía (P18-FR-1359) and the Ministry of Science and Innovation (PID2019-103924GB-I00), Spain]
SFRR-E PP 2.10:
Metabolomics- and frailty-related signature of lipophilic micronutrients in age-related diseases
Prof. Dr. Dr. Maria Cristina Polidori Nelles | Universität zu Köln | Germany
There have been several studies that have tried to build a ‘biological age’ predictor using metabolomics data. The conclusion from these studies is that metabolomics-based ‘biological age’ predictors are less capable of predicting chronological age in comparison to, for example, epigenetic-based ‘clocks’, but they are still able to capture part of an individual’s ‘biological age’. As healthy nutrition and antioxidant micronutrients are crucial in maintaining health and robustness, we aimed at identifying metabolites and lipophilic micronutrients associated with age-related diseases and their outcomes (mortality, multimorbidity, general health, physical and multidimensional (pre-)frailty and nursing needs). To do so, within a large prospective study with one year follow up in the Emergency Department (ED) (>1,000 participants until time of submission), a blood withdrawal and a deep clinical phenotyping were obtained from a subgroup of 265 patients (140 M, age range 65-96 yeras). High‐throughput NMR-spectroscopy metabolomic analyses (Nightingale Health Ltd., Helsinki, Finland) and HPLC measurements of carotenoids, retinol and tocopherols were performed in serum as well as the comprehensive geriatric assessment (CGA) – based Multidimensional Prognostic Index (MPI) as a surrogate marker of biological age. The simultaneous quantification of routine lipids, lipoprotein subclass profiling with lipid concentrations within 14 subclasses, fatty acid composition, and various low-molecular metabolites, including amino acids, ketone bodies, and gluconeogenesis-related metabolites yielded the preliminary results of a differential association of specific carotenoids and tocopherols with lipid classes. Furthermore, selected carotenoids appear to be preferentially associated to the metabolomics mortality score and selected geriatric syndromes and comorbidities. Although analyses are ongoing at the time of submission and the final results will be presented at the Redox Biology Congress, preliminary observations suggest a metabolomics and frailty signature of lipophilic micronutrients which may disclose pathophysiological mechanisms of age-related diseases and new therapeutic options.