Interactions between the gut microbiome, associated metabolites and the manifestation and progression of heart failure with preserved ejection fraction in ZSF1 rats.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is associated with systemic inflammation, obesity, metabolic syndrome, and gut microbiome changes. Increased trimethylamine-N-oxide (TMAO) levels are predictive for mortality in HFpEF. The TMAO precursor trimethylamine (TMA) is synthesized by the intestinal microbiome, crosses the intestinal barrier and is metabolized to TMAO by hepatic flavin-containing monooxygenases (FMO). The intricate interactions of microbiome alterations and TMAO in relation to HFpEF manifestation and progression are analyzed here. METHODS: Healthy lean (L-ZSF1, n = 12) and obese ZSF1 rats with HFpEF (O-ZSF1, n = 12) were studied. HFpEF was confirmed by transthoracic echocardiography, invasive hemodynamic measurements, and detection of N-terminal pro-brain natriuretic peptide (NT-proBNP). TMAO, carnitine, symmetric dimethylarginine (SDMA), and amino acids were measured using mass-spectrometry. The intestinal epithelial barrier was analyzed by immunohistochemistry, in-vitro impedance measurements and determination of plasma lipopolysaccharide via ELISA. Hepatic FMO3 quantity was determined by Western blot. The fecal microbiome at the age of 8, 13 and 20 weeks was assessed using 16s rRNA amplicon sequencing. RESULTS: Increased levels of TMAO (+ 54%), carnitine (+ 46%) and the cardiac stress marker NT-proBNP (+ 25%) as well as a pronounced amino acid imbalance were observed in obese rats with HFpEF. SDMA levels in O-ZSF1 were comparable to L-ZSF1, indicating stable kidney function. Anatomy and zonula occludens protein density in the intestinal epithelium remained unchanged, but both impedance measurements and increased levels of LPS indicated an impaired epithelial barrier function. FMO3 was decreased (- 20%) in the enlarged, but histologically normal livers of O-ZSF1. Alpha diversity, as indicated by the Shannon diversity index, was comparable at 8 weeks of age, but decreased by 13 weeks of age, when HFpEF manifests in O-ZSF1. Bray-Curtis dissimilarity (Beta-Diversity) was shown to be effective in differentiating L-ZSF1 from O-ZSF1 at 20 weeks of age. Members of the microbial families Lactobacillaceae, Ruminococcaceae, Erysipelotrichaceae and Lachnospiraceae were significantly differentially abundant in O-ZSF1 and L-ZSF1 rats. CONCLUSIONS: In the ZSF1 HFpEF rat model, increased dietary intake is associated with alterations in gut microbiome composition and bacterial metabolites, an impaired intestinal barrier, and changes in pro-inflammatory and health-predictive metabolic profiles. HFpEF as well as its most common comorbidities obesity and metabolic syndrome and the alterations described here evolve in parallel and are likely to be interrelated and mutually reinforcing. Dietary adaption may have a positive impact on all entities.

Elastin-derived peptides (EDPs) affect gene and protein expression in human mesenchymal stem cells (hMSCs) - preliminary study.

During the aging process, elastin is degraded and the level of elastin-derived peptides (EDPs) successively increases. The main peptide released from elastin during its degradation is a peptide with the VGVAPG sequence. To date, several papers have described that EDPs or elastin-like peptides (ELPs) affect human mesenchymal stem cells (hMSCs) derived from different tissues. Unfortunately, despite the described effect of EDPs or ELPs on the hMSC differentiation process, the mechanism of action of these peptides has not been elucidated. Therefore, the aim of the present study was to evaluate the impact of the VGVAPG and VVGPGA peptides on the hMSC stemness marker and elucidation of the mechanism of action of these peptides. Our data show that both studied peptides (VGVAPG and VVGPGA) act with the involvement of ERK1/2 and c-SRC kinases. However, their mechanism of activation is probably different in hMSCs derived from adipose tissue. Both studied peptides increase the KI67 protein level in hMSCs, but this is not accompanied with cell proliferation. Moreover, the changes in the NANOG and c-MYC protein expression and in the SOX2 and POU5F1 mRNA expression suggest that EDPs reduced the hMSC stemness properties and could initiate cell differentiation. The initiation of differentiation was evidenced by changes in the expression of AhR and PPARγ protein as well as specific genes (ACTB, TUBB3) and proteins (ß-actin, RhoA) involved in cytoskeleton remodeling. Our data suggest that the presence of EDPs in tissue can initiate hMSC differentiation into more tissue-specific cells.

Role of Ciminalum-4-thiazolidinone Hybrids in Molecular NF-κB Dependent Pathways.

A range of hybrid molecules incorporating the ciminalum moiety in the thiazolidinone ring demonstrate significant anticancer and antimicrobial properties. Therefore, the aim of our study was to evaluate the properties and mechanism of action of two 4-thiazolidinone-based derivatives, i.e., 3-{5-[(Z,2Z)-2-chloro-3-(4-nitrophenyl)-2-propenylidene]-4-oxo-2-thioxothiazolidin-3-yl}propanoic acid (Les-45) and 5-[2-chloro-3-(4-nitrophenyl)-2-propenylidene]-2-(3-hydroxyphenylamino)thiazol-4(5H)-one (Les-247). In our study, we analyzed the impact of Les-45 and Les-247 on metabolic activity, caspase-3 activity, and the expression of genes and proteins related to inflammatory and antioxidant defenses and cytoskeleton rearrangement in healthy human fibroblasts (BJ) and a human lung carcinoma cell line (A549). The cells were exposed to increasing concentrations (1 nM to 100 µM) of the studied compounds for 24 h and 48 h. A decrease in the metabolic activity in the BJ and A549 cell lines was induced by both compounds at a concentration range from 10 to 100 µM. Both compounds decreased the mRNA expression of NRF2 (nuclear factor erythroid 2-related factor 2) and ß-actin in the BJ cells. Interestingly, a significant decrease in the level of NF-κB gene and protein expression was detected in the BJ cell line, suggesting a direct impact of the studied compounds on the inhibition of inflammation. However, more studies are needed due to the ability of Les-45 and Les-247 to interfere with the tubulin/actin cytoskeleton, i.e., a critical system existing in eukaryotic cells.

Engagement of specific intracellular pathways in the inflammation-based reprotoxicity effect of small-size silver nanoparticles on spermatogonia and spermatocytes invitro cell models.

Male infertility is a serious ongoing problem, whose causes have not yet been clearly identified. However, since human exposure to silver nanoparticles (AgNPs) has recently increased due to their beneficial properties, the present study aimed to determine the impact of small-size AgNPs on mouse spermatogonia (GC-1 spg) and spermatocytes [GC-2 spd(ts)] in vitro models as well as the ability of these nanostructures to induce inflammation. The results showed a significant dose- and time-dependent decrease in the metabolic activity in both cell models, which was correlated with an increase in the intracellular ROS level. Moreover, increased activity of caspase-9 and -3, together with enhanced expression of CASP3 and p(S15)-p53 proteins, was detected. Further studies indicated a decrease in ΔΨm after the AgNP-treatment, which proves induction of apoptosis with engagement of an intrinsic pathway. The PARP1 protein expression, the activity and protein expression of antioxidant enzymes, the GSH level, and the increased level of p-ERK1/2 indicate not only the engagement of DNA damage but also the occurrence of oxidative stress. The small-size AgNPs were able to induce inflammation, proved by increased protein expression of NF-κB, p-IκBα, and NLRP3, which indicate damage to spermatogonia and spermatocyte cells. Moreover, the PGC-1α/PPARγ and NRF2/Keap1 pathways were engaged in the observed effect. The spermatogonial cells were characterized by a stronger inflammation-based response to AgNPs, which may be correlated with the TNFα/TRAF2-based pathway. Summarizing, the obtained results prove that AgNPs impair the function of testis-derived cells by inducing the redox imbalance and inflammation process; therefore, these NPs should be carefully implemented in the human environment.

Triclosan affects steroidogenesis in mouse primary astrocytes in vitro with engagement of Sirtuin 1 and 3.

Triclosan (TCS) is a widely used antimicrobial, antifungal, and antiviral agent. To date, it has been reported that TCS can enter the human body and disrupt hormonal homeostasis. Therefore, the aim of our paper was to evaluate the impact of TCS on astrocytes, i.e. a crucial population of cells responsible for steroid hormone production. Our data showed that, in mouse primary astrocyte cultures, TCS can act as an endocrine disrupting chemical through destabilization of the production or secretion of progesterone (P4), testosterone (T), and estradiol (E2). TCS affects the mRNA expression of enzymes involved in neurosteroidogenesis, such as Cyp17a1, 17ß-Hsd, and Cyp19a1. Our data showed that a partial PPARγ agonist (honokiol) prevented changes in Cyp17a1 mRNA expression caused by TCS. Similarly, honokiol inhibited TCS-stimulated P4 release. However, rosiglitazone (classic PPARγ agonist) or GW9662 (PPARγ antagonist) had a much stronger effect. Therefore, we believe that the changes observed in the P4, T, and E2 levels are a result of dysregulation of the activity of the aforementioned enzymes, whose expression can be affected by TCS through a Pparγ-dependent pathway. TCS was found to decrease the aryl hydrocarbon receptor (AhR) and Sirtuin 3 protein levels, which may be the result of the activation of the these proteins. Since our study showed dysregulation of the production or secretion of neurosteroids in astrocytes, it can be concluded that TCS reaching the brain may contribute to the development of neurodegenerative diseases in which an abnormal amount of neurosteroids is observed.

Elastin-derived peptides (EDPs) as a potential pro-malignancy factor in human leukemia cell lines.

The extracellular matrix (ECM) is currently considered to be an important factor influencing the migration and progression of cancer cells. Therefore, the aim of our study was to investigate the mechanism of action of elastin-derived peptides in cancerous cells derived from the immunological system, i.e., HL-60, K562, and MEG-A2 cell lines. Moreover, an attempt to clarify the involvement of c-SRC kinase in EDP mechanism of action was also undertaken. Our data show that the VGVAPG and VVGPGA peptides are not toxic in the studied cell lines. Moreover, due to the involvement of KI67 and PCNA proteins in the cell cycle and proliferation, we can assume that neither peptide stimulates cell proliferation. Our data suggest that both peptides could initiate the differentiation process in all the studied cell lines. However, due to the different origins (HL-60 and K562-leukemic cell line vs. MEG-A2-megakaryoblastic origin) of the cell lines, the mechanism may differ. The increase in the ELANE mRNA expression noted in our experiments may also suggest enhancement of the migration of the tested cells. However, more research is needed to fully explain the mechanism of action of the VGVAPG and VVGPGA peptides in the HL-60, K562, and MEG-A2 cell lines. HIGHLIGHTS: • VGVAPG and VVGPGA peptides do not affect the metabolic activity of HL-60, K562, and MEG-A2 cells. • mTOR and PPARγ proteins are involved in the mechanism of action of VGVAPG and VVGPGA peptides. • Both peptides may initiate differentiation in HL-60, K562, and MEG-A2 cell lines.

Role of 4-Thiazolidinone-Pyrazoline/Indoline Hybrids Les-4369 and Les-3467 in BJ and A549 Cell Lines.

Cancer is one of the most important problems of modern societies. Recently, studies have reported the anticancer properties of rosiglitazone related to its ability to bind peroxisome proliferator receptor γ (PPARγ), which has various effects on cancer and can inhibit cell proliferation. In this study, we investigated the effect of new 4-thiazolidinone (4-TZD) hybrids Les-4369 and Les-3467 and their effect on reactive oxygen species (ROS) production, metabolic activity, lactate dehydrogenase (LDH) release, caspase-3 activity, and gene and protein expression in human foreskin fibroblast (BJ) cells and lung adenocarcinoma (A549) cells. The ROS production and caspase-3 activity were mainly increased in the micromolar concentrations of the studied compounds in both cell lines. Les-3467 and Les-4369 increased the mRNA expression of PPARG, P53 (tumor protein P53), and ATM (ATM serine/threonine kinase) in the BJ cells, while the mRNA expression of these genes (except PPARG) was mainly decreased in the A549 cells treated with both of the tested compounds. Our results indicate a decrease in the protein expression of AhR, PPARγ, and PARP-1 in the BJ cells exposed to 1 µM Les-3467 and Les-4369. In the A549 cells, the protein expression of AhR, PPARγ, and PARP-1 increased in the treatment with 1 µM Les-3467 and Les-4369. We have also shown the PPARγ modulatory properties of Les-3467 and Les-4369. However, both compounds prove weak anticancer properties evidenced by their action at high concentrations and non-selective effects against BJ and A549 cells.

Interaction Between Aging-Related Elastin-Derived Peptide (VGVAPG) and Sirtuin 2 and its Impact on Functions of Human Neuron Cells in an In Vitro Model.

Elastin is a stable protein present in many tissues, including brain tissues, and is one of the most long-life proteins with a half-life of approximately 70 years. The peptide with a Val-Gly-Val-Ala-Pro-Gly (VGVAPG) amino acid sequence is released during elastin decay, which correlates with aging-related neurodegeneration. A recent study has shown enhanced protein expression of Sirtuin 2 (SIRT2 - one of the redox homeostatic factors) in aged rodent brains, while the correlation between VGVAPG and SIRT2 has never been evaluated so far. Therefore, the study aimed to determine the impact of the VGVAPG hexapeptide on SIRT2 and neuronal functions in differentiated SH-SY5Y cells at the gene and protein expression levels. The present results showed that VGVAPG caused a 52.69% decrease in the level of reactive oxygen species (ROS), as in the case of neurons treated with AGK2 (Sirtuin 2 inhibitor) after 24h and 48h. Furthermore, a decrease in superoxide dismutase (SOD) activity was observed. The SIRT2 gene expression was found to fluctuate after 6h and 24h as a result of the exposure to the VGVAPG peptide. In turn, a decrease in the PPARγ, P53, SOD2, and CAT mRNA expression was shown in VGVAPG-treated cells. Additionally, an increase in the Sirtuin 2 protein expression was recorded after 24h and 48h in the VGVAPG peptide-treated neurons. Last but not least, the decrease in the level of acetylation of α-tubulin after the hexapeptide treatment was correlated with shortening of neurites, which may indicate the destabilization of the microtubule and ROS-independent induction of neurodegeneration.

Comparison of the Antioxidant and Cytoprotective Properties of Extracts from Different Cultivars of Cornus mas L.

Cornus mas L. is a rich source of vitamin C and polyphenols. Due to their health-benefit properties, C. mas L. extracts have been used in, e.g., dermatology and cosmetology, and as a food supplement. Peroxisome proliferator-activated receptor gamma (PPARγ) and its co-activator (PGC-1α) are now suspected to be the main target of active substances from C. mass extracts, especially polyphenols. Moreover, the PPARγ pathway is involved in the development of different diseases, such as type 2 diabetes mellitus (DM2), cancers, skin irritation, and inflammation. Therefore, the aim of the present study was to evaluate the PPARγ pathway activation by the most popular water and ethanol extracts from specific C. mas L. cultivars in an in vitro model of the human normal fibroblast (BJ) cell line. We analyzed the content of biologically active compounds in the extracts using the UPLC-DAD-MS technique and revealed the presence of many polyphenols, including gallic, quinic, protocatechuic, chlorogenic, and ellagic acids as well as iridoids, with loganic acid being the predominant component. In addition, the extracts contained cyanidin 3-O-galactoside, pelargonidin 3-O-glucoside, and quercetin 3-glucuronide. The water-ethanol dark red extract (DRE) showed the strongest antioxidant activity. Cytotoxicity was assessed in a normal skin cell line, and positive effects of all the extracts with concentrations ranging from 10 to 1000 µg/mL on the cells were shown. Our data show that the studied extracts activate the PPARγ/PGC-1α molecular pathway in BJ cells and, through this mechanism, initiate antioxidant response. Moreover, the activation of this molecular pathway may increase insulin sensitivity in DM2 and reduce skin irritation.

Current state of knowledge of triclosan (TCS)-dependent reactive oxygen species (ROS) production.

Triclosan (TCS) is widely used in a number of industrial and personal care products. This molecule can induce reactive oxygen species (ROS) production in various cell types, which results in diverse types of cell responses. Therefore, the aim of the present study was to summarize the current state of knowledge of TCS-dependent ROS production and the influence of TCS on antioxidant enzymes and pathways. To date, the TCS mechanism of action has been widely investigated in non-mammalian organisms that may be exposed to contaminated water and soil, but there are also in vivo and in vitro studies on plants, algae, mammalians, and humans. This literature review has revealed that mammalian organisms are more resistant to TCS than non-mammalian organisms and, to obtain a toxic effect, the effective TCS dose must be significantly higher. The TCS-dependent increase in the ROS level causes damage to DNA, protein, and lipids, which together with general oxidative stress leads to cell apoptosis or necrosis and, in the case of cancer cells, faster oncogenesis and even initiation of oncogenic transformation in normal human cells. The review presents the direct and indirect TCS action through different receptor pathways.

The elastin-derived peptide (VGVAPG) activates autophagy in neuroblastoma (SH-SY5Y) cells via peroxisome proliferator-activated receptor gamma (PPARγ).

Autophagy is a self-degradative process important for balancing the sources of energy and involved in the development of Alzheimer's disease (AD). To date, a number of papers have shown that elastin-derived peptides (EDPs) affect the expression and activation of peroxisome proliferator-activated receptor gamma (PPARγ), which is crucial for the development of AD and autophagy initiation. Therefore, the aim of the present study was to determine whether EDPs with a Val-Gly-Val-Ala-Pro-Gly (VGVAPG) amino acid sequence activate the autophagic process in undifferentiated SH-SY5Y human neuroblastoma cells. Our study is the first to show that EDPs with the VGVAPG sequence initiate the autophagy process in the undifferentiated SH-SY5Y cell line exhibiting a number of features of normal neuroblasts. In particular, we observed in our study that VGAVPG peptide increased ULK1, AKT, PPARγ, and LC3B protein expression. Moreover, our experiments with the agonist (rosiglitazone) and antagonist (GW9662) of PPARγ confirm that the studied EDP acts through the PPARγ pathway affecting mTOR and finally autophagy. Some studies have shown that autophagy disturbances are involved in the development of AD. Therefore, we believe that our study will provide new evidence of the possible involvement of EDPs (especially VGVAPG) in the development of AD.

Results of systematic patient outcome monitoring: Does post-dilatation during angiography-guided percutaneous coronary intervention improve clinical outcomes?

OBJECTIVES: This study evaluates clinical outcomes after implementing a liberal post-dilatation strategy during PCI. BACKGROUND: Post-dilatation after percutaneous coronary intervention (PCI) is performed to achieve optimal stent expansion and reduce complications. However, its prognostic effects are unclear and conflicting. METHODS: This study is a pre-post-intervention analysis of two cohorts, before (2015-2017) and after (2018-2020) implementation of a liberal post-dilatation strategy. The primary end point consisted of major adverse cardiovascular events (MACE) at 30 days. Secondary end points consisted of the individual components of the primary end point as well as 1 year mortality and target vessel revascularization. RESULTS: A total of 10,153 patients were included: 5,383 in the pre-cohort and 4,770 in the post-cohort. The 30-day MACE was 5.00% in the pre-cohort and 4.09% in the post-cohort (p = 0.008; OR 0.75 (CI 0.61-0.93)). There was a significant difference between the pre- and post-cohort in 30-day mortality, respectively, 2.91% and 2.25% (p = .01; OR 0.70 (CI 0.53-0.93)), and MI at 30 days, 1.17% versus 0.59% (p = .003; OR 0.49 (CI 0.31-0.78)). At 1 year, there was a significant difference in mortality between the pre-cohort, 5.84%, and post-cohort, 5.19% (p = .02; OR 0.79 (CI 0.66-0.96)). CONCLUSIONS: A liberal post-dilatation strategy after PCI was associated with a significant decrease in 30-day MACE, 30-day MI, 30-day mortality, and 1-year mortality. Future studies are warranted to validate the causality between post-dilatation and improvement of clinical outcomes.

Curcuminoid Chalcones: Synthesis and Biological Activity against the Human Colon Carcinoma (Caco-2) Cell Line.

BACKGROUND: There are many current scientific reports on the synthesis of various derivatives modelled on the structure of known small-molecular and natural bioactive compounds. Curcuminoid chalcones are an innovative class of compounds with significant therapeutic potential against various diseases and they perfectly fit into the current trends in the search for new biologically active substances. AIM: The aim of this study was to design and synthesise a series of curcuminoid chalcones. OBJECTIVE: The objective of this scientific paper was to synthesise twelve curcuminoid chalcones and confirm their structures using spectral methods. Additionally, the biological activity of three of the synthesised compounds was evaluated using various assays, and their anticancer properties and toxicity were studied. METHODS: The proposed derivatives were obtained via the Claisen-Schmidt reaction of selected acetophenones and aldehydes in various conditions using both classical methods: the solutions and solvent-free microwave (MW) or ultrasound (US) variants. The most optimal synthetic method for the selected curcuminoid chalcones was the classical Claisen-Schmidt condensation in an alkaline (NaOH) medium. Spectral methods were used to confirm the structures of the compounds. The resazurin reduction assay, caspase-3 activity assay, and RT-qPCR method were performed, followed by measurements of the intracellular reactive oxygen species (ROS) level and the lactate dehydrogenase (LDH) release level. RESULTS: Twelve designed curcuminoid chalcones were successfully synthesized and structurally confirmed by NMR, MS, and IR spectroscopy. Examination of the anticancer activity was carried out for the three most interesting chalcone products. CONCLUSION: The results suggested that compound 3a increased the metabolism and/or proliferation of the human colon carcinoma (Caco-2) cell line, while compounds 3b and 3f showed significant toxicity against the Caco-2 cell line. Overall, the preliminary results suggested that compound 3b exhibited the most favorable anticancer activity.

Dual mechanism of silver nanoparticle-mediated upregulation of adipogenesis in mouse fibroblasts (3T3-L1) in vitro.

Silver nanoparticles (AgNPs) are widespread in the environment due to the increase in their application e.g. in medicine as part of hard-to-heal wound dressings. Many studies have revealed easy diffusion of AgNPs into deep skin layers through damaged epidermis and contact with e.g. fibroblasts. Therefore, the aim of this study was to evaluate the impact of small-size AgNPs (10 nm) in ppm concentrations on the adipogenesis process in mouse embryo fibroblasts (3T3-L1). The results showed a decrease in the metabolic activity, followed by an increase in the reactive oxygen species (ROS) level in a dose- and time-dependent manner (0-20 ppm). The increased caspase-3 activity was observed only at the highest concentration (20 ppm) of AgNPs. Further analysis showed the ability of the tested NPs to increase the lipid accumulation in adipocytes, similar to ROSI [peroxisome proliferator-activated receptor gamma (PPARγ) agonist], measured by Oil-Red-O staining. Moreover, the analyses evidenced the ability of AgNPs to increase the lipoxygenase activity and malondialdehyde levels, which is probably based on ROS-dependent enhancement of lipid hydroperoxidation. Lastly, a significant increase in the PPARγ, Adiponectin, Resistin, Vegf, and Serpine mRNA expression was shown 6 h after the induction of the differentiation process. Based on the obtained results, it can be concluded that small-size AgNPs increase adipogenesis via ROS- and PPARγ-based mechanisms with potential engagement of crosstalk with the aryl hydrocarbon receptor, which is important due to the widespread application of AgNPs in medicine. However, more studies are needed to elucidate the full mechanism of these NPs in the tested cell model in depth.

Involvement of the aryl hydrocarbon receptor (AhR) in the mechanism of action of elastin-derived peptide (VGVAPG) and its impact on neurosteroidogenesis.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor from the family of basic helix-loop-helix transcription factors. Several studies have indicated an important role of AhR signaling pathways in senescence, aging, and neurodegenerative diseases. During aging, elastin is degraded and elastin-derived peptides (EDPs) are formed. EDPs have been detected in human blood, serum, and cerebrospinal fluid. Literature data suggest a role of EDPs in the development of neurodegenerative diseases. However, the impact of EDPs on the AhR signaling pathway has never been investigated. Therefore, the aim of our paper was to study the role of AhR in the mechanism of action of the VGVAPG peptide (one of the EDPs) in mouse primary astrocytes in vitro. Our experiments have shown that AhR plays an important role in the EDP mechanism of action in a model of mouse primary astrocytes. Moreover, due to the involvement of Sirt3, Pparγ, AhR, Glb1, Nf-κb1, Ece1, Ide, and Nepr genes and the production and release of neurosteroids, VGVAPG can accelerate the development of neurodegenerative diseases in which the proper metabolism of astrocytes is crucial. Furthermore, our studies have proved that AhR is likely involved in the co-control of the Sirt1, Glb1, Nf-κb1, Ece1, and Nepr expression in astrocytes.

Pyrrolidinedione-thiazolidinone hybrid molecules with potent cytotoxic effect in squamous cell carcinoma SCC-15 cells.

The hybrid heterocyclic molecules are perspective materials in the development of anticancer drugs. Here, the pyrrolidinedione-thiazolidinone hybrid molecules were designed as potent anticancer agents. This study aimed to investigate the cytotoxic effect of three derivatives 1-(4-hydroxyphenyl)-, 1-(4-chlorophenyl)- and 1-(4-bromophenyl)-3-[5-[2-chloro-3-(4-nitrophenyl)prop-2-enylidene]-4-oxo-2-thioxothiazolidine-3-yl]pyrrolidine-2,5-diones (Les-6287, Les-6294, and Les-6328, respectively), their effect on the production of the reactive oxygen species (ROS), apoptosis induction, and expression of genes - PPARγ, AHR, and NRFL2 - whose products are important in metabolism in human tongue squamous cell carcinoma cells of SCC-15 line. The results of resazurin reduction and lactate dehydrogenase (LDH) release assays proved the toxicity of the tested derivatives for the SCC-15 cells. Les-6287, Les-6294, and Les-6328 inhibited the viability of SCC-15 cells with the half-maximal effective concentration (EC50) in the range of 10.18-32.75 µM at 24 and 48 h treatment. These derivatives reduced the metabolism of SCC-15 cells with the half-maximal inhibitory concentration (IC50) of 6.72-39.85 µM at 24 and 48 h treatment. Les-6287, Les-6294, and Les-6328 reduced the metabolism of normal human keratinocytes of HaCaT line murine fibroblasts of Balb/c 3T3 line to a lesser extent. The compounds used in a range from 50 to 100 µM concentrations decreased ROS production in the SCC-15 cells. The derivatives Les-6287 and Les-6328 decreased the level of expression of mRNA of PPARγ, AHR, and NRFL2 genes in these cells at PPARγ siRNA knockdown and without it. Thus, the anticancer effect of studied hybrid pyrrolidinedione-thiazolidinones in the SCC-15 carcinoma cells is accompanied by a reduction of their metabolic activity and ROS level, and increase in caspase 3 activity. However, these changes are not the result of direct interaction of Les-6287, Les-6294, and Les-6328 with the PPARγ molecule.

Involvement of the peroxisome proliferator-activated receptor gamma (Pparγ) and matrix metalloproteinases-2 and -9 (Mmp-2 and -9) in the mechanism of action of di(2-ethylhexyl)phthalate (DEHP) in cultured mouse brain astrocytes and neurons.

Di(2-ethylhexyl)phthalate (DEHP) is one of the most widely used phthalates in industry. It has been shown that, after entering the body, DEHP has the ability to cross the blood-placenta and blood-brain barriers. One of the proposed mechanisms of action of DEHP is the activation of peroxisome proliferator-activated receptors (PPARs). Many different functions of PPARγ in cells have been demonstrated, one of which is the modulation of the activation of matrix metalloproteinases (MMPs). The aim of this study was to investigate the role of Pparγ, Mmp-2, and Mmp-9 in the mechanism of action of DEHP. The experiments were performed on in vitro primary murine neurons and astrocytes. The results showed that DEHP has a pro-apototic effect on neurons, causing an increase in caspase-3 activity and in the number of apoptotic bodies. However, in astrocytes, the increase in caspase-3 activity was not related to the apoptosis process, as no increase in the formation of apoptotic bodies was observed. Moreover, DEHP increased the proliferation of astrocytes, which was confirmed by the increase in the amount and expression of the Ki-67 protein. In astrocytes, DEHP decreased the expression of the Pparγ and Mmp-9 proteins but increased the expression of the Mmp-2 protein. In DEHP neurons, it increased the expression of the Pparγ protein but decreased the expression of the Mmp-2 and Mmp-9 proteins.

Suppression of sonic hedgehog pathway-based proliferation in glioblastoma cells by small-size silver nanoparticles in vitro.

Glioblastomas (GBs) are one of the most aggressive and invasive intracranial cancers. Recently, it has been postulated that, among other factors, the hedgehog (HH) pathway may be a key factor in this phenomenon. Moreover, it has been reported that small-size silver nanoparticles (AgNPs) are characterized by a high cytotoxic effect towards GBs. However, their effect on the sonic hedgehog (SHH) pathway has never been demonstrated in any cancer cells. Therefore, the aim of the present study was to evaluate the impact of the anti-proliferative properties of 5-nm AgNPs on the SHH pathway in the GB cell line (U-87MG) in vitro. The results showed a time- and dose-dependent decrease in the metabolic activity in the U-87MG cells treated with AgNPs, with IC50 reaching 30.41 and 21.16 µg/mL after 24 h and 48 h, respectively, followed by an increase in the intracellular reactive oxygen species (ROS) level. The co-treatment of the cells with AgNPs and Robotnikinin (SHH inhibitor) abolished and/or strengthened the effect of AgNPs, especially on the SHH mRNA levels and on the PCNA, PTCH1, Gli1, and SUFU protein levels. Interestingly, no changes in the level of ERK1/2, Akt, and SRC kinase protein expression were detected, suggesting a direct impact of AgNPs and/or ROS on the inhibition of the canonical SHH pathway. However, more studies are needed due to the increase in the mTOR protein expression after the treatment of the cells with AgNPs, as in the Robotnikinin treatment. In conclusion, small-size AgNPs are able to inhibit the proliferation of GB cells in vitro by suppressing the canonical SHH pathway.

Machine learning-based prediction of in-hospital death for patients with takotsubo syndrome: The InterTAK-ML model.

AIMS: Takotsubo syndrome (TTS) is associated with a substantial rate of adverse events. We sought to design a machine learning (ML)-based model to predict the risk of in-hospital death and to perform a clustering of TTS patients to identify different risk profiles. METHODS AND RESULTS: A ridge logistic regression-based ML model for predicting in-hospital death was developed on 3482 TTS patients from the International Takotsubo (InterTAK) Registry, randomly split in a train and an internal validation cohort (75% and 25% of the sample size, respectively) and evaluated in an external validation cohort (1037 patients). Thirty-one clinically relevant variables were included in the prediction model. Model performance represented the primary endpoint and was assessed according to area under the curve (AUC), sensitivity and specificity. As secondary endpoint, a K-medoids clustering algorithm was designed to stratify patients into phenotypic groups based on the 10 most relevant features emerging from the main model. The overall incidence of in-hospital death was 5.2%. The InterTAK-ML model showed an AUC of 0.89 (0.85-0.92), a sensitivity of 0.85 (0.78-0.95) and a specificity of 0.76 (0.74-0.79) in the internal validation cohort and an AUC of 0.82 (0.73-0.91), a sensitivity of 0.74 (0.61-0.87) and a specificity of 0.79 (0.77-0.81) in the external cohort for in-hospital death prediction. By exploiting the 10 variables showing the highest feature importance, TTS patients were clustered into six groups associated with different risks of in-hospital death (28.8% vs. 15.5% vs. 5.4% vs. 1.0.8% vs. 0.5%) which were consistent also in the external cohort. CONCLUSION: A ML-based approach for the identification of TTS patients at risk of adverse short-term prognosis is feasible and effective. The InterTAK-ML model showed unprecedented discriminative capability for the prediction of in-hospital death.