The Molecular Mechanisms in Senescent Cells Induced by Natural Aging and Ionizing Radiation.

This review discusses the relationship between cellular senescence and radiation exposure. Given the wide range of ionizing radiation sources encountered by people in professional and medical spheres, as well as the influence of natural background radiation, the question of the effect of radiation on biological processes, particularly on aging processes, remains highly relevant. The parallel relationship between natural and radiation-induced cellular senescence reveals the common aspects underlying these processes. Based on recent scientific data, the key points of the effects of ionizing radiation on cellular processes associated with aging, such as genome instability, mitochondrial dysfunction, altered expression of miRNAs, epigenetic profile, and manifestation of the senescence-associated secretory phenotype (SASP), are discussed. Unraveling the molecular mechanisms of cellular senescence can make a valuable contribution to the understanding of the molecular genetic basis of age-associated diseases in the context of environmental exposure.

Plasma microRNA Profiling in Type 2 Diabetes Mellitus: A Pilot Study.

Type 2 diabetes mellitus (T2D) is a chronic metabolic disease characterized by insulin resistance and ß-cell dysfunction and leading to many micro- and macrovascular complications. In this study we analyzed the circulating miRNA expression profiles in plasma samples from 44 patients with T2D and 22 healthy individuals using next generation sequencing and detected 229 differentially expressed miRNAs. An increased level of miR-5588-5p, miR-125b-2-3p, miR-1284, and a reduced level of miR-496 in T2D patients was verified. We also compared the expression landscapes in the same group of patients depending on body mass index and identified differential expression of miR-144-3p and miR-99a-5p in obese individuals. Identification and functional analysis of putative target genes was performed for miR-5588-5p, miR-125b-2-3p, miR-1284, and miR-496, showing chromatin modifying enzymes and apoptotic genes being among the significantly enriched pathways.

Molecular and Cellular Mechanism of Action of Chrysotile Asbestos in MRC5 Cell Line.

Asbestos is a known carcinogen; however, the influence of chrysotile asbestos on the development of tumor-related diseases remains a subject of intense debate within the scientific community. To analyze the effect of asbestos, we conducted a study using the MRC5 cell line. We were able to demonstrate that chrysotile asbestos stimulated the production of reactive oxygen species (ROS), leading to cell death and DNA damage in the MRC5 cell line, using various techniques such as ROS measurement, comet assay, MTT assay, and qPCR. In addition, we found that chrysotile asbestos treatment significantly increased extracellular mitochondrial DNA levels in the culture medium and induced significant changes in the expression profile of several miRNAs, which was the first of its kind. Thus, our research highlights the importance of studying the effects of chrysotile asbestos on human health and reveals multiple adverse effects of chrysotile asbestos.

Molecular and immunological changes in blood of rats exposed to various doses of asbestos dust.

BACKGROUND: Asbestos-related diseases are a group of diseases resulting from the inhalation of asbestos fibres and their subsequent deposition in the lung parenchyma, which causes the development of inflammatory and fibrotic processes in the respiratory system. Cases of the disease often occur in the practice of doctors. AIMS: The purpose of the study was to examine the level of circulating-free mitochondrial DNA (cf mtDNA), pro-inflammatory cytokines, immunological status and structural changes in the lung of rats exposed to various doses of asbestos dust. METHODS: Immune monitoring was performed using the peripheral blood samples of 40 male Wistar rats exposed and unexposed to asbestos dust. cf mtDNA copy numbers were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and cytokines were determined using a rat Enzyme Linked Immuno Sorbent Assay (ELISA) kit. RESULTS: A comprehensive assessment of the histopathological study performed under exposure to asbestos at a dose of 25 mg and 50 mg showed the presence of pronounced structural defects in the lung tissue of laboratory rats. The level of cf mtDNA in plasma of rats exposed to asbestos at a dose of 25 mg was reliably higher than that of control rats, and animals exposed to asbestos at a dose of 50 mg. The highest levels of pro-inflammatory cytokines IL-6 and TNF-a were also observed after asbestos dusting at a dose of 25 mg. CONCLUSIONS: According to the results of the immunological status obtained, the decrease in the levels of pro-inflammatory cytokines in the blood plasma at 50 mg is due to the immunosuppressive effect in the rat immune system at this dose. A positive correlation was found between TNF-a level and copy numbers of cf mtDNA at a dose of 25 mg.

The Role of Mitochondrial miRNAs in the Development of Radon-Induced Lung Cancer.

MicroRNAs are short, non-coding RNA molecules regulating gene expression by inhibiting the translation of messenger RNA (mRNA) or leading to degradation. The miRNAs are encoded in the nuclear genome and exported to the cytosol. However, miRNAs have been found in mitochondria and are probably derived from mitochondrial DNA. These miRNAs are able to directly regulate mitochondrial genes and mitochondrial activity. Mitochondrial dysfunction is the cause of many diseases, including cancer. In this review, we consider the role of mitochondrial miRNAs in the pathogenesis of lung cancer with particular reference to radon exposure.

The level of free-circulating mtDNA in patients with radon-induced lung cancer.

OBJECTIVE: According to the World Health Organization, radon is the second leading cause of lung cancer after smoking. Cell free circulating mitochondrial DNA (cf mtDNA) have been used not only as a biomarker of carcinogenesis but also as a biomarker of exposure to radiation, but nothing is known about changes in the level of cf mtDNA following radon exposure. Therefore, the purpose of this study was to estimate the cf mtDNA copy number as a biomarker of the response to radon exposure in lung cancer pathogenesis. METHODS: 207 subjects were examined including 41 radon-exposed lung cancer patients, 40 lung cancer patients without radon exposure and 126 healthy controls exposed/not exposed to high level of radon. Total cell free circulating DNA from blood samples was extracted and used to detect cell free circulating mitochondrial DNA copy number by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Our data indicate that the level of cf mtDNA in the radon-induced lung cancer patients was significantly higher than that of the other study participants. There was a significant difference in the level of cf mtDNA in the blood plasma of healthy volunteers exposed and not exposed to high doses of radon. Moreover, in healthy volunteers living in areas with high radon levels, the mtDNA copy number was higher than that in patients with lung cancer who were not exposed to high doses of radon. CONCLUSION: Our study provides evidence for a possible role of cf mtDNA as a promising biomarker of lung cancer induced by exposure to high dose of radon.

The effectiveness of resveratrol in treatment of PCOS on the basis of experimental model in rats.

PURPOSE OF THE STUDY: Polycystic ovary syndrome (PCOS) is a widespread endocrine disorder in women of reproductive age. Further research is required to justify new directions of effective targeted therapy of this condition. Resveratrol possesses anti-inflammatory, antioxidant and antidiabetic properties. The purpose of this study was to evaluate the potential effectiveness of resveratrol in PCOS based on the created model of this disease in Wistar rats. MATERIALS AND METHODS: The PCOS model was created by oral administration of letrozole to female Wistar rats.. The animals received resveratrol at a dosage of 20 mg/kg and 30 mg/kg for the next 30 days. Then ovariectomy was performed for histological confirmation of the effectiveness of resveratrol in the treatment of PCOS. Regularity of estrous cycle, animal's body mass and the level of soluble receptors for advanced glycation end products (sRAGE) in the blood of rats were also evaluated in dynamics. RESULTS: The study revealed that administration of resveratrol leads to dose-dependent restoration of normal morphology of ovarian tissue, normalizes regularity of estrous cycle and decreases body weight of rats with PCOS. CONCLUSION: The results obtained in rats suggest that resveratrol may be a promising agent for the treatment of PCOS in women.

Involvement of Circulating Cell-Free Mitochondrial DNA and Proinflammatory Cytokines in Pathogenesis of Chronic Obstructive Pulmonary Disease and Lung Cancer.

OBJECTIVE: Circulating cell-free mitochondrial DNA (cf-MtDNA) has been reported in patients with chronic obstructive pulmonary disease (COPD) and lung cancers. However, inter-relationships among the three biological events have not been well-characterized. Therefore, our investigation was conducted to better understand the role of cf-MtDNA on pathogenesis of the two diseases. METHODS: Plasma samples were collected from 64 non-small cell lung cancer (NSCLC) patients (before therapy), 45 patients with COPD and 62 healthy individuals. cf-MtDNA copy numbers were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and cytokines were determined using a human ELISA kit. RESULTS: Our data indicate that smoking statuses of the patients and controls were significantly associated with increased cf-MtDNA in plasma samples. Furthermore, NSCLC patients had significantly higher cf-MtDNA copy numbers than COPD patients (p < 0.03) and normal controls (p < 0.02), together with elevated proinflammatory cytokines over the controls (p < 0.05). Our  study shows that the copy numbers for the NSCLC patients were positively associated with their subsequent metastasis but inversely associated with their overall survival.  Conclusion: Our study indicates certain lung injury (e.g., from cigarette smoking) was responsible for the release of cf-MtDNA and proinflammatory cytokines into plasmas among our patients and controls. The increase in cf-MtDNA copy numbers was significantly associated with the development of both COPD and NSCLC, with increase in interleukin 6, and from our 5-year follow-up, with poor prognosis among the NSCLC patients. Therefore, with further validation, cf-MtDNA can be considered for use as diagnostic and prognostic biomarkers for NSCLC.

The Plasma Levels of hsa-miR-19b-3p, hsa-miR-125b-5p, and hsamiR- 320c in Patients with Asthma, COPD and Asthma-COPD Overlap Syndrome (ACOS).

BACKGROUND: Bronchial Asthma (BA) and Chronic Obstructive Pulmonary Disease (COPD) are chronic airway inflammation diseases. In recent years, patients with signs of both BA and COPD have been assigned to a separate group as Asthma-COPD Overlap Syndrome (ACOS). Free-circulating plasma microRNAs are considered as potential biomarkers of pulmonology diseases, including BA, COPD, and ACOS. OBJECTIVE: This study aimed to investigate the expression level of free-circulating plasma microRNAs, hsa-miR-19b-3p, hsa-miR-125b-5p, and hsa-miR-320c in patients with BA, COPD and ACOS for the detection and validation of new microRNAs as biomarkers for chronic lung diseases. METHODS: The relative expression levels of 720 microRNAs were evaluated by Real Time-Polymerase Chain Reaction (RT-PCR) in patients with COPD and BA. Three upregulated microRNAs (hsa-miR-19b-3p, hsa-miR-125b-5p and hsa-miR-320c) were selected for further study. The obtained data were analyzed using the microRNA PCR Array Data Analysis tool. The sensitivity and specificity were estimated using the area under the Receiver Operating Characteristics curve (ROC). RESULTS: The expression level of free-circulating hsa-miR-19b-3p was decreased in the blood plasma of patients with BA and ACOS, and increased in patients with COPD. hsa-miR-125b-5p was downregulated in the blood plasma of patients with COPD and upregulated in patients with BA and ACOS. hsa-miR-320c was downregulated in the blood plasma of patients with BA, and upregulated in patients with COPD and ACOS. The ROC curves of patients with BA for hsa-miR-19b-3p, patients with ACOS for hsa-miR-125b-5p, and patients with COPD for hsa-miR-320c revealed the probability of them as valuable biomarkers with AUCs of 0.824, 0.825, and 0.855, respectively. CONCLUSION: Our study revealed three promising biomarkers for the diagnosis of COPD, BA and ACOS.

Role of microRNAs in Lung Carcinogenesis Induced by Asbestos.

MicroRNAs are a class of small noncoding endogenous RNAs 19-25 nucleotides long, which play an important role in the post-transcriptional regulation of gene expression by targeting mRNA targets with subsequent repression of translation. MicroRNAs are involved in the pathogenesis of numerous diseases, including cancer. Lung cancer is the leading cause of cancer death in the world. Lung cancer is usually associated with tobacco smoking. However, about 25% of lung cancer cases occur in people who have never smoked. According to the International Agency for Research on Cancer, asbestos has been classified as one of the cancerogenic factors for lung cancer. The mechanism of malignant transformation under the influence of asbestos is associated with the genotoxic effect of reactive oxygen species, which initiate the processes of DNA damage in the cell. However, epigenetic mechanisms such as changes in the microRNA expression profile may also be implicated in the pathogenesis of asbestos-induced lung cancer. Numerous studies have shown that microRNAs can serve as a biomarker of the effects of various adverse environmental factors on the human body. This review examines the role of microRNAs, the expression profile of which changes upon exposure to asbestos, in key processes of carcinogenesis, such as proliferation, cell survival, metastasis, neo-angiogenesis, and immune response avoidance.

Study of the Potential of the Capsule Shell Based on Natural Polysaccharides in Targeted Delivery of the L-Phenylalanine Ammonia-Lyase Enzyme Preparation.

The treatment of classical phenylketonuria is currently represented by many new methods of disease management. A promising method is the use of the enzyme L-phenylalanine ammonia-lyase (PAL) in various forms. The widespread use of enzyme preparations in therapy is limited by a lack of understanding of the mechanisms and systems of the targeted transport of PAL into certain organs and tissues as a result of the incorporation of a drug into the carrier. To ensure the stability of enzymes during the delivery process, encapsulation is preferable, which, as a rule, ensures the preservation of the qualitative characteristics of the enzymes orally applied to the environmental effects of the gastrointestinal tract (acidity, temperature, oxidation, etc.). Capsule preparations showed sufficient stability in the model gastric fluids and sustained release of the drug in the simulated intestinal fluid. Currently, there is a wide range of polymers used for encapsulation. The use of natural sources in the production technology of capsule systems improves bioavailability, controls the release, and prolongs the half-life of active substances. The advantage of this method is that the used enzyme is completely protected by the cell membranes of the capsules, which preserve its stability in the aggressive environment of the gastrointestinal tract. Capsules were obtained on the basis of compositions of hydrocolloids of plant origin. The potential of the developed capsules for targeted delivery of the enzyme preparation was studied. The degradation of the encapsulated form of the PAL enzyme preparation was studied in vitro in model bio-relevant media simulating the gastric and intestinal environment. The dynamics of the breakdown of the capsule shell allow us to expect that the release of L-phenylalanine ammonia-lyase from capsules based on plant hydrocolloids will occur no earlier than reaching the upper intestines, where the interaction with the protein components of the consumed food products to neutralize phenylalanine should occur.

Radon Biomonitoring and microRNA in Lung Cancer.

Radon is the number one cause of lung cancer in non-smokers. microRNA expression in human bronchial epithelium cells is altered by radon, with particular reference to upregulation of miR-16, miR-15, miR-23, miR-19, miR-125, and downregulation of let-7, miR-194, miR-373, miR-124, miR-146, miR-369, and miR-652. These alterations alter cell cycle, oxidative stress, inflammation, oncogene suppression, and malignant transformation. Also DNA methylation is altered as a consequence of miR-29 modification induced by radon. Indeed miR-29 targets DNA methyltransferases causing inhibition of CpG sites methylation. Massive microRNA dysregulation occurs in the lung due to radon expose and is functionally related with the resulting lung damage. However, in humans this massive lung microRNA alterations only barely reflect onto blood microRNAs. Indeed, blood miR-19 was not found altered in radon-exposed subjects. Thus, microRNAs are massively dysregulated in experimental models of radon lung carcinogenesis. In humans these events are initially adaptive being aimed at inhibiting neoplastic transformation. Only in case of long-term exposure to radon, microRNA alterations lead towards cancer development. Accordingly, it is difficult in human to establish a microRNA signature reflecting radon exposure. Additional studies are required to understand the role of microRNAs in pathogenesis of radon-induced lung cancer.

miR-19 in blood plasma reflects lung cancer occurrence but is not specifically associated with radon exposure.

Radon is one of the most powerful carcinogens, particularly in terms of lung cancer onset and development. miRNAs may be considered not only as markers of the ongoing tumorigenesis but also as a hallmark of exposure to radiation, including radon and its progeny. Therefore, the purpose of the present study was to estimate the value of plasma miR-19b-3p level as the prospective marker of the response to radon exposure in lung cancer pathogenesis. A total of 136 subjects were examined, including 49 radon-exposed patients with lung cancer, 37 patients with lung cancer without radon exposure and 50 age/sex matched healthy controls. Total RNA from blood samples was extracted and used to detect miR-19b-3p expression via reverse transcription quantitative-polymerase chain reaction. The 2-ΔΔCq method was used to quantify the amount of relative miRNA. The plasma level of p53 protein was determined using a Human p53 ELISA kit. Plasma miR-19b-3p level was significantly higher in the patients with lung cancer groups, compared with the healthy control group (P<0.0001). No other statistically significant differences were determined in the expression level of plasma miR-19b-3p between patients diagnosed with lung cancer exposed to radon and not exposed to radon. The expression level of free circulating miR-19b-3p was higher in the group of non-smoking patients with lung cancer, compared with smokers with lung cancer. The miR-19b-3p was 1.4-fold higher in non-smokers than in smokers (P<0.05). No association between plasma levels of p53 protein and miR-19b-3p freely circulating in patients with lung cancer was observed. No other statistically significant differences were determined in the plasma p53 protein level between patients diagnosed with lung cancer exposed and not exposed to radon. These results indicated that detection of miR-19b-3p levels in plasma potentially could be exploited as a noninvasive method for the lung cancer diagnostics. However, this miRNA is not suitable as the precise marker for radon impact.

The health effects of radon and uranium on the population of Kazakhstan.

The radioactive contamination is a significant factor affecting the environment and human health. Radon and its decay products are the major contributors to human exposure from natural radiation sources. World Health Organization has identified the chronic residential exposure to radon and its decay products as the second cause of lung cancer after tobacco consumption and also as the main risk-factor in never smokers. The high levels of radon are observed in the North and East areas of Kazakhstan because of the natural radiation sources and the long-term and large-scale mining of uranium. The genotoxic effects of radon on population of Kazakhstan are poorly understood, in spite of the fact that many regions of the country contain the high levels of radon. Studies elucidating potential health risk among population exposed to radon and genotoxic effect of radon in Kazakhstan are very limited or they have never been addressed in some areas. In this review, we are presenting available data on the residential radon exposure of humans in uranium mining and milling areas in the North and East areas of Kazakhstan.

Autoregulation of the mechanistic target of rapamycin (mTOR) complex 2 integrity is controlled by an ATP-dependent mechanism.

Nutrients are essential for living organisms because they fuel biological processes in cells. Cells monitor nutrient abundance and coordinate a ratio of anabolic and catabolic reactions. Mechanistic target of rapamycin (mTOR) signaling is the essential nutrient-sensing pathway that controls anabolic processes in cells. The central component of this pathway is mTOR, a highly conserved and essential protein kinase that exists in two distinct functional complexes. The nutrient-sensitive mTOR complex 1 (mTORC1) controls cell growth and cell size by phosphorylation of the regulators of protein synthesis S6K1 and 4EBP1, whereas its second complex, mTORC2, regulates cell proliferation by functioning as the regulatory kinase of Akt and other members of the AGC kinase family. The regulation of mTORC2 remains poorly characterized. Our study shows that the cellular ATP balance controls a basal kinase activity of mTORC2 that maintains the integrity of mTORC2 and phosphorylation of Akt on the turn motif Thr-450 site. We found that mTOR stabilizes SIN1 by phosphorylation of its hydrophobic and conserved Ser-260 site to maintain the integrity of mTORC2. The optimal kinase activity of mTORC2 requires a concentration of ATP above 1.2 mM and makes this kinase complex highly sensitive to ATP depletion. We found that not amino acid but glucose deprivation of cells or acute ATP depletion prevented the mTOR-dependent phosphorylation of SIN1 on Ser-260 and Akt on Thr-450. In a low glucose medium, the cells carrying a substitution of SIN1 with its phosphomimetic mutant show an increased rate of cell proliferation related to a higher abundance of mTORC2 and phosphorylation of Akt. Thus, the homeostatic ATP sensor mTOR controls the integrity of mTORC2 and phosphorylation of Akt on the turn motif site.

Isolation of the mTOR complexes by affinity purification.

The mammalian Target Of Rapamycin (mTOR) protein is a central component of the essential and highly conserved signaling pathway that emerged as a critical effector in regulation of cell physiology. Biochemical studies defined mTOR as the protein kinase that exists at least in two distinct complexes. The first complex has been characterized as the nutrient-sensitive mTOR complex 1 that controls cell growth and cell size by regulating protein synthesis and autophagy. The second complex of mTOR has been defined as the component of growth factor signaling that functions as a major regulatory kinase of Akt/PKB. Here, we provide the detailed methods how to purify the functional complexes of mTOR by affinity purification. In the first part, we describe the purification of the distinct mTOR complexes by immunoprecipitation. Purification of the soluble mTOR complexes is explained in the second part of this chapter.