Implication of integrin α5ß1 in senescence of SK-Mel-147 human melanoma cells.

Downregulation of α5ß1 integrin in the SK-Mel-147 human melanoma culture model sharply inhibits the phenotypic manifestations of tumor progression: cell proliferation and clonal activity. This was accompanied by a 2-3-fold increase in the content of SA-ß-Gal positive cells thus indicating an increase in the cellular senescence phenotype. These changes were accompanied by a significant increase in the activity of p53 and p21 tumor suppressors and components of the PI3K/Akt/mTOR/p70 signaling pathway. Pharmacological inhibition of mTORC1 reduced the content of SA-ß-Gal positive cells in the population of α5ß1-deficient SK-Mel-147 cells. A similar effect was observed with pharmacological and genetic inhibition of the activity of Akt1, one of the three Akt protein kinase isoenzymes; suppression of other Akt isozymes did not affect melanoma cell senescence. The results presented in this work and previously obtained indicate that α5ß1 shares with other integrins of the ß1 family the function of cell protection from senescence. This function is realized via regulation of the PI3K/Akt1/mTOR signaling pathway, in which Akt1 exhibits a non-canonical activity.

The effect of renalase-derived peptides on viability of HepG2 and PC3 cells.

Renalase (RNLS) is a recently discovered protein, which plays different roles inside and outside cells. Intracellular RNLS is a FAD-dependent oxidoreductase (EC 1.6.3.5), while extracellular RNLS lacks its N-terminal peptide, FAD cofactor, and exhibits various protective effects in a non-catalytic manner. Certain evidence exists, that plasma/serum RNLS is not an intact protein secreted into the extracellular space, and exogenous recombinant RNLS is effectively degraded during short-term incubation with human plasma samples. Some synthetic analogues of the RNLS sequence (e.g. the Desir's peptide RP-220, a 20-mer peptide corresponding to the RNLS sequence 220-239) have effects on cell survival. This suggests that RNLS-derived peptides, formed during proteolytic processing, may have own biological activity. Based on results of a recent bioinformatics analysis of potential cleavage sites of RNLS (Fedchenko et al., Medical Hypotheses, 2022) we have investigated the effect of four RNLS-derived peptides as well as RP-220 and its fragment (RP-224) on the viability of two cancer cell lines: HepG2 (human hepatoma) and PC3 (prostate cancer). Two RNLS-derived peptides (RP-207 and RP-220) decreased the viability of HepG2 cells in a concentration dependent manner. The most pronounced and statistically significant effect (30-40% inhibition of cell growth) was observed at 50 µM concentration of each peptide. In the experiments with PC3 cells five of six RNLS-derived peptides had a significant impact on the cell viability. RP-220 and RP-224 decreased cell viability; however, no concentration dependence of this effect was observed in the range of concentrations studied (1-50 µM). Three other RNLS-derived peptides (RP-207, RP-233, and RP-265) increased viability of PC3 cells by 20-30%, but no concentration-dependence of this effect was found. Data obtained suggest that some RNLS-derived peptides may influence the viability of various cells and manifestation and direction of the effect (increase of decrease of the cell viability) is cell-type-specific.

Prospects for the Use of Marine Sulfated Fucose-Rich Polysaccharides in Treatment and Prevention of COVID-19 and Post-COVID-19 Syndrome.

Symptoms of the new coronavirus infection that appeared in 2019 (COVID-19) range from low fever and fatigue to acute pneumonia and multiple organ failure. The clinical picture of COVID-19 is heterogeneous and involves most physiological systems; therefore, drugs with a wide spectrum of mechanism of action are required. The choice of the treatment strategy for post-COVID-19 syndrome is still a challenge to be resolved. Polysaccharides with a high fucose content derived from seaweed and marine animals can form the basis for the subsequent development of promising agents for the treatment of COVID-19 and post-COVID-19 syndrome. This class of biopolymers is characterized by a variety of biological activities, including antiviral, antithrombotic, anticoagulant, hemo-stimulating, anti-inflammatory and immune-regulatory. Low molecular weight derivatives of these polysaccharides, as well as synthetic oligosaccharides with a sufficient amount and sulfation type may be considered as the most promising compounds due to their better bioavailability, which undoubtedly increases their therapeutic potential.

Erratum to: Prospects for the Use of Marine Sulfated Fucose-Rich Polysaccharides in Treatment and Prevention of COVID-19 and Post-COVID-19 Syndrome.

[This corrects the article DOI: 10.1134/S1068162022060152.].

Composite phospholipid-gold nanoparticles with targeted fragment for tumor imaging.

Gold nanoparticles and their conjugates have significant potential in the field of diagnosis of various diseases due to their SPR, which enhances light scattering and absorption. Conjugates of gold nanoparticles with various ligands can be used for imaging biomolecules or detecting malignant neoplasms at an early stage. This study focuses on the construction of composite (or hybrid) phospholipid-gold nanoparticles using soy phosphatidylcholine and a targeted ligand (folic acid derivative) to attach specific targeting properties. According to the method of dynamic light scattering, the diameter of the obtained nanoparticles was less than 100 nm, the results of the MTT test indicated their moderate cytotoxicity. In vitro and in vivo experiments showed a significant increase in the accumulation of phospholipid-gold nanoparticles with a targeted fragment compared to those without a targeted fragment both in HeLa cells and in a tumor (in BDF mice with an injected LLC tumor). The resulting nanoparticles are suitable for specific delivery into tumor cells and visualization of various malignant neoplasms, including at early stages, due to the increased expression of the folate receptor characteristic of cells of a wide range of tumors.

[Targeted drug delivery system for doxorubicin based on a specific peptide and phospholipid nanoparticles]. / Sistema adresnoi dostavki dlia doksorubitsina na osnove spetsificheskogo peptida i fosfolipidnykh nanochastits.

Doxorubicin is one of the widely known and frequently used chemotherapy drugs for the treatment of various types of cancer, the use of which is difficult due to its high cardiotoxicity. Targeted drug delivery systems are being developed to reduce side effects. One of the promising components as vector molecules (ligands) are NGR-containing peptides that are affinity for the CD13 receptor, which is expressed on the surface of many tumor cells and tumor blood vessels. Previously, a method was developed for preparing a composition of doxorubicin embedded in phospholipid nanoparticles with a targeted fragment in the form of an ultrafine emulsion. The resulting composition was characterized by a small particle size (less than 40 nm) and a high degree of incorporation of doxorubicin (about 93%) into transport nanoparticles. When assessing the penetrating ability and the degree of binding to the surface of fibrosarcoma cells (HT-1080), it was shown that when the composition with the targeted fragment was added to the cells, the level of doxorubicin was almost 2 times higher than that of the liposomal form of doxorubicin, i.e. the drug in the system with the targeted peptide penetrated the cell better. At the same time, on the control line of breast adenocarcinoma cells (MCF-7), which do not express the CD13 receptor on the surface, there was not significant difference in the level of doxorubicin in the cells. The data obtained allow us to draw preliminary conclusions about the prospects of targeted delivery of doxorubicin to tumor cells when using a peptide conjugate containing an NGR motif and the further need for its comprehensive study.

Effect of Cell-Penetrating Arginine Peptide on Interaction of Photosensitizer Chlorin e6 Incorporated into Phospholipid Nanoparticles with Tumor Cells.

We studied the possibility of increasing the efficiency of photodynamic therapy by improving delivery of photosensitizers chlorin e6 into tumor cells. Previous studies showed that incorporation of chlorin e6 onto phospholipid nanoparticles with a diameter <20 nm reduces its cytotoxicity due to accelerated elimination from organs [8]. A heptapeptide R7 synthesized and added to this combination promoted internalization of chlorin e6 into HepG2 cells in comparison with initial nanoparticles without peptide R7. The observed effect of peptide R7 can be explained by activation of endocytosis and/or macropinocytosis (bearing in mind the interaction of arginine with carboxyl groups of e6. The development of this transporting system is a promising trend in photodynamic therapy of cancer diseases.

Implication of Integrin α2ß1 in Proliferation and Invasion of Human Breast Carcinoma and Melanoma Cells: Noncanonical Function of Akt Protein Kinase.

Blocking the expression of integrin α2ß1, which was accomplished by transduction of α2-specific shRNA, resulted in significant inhibition of proliferation and clonal activity in human MCF-7 breast carcinoma and SK-Mel-147 melanoma cells. Along with these changes, deprivation of α2ß1 caused a sharp decrease in melanoma cell invasion in vitro. Analysis of integrin-mediating signal pathways that control cell behavior revealed a significant increase in activity of Akt protein kinase in response to depletion of α2ß1. The increase in Akt activity that accompanies a suppressive effect on cell invasion contradicts well-known Akt function aimed at stimulation of tumor progression. This contradiction could be explained by the "reversed" (noncanonical) role played by Akt in some cells that consists in suppression rather than promotion of invasive phenotype. To test this suggestion, the effects of Akt inhibitors on invasive activity of SK-Mel-147 cells were investigated. If the above suggestion is true, then inhibition of Akt in cells depleted of α2ß1 should result in the restoration of their invasive activity. It appeared that treatment with LY294002, which inhibits all Akt isoforms (Akt1, Akt2, Akt3), not only failed to restore the invasive phenotype of melanoma cells but further attenuated their invasive activity. However, treatment of the cells with an Akt1-specific inhibitor significantly increased their invasion. Thus, the stimulating effect of α2ß1 integrin on invasion of melanoma cells is realized through a mechanism based on inhibition of one of the Akt isoforms, which in these cells exhibits a noncanonical function consisting in suppression of invasion.

Hyperexpression of Integrin α5ß1 Promotes Resistance of MCF-7 Human Breast Carcinoma Cells to Doxorubicin via ERK Protein Kinase Down-regulation.

In MCF-7 human breast carcinoma cells, α5ß1 integrin hyperexpression, which was accomplished by transduction of a full-length α5 integrin cDNA, increased by about 50-70% the number of cells, survived during 48-72 h cell treatment with doxorubicin. Up-regulation of α5ß1 reduced the level of the apoptogenic p53 protein and p21 cell cycle inhibitor, but enhanced the activity of Akt and mTOR protein kinases. In addition to these findings, we observed a significant decrease in the activity of both isoforms of phosphokinase Erk1/2, which is known to play a key role in cell viability pathways, including pathways alleviating stress damages caused by distinct antitumor drugs. Diminished Erk activity accompanying the rise of drug resistance can be explained by an "atypical" function of this kinase, which, in the cells studied, promotes an enhanced rather than reduced sensitivity to doxorubicin. To verify this suggestion, the effect of a specific Erk inhibitor, PD98059, on the resistance to doxorubicin of control and α5 cDNA-transduced MCF-7 cells was investigated. The data showed that suppression of Erk activity increased the resistance of control cells (transduced with an "empty" vector) to a level higher than that demonstrated by the α5 cDNA-transduced cells. The highest level of resistance was observed in α5ß1-trancduced cells treated with PD98059. Akt and mTOR kinase inhibitors had little if any effect on doxorubicin resistance of α5 cDNA-transduced MCF-7 cells. The data show for the first time that integrin α5ß1 can stimulate drug resistance of tumor cells through a mechanism based on the inhibition of protein kinase Erk. From a more general view, the results of this investigation suggest that signal protein kinases can perform in tumor cells "non-canonical" functions, opposite to those, which are the basis for using kinase inhibitors in targeted cancer therapy. It follows that if a protein kinase is supposed to be used as a target for such therapy, it is important to explore its features in the particular tumor prior to the onset of treatment.

[Implication of integrin alpha5beta1 signal pathways in proliferation and apoptosis of MCF-7/Dox human breast carcinoma cells]. / Razlichiia mezhdu integrinom al'fa5beta1i retseptorom EGRF v signal'nykh putiakh, kontroliruiushchikh proliferatsiiu i apoptoz kletokMCF-7/Dox kartsinomy molochnoi zhelezy cheloveka.

In MCF-7/Dox human breast carcinoma cells, down-regulation of integrin alpha5beta1 and inhibition of epidermal growth factor receptor (EGFR) markedly reduced rates of cell proliferation. Mitotic cycle analysis showed that alpha5beta1 down-regulation resulted in cell cycle arrest at the S phase, followed by a significant increase in the population of apoptotic cells (subG1 population). Inhibition of EGFR activity also caused cell cycle arrest at the S-phase but without any increase in the subG1 population. Down-regulation of alpha5beta1 and EGFR inhibition resulted in a significant decrease of cell content of the active (phosphorylated) forms of FAK and Erk protein kinases. The data obtained suggest that alpha5beta1 integrin is implicated in cell growth control via inhibition of apoptotic cell death and through EGFR activation.

Pharmacological targeting of guanosine monophosphate synthase suppresses melanoma cell invasion and tumorigenicity.

Malignant melanoma possesses one of the highest metastatic potentials among human cancers. Acquisition of invasive phenotypes is a prerequisite for melanoma metastases. Elucidation of the molecular mechanisms underlying melanoma invasion will greatly enhance the design of novel agents for melanoma therapeutic intervention. Here, we report that guanosine monophosphate synthase (GMPS), an enzyme required for the de novo biosynthesis of GMP, has a major role in invasion and tumorigenicity of cells derived from either BRAF(V600E) or NRAS(Q61R) human metastatic melanomas. Moreover, GMPS levels are increased in metastatic human melanoma specimens compared with primary melanomas arguing that GMPS is an attractive candidate for anti-melanoma therapy. Accordingly, for the first time we demonstrate that angustmycin A, a nucleoside-analog inhibitor of GMPS produced by Streptomyces hygroscopius efficiently suppresses melanoma cell invasion in vitro and tumorigenicity in immunocompromised mice. Our data identify GMPS as a powerful driver of melanoma cell invasion and warrant further investigation of angustmycin A as a novel anti-melanoma agent.

Implication of α2ß1 integrin in anoikis of MCF-7 human breast carcinoma cells.

Silencing of α2ß1 integrin expression significantly promoted anchorage-dependent apoptosis (anoikis) and drastically reduced clonal activity of MCF-7 human breast carcinoma cells. Depletion of α2ß1 enhanced the production of apoptotic protein p53 and of inhibitor of cyclin-dependent protein kinases, p27, while downregulating antiapoptotic protein Bcl-2 and multifunctional protein cMyc. Blocking the expression of α2ß1 had no effect on activity of protein kinase Akt, but it sharply increased the kinase activity of Erk1/2. Pharmacological inhibition of Erk1/2 had a minor effect on anoikis of control cells, while it reduced anoikis of cells with downregulated α2ß1 to the level of control cells. The data show for the first time that integrin α2ß1 is implicated in the protection of tumor cells from anoikis through a mechanism based on the inhibition of protein kinase Erk.

[Integrins as a potential target for targeted anticancer therapy].

The review briefly summarizes information of structure of integrins and their involvement in the development and malignant progression of tumors. Special attention is paid to approaches based on modification of functional properties of integrins that prevent/antagonize tumor growth and progression; these approaches developed in modem experimental biology have certain perspective in clinical application.

[Molecular modeling of interaction of 17(20)Z- and 17(20)E-pregna-5,17(20)-dien-21-oyl amides with the nuclear receptor LXRbeta].

Aiming the search of novel regulators of lipid metabolism and their potential targets, in this study we performed molecular modeling of eight isomeric 17(20)Z- and 17(20)E-pregna-5,17(20)-dien-21-oyl amides differing in structure of the amide moiety. Analysis of the low energy conformers revealed that all 17(20)E-isomers had three main energy minima (corresponding to values of the dihedral angle theta20,21 (C17 = C20-C21 = 0) to approximately 0 degrees, to approximately 120 degrees and to approximately 240 degrees), the most occupied minimum was found to correspond to theta20,21 to approximately 0 degrees; while 17(20)Z-isomers had either one or two pools of low energy conformations. Molecular docking of these compounds to the ligand-binding site of the nuclear receptor LXRbeta (a potential target) indicates high probability of binding for E-isomers and the absence of that for Z-isomers. Results of the molecular modeling were confirmed by an experiment in which stimulation of triglyceride biosynthesis in Hep G2 cells in the presence of 17(20)E-3beta-hydroxypregna-5,17(20)-dien-21-oyl (hydroxyethyl)amide was demonstrated.

[Implication of integrin alpha5beta1 in human breast carcinoma apoptosis and drug resistance].

Doxorubicin-resistant MCF-7Dox line, which is a derivative of the drug-sensitive MCF-7 human breast carcinoma line, differs from the latter by a strongly reduced expression of the alpha2beta1 integrin and a highly increased expression of the alpha5beta1 receptor. Silencing of this integrin in the MCF-7Dox cells by transfection with alpha5-specific siRNA markedly stimulated anoikis and increased sensitivity of the cells to doxorubicin. Alpha5beta1 silencing also leads to significant inhibition of the activity of kinases Akt and Erk2 in MCF-7Dox cells. Our results suggest that integrins alpha5beta1-induced signals, controlling distinct aspects of cell behavior, are conducted through the common signal pathways.

[Anticoagulant activity of fucoidans from brown algae].

The anticoagulant activity of polysaccharide fucoidans from 11 species of brown algae was studied. The anticoagulant activity was measured by the activated partial thromboplastin time (APTT), prothrombin time and thrombin time. Inhibitory action of fucoidans varied significantly from one species to another. Fucoidans from Laminaria saccharina and Fucus distichus showed high anticoagulant activities, while fucoidans from Cladosiphon okamuranus and Analipus japonicus were almost inactive. The fucoidan inhibitory effect on thrombin and factor Xa in the presence or in the absence of natural thrombin inhibitor, antithrombin III (AT III), was also studied. In contrast to the best studied anticoagulant heparin the most of the fucoidans inhibited thrombin in the absence of AT III. In the presence of AT III inhibitory effect of fucoidans was increased considerably. Unlike heparin, the effect of fucoidans on factor Xa was very weak in the presence of AT III and was not observed in the absence of AT III. The correlation between the anticoagulant activities of this series of fucoidans and their anti-inflammatory action, studied by us earlier, was not found. It is expected that two these types of fucoidan activities depend on different structural features of fucoidans. These findings show the possibility to obtain fucoidans with high anti-inflammatory action and with low anticoagulant activity. Anticoagulant activity of the fucoidans did not depend on the content of fucose, the other neutral sugars and sulfates in the preparations, and also on the structure of the backbone of molecule. Taken together, these results indicate on prospects of fucoidan study as potential therapeutic agents.

Implication of alpha5beta1 integrin in invasion of drug-resistant MCF-7/ADR breast carcinoma cells: a role for MMP-2 collagenase.

Expression of alpha5beta1 integrin in the drug-resistant MCF-7/ADR breast carcinoma cells was inhibited by treatment of these cells with alpha5-specific siRNA. The decrease of alpha5beta1 expression resulted in a sharp decrease of expression of MMP-2 collagenase and inhibition of invasion activity of these cells in vitro. Similar decrease of invasion was also observed during inhibition of MMP-2 expression by treatment of these cells with MMP-2-specific siRNA. Inhibition of alpha5beta1 expression was also accompanied by significant decrease in cell content of active (phosphorylated) forms of signal protein kinases Akt and Erk1/2. Inhibition of activity of these kinases by treatment of cells with PI-3K/Akt-specific inhibitor LY294002 or Erk-specific inhibitor PD98059 resulted in inhibition of MMP-2 expression and the decrease of invasion in vitro. These data suggest that alpha5beta1 controls invasion ability of these cells by regulating expression of MMP-2, which involves PI-3K and Erk1/2 protein kinase signaling.

[Cytotoxic derivatives of (22R,23R)-22,23-dihydroxystigmastane].

(22R,23R)-22,23-dihydroxystigmast-4-en-3-one, (22R,23R)-22,23-dihydroxystigmast-4-en-3,6-dione, (22R,23R)-3beta,5alpha,6beta,22,23-pentahydroxystigmastane, (22R,23R)-5alpha,6alpha-oxido-3beta,22,23-trihydroxystigmastane, (22R,23R)-5beta,6beta-oxido-3beta,22,23-trihydroxystigmastane, and (22R,23R)-3beta,6beta,22,23-tetrahydroxystigmast-4-ene were synthesized. Their cytotoxicities were comparatively studied using the MCF-7 line of carcinoma cells of human mammary gland and cells of human hepatoma of the Hep G2 line.

[Delta5-7-Ketosterols with modified side chain: the synthesis and the effects on viability and cholesterol biosynthesis in Hep G2 cells].

(22E)-3beta-Hydroxysitosta-5,22-dien-7-one, (22R, 23R)-3beta,22,23-trihydroxysitost-5-en-7-one, and (22R, 23R)-3beta-hydroxy-22,23-isopropylidenedioxysitost-5-en-7-one were synthesized. The cytotoxicity and effects on cholesterol biosynthesis of the resulting 7-ketosterols, 7-ketocholesterol, and (22S,23S)-3beta-hydroxy-22,23-oxidositost-5-en-7-one were studied in hepatoblastoma Hep G2 cells.

The role of beta1 integrin subfamily in anchorage-dependent apoptosis of breast carcinoma cells differing in multidrug resistance.

Integrin expression was investigated in MCF-7 human breast adenocarcinoma line and in the MCF-7Dox line, which was selected from MCF-7 by a resistance to multiple antitumor drugs (MDR). We have shown that acquisition of MDR was accompanied by a drastically reduced expression of some integrins of the beta1-subfamily (alpha2beta1, alpha3beta1, alpha6beta1) and of alpha vbeta5 intergin in the adenocarcinoma cells. In contrast, expression of alpha5beta1 integrin was markedly increased in the MDR cells. Along with multiple antitumor drug resistance, MCF-7Dox cells demonstrate elevated resistance to anchorage-dependent apoptosis (anoikis) and enhanced in vitro invasive activity. To elucidate the implication of beta1-integrins in the above phenotypic modifications, the effect of beta1-integrin signaling was assayed. Stimulation of beta1-mediated signaling was accomplished by treating of the cells with antibodies to the beta1-subunit common for members of the beta1-subfamily. These data show that activation of beta1-integrin signaling markedly upregulated anoikis of the adenocarcinoma cells.