Olga A. Bystrova

Superparamagnetic iron oxide nanoparticles conjugated with epidermal growth factor (SPION-EGF) for targeting brain tumors.

Superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with recombinant human epidermal growth factor (SPION–EGF) were studied as a potential agent for magnetic resonance imaging contrast enhancement of malignant brain tumors. Synthesized conjugates were characterized by transmission electron microscopy, dynamic light scattering, and nuclear magnetic resonance relaxometry. The interaction of SPION–EGF conjugates with cells was analyzed in a C6 glioma cell culture. The distribution of the nanoparticles and their accumulation in tumors were assessed by magnetic resonance imaging in an orthotopic model of C6 gliomas. SPION–EGF nanosuspensions had the properties of a negative contrast agent with high coefficients of relaxation efficiency. In vitro studies of SPION–EGF nanoparticles showed high intracellular incorporation and the absence of a toxic influence on C6 cell viability and proliferation. Intravenous administration of SPION–EGF conjugates in animals provided receptor-mediated targeted delivery across the blood–brain barrier and tumor retention of the nanoparticles; this was more efficient than with unconjugated SPIONs. The accumulation of conjugates in the glioma was revealed as hypotensive zones on T2-weighted images with a twofold reduction in T2 relaxation time in comparison to unconjugated SPIONs (P<0.001). SPION–EGF conjugates provide targeted delivery and efficient magnetic resonance contrast enhancement of EGFR-overexpressing C6 gliomas.

Tumor targeting using magnetic nanoparticle Hsp70 conjugate in a model of C6 glioma

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSION 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.

The presence of ANP in rat peritoneal mast cells

ABSTRACTAtrial natriuretic peptide (ANP) is an important component of the natriuretic peptide system. A great role in many regulatory systems is played by mast cells. Meanwhile involvement of these cells in ANP activity is poorly studied. In this work, we have shown the presence of ANP in rat peritoneal mast cells. Pure fraction of mast cells was obtained by separation of rat peritoneal cells on a Percoll density gradient. By Western blotting, two ANP-immunoreactive proteins of molecular masses of 2.5 kDa and 16.9 kDa were detected in lysates from these mast cells. Electron microscope immunogold labeling has revealed the presence of ANP-immunoreactive material in storage, secreting and released granules of mast cells. Our findings indicate the rat peritoneal mast cells to contain both ANP prohormone and ANP. These both peptides are located in mast cell secretory granules and released by mechanism of degranulation. It is discussed that many mast cell functions might be due to production of natriuretic peptides by these cells.

Hsp70 in the atrial neuroendocrine units of the snail, Achatina fulica

Heat shock proteins (Hsps) are evolutionary conserved peptides well known as molecular chaperones and stress proteins. Elevated levels of extracellular Hsps in blood plasma have been observed during the stress responses and some diseases. Information on the cellular sources of extracellular Hsps and mechanisms regulating their release is still scanty. Here we showed the presence and localization of Hsp70 in the neuroendocrine system in the atrium of the snail, Achatina fulica. The occurrence of the peptide in snail atrium lysate was detected by Western blot analysis. Immunoperoxidase and immunogold staining demonstrated that Hsp70‐immunoreactivity is mainly confined to the peculiar atrial neuroendocrine units which are formed by nerve fibers tightly contacted with large granular cells. Immunolabelling intensity differed in morphologically distinct types of secretory granules in the granular cells. The pictures of exocytosis of Hsp70‐immunolabeled granules from the granular cells were observed. In nerve bundles, axon profiles with Hsp70‐immunoreactive and those with non‐immunoreactive neurosecretory granules were found. In addition, Hsp70‐like material was also revealed in the granules of glia‐interstitial cells that accompanied nerve fibers. Our findings provide an immuno‐morphological basis for a role of Hsp70 in the functioning of the neuroendocrine system in the snail heart, and show that the atrial granular cells are a probable source of extracellular Hsp70 in the snail hemolymph.

Atrial granular cells of the snail Achatina fulica release proteins into hemolymph after stimulation of the heart nerve

SUMMARY The atrium of the gastropod mollusc Achatina fulica receives rich innervation and contains numerous granular cells (GCs). We studied the atrial innervation and discovered that axon profiles typical in appearance of peptidergic neurons form close unspecialized membrane contacts with GCs. Then, we investigated, at both morphological and biochemical levels, the effect of electrical stimulation of the heart nerve on GCs of Achatina heart perfused in situ. The ultrastructural study demonstrated changes in granule morphology consistent with secretion. These events included alteration of granule content, intracellular granule fusion and formation of complex degranulation channels, within which the granule matrix solubilized. It was shown that electrical stimulation resulted in a significant increase of the total protein concentration in the perfusate. Furthermore, SDS-PAGE analysis of the perfusate revealed three new proteins with molecular masses of 16, 22, and 57 kDa. Affinity-purified polyclonal antibodies against the 16 kDa protein were obtained; the whole-mount immunofluorescence technique revealed the presence of this protein in the granules of atrial GCs. In GCs of the stimulated atrium, a progressive loss of their granular content was observed. The results suggest that the central nervous system can modulate the secretory activity of the atrial GCs through non-synaptic pathways.

The process of granule exocytosis in non-stimulated atrial granular cells of the snail, Achatina achatina: an ultrastructural, histochemical and immunocytochemical study.

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.

mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells

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.

Undifferentiated Cells in the Snail Myocardium Are Capable of DNA Synthesis and Myodifferentiation

Cellular mechanisms of heart-muscle growth in the snail Achatina fulica have been studied using cytophotometry and electron microscopic autoradiography. Cytophotometric DNA measurements showed that the snail cardiomyocytes are mononucleated cells with diploid nuclei. Ultrastructural analysis of the snail myocardium revealed that, in addition to mature myocytes, it contains small roundish undifferentiated cells (UCs) and poorly differentiated muscle cells. EM autoradiography detected silver grains over the nuclei of UCs 2 h after injection of tritiated thymidine ([3H]Tdr), while the nuclei of both mature and poorly differentiated myocytes remained unlabeled. In EM autographs of the myocardial tissue fixed 14 days after [3H]Tdr administration, labeled myonuclei were evident, which may suggest some myodifferentiation of prelabeled UCs. Many labeled UCs persist for 14 days after a single [3H]Tdr injection, suggesting that not all UCs undergo myodifferentiation after passing through the cell cycle, and that those that do not can enter the next cycle. UCs in the snail myocardium presumably provide not only reserve but also stem cells for myocytes. Thus, the heart muscle of the adult snail consists of mononucleated diploid myocytes with blocked proliferative activity and a renewable population of precursor myogenic cells. The results obtained suggest that the growth of this muscle involves a myoblastic mechanism of myogenesis; this mechanism differs from that of vertebrate cardiac muscle growth, which is non-myoblastic—that is, based on proliferation or polyploidization of cardiomyocytes. Evolutionary aspects of cellular mechanisms of the heart-muscle growth are discussed.

Localization of heat shock protein 70 in rat mast cells

Heat shock proteins (Hsp) are intracellular chaperons, as well as extracellular molecules with immunomodulatory and signaling functions engaged in adaptation to stress on the cellular and organism levels. The presence of Hsp in secretory granules of mast cells (MCs) may be correlated with mast cells’ active participation in adaptation to stress. Using immunoelectron microscopy, we showed that Hsp70 was localized in secretory granules of rat pericardial and peritoneal mast cells. Localization of Hsp70 in rat peritoneal mast cells isolated by Percoll density gradient centrifugation was confirmed by immunoblotting. The possible involvement of mast cells in production of extracellular Hsp70, as well as Hsp70 functions inside the mast cells, is discussed.

Targeted elimination of senescent Ras-transformed cells by suppression of MEK/ERK pathway

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 E1a+cHa-Ras-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 hresults 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.