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Objective: CD73 (ecto-5′-nucleotidase, NT5E), a cell-surface enzyme converting 5′-AMP to adenosine, is crucial for cancer progression. However, its role in the tumorigenesis process remains mostly obscure. We aimed to demonstrate CD73's role in breast cancer (BC) tumorigenesis through metabolic rewiring of fatty acid metabolism, a process recently indicated to be regulated by BC major prognostic markers, hormone receptors (HR) for estrogen (ER), and progesterone (PR). Methods: A murine model of chemically induced mammary gland tumorigenesis was applied to analyze CD73 knock-out (KO)-induced changes at the transcriptome (RNA-seq), proteome (IHC, WB), and lipidome (GC-EI-MS) levels. CD73 KO-induced changes were correlated with scRNA-seq and bulk RNA-seq data for human breast tissues and BCs from public collections and confirmed at the proteome level with IHC or WB analysis of BC tissue microarrays and cell lines. Results: CD73 KO delayed the onset of HR/PR-negative mammary tumors in a murine model. This delay correlated with increased expression of genes related to biosynthesis and β-oxidation of fatty acids (FAs) in the CD73 KO group at the initiation stage. STRING analysis based on RNA-seq data indicated an interplay between CD73 KO, up-regulated expression of PR-coding gene, and DEGs involved in FA metabolism, with PPARγ, a main regulator of FA synthesis, as a main connective node. In epithelial cells of mammary glands, PPARγ expression correlated with CD73 at the RNA level. With cancer progression, CD73 KO increased the levels of PUFAn3/6 (polyunsaturated omega 3/6 FAs), known ligands of PPARγ and target for lipid peroxidation, which may lead to oxidative DNA damage. It correlated with the downregulation of genes involved in cellular stress response (Mlh1, Gsta3), PR–or CD73-dependent changes in the intracellular ROS levels and expression or activation of proteins involved in DNA repair or oxidative stress response in mammary tumor or human BC cell lines, increased tumor mutational burden (TMB) and genomic instability markers in CD73 low HR-negative human BCs, and the prolonged onset of tumors in the CD73 KO HR/PR-negative group. Conclusions: CD73 has a significant role in tumorigenesis driving the reprogramming of lipid metabolism through the regulatory loop with PR and PPARγ in epithelial cells of mammary glands. Low CD73 expression/CD73 KO might enhance mutational burden by disrupting this regulatory loop, delaying the onset of HR-negative tumors. Our results support combining therapy targeting the CD73-adenosine axis and tumor lipidome against HR-negative tumors, especially at their earliest developmental stage.

IntroductionRecent years, microbiota-associated aspects have been analysed in multiple disorders regarding cancers. Existing evidence pints that gut microorganisms might take part in tumour origin and therapy efficacy. Nevertheless, to date, data on faecal metabolomics in cancer patients is still strongly limited. Therefore, we aimed to analyse gut untargeted metabolome in gastrointestinal cancer patients (i.e., gastric and colorectal cancer).Patients and methodsThere were 12 patients with either gastric (n=4) or colorectal cancer (n=8) enrolled and 8 analysed (n=4 each). Stool samples were collected prior to anti-cancer treatments. Untargeted metabolomics analyses were conducted by means of mass spectrometry.ResultsA plethora of metabolites in cancer patients we analysed were noted, with higher homogenity in case of gastric cancer patients. We found that the level of Deoxyguanosine,m/z 266.091,[M-H]-, Uridine,m/z 245.075,[M+H]+, Deoxyguanosine,m/z 268.104,[M]+, 3-Indoleacetic acid,m/z 176.07,[M+H]+, Indoxyl,m/z 132.031,[M-H]-, L-Phenylalanine,m/z 164.073,[M-H]-, L-Methionine,m/z 150.058,[M+NH4]+, was significantly higher in colorectal cancer patients and Ethyl hydrogen malonate,m/z 133.031,[M+H]+ in gastric cancer.ConclusionThe overall insights into untargeted metabolomics showed that most often higher levels of analysed metabolites were detected in colorectal cancer patients compared to gastric cancer patients. The link between gut metabolome and both local and distal metastasis might exist, however it requires confirmation in further multi-centre studies regarding larger sample size.

Head and neck squamous cell carcinoma (HNSCC) exhibits significant genetic heterogeneity and primarily concerns the oral cavity and oropharynx. These cancers occur more frequently in men with a 5-year survival rate below 50%. Major risk factors include human papilloma virus (HPV) (notably type 16), Epstein–Barr virus, tobacco, alcohol, and poor oral hygiene with approximately 4.5% of global cancers linked to HPV. Notably, differences in the microbiome between healthy individuals and patients with head and neck cancers (HNCs) have been identified. Recent studies highlight the significance of certain oral microbes in risk assessment and the potential of the microbiome as a biomarker for HNCs. Additionally, role of the microbiome in metastasis has been acknowledged. Treatment for HNCs includes local methods, such as surgery and radiotherapy, and systemic approaches, such as immunotherapy. Numerous side effects accompany these treatments. Emerging research suggests the beneficial role of preoperative immunonutrition and probiotics in patient outcomes, emphasizing the influence of the microbiome on treatment efficacy. This review explores the reciprocal effects of HNC treatment and the gut microbiome using radiotherapy, brachytherapy, surgery, immunotherapy, and chemotherapy.

ABSTRACTIntestinal bacteria are equipped with an enzyme apparatus that is involved in the active biotransformation of xenobiotics, including drugs. Pharmacomicrobiomics, a new area of pharmacology, analyses interactions between bacteria and xenobiotics. However, there is another side to the coin. Pharmacotherapeutic agents can significantly modify the microbiota, which consequently affects their efficacy. In this review, we comprehensively gathered scientific evidence on the interplay between anticancer therapies and gut microbes. We also underlined how such interactions might impact the host response to a given therapy. We discuss the possibility of modulating the gut microbiota to increase the effectiveness/decrease the incidence of adverse events during tumor therapy. The anticipation of the future brings new evidence that gut microbiota is a target of interest to increase the efficacy of therapy.

Abstract Nitric oxide (NO) is one of the most important molecules released by endothelial cells, and its antiatherogenic properties support cardiovascular homeostasis. Diminished NO bioavailability is a common hallmark of endothelial dysfunction underlying the pathogenesis of the cardiovascular disease. Vascular NO is synthesized by endothelial nitric oxide synthase (eNOS) from the substrate L-arginine (L-Arg), with tetrahydrobiopterin (BH4) as an essential cofactor. Cardiovascular risk factors such as diabetes, dyslipidemia, hypertension, aging, or smoking increase vascular oxidative stress that strongly affects eNOS activity and leads to eNOS uncoupling. Uncoupled eNOS produces superoxide anion (O2 −) instead of NO, thus becoming a source of harmful free radicals exacerbating the oxidative stress further. eNOS uncoupling is thought to be one of the major underlying causes of endothelial dysfunction observed in the pathogenesis of vascular diseases. Here, we discuss the main mechanisms of eNOS uncoupling, including oxidative depletion of the critical eNOS cofactor BH4, deficiency of eNOS substrate L-Arg, or accumulation of its analog asymmetrical dimethylarginine (ADMA), and eNOS S-glutathionylation. Moreover, potential therapeutic approaches that prevent eNOS uncoupling by improving cofactor availability, restoration of L-Arg/ADMA ratio, or modulation of eNOS S-glutathionylation are briefly outlined.

The gut microbiome has been increasingly understood to play a critical role in carcinogenesis and cancer disease progression. The most recent research advancements have shown that different tools of microbiota manipulation contribute to gut microbiome–immune–oncology axis modulation, offering exciting opportunities for targeted interventions aimed at improving the efficacy of established anti-cancer therapy. Postbiotics are a new entry among the biotics showing beneficial effects on human health while not requiring living cells to obtain the health effect and therefore not subjected to food safety rules for live microorganisms. Postbiotics are recently defined as the “preparation of inanimate microorganisms and/or their components that confers a health benefit on the host” and have gradually become the focus of the scientific community. Since the beginning of research on this topic, numerous studies about postbiotics have been proven to strengthen the gut barrier, reduce inflammation, and promote antimicrobial activity. However, research on the potential application of cancer therapy is still at the early stages of its efforts to uncover all the secrets surrounding postbiotics. This review aims to increase our understanding of the anti-cancer effect of postbiotics throughout a “bibliographic journey” on the biological activity of their components, including exopolysaccharides, cell wall fragments, tryptophan metabolites, enzymes, bacterial lysates, extracellular vesicles, and short-chain fatty acids, highlighting their perspective as a new supportive therapeutic method of treatment and identifying the literature gaps where further research is needed.

The role of endothelial nitric oxide synthase (eNOS) in the regulation of a variety of biological processes is well established, and its dysfunction contributes to brain pathologies, including schizophrenia or Alzheimer’s disease (AD). Positive allosteric modulators (PAMs) of metabotropic glutamate (mGlu) receptors were shown to be effective procognitive compounds, but little is known about their impact on eNOS expression and stability. Here, we investigated the influence of the acute and chronic administration of LY487379 or CDPPB (mGlu2 and mGlu5 PAMs), on eNOS expression in the mouse brain and the effect of the joint administration of the ligands with nitric oxide (NO) releasers, spermineNONOate or DETANONOate, in different combinations of doses, on MK-801- or scopolamine-induced amnesia in the novel object recognition (NOR) test. Our results indicate that both compounds provoked eNOS monomer formation, and CDPPB at a dose of 5 mg/kg exaggerated the effect of MK-801 or scopolamine. The coadministration of spermineNONOate or DETANONOate enhanced the antiamnesic effect of CDPPB or LY487379. The best activity was observed for ineffective or moderate dose combinations. The results indicate that treatment with mGluR2 and mGluR5 PAMs may be burdened with the risk of promoting eNOS uncoupling through the induction of dimer dissociation. Administration of the lowest possible doses of the compounds with NO• donors, which themselves have procognitive efficacy, may be proposed for the treatment of schizophrenia or AD.

The association between cadmium and breast cancer remains unexplained due to inconsistent epidemiological data and unknown underlying mechanisms. This study aimed to assess the relationship between environmental exposure to cadmium and the Warburg effect in breast cancer and, thus, its possible interference with breast cancer treatment. The observational study in two groups of breast cancer patients indicated a positive correlation between urinary cadmium concentration and tumor expression of HIF1A (a master regulator of the Warburg effect). Further explanatory research in MCF-7 cells showed no impact of cadmium exposure on molecular and biochemical markers of the Warburg effect. However, long-term exposure to a low and environmentally relevant concentration of cadmium led to the accumulation of the metal in MCF-7 cells and decreased their sensitivity to tamoxifen. To conclude, the association between cadmium and the Warburg effect was suggested in the observational study, although not confirmed in vitro. Nevertheless, cadmium seems to interfere with tamoxifen treatment which deserves further investigation in terms of its possible implication in intrinsic resistance to hormone therapy.

Microbiome is a keystone polymicrobial community that coexist with human body in a beneficial relationship. These microorganisms enable the human body to maintain homeostasis and take part in mechanisms of defense against infection and in the absorption of nutrients. Even though microbiome is involved in physiologic processes that are beneficial to host health, it may also cause serious detrimental issues. Additionally, it has been proven that bacteria can migrate to other human body compartments and colonize them even although significant structural differences with the area of origin exist. Such migrations have been clearly observed when the causes of genesis and progression of colorectal cancer (CRC) have been investigated. It has been demonstrated that the oral microbiome is capable of penetrating into the large intestine and cause impairments leading to dysbiosis and stimulation of cancerogenic processes. The main actors of such events seem to be oral pathogenic bacteria belonging to the red and orange complex (regarding classification of bacteria in the context of periodontal diseases), such as Porphyromonas gingivalis and Fusobacterium nucleatum respectively, which are characterized by significant amount of cancerogenic virulence factors. Further examination of oral microbiome and its impact on CRC may be crucial on early detection of this disease and would allow its use as a precise non-invasive biomarker.

Efficient brain function requires as much as 20% of the total oxygen intake to support normal neuronal cell function. This level of oxygen usage, however, leads to the generation of free radicals, and thus can lead to oxidative stress and potentially to age-related cognitive decay and even neurodegenerative diseases. The regulation of this system requires a complex monitoring network to maintain proper oxygen homeostasis. Furthermore, the high content of mitochondria in the brain has elevated glucose demands, and thus requires a normal redox balance. Maintaining this is mediated by adaptive stress response pathways that permit cells to survive oxidative stress and to minimize cellular damage. These stress pathways rely on the proper function of the endoplasmic reticulum (ER) and the activation of the unfolded protein response (UPR), a cellular pathway responsible for normal ER function and cell survival. Interestingly, the UPR has two opposing signaling pathways, one that promotes cell survival and one that induces apoptosis. In this narrative review, we discuss the opposing roles of the UPR signaling pathways and how a better understanding of these stress pathways could potentially allow for the development of effective strategies to prevent age-related cognitive decay as well as treat neurodegenerative diseases.

The Morris water maze (MWM) is regarded as one of the most popular tests for detecting spatial memory in rodents. Long-term potentiation and cGMP synthesis seem to be among the crucial factors involved in this type of learning. Muscarinic (M1, M4, and M5 receptors) and metabotropic glutamate (mGlu) receptors are important targets in the search for antipsychotic drugs with the potency to treat cognitive disabilities associated with the disorder. Here, we show that muscarinic receptor activators (VU0357017, VU0152100, and VU0238429) and an mGlu2 receptor activator, LY487379, dose-dependently prevented the development of cognitive disorders as a result of MK-801 administration in the MWM. The dose-ranges of the compounds were as follows: VU0357017, 0.25, 0.5, and 1 mg/kg; VU0152100, 0.05, 0.25, and 1 mg/kg; VU0238429, 1, 5, and 20 mg/kg; and LY487379, 0.5, 3, and 5 mg/kg. The co-administration of LY487379 with each of the individual muscarinic receptor ligands showed no synergistic effect, which contradicts the results obtained earlier in the novel object recognition (NOR) test. MWM learning resulted in increased cGMP synthesis, both in the cortex and hippocampi, when compared to that in intact animals, which was prevented by MK-801 administration. The investigated compounds at the highest doses reversed this MK-801-induced effect. Neither the procedure nor the treatment resulted in changes in GluN2B-NMDA expression.

Endurance athletes have an increased risk of atrial remodeling and atrial arrhythmias. However, data regarding atrial adaptation to physical exercise in non-elite athletes are limited. Even less is known about atrial performance in women. We aimed to elucidate exercise-induced changes in atrial morphology and function in female amateur marathon runners using three-dimensional (3D) echocardiography and two-dimensional (2D) speckle tracking echocardiography (STE). The study group consisted of 27 female (40 ± 7 years) amateur athletes. Right (RA) and left atrial (LA) measures were assessed three times: 2–3 weeks before the marathon (stage 1), immediately after the run (stage 2), and 2 weeks after the competition (stage 3). Directly after the marathon, a remarkable RA dilatation, as assessed by RA maximal volume (RAVmax, 31.3 ± 6.8 vs. 35.0 ± 7.0 ml/m2; p = 0.008), with concomitant increase in RA contractile function [RA active emptying fraction (RA active EF), 27.7 ± 8.6 vs. 35.0 ± 12.1%; p = 0.014; RA peak atrial contraction strain (RA PACS) 13.8 ± 1.8 vs. 15.6 ± 2.5%; p = 0.016] was noticed. There were no significant changes in LA volumes between stages, while LA active EF (34.3 ± 6.4 vs. 39.4 ± 8.6%; p = 0.020), along with LA PACS (12.8 ± 2.1 vs. 14.9 ± 2.7%; p = 0.002), increased post race. After the race, an increase in right ventricular (RV) dimensions (RV end-diastolic volume index, 48.8 ± 11.0 vs. 60.0 ± 11.1 ml/m2; p = 0.001) and a decrease in RV function (RV ejection fraction, 54.9 ± 6.3 vs. 49.1 ± 6.3%; p = 0.006) were observed. The magnitude of post-race RV dilatation was correlated with peak RA longitudinal strain deterioration (r = −0.56, p = 0.032). The measured parameters did not differ between stages 1 and 3. In female amateur athletes, apart from RV enlargement and dysfunction, marathon running promotes transient biatrial remodeling, with more pronounced changes in the RA. Post-race RA dilatation and increment of the active contraction force of both atria are observed. However, RA reservoir function diminishes in those with post-race RV dilation.

Abstract Background Molecular imaging with molecularly targeted probes is a powerful tool for studying the spatio-temporal interactions between complex biological processes. The pivotal role of the receptor for advanced glycation end products (RAGE), and its involvement in numerous pathological processes, aroused the demand for RAGE-targeted imaging in various diseases. In the present study, we evaluated the use of a diagnostic imaging agent for RAGE quantification in an animal model of peripheral artery disease, a multimodal dual-labeled probe targeted at RAGE (MMIA-CML). Methods PAMAM dendrimer was conjugated with Nε-carboxymethyl-lysine (CML) modified albumin to synthesize the RAGE-targeted probe. A control untargeted agent carried native non-modified human albumin (HSA). Bifunctional p-SCN-Bn-NOTA was used to conjugate the 64Cu radioisotope. Surgical right femoral artery ligation was performed on C57BL/6 male mice. One week after femoral artery ligation, mice were injected with MMIA-CML or MMIA-HSA labeled with 64Cu radioisotope and 60 min later in vivo microPET-CT imaging was performed. Immediately after PET imaging studies, the murine hindlimb muscle tissues were excised and prepared for gene and protein expression analysis. RAGE gene and protein expression was assessed using real-time qPCR and Western blot technique respectively. To visualize RAGE expression in excised tissues, microscopic fluorescence imaging was performed using RAGE-specific antibodies and RAGE-targeted and -control MMIA. Results Animals subjected to PET imaging exhibited greater MMIA-CML uptake in ischemic hindlimbs than non-ischemic hindlimbs. We observed a high correlation between fluorescent signal detection and radioactivity measurement. Significant RAGE gene and protein overexpression were observed in ischemic hindlimbs compared to non-ischemic hindlimbs at one week after surgical ligation. Fluorescence microscopic staining revealed significantly increased uptake of RAGE-targeted nanoparticles in both ischemic and non-ischemic muscle tissues compared to the control probe but at a higher level in ischemic hindlimbs. Ischemic tissue exhibited explicit RAGE dyeing following anti-RAGE antibody and high colocalization with the MMIA-CML targeted at RAGE. Conclusions The present results indicate increased expression of RAGE in the ischemic hindlimb and enable the use of multimodal nanoparticles in both in vitro and in vivo experimental models, creating the possibility for imaging structural and functional changes with a RAGE-targeted tracer.

Abstract Osteosarcoma (OS) is one of the most malignant tumors of childhood and adolescence. Research on mitochondrial dynamics (fusion/fission) and biogenesis has received much attention in last few years, as they are crucial for death of cancer cells. Specifically, it was shown that increased expression of the cytoplasmic dynamin-related protein 1 (Drp1) triggers mitochondrial fission (division), which activates BAX and downstream intrinsic apoptosis, effectively inhibiting OS growth. In the presented study, human OS cells (metastatic 143B OS cell line) were incubated with 2-methoxyestradiol (2-ME) at both physiologically and pharmacologically relevant concentrations. Cell viability was determined by the MTT assay. Confocal microscopy and western blot methods were applied to examine changes in Drp1 and BAX protein levels. Mitochondrial Division Inhibitor 1, MDIVI-1, was used in the study to further examine the role of Drp1 in 2-ME-mediated mechanism of action. To determine quantitative and qualitative changes in mitochondria, electron microscopy was used. 2-ME at all used concentrations increased mitochondrial fission and induced autophagy in OS cells. At the concentration of 1 µM 2-ME increased the area density of mitochondria in OS cells. Subsequent, upregulated expression of Drp1 and BAX proteins by 2-ME strongly suggests the activation of the intrinsic apoptosis pathway. We further observed 2-ME-mediated regulation of glycolytic state of OS cells. Therefore, we suggest that changes of mitochondrial dynamics may represent a novel mechanism of anticancer action of 2-ME. This finding may open new approaches to improve the efficacy of chemotherapy in the treatment of OS, however, it has to be confirmed by in vivo studies.

Abstract Background Mastocytosis is a clinically heterogeneous, usually acquired disease of the mast cells with a survival time that depends on the onset of the disease and ranges from skin‐limited to systemic disease, including indolent and more aggressive variants. The crucial element in pathogenesis is the presence of oncogenic KIT somatic mutation D816V. Further epigenetic alterations are responsible for regulating the expression of genes. It is essential to identify indicators of disease progression, and the specific clinical picture to establish an appropriate therapeutic strategy. Objective The aim of this study was to analyze the relation of mastocytosis symptoms and epigenetic changes, and to identify epigenetic predictors of the disease. Methods Global DNA methylation profile analysis was performed in peripheral blood collected from 73 patients with indolent systemic mastocytosis (ISM) and 43 healthy adult volunteers. Levels of 5‐methylcytosine (5‐mC) and 5‐hydroxymethylcytosine (5‐hmC) were determined using an ELISA‐based method, while the methylation of the Alu and LINE‐1 repeats were assayed with the quantitative methylation‐specific PCR technique. A questionnaire interview was conducted among the study participants to collect data on possible epigenetic modifiers. Additionally, the methylation profile was compared between three human mast cell lines: ROSA KIT D816V, ROSA KIT WT, and HMC‐1.1 KIT V560G, in order to assess the association between KIT mutations and methylation profile. Results A significantly lower level of DNA hydroxymethylation (5‐hmC) in the blood was found in patients with ISM as compared to the controls (0.022% vs. 0.042%, p = 0.0001). Differences in the markers of global DNA methylation (5‐mC, Alu, LINE‐1) were not statistically significant, although they did indicate generally higher DNA methylation in patients with mastocytosis. The 5‐hmC level was significantly associated with allergy (p = 0.011) in patients with ISM, showing a higher level of 5‐hmC in patients with allergy as compared to patients without allergy. The in vitro study revealed significant differences between the studied cell lines at the level of 5‐mC, Alu, and LINE‐1. Conclusions This study confirms that epigenetic changes are involved in mastocytosis, and suggests that allergy may be an important epigenetic modifier of the disease. A possible association between KIT mutations and methylation status observed in human mast cell lines requires further investigation in human studies. Clinical Implications Epigenetic alterations are involved in mastocytosis pathology. The possible role of allergy as an important epigenetic modifier suggests the more impaired function of mast cells in ISM patients without allergy. Capsule summary Decreased DNA demethylation in the blood DNA of patients with ISM confirms that epigenetic alterations are involved in mastocytosis pathology. We observed a possible role of allergy as an important epigenetic modifier. There is a possible association between KIT mutations and the methylation status observed in human mast cell lines.

Gliomas are the most prevalent primary tumors of the central nervous system (CNS), accounting for over fifty percent of all primary intracranial neoplasms. Glioblastoma (GBM) is the most prevalent form of malignant glioma and is often incurable. The main distinguishing trait of GBM is the presence of hypoxic regions accompanied by enhanced angiogenesis. 2-Methoxyestradiol (2-ME) is a well-established antiangiogenic and antiproliferative drug. In current clinical studies, 2-ME, known as Panzem, was examined for breast, ovarian, prostate, and multiple myeloma. The SW1088 grade III glioma cell line was treated with pharmacological and physiological doses of 2-ME. The induction of apoptosis and necrosis, oxidative stress, cell cycle arrest, and mitochondrial membrane potential were established by flow cytometry. Confocal microscopy was used to detect DNA damage. The Western blot technique determined the level of nitric oxide synthase and heat shock proteins. Here, for the first time, 2-ME is shown to induce nitro-oxidative stress with the concomitant modulation of heat shock proteins (HSPs) in the SW1088 grade III glioma cell line. Crucial therapeutic strategies for GMB should address both cell proliferation and angiogenesis, and due to the above, 2-ME seems to be a perfect candidate for GBM therapy.

Urolithins are bioactive compounds generated in human and animal intestines because of the bacterial metabolism of dietary ellagitannins (and their constituent, ellagic acid). Due to their multidirectional effects, including anti-inflammatory, antioxidant, anti-cancer, neuroprotective, and antiglycative properties, urolithins are potential novel therapeutic agents. In this study, while considering the future possibility of using urolithins to improve podocyte function in diabetes, we assessed the results of exposing mouse podocytes cultured in normal (NG, 5.5 mM) and high (HG, 25 mM) glucose concentrations to urolithin A (UA) and urolithin B (UB). Podocytes metabolized UA to form glucuronides in a time-dependent manner; however, in HG conditions, the metabolism was lower than in NG conditions. In HG milieu, UA improved podocyte viability more efficiently than UB and reduced the reactive oxygen species level. Both types of urolithins showed cytotoxic activity at high (100 µM) concentration. The UA upregulated total and surface nephrin expression, which was paralleled by enhanced nephrin internalization. Regulation of nephrin turnover was independent of ambient glucose concentration. We conclude that UA affects podocytes in different metabolic and functional aspects. With respect to its pro-survival effects in HG-induced toxicity, UA could be considered as a potent therapeutic candidate against diabetic podocytopathy.

2-methoxyestradiol (2-ME) is a physiological anticancer compound, metabolite of 17β-estradiol. Previously, our group evidenced that from mechanistic point of view one of anticancer mechanisms of action of 2-ME is specific induction and nuclear hijacking of neuronal nitric oxide synthase (nNOS), resulting in local generation of nitro-oxidative stress and finally, cancer cell death.The current study aims to establish the substantial mechanism of generation of reactive nitrogen species by 2-ME. We further achieved to identify the specific reactive nitrogen species involved in DNA-damaging mechanism of 2-ME.The study was performed using metastatic osteosarcoma 143B cells. We detected the release of biologically active (free) nitric oxide (•NO) with concurrent measurements of peroxynitrite (ONOO−) in real time in a single cell of 143B cell line by using •NO/ONOO− sensitive microsensors after stimulation with calcium ionophore. Detection of nitrogen dioxide (•NO2) and determination of chemical rate constants were carried out by a stopped-flow technique. The affinity of reactive nitrogen species toward the guanine base of DNA was evaluated by density functional theory calculations. Expression and localization of nuclear factor NF-kB was determined using imaging cytometry, while cell viability assay was evaluated by MTT assay.Herein, we presented that 2-ME triggers pro-apoptotic signalling cascade by increasing cellular reactive nitrogen species overproduction – a result of enzymatic uncoupling of increased nNOS protein levels. In particular, we proved that ONOO− and •NO2 directly formed from peroxynitrous acid (ONOOH) and/or by auto-oxidation of •NO, are inducers of DNA damage in anticancer mechanism of 2-ME. Specifically, the affinity of reactive nitrogen species toward the guanine base of DNA, evaluated by density functional theory calculations, decreased in the order: ONOOH > ONOO− > •NO2 > •NO.Therefore, we propose to consider the specific inducers of nNOS as an effective tool in the field of chemotherapy.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. The initial stage of CVDs is characterized by endothelial dysfunction, defined as the limited bioavailability of nitric oxide (NO). Thus, any factors that interfere with the synthesis or metabolism of NO in endothelial cells are involved in CVD pathogenesis. It is well established that hypoxia is both the triggering factor as well as the accompanying factor in cardiovascular disease, and diminished tissue oxygen levels have been reported to influence endothelial NO bioavailability. In endothelial cells, NO is produced by endothelial nitric oxide synthase (eNOS) from L-Arg, with tetrahydrobiopterin (BH4) as an essential cofactor. Here, we discuss the mechanisms by which hypoxia affects NO bioavailability, including regulation of eNOS expression and activity. What is particularly important is the fact that hypoxia contributes to the depletion of cofactor BH4 and deficiency of substrate L-Arg, and thus elicits eNOS uncoupling—a state in which the enzyme produces superoxide instead of NO. eNOS uncoupling and the resulting oxidative stress is the major driver of endothelial dysfunction and atherogenesis. Moreover, hypoxia induces impairment in mitochondrial respiration and endothelial cell activation; thus, oxidative stress and inflammation, along with the hypoxic response, contribute to the development of endothelial dysfunction.

Brain ischemia is one of the leading causes of disability and mortality worldwide. Nitric oxide (NO•), a molecule that is involved in the regulation of proper blood flow, vasodilation, neuronal and glial activity constitutes the crucial factor that contributes to the development of pathological changes after stroke. One of the early consequences of a sudden interruption in the cerebral blood flow is the massive production of reactive oxygen and nitrogen species (ROS/RNS) in neurons due to NO• synthase uncoupling, which leads to neurotoxicity. Progression of apoptotic or necrotic neuronal damage activates reactive astrocytes and attracts microglia or lymphocytes to migrate to place of inflammation. Those inflammatory cells start to produce large amounts of inflammatory proteins, including pathological, inducible form of NOS (iNOS), which generates nitrosative stress that further contributes to brain tissue damage, forming vicious circle of detrimental processes in the late stage of ischemia. S-nitrosylation, hypoxia-inducible factor 1α (HIF-1α) and HIF-1α-dependent genes activated in reactive astrocytes play essential roles in this process. The review summarizes the roles of NO•-dependent pathways in the early and late aftermath of stroke and treatments based on the stimulation or inhibition of particular NO• synthases and the stabilization of HIF-1α activity.

Abstract Cyclic peptides containing the Arg-Gly-Asp (RGD) sequence have been shown to specifically bind the angiogenesis biomarker α V β 3 integrin. We report the synthesis, chemical characterization, and biological evaluation of two novel dimeric cyclic RGD-based molecular probes for the targeted imaging of α V β 3 activity (a radiolabeled version, 64Cu-NOTA-PEG4-cRGD2, for PET imaging, and a fluorescent version, FITC-PEG4-cRGD2, for in vitro work). We investigated the performance of this probe at the receptor, cell, organ, and whole-body levels, including its use to detect diabetes associated impairment of ischemia-induced myocardial angiogenesis. Both versions of the probe were found to be stable, demonstrated fast receptor association constants, and showed high specificity for α V β 3 in HUVECs (K d ~ 35 nM). Dynamic PET-CT imaging indicated rapid blood clearance via kidney filtration, and accumulation within α V β 3-positive infarcted myocardium. 64Cu-NOTA-PEG4-cRGD2 demonstrated a favorable biodistribution, slow washout, and excellent performance with respect to the quality of the PET-CT images obtained. Importantly, the ratio of probe uptake in infarcted heart tissue compared to normal tissue was significantly higher in non-diabetic rats than in diabetic ones. Overall, our probes are promising agents for non-invasive quantitative imaging of α V β 3 expression, both in vitro and in vivo.

Introduction : Mammographic screening results in diagnosis of less advanced breast cancer (BC). A meta-analysis of randomized clinical trials confirmed that BC screening reduces mortality. In 2007, the National Breast Cancer Screening Program (NBCSP) was established in Poland with the crucial aim of reducing mortality from BC. The purpose of this study was to assess the impact of participation in the NBCSP on prognosis. Material and methods : A single institution, non-randomized retrospective study was undertaken. The study population comprised 643 patients with BC treated in the Department of Surgical Oncology (DSO) at the Medical University of Gdansk over a 4-year period, from 01.01.2007 until 31.12.2010. Patients were divided into two groups: group A – patients who participated in the NBCSP (n = 238, 37.0%); and group B – patients who did not participate in the NBCSP (n = 405, 63.0%). Results : Statistical analysis revealed that group A displayed a less advanced AJCC stage (more patients in AJCC stage I, p = 0.002), lower tumor diameter (more patients with pT1, p = 0.006, and pT < 15 mm, p = 0.008) and a lower incidence of metastases to axillary lymph nodes (more patients with pNO, p = 0.01). From 2009 to 2010 the NBCSP revealed a statistically significant benefit – significantly more patients in stage 0 + I (60.7% vs. 48.8%, p = 0.018) and with tumors pT < 15 mm (48.8% vs. 35.1%, p = 0.011) were observed in group A. Conclusions : The study results revealed the beneficial impact of the NBCSP. Superior prognostic factors and favorable staging were observed in women who participated in the NBCSP.

Gender differences in the burden of cardiovascular disease (CVD) have been observed worldwide. In this study, plasmatic levels of trimethylamine (TMA) and blood oxidative biomarkers have been evaluated in 358 men (89 controls and 269 CVD patients) and 189 women (64 control and 125 CVD patients). The fluorescence technique was applied to determine erythrocyte membrane fluidity using 1,6-diphenyl-1,3,5-hexatriene (DPH) and Laurdan, while lipid hydroperoxides were assessed by diphenyl−1-pyrenylphosphine (DPPP). Results show that levels of plasmatic TMA were higher in healthy men with respect to healthy women (p = 0.0001). Significantly lower TMA was observed in male CVD patients (0.609 ± 0.104 μM) compared to healthy male controls (0.680 ± 0.118 μM) (p < 0.001), while higher levels of TMA were measured in female CVD patients (0.595 ± 0.115 μM) with respect to female controls (0.529 ± 0.073 μM) (p < 0.001). DPPP was significantly higher in healthy control men than in women (p < 0.001). Male CVD patients displayed a lower value of DPPP (2777 ± 1924) compared to healthy controls (5528 ± 2222) (p < 0.001), while no significant changes were measured in females with or without CVD (p > 0.05). Membrane fluidity was significantly higher (p < 0.001) in the hydrophobic bilayer only in control male subjects. In conclusion, gender differences were observed in blood oxidative biomarkers, and DPPP value might be suggested as a biomarker predictive of CVD only in men.

Breast cancer has a multifactorial etiology. One of the supposed and novel mechanisms is an alteration of circadian gene expression. Circadian genes play a crucial role in many physiological processes. These processes, such as genomic stability, DNA repair mechanism and apoptosis, are frequently disrupted in breast tumors. To assess the significance of circadian gene expression in breast cancer, we carried out an analysis of CLOCK, BMAL1, NPAS2, PER1, PER2, PER3 and CRY1, CRY2, TIMELESS, CSNK1E expression by the use of the quantitative Real-Time PCR technique in tumor tissue and non-tumor adjacent normal tissue sampled from 107 women with a newly diagnosed disease. The obtained data were compared to the clinical and histopathological features. PER1, PER2, PER3, CRY2 were found to be significantly down-expressed, while CLOCK, TIMELESS were over-expressed in the studied tumor samples compared to the non-tumor samples. Only gene expression of CRY1 was significantly down-regulated with progression according to the TNM classification. We found significantly decreased expression of CRY2, PER1, PER2 genes in the ER/PR negative breast tumors compared to the ER/PR positive tumors. Additionally, expression of CRY2, NPAS2 genes had a decreased level in the poorly differentiated tumors in comparison with the well and moderately differentiated ones. Our results indicate that circadian gene expression is altered in breast cancer tissue, which confirms previous observations from various animal and in vitro studies.

The mitochondrial β-oxidation of fatty acids is a complex catabolic pathway. One of the enzymes of this pathway is the heterooctameric mitochondrial trifunctional protein (MTP), composed of four α- and β-subunits. Mutations in MTP genes (HADHA and HADHB), both located on chromosome 2p23, cause MTP deficiency, a rare autosomal recessive metabolic disorder characterized by decreased activity of MTP. The most common MTP mutation is long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency caused by the c.1528G>C (rs137852769, p.Glu510Gln) substitution in exon 15 of the HADHA gene.We analyzed the frequency of genetic variants in the HADHA gene in the adults of Kashubian origin from North Poland and compared this data in other Polish provinces.We found a significantly higher frequency of HDHA c.1528G>C (rs137852769, p.Glu510Gln) carriers among Kashubians (1/57) compared to subjects from other regions of Poland (1/187). We found higher frequency of c.652G>C (rs71441018, pVal218Leu) polymorphism in the HADHA gene within population of Silesia, southern Poland (1/107) compared to other regions.Our study indicate described high frequency of c.1528G>C variant of HADHA gene in Kashubian population, suggesting the founder effect. For the first time we have found high frequency of rs71441018 in the South Poland Silesian population.