Co-delivery of paclitaxel and etoposide prodrug by human serum albumin and PLGA nanoparticles: synergistic cytotoxicity in brain tumour cells.

The aims of this study were to develop co-delivery systems of paclitaxel (PTX) and etoposide prodrug (4'-O-benzyloxycarbonyl-etoposide, ETP-cbz) based on non-cross-linked human serum albumin (HSA) and poly(lactide-co-glycolide) nanoparticles and to evaluate the synergistic potential of these drugs in vitro. The nanoformulations were prepared by the high-pressure homogenisation technique and characterised using DLS, TEM, SEM, AFM, HPLC, CZE, in-vitro release, and cytotoxicity in human and murine glioma cells. All nanoparticles had 90-150 nm in size and negative ζ-potentials. The Neuro2A cells were the most sensitive to both HSA- and PLGA-based co-delivery systems (IC50 0.024 µM and 0.053 µM, respectively). The drugs' synergistic effect (combination index < 0.9) was observed in the GL261 cells for both types of co-delivery formulations and in the Neuro2A cells for the HSA-based system. These nanodelivery systems may be useful to improve combination chemotherapy for brain tumour treatment. To our knowledge, this is the first report describing the non-cross-linked HSA-based co-delivery nanosuspension which was prepared using nab™ technology.

The Molecular Mechanisms of Oleanane Aldehyde-ß-enone Cytotoxicity against Doxorubicin-Resistant Cancer Cells.

Oleanane aldehyde-ß-enone (OA), being the semi-synthetic derivative of the triterpenoid betulin, effectively inhibits the proliferation of HBL-100 and K562 cancer cells (IC50 0.47-0.53 µM), as well as the proliferation of their resistant subclones with high P-gp expression HBL-100/Dox, K562/i-S9 and K562/i-S9_Dox (IC50 0.45-1.24 µM). A molecular docking study, rhodamine efflux test, synergistic test with Dox, and ABC transporter gene expression were used to investigate the ability of OA to act as a P-gp substrate or inhibitor against Dox-resistant cells. We noted a trend toward a decrease in ABCB1, ABCC1 and ABCG2 expression in HBL-100 cells treated with OA. The in silico and in vitro methods suggested that OA is neither a direct inhibitor nor a competitive substrate of P-gp in overexpressing P-gp cancer cells. Thus, OA is able to overcome cellular resistance and can accumulate in Dox-resistant cells to realize toxic effects. The set of experiments suggested that OA toxic action can be attributed to activating intrinsic/extrinsic or only intrinsic apoptosis pathways in Dox-sensitive and Dox-resistant cancer cells, respectively. The cytotoxicity of OA in resistant cells is likely mediated by a mitochondrial cell death pathway, as demonstrated by positive staining with Annexin V-FITC, an increasing number of cells in the subG0/G1 phase, reactive oxygen species generation, mitochondrial dysfunction, cytochrome c migration and caspases-9,-6 activation.

Role of YB-1 Protein in Inflammation.

This review discusses the role of the multifunctional DNA/RNA-binding protein YB-1 in inflammation. YB-1 performs multiple functions in the cell depending on its location: it acts as transcriptional factor for many genes in the nucleus, regulates translation and stability of mRNA in the cytoplasm, and becomes a paracrine factor when secreted from the cells. The review presents the data on the YB-1-mediated regulation of inflammation-associated genes, as well as results of studies on the YB-1 role in animal model of various inflammatory diseases, such as glomerulonephritis, tubulointerstitial fibrosis, and bacterial sepsis, and on the YB-1 expression in different human diseases associated with inflammatory processes in kidney, liver, and endometrium. The last section of the review presents several approaches to the regulation of YB-1 with small molecules in the treatment of inflammatory diseases.

Natural Phaeosphaeride A Derivatives Overcome Drug Resistance of Tumor Cells and Modulate Signaling Pathways.

In the present study, natural phaeosphaeride A (PPA) derivatives are synthesized. Anti-tumor studies are carried out on the PC3, K562, HCT-116, THP-1, MCF-7, A549, NCI-H929, Jurkat, and RPMI8226 tumor cell lines, and on the human embryonic kidney (HEK293) cell line. All the compounds synthesized turned out to have better efficacy than PPA towards the tumor cell lines listed. Among them, three compounds exhibited an ability to overcome the drug resistance of tumor cells associated with the overexpression of the P-glycoprotein by modulating the work of this transporter. Luminex xMAP technology was used to assess the effect of five synthesized compounds on the activation of intracellular kinase cascades in A431 cells. MILLIPLEX MAP Multi-Pathway Magnetic Bead 9-Plex was used, which allowed for the simultaneous detection of the following nine phosphorylated protein markers of the main intracellular signaling pathways: a universal transcription factor that controls the expression of immune-response genes, apoptosis and cell cycle NFκB (pS536); cAMP-dependent transcription factor (CREB (pS133); mitogen-activated kinase p38 (pT180/pY182); stress-activated protein kinase JNK (pT183/pY185); ribosomal SK; transcription factors STAT3 (pS727) and STAT5A/B (pY694/699); protein kinase B (Akt) (pS473); and kinase regulated by extracellular signals ERK1/2 (pT185/pY187). The effect of various concentrations of PPA derivatives on the cell culture was studied using xCelligence RTCA equipment. The compounds were found to modulate JNK, ERK1/2, and p38 signaling pathways. The set of activated kinase cascades suggests that oxidative stress is the main probable mechanism of the toxic action of PPA derivatives.

Soft Tissue Sarcoma Study: Association of Genetic Alterations in the Apoptosis Pathways with Chemoresistance to Doxorubicin.

Soft tissue sarcomas (STS) are heterogeneous cancers with more than 100 histological subtypes, different in molecular alterations, which make its personalized therapy very complex. Gold standard of chemotherapy for advanced STS includes combinations of Doxorubicin and Ifosfamide or Gemcitabine and Docetaxel. Chemotherapy is efficient for less than 50% of patients and it is followed by a fast development of drug resistance. Our study was directed to the search of genetic alterations in cancer cells associated with chemoresistance of undifferentiated pleomorphic and synovial sarcomas to the abovementioned genotoxic drugs. We analyzed chemoresistance of cancer cells in vitro using primary STS cultures and performed genetic analysis for the components of apoptotic signaling. In 27% of tumors, we revealed alterations in TP53, ATM, PIK3CB, PIK3R1, NTRK1, and CSF2RB. Cells from STS specimens with found genetic alterations were resistant to Dox, excluding the only one case when TP53 mutation resulted in the substitution Leu344Arg associated with partial oligomerization loss and did not cause total loss of TP53 function. Significant association between alterations in the components of apoptosis signaling and chemoresistance to Dox was found. Our data are important to elaborate further the therapeutic strategy for STS patients with alterations in apoptotic signaling.

Analysis of Multiple Drug Resistance Mechanism in Different Types of Soft Tissue Sarcomas: Assessment of the Expression of ABC-Transporters, MVP, YB-1, and Analysis of Their Correlation with Chemosensitivity of Cancer Cells.

Chemotherapy of soft tissue sarcomas (STS) is restricted by low chemosensitivity and multiple drug resistance (MDR). The purpose of our study was the analysis of MDR mechanism in different types of STS. We assessed the expression of ABC-transporters, MVP, YB-1, and analyzed their correlation with chemosensitivity of cancer cells. STS specimens were obtained from 70 patients without metastatic disease (2018-2020). Expression level of MDR-associated genes was estimated by qRT-PCR and cytofluorimetry. Mutations in ABC-transporter genes were captured by exome sequencing. Chemosensitivity (SI) of STS to doxorubicin (Dox), ifosfamide (Ifo), gemcitabine (Gem), and docetaxel (Doc) was analyzed in vitro. We found strong correlation in ABCB1, ABCC1, and ABCG2 expression. We demonstrated strong negative correlations in ABCB1 and ABCG2 expression with SI (Doc) and SI (Doc + Gem), and positive correlation of MVP expression with SI (Doc) and SI (Doc + Gem) in undifferentiated pleomorphic sarcoma. Pgp expression was shown in 5 out of 44 STS samples with prevalence of synovial sarcoma relapses and it is strongly correlated with SI (Gem). Mutations in MDR-associated genes were rarely found. Overall, STS demonstrated high heterogeneity in chemosensitivity that makes reasonable in vitro chemosensitivity testing to improve personalized STS therapy, and classic ABC-transporters are not obviously involved in MDR appearance.

Triterpenoids with modified A-ring as modulators of P-gp-dependent drug-resistance in cancer cells.

Semi-synthetic A-cycle modified triterpenic derivatives with A-cycle condensed with a heterocyclic fragment (compound 1) and fragmented A-ring (compound 2) were tested for cytotoxicity against several tumor cell cultures and doxorubicin (Dox)-resistant cell lines. The equal cytotoxicity of the tested compounds to the parental tumor cell lines (HBL-100, K562) and their resistant subclones (HBL-100/Dox, K562/i-S9) was revealed. The overexpression of ABCB1 (MDR1) gene and P-glycoprotein (P-gp) was confirmed for both resistant subclones of tumor cells. Compounds 1 and 2 were shown to inhibit the ABC-transporter gene expression (MDR1, MRP, MVP, and BCRP) and the transport of well-known P-gp substrate Rhodamine 123 from resistant cells. The docking of triterpenoids 1 and 2 into the drug binding site of P-gp revealed a similarity between the conformation of the tested triterpenoids and that of classical inhibitor verapamil, thus assuming these compounds to be more likely the inhibitors than the substrates of P-gp. Any tested triterpenic derivatives, when combined at non-toxic concentrations with doxorubicin, improved cytotoxic effect of the therapeutic drug against resistant subclones of tumor cells.

Towards lead compounds as anti-cancer agents via new phaeosphaeride A derivatives.

New derivatives of phaeosphaeride A (PPA) were synthesized and characterized. Anti-tumor studies were carried out on the U937, HCT-116, PC3, MCF-7, A549, К562, NCI-H929, Jurkat, THP-1, RPMI8228 tumor cell lines, and on the HEF cell line. All the compounds synthesized were found to have better efficacy than PPA towards the tumor cell lines mentioned. Compound 6 (IC50 = 0.59 ±â€¯0.27 µM) was observed to be 11 times more active than PPA (IC50 = 6.5 ±â€¯0.30 µM) towards the NCI-H929 cell line, with a therapeutic index of 18. Compound 6 was determined to be over half and 16 times more active than etoposide towards the NCI-H929 (IC50 = 0.9 ±â€¯0.05 µM) and A549 (IC50 = 100 ±â€¯7.0 µM) cell lines, respectively.

Synthesis and Biological Evaluation of Phaeosphaeride A Derivatives as Antitumor Agents.

New derivatives of phaeosphaeride A (PPA) were synthesized and characterized. Anti-tumor activity studies were carried out on the HCT-116, PC3, MCF-7, A549, К562, NCI-Н929, Jurkat, THP-1, RPMI8228 tumor cell lines, and on the HEF cell line. All of the compounds synthesized were found to have better efficacy than PPA towards the tumor cell lines mentioned. Compound 6 was potent against six cancer cell lines, HCT-116, PC-3, K562, NCI-H929, Jurkat, and RPMI8226, showing a 47, 13.5, 16, 4, 1.5, and 7-fold increase in anticancer activity comparative to those of etoposide, respectively. Compound 1 possessed selectivity toward the NCI-H929 cell line (IC50 = 1.35 ± 0.69 µM), while product 7 was selective against three cancer cell lines, HCT-116, MCF-7, and NCI-H929, each having IC50 values of 1.65 µM, 1.80 µM and 2.00 µM, respectively.

"Dry mouth" and "Flammer" syndromes-neglected risks in adolescents and new concepts by predictive, preventive and personalised approach.

BACKGROUND: "Dry mouth" syndrome (chronic hyposalivation) can be caused by a number of pathophysiological conditions such as acute and chronic stress exposure, abnormal body weight (both too high and too low ones), eating disorders (such as anorexia nervosa), metabolic syndrome(s), Sjögren's and Sicca syndromes, drugs and head/neck radiotherapy application. In turn, the chronic hyposalivation as a suboptimal health condition significantly reduces quality of life, may indicate a systemic dehydration, provokes and contributes to a number of pathologies such as a strongly compromised protection of the oral cavity, chronic infections and inflammatory processes, periodontitis, voice and digestive disorders. Consequently, "dry mouth" syndrome might be extremely useful as an indicator for an in-depth diagnostics of both-co-existing and snowballing health-threating conditions. However, predictive diagnostics, targeted prevention and personalisation of treatments are evidently underdeveloped for individuals at high risk suffering from the "dry mouth" syndrome. WORKING HYPOTHESIS AND METHODOLOGY: In the current study, we have hypothesised that individuals demonstrating "Flammer syndrome" (FS) phenotype may suffer from the "dry mouth" syndrome more frequently, due to disturbed microcirculation, psychological factors (obsessional personality/perfectionism), and diminished feeling of thirst with consequently insufficient daily liquid intake potentially resulting in the systemic dehydration with individually pronounced level of severity. If confirmed, FS phenotyping linked to the chronic hyposalivation might be predictive for individuals at risk identified by innovative screening programmes. To verify the working hypothesis, healthy individuals (negative control group) versus individuals with evident hyposalivation as well as patients diagnosed with periodontitis (positive control group) observed and treated at the dental clinic were investigated. The degree to which an individual is affected by hyposalivation was determined by the Bother xerostomia Index utilising a questionnaire of 10 issue-specific items and monitoring of a typically matt roof of the mouth in dental practice. An extent to which individuals included in the study are the carriers of the FS phenotype was estimated by the specialised 15-item questionnaire. RESULTS AND CONCLUSIONS: For both-the target group (hyposalivation) and positive control group (periodontitis)-FS phenotype was demonstrated to be more specific compared to the disease-free (negative control) group. Moreover, self-reports provided by interviewed adolescents of the target group frequently recorded remarkable discomfort related to "dry mouth" syndrome, acute and chronic otorhinolaryngological infections and even delayed wound healing. Further, interviewed adolescents do worry about the symptoms which might be indicative for potential diseases; they are also amazed that too little attention is currently paid to the issue by caregivers. In conclusion, FS questionnaire linked to the "dry mouth" syndrome is strongly recommended for application in primary healthcare. Consequently, targeted preventive measures can be triggered early in life. For example, traditional, complementary and alternative medicine demonstrates positive therapeutic effects in individuals suffering from xerostomia. For in-depth diagnostics, epi/genetic regulations involved into pathophysiologic mechanisms of hyposalivation in FS-affected individuals should be thoroughly investigated at molecular level. Identified biomarker panels might be of great clinical utility for predictive diagnostics and patient stratification that, further, would sufficiently improve personalised care to the patient.

Efficiency of dental caries prevention with the use of polymer-based toothpastes modified by the electromagnetic field.

The most important problems of modern dentistry cover not only the early diagnosis but also prevention of dental caries, including the one based on the use of various hygienic products and therapeutic agents. Normally, the processes of re- and demineralization in the oral cavity are in a state of dynamic. However, in the presence of cariogenic factors, the balance is shifted towards demineralization. In the event of initial caries, enamel remineralization is possible only to a certain extent, and specifically, for a reversible degree of carious lesion. There was an idea about structural changes in polymer-based consumable dental materials including toothpastes. The purpose of the study is to increase the efficiency of dental caries prevention based on the activation of metabolic processes in dental hard tissues with the use of R.O.C.S. Active Calcium toothpastes (Russian-Swiss "DRC-Group" company) modified by an electromagnetic field. As a result of the experimental part of the study, certain changes in the properties of the R.O.C.S. Active Calcium toothpastes under the electromagnetic field influence including microstructural transformation of the toothpaste (an increase in the size of the polymer matrix particles and a decrease in the distance between them confirmed by the electron microscopy findings), confirmed by the transmission electron microscopy results. The investigations with the help of X-ray microanalysis made it possible to clarify the chemical composition of the surface layer of the tooth enamel, for the first time to reveal in detail the concentration of micro- and macroelements of organic and inorganic nature, to trace the changes occurring under controlled tooth cleaning with the R.O.C.S. Active Calcium toothpaste treated with an electromagnetic field. The obtained results promote the further studies of laws in change of physical and mechanical properties of treatment-and-prophylactic means under the electromagnetic field influence in respect of dental caries preventive maintenance efficiency increase.

Clinical and laboratory evaluation of microstructural changes in the physical, mechanical and chemical properties of dental filling materials under the influence of an electromagnetic field.

Restorative filling materials used for dental caries prevention and treatment consist of various components including monomers or oligomers, which play a significant role in forming the main structure of these materials, as well as in characterising their physical, mechanical and chemical properties. The necessity for the development and improvement of structural characteristics of polymeric dental filling materials intended for caries prevention and their life duration increase served as the initiating factor of our research. According to the research purpose and challenges, we studied the changes in the physical, mechanical and chemical properties of composite filling materials with and without electromagnetic field influence. The investigations in vivo include the study of microstructural features of polymeric filling materials by scanning electron microscopy (SEM) and the investigations in vitro include the study of sealed and extracted human teeth chips by using X-ray spectral analysis. We also evaluated the changes in the strength characteristics of dental filling materials with and without electromagnetic field influence. The analysis of the obtained data indicates the presence of structural changes in polymeric dental filling materials, including the material microstructure condensation confirmed by the SEM results, an increase in the strength and adhesion characteristics and certain regularities of the chemical elemental composition concentration change in the area of hard tooth tissue and dental filling material. These scientific data will provide tooth caries prevention and promote the increase of treatment quality.

The role of etiopathogenetic aspects in prediction and prevention of discontinuous-hemorrhagic (Mallory-Weiss) syndrome.

The article contains an overview of the literature on Mallory-Weiss syndrome. It analyzes numerous etiological factors, provides new insights into the pathogenesis of the disease, gives a description of a previously unknown dependence of discontinuous-hemorrhagic syndrome on the topographic and structural features of the cardioesophageal area of the digestive tract, and gives scientific credence to methods of prediction, prevention, and treatment of the syndrome with complex involvement of granular sorbents.

Age-related differences of tooth enamel morphochemistry in health and dental caries.

Currently, dental caries is the main reason of patient visits to dentists. A great deal of scientific work is dedicated to the study of enamel caries. The reason for this is the necessity for more detailed study of the pathogenesis of dental caries and other pathological processes occurring in tooth enamel. The application of modern high-technological methods of research has made it possible to study enamel structure in detail. Hard dental tissues are composed of organic and inorganic components and water. The organic substance consists of proteins, lipids, and carbohydrates. At different ages, caries intensity may vary. The carious process often develops during the first months after the tooth eruption but much less in adulthood and old age. These processes are mainly associated with the mechanisms of ionic exchange between the oral cavity and hard dental tissues. Different groups of teeth are differently affected by the carious process. Previous studies have revealed that age is an important factor influencing on the structure and chemical composition of hard dental tissues. Various recent works at the Department of Therapeutic Dentistry of Voronezh N.N. Burdenko State Medical Academy (VSMA) have studied not only the structural-morphological features of the enamel in children and adults but also the level of metabolic processes inside it. As a result of aging, teeth change both the enamel structure and the level of its ionic processes, but unfortunately, these changes have not been well characterized in teeth with conventional age-specific differences.

Predictive research methods of enamel and dentine for initial caries detection.

Currently, various research methods of enamel and dentine for precautionary diagnostics of initial caries forms are developed; however, the vast majority of these do not provide objective criteria of caries diagnostics or are very difficult to perform. Therefore, the search of diagnostics and enamel research methods, which will allow predicting caries emergence and to carry out personalised prevention of this pathology, is necessary. In this review, modern diagnostic methods that allow understanding the main aspects of caries process, assess the risk of its development, and also suggest the possibility of emergency prevention of caries progression in the nearest future are presented.

On the possibility of using polycrystalline material in the development of structure-based generic assays.

The discovery of ligands that bind specifically to a targeted protein benefits from the development of generic assays for high-throughput screening of a library of chemicals. Protein powder diffraction (PPD) has been proposed as a potential method for use as a structure-based assay for high-throughput screening applications. Building on this effort, powder samples of bound/unbound states of soluble hen-egg white lysozyme precipitated with sodium chloride were compared. The correlation coefficients calculated between the raw diffraction profiles were consistent with the known binding properties of the ligands and suggested that the PPD approach can be used even prior to a full description using stereochemically restrained Rietveld refinement.

Structure of acostatin, a dimeric disintegrin from Southern copperhead (Agkistrodon contortrix contortrix), at 1.7 A resolution.

Disintegrins are a family of small (4-14 kDa) proteins that bind to another class of proteins, integrins. Therefore, as integrin inhibitors, they can be exploited as anticancer and antiplatelet agents. Acostatin, an alphabeta heterodimeric disintegrin, has been isolated from the venom of Southern copperhead (Agkistrodon contortrix contortrix). The three-dimensional structure of acostatin has been determined by macromolecular crystallography using the molecular-replacement method. The asymmetric unit of the acostatin crystals consists of two heterodimers. The structure has been refined to an R(work) and R(free) of 18.6% and 21.5%, respectively, using all data in the 20-1.7 A resolution range. The structure of all subunits is similar and is well ordered into N-terminal and C-terminal clusters with four intramolecular disulfide bonds. The overall fold consists of short beta-sheets, each of which is formed by a pair of antiparallel beta-strands connected by beta-turns and flexible loops of different lengths. Conformational flexibility is found in the RGD loops and in the C-terminal segment. The interaction of two N-terminal clusters via two intermolecular disulfide bridges anchors the alphabeta chains of the acostatin dimers. The C-terminal clusters of the heterodimer project in opposite directions and form a larger angle between them in comparison with other dimeric disintegrins. Extensive interactions are observed between two heterodimers, revealing an alphabetabetaalpha acostatin tetramer. Further experiments are required to identify whether the alphabetabetaalpha acostatin complex plays a functional role in vivo.

Using barium ions for heavy-atom derivatization and phasing of xylanase II from Trichoderma longibrachiatum.

This paper describes the use of barium chloride to produce a heavy-atom derivative of xylanase II crystals from Trichoderma longibrachiatum, which was obtained either by cocrystallization or soaking. SAD phasing led to interpretable electron-density maps that allowed unambiguous chain tracing. In the best case, with a data set collected at 9.5 keV, 88% of the residues were built, with 83% of the side chains assigned. The barium ions are found to mainly interact with main-chain carbonyl groups and water molecules. It is suggested that barium ions could also be used as a potential anomalous scatterer in the quick cryosoaking procedure for phasing.

Computational study of the thermochemistry of organophosphorus(III) compounds.

The enthalpies of formation of organophosphorus(III) compounds have been calculated at the G3X, G3X(MP2), and B3LYP/6-311+G(3df,2p)//B3LYP/6-31G(d,p) levels of theory using the atomization energy procedure and the method of isodesmic reactions. The Delta f H298 degree values for 50 relatively large molecules with up to 10 non-hydrogen atoms, such as P(CH3)3, P(C2H5)3, P(OCH3)3, n-C4H9OPCl2, [(CH3)2N]2PCl, (C2H5)2NPCl2, and [(CH3)2N]2PCN, have been calculated directly from the G3X atomization energies. A good agreement between the known experimental values and G3X results for 14 compounds provides support to our predictions for remaining species whose experimental enthalpies of formation are unknown or known with relatively large uncertainties. On the basis of our calculations and sometimes conflicting experimental data a set of internally consistent enthalpies of formation has been recommended for organophosphorus(III) compounds. Our computational results call into question the experimental enthalpies of formation of P(C2H5)3 and P(n-C4H9)3. From comparison with most reliable experimental data, the accuracy of the theoretical enthalpies of formation is estimated as ranging from 5 to 10 kJ/mol. The recommended Delta f H298 degree values were used to derive the group additivity values (GAVs) for 45 groups involving the phosphorus(III) atom. These GAVs significantly extend the applicability of Benson's group additivity method and may be used to estimate the enthalpies of formation of larger organophosphorus(III) compounds, where high level quantum chemical calculations are impracticable.

Structure of TonB in complex with FhuA, E. coli outer membrane receptor.

The cytoplasmic membrane protein TonB spans the periplasm of the Gram-negative bacterial cell envelope, contacts cognate outer membrane receptors, and facilitates siderophore transport. The outer membrane receptor FhuA from Escherichia coli mediates TonB-dependent import of ferrichrome. We report the 3.3 angstrom resolution crystal structure of the TonB carboxyl-terminal domain in complex with FhuA. TonB contacts stabilize FhuA's amino-terminal residues, including those of the consensus Ton box sequence that form an interprotein beta sheet with TonB through strand exchange. The highly conserved TonB residue arginine-166 is oriented to form multiple contacts with the FhuA cork, the globular domain enclosed by the beta barrel.