The Ras-Raf-MEK-ERK pathway plays a central role in tumorigenesis and is a target for anticancer therapy. The successful strategy based on the activation of cell death in Ras-expressing cells is ...associated with the suppression of kinases involved in Ras pathway. However, activation of cytoprotective autophagy overcomes antiproliferative effect of the inhibitors and develops drug resistance. We studied whether cellular senescence induced by HDAC inhibitor sodium butyrate in
-transformed rat embryo fibroblasts (ERas) and A549 human Ki-Ras mutated lung adenocarcinoma cells would enhance the tumor suppressor effect of MEK/ERK inhibition. Treatment of control ERas cells with PD0325901 for 24 h results in mitochondria damage and apoptotic death of a part of cellular population. However, the activation of AMPK-dependent autophagy overcomes pro-apoptotic effects of MEK/ERK inhibitor and results in restoration of the mitochondria and rescue of viability. Senescent ERas cells do not develop cytoprotective autophagy upon inhibition of MEK/ERK pathway due to spatial dissociation of lysosomes and autophagosomes in the senescent cells. Senescent cells are unable to form the autophagolysosomes and to remove the damaged mitochondria resulting in apoptotic death. Our data show that suppression of MEK/ERK pathway in senescent cells provides a new strategy for elimination of Ras-expressing cells.
Imaging techniques such as computed tomographies (CT) play a major role in clinical imaging and diagnosis of malignant lesions. In recent years, metal nanoparticle platforms enabled effective payload ...delivery for several imaging techniques. Due to the possibility of surface modification, metal nanoparticles are predestined to facilitate molecular tumor targeting. In this work, we demonstrate the feasibility of anti-plasma membrane Heat shock protein 70 (Hsp70) antibody functionalized gold nanoparticles (cmHsp70.1-AuNPs) for tumor-specific multimodal imaging. Membrane-associated Hsp70 is exclusively presented on the plasma membrane of malignant cells of multiple tumor entities but not on corresponding normal cells, predestining this target for a tumor-selective in vivo imaging. In vitro microscopic analysis revealed the presence of cmHsp70.1-AuNPs in the cytosol of tumor cell lines after internalization via the endo-lysosomal pathway. In preclinical models, the biodistribution as well as the intratumoral enrichment of AuNPs were examined 24 h after i.v. injection in tumor-bearing mice. In parallel to spectral CT analysis, histological analysis confirmed the presence of AuNPs within tumor cells. In contrast to control AuNPs, a significant enrichment of cmHsp70.1-AuNPs has been detected selectively inside tumor cells in different tumor mouse models. Furthermore, a machine-learning approach was developed to analyze AuNP accumulations in tumor tissues and organs. In summary, utilizing mHsp70 on tumor cells as a target for the guidance of cmHsp70.1-AuNPs facilitates an enrichment and uniform distribution of nanoparticles in mHsp70-expressing tumor cells that enables various microscopic imaging techniques and spectral-CT-based tumor delineation in vivo.
G-protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. They modulate key physiological functions and are required in diverse developmental processes including ...embryogenesis, but their role in pluripotency maintenance and acquisition during the reprogramming towards hiPSCs draws little attention. Meanwhile, it is known that more than 106 GPCRs are overexpressed in human pluripotent stem cells (hPSCs). Previously, to identify novel effectors of reprogramming, we performed a high-throughput RNA interference (RNAi) screening assay and identified adhesion GPCR, GPR123, as a potential reprogramming effector. Its role has not been explored before. Herein, by employing
RNAi we addressed the role of GPR123 for hPSCs. The suppression of
in hPSCs leads to the loss of pluripotency and differentiation, impacted colony morphology, accumulation of cells at the G2 phase of the cell cycle, and absence of the scratch closure. Application of the
RNAi at the initiation stage of reprogramming leads to a decrease in the percentage of the "true" hiPSC colonies, a drop in E-cadherin expression, a decrease in the percentage of NANOG+ nuclei, and the absence of actin cytoskeleton remodeling. Together this leads to the absence of the alkaline-phosphatase-positive hiPSCs colonies on the 18th day of the reprogramming process. Overall, these data indicate for the first time the essential role of GPR123 in the maintenance and acquisition of pluripotency.
Autophagy is conservative catabolic process that degrades organelles, in particular, mitochondria, and misfolded proteins within the lysosomes, thus maintaining cellular viability. Despite the close ...relationship between mitochondrial dysfunction and cellular senescence, it is unclear how mitochondria damage can induce autophagy in senescent cells. We show that MEK/ERK suppression induces mitochondria damage followed by apoptosis of senescent Ras-expressing cells. To understand the role of persistent mTORC1 signaling in breaking the cAMPK-induced autophagy caused by mitochondrial damage, we inhibited mTORС1 with low concentrations of pp242. mTORC1 suppression neither restores the AMPK-induced autophagy nor decreases the level of apoptosis upon MEK/ERK inhibition. We discovered the existence of an alternative autophagy-like way that partially increases the viability of senescent cells under suppressed mTORC1. The pp242-treated cells survive due to formation of the non-autophagous LC3-negative vacuoles, which contain the damaged mitochondria and lysosomes with the following excretion the content from the cell. MEK/ERK activity is required to implement this process in senescent cells. Senescent cells exhibit distinctive spatial distribution of organelles and proteins that provides uncoupling of final participants of autophagy. We show that this feature stops the process of cytoprotective autophagy in response to MEK/ERK suppression, thus allowing selective elimination of senescent Ras-expressing cells.
Superparamagnetic iron oxide nanoparticles (SPIONs), due to their unique magnetic properties, have the ability to function both as magnetic resonance (MR) contrast agents, and can be used for ...thermotherapy. SPIONs conjugated to the heat shock protein Hsp70 that selectively binds to the CD40 receptor present on glioma cells, could be used for MR contrast enhancement of experimental C6 glioma.
The magnetic properties of the Hsp70-SPIONs were measured by NMR relaxometry method. The uptake of nanoparticles was assessed on the C6 glioma cells by confocal and electron microscopes. The tumor selectivity of Hsp70-SPIONs being intravenously administered was analyzed in the experimental model of C6 glioma in the MRI scanner.
Hsp70-SPIONs relaxivity corresponded to the properties of negative contrast agents with a hypointensive change of resonance signal in MR imaging. A significant accumulation of the Hsp70-SPIONs but not the non-conjugated nanoparticles was observed by confocal microscopy within C6 cells. Negative contrast tumor enhancement in the T2-weighted MR images was higher in the case of Hsp70-SPIONs in comparison to non-modified SPIONs. Histological analysis of the brain sections confirmed the retention of the Hsp70-SPIONs in the glioma tumor but not in the adjacent normal brain tissues.
The study demonstrated that Hsp70-SPION conjugate intravenously administered in C6 glioma model accumulated in the tumors and enhanced the contrast of their MR images.
Introduction. There is an acute shortage of labor resources in the modern labor market, especially with engineering qualifications. A significant part of university students do not reach the stage of ...obtaining a diploma. Efficient spending of federal budget funds for training of high-class technical specialists becomes extremely necessary. The purpose of this study is to identify the main strategies of choosing a university among first-year students participating in the “Priority-2030ˮ program. Materials and Methods. The goals and objectives of this descriptive study suggest obtaining a more complete understanding of professional and educational applicants’ strategies when choosing technical universities. The questionnaire method implemented in the form of an online survey was used as the main method of collecting primary empirical data. The survey was conducted in May 2023 (n = 1,888). Results. The main motives and factors influencing the professional and educational strategy of choosing a higher educational institution are determined: the characteristics of the university (its type and academic authority, position in university rankings, its transport accessibility and the availability of dormitories); prospects for further employment in the speciality received; the financial capabilities of the applicantʼs family, the main trajectories of admission to the university. The most relevant aspects of the attractiveness of the university for applicants are formulated. Discussion and Conclusion. The authorʼs conclusions contribute to the development of scientific approaches to the study of trends in the development of higher education, the theory and practice of organizing the educational process in educational organizations, the problems of professional self-determination and career guidance, methods of supporting studentsʼ professional choice. The materials of the article can be useful to Russian universities in developing an effective system of career guidance.
This paper contributes to the research on corporate governance by predicting the effects of European takeover regulation. In particular, we investigate whether the recent reforms of takeover ...regulation in Europe are leading to a harmonization of the national legislations. With the help of 150 corporate governance lawyers from 30 European countries, we collected the main changes in takeover regulation. We assess whether a process of convergence towards the Anglo-(American) corporate governance system has been started and we find that this is the case. We make predictions as to the consequences of the reforms for ownership and control. However, we find that while in some countries the adoption of a unified takeover code may result in dispersed ownership, in others it may further consolidate the blockholder-based system.
mTOR is a critical target for controlling cell cycle progression, senescence and cell death in mammalian cancer cells. Here we studied the role of mTOR-dependent autophagy in implementating the ...antiprolifrative effect of mTORC1-specific inhibitor rapamycin and ATP-competitive mTOR kinase inhibitor pp242. We carried out a comprehensive analysis of pp242- and rapamycin-induced autophagy in ERas tumor cells. Rapamycin exerts cytostatic effect on ERas tumor cells, thus causing a temporary and reversible cell cycle arrest, activation of non-selective autophagy not accompanied by cell death. The rapamycin-treated cells are able to continue proliferation after drug removal. The ATP-competitive mTORC1/mTORC2 kinase inhibitor pp242 is highly cytotoxic by suppressing the function of mTORC1-4EBP1 axis and mTORC1-dependent phosphorylation of mTORC1 target--ULK1-Ser757 (Atg1). In contrast to rapamycin, pp242 activates the selective autophagy targeting mitochondria (mitophagy). The pp242-induced mitophagy is accompanied by accumulation of LC3 and conversion of LC3-I form to LC3-II. However reduced degradation of p62/SQSTM indicates abnormal flux of autophagic process. According to transmission electron microscopy data, short-term pp242-treated ERas cells exhibit numerous heavily damaged mitochondria, which are included in single membrane-bound autophagic/autolysophagic vacuoles (mitophagy). Despite the lack of typical for apoptosis features, ERas-treated cells with induced mitophagy revealed the activation of caspase 3, 9 and nucleosomal DNA fragmentation. Thus, pp242 activates autophagy with suppressed later stages, leading to impaired recycling and accumulation of dysfunctional mitochondria and cell death. Better understanding of how autophagy determines the fate of a cell--survival or cell death, can help to development of new strategy for cancer therapy.
Abundant secretory granular cells (GCs) in the Giant African land snail atrium harbor a range of bioactive substances and undergo rapid total degranulation in response to stimulation of the cardiac ...nerve or stressful influences. Here we have analyzed exocytotic events in the non-stimulated GCs. It was shown that the GCs contain three major distinct types of granules that differ histochemically, immunocytochemically and ultrastructurally, each performing specific functions. The type I granules characteristically filled with electron-lucent homogeneous materials exhibit intense immunoreactivity for bioactive proteins and therefore are considered to be storage granules. Histochemistry using vital staining with Acridine Orange and Gomori acid phosphatase technique has revealed lysosomal-related nature of the electron-dense type II granules. Digestion remnants appearing as fine filamentous materials fill the type III granules. Only the type III granules fuse together and with the plasma membrane form degranulation channels and surface pores, through which the debris is removed from the cell. The finding of granules exhibiting intermediate ultrastructural, histochemical and immunocytochemical features suggests that the major granule types represent most stable states along a granule empting continuum. Thus, under physiological conditions, the GCs continuously produce secretory proteins and so maintain readiness for stress-response, but use protein degradation machinery to prevent massive release of these bioactive substances into hemolymph.
Here we focused on the dynamic processes in the snail at different time after short-term hypergravity load (STHL) by monitoring the state of neuroendocrine and immune systems, the nucleic acid ...synthesis levels in the atrial cells, and the behaviour of the atrial granular cells (GCs). We observed that immediately after centrifugation (14
g
for 15 min) in the snail haemolymph concentration of dopamine and noradrenaline (measured by high-performance liquid chromatography) and the number of circulating haemocytes and their proliferative activity (estimated by the direct cell counting and 3Hthymidine incorporation, respectively) increased significantly, whereas the concentration of adrenaline decreased. Twenty-four hours after STHL, the levels of catecholamines and haemocytes returned to their control values. In the atrial epicardial and endothelial cells, a notable drop of transcription activity (evaluated by 3Huridine autoradiography) from the baseline in the immediate post-STHL period was followed by its gradual increase reaching a maximum at the day 5 and subsequent decrease to control value by the day 10. In endothelial cells, DNA-synthesizing activity (evaluated by 3Hthymidine autoradiography) equal to zero before and just after STHL, increased significantly at the day 5, and decreased by the day 10. The atrial GCs underwent total degranulation. Formed as a result small ungranulated cells exhibited DNA synthesis. Afterwards, most probably, the GCs divided and regranulated. One month after STHL the GC population had been restored. Overall, STHL has triggered an immediate reaction of the neuroendocrine and immune systems and initiated long-lasting processes at a cellular level, which included alterations in activity of nucleic acid syntheses in the epicardial and endothelial cells and remodelling of the GC population in the atrium.
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