Suppression of the Antioxidant System and PI3K/Akt/mTOR Signaling Pathway in Cisplatin-Resistant Cancer Cells by Quercetin.

We studied the effect of quercetin on ovarian adenocarcinoma SKOV-3 cell line and isogenic subline SKOV-3/CDDP resistant to the anticancer drug cisplatin. It was found that in resistant cells, quercetin in a concentration of 100 µM that causes a decrease in the cell viability suppressed the expression of genes encoding the key antioxidant enzymes (SOD2, CAT, GPX1, and HO-1), transcription factor Nrf2, and kinases of the PI3K/Akt/mTOR signaling pathway. In parental cells, quercetin, on the contrary, increased the expression of these genes. The results confirm the redox-dependent regulation induced by quercetin and its opposite nature in cisplatin-sensitive and cisplatin-resistant cancer cells.

Suppression of PI3K/Akt/mTOR Signaling Pathway and Antioxidant System and Reversal of Cancer Cells Resistance to Cisplatin under the Effect of Curcumin.

The effect of curcumin on the resistance of SKOV-3 human ovarian adenocarcinoma cells to cisplatin was studied. It was found that curcumin induced "reversal" of cancer cells resistance, which was associated with suppression of the expression of genes encoding the key antioxidant enzymes (SOD1, SOD2, CAT, GPX1, and HO-1) and transcription factor Nrf2 and a decrease in the expression of genes encoding kinases of the PI3K/Akt/mTOR signaling pathway. The obtained results confirm the role of redox-dependent regulation in the "reversal" of cancer cells resistance to cisplatin.

[Mechanisms of development of side effects and drug resistance to asparaginase and ways to overcome them]. / Mekhanizmy razvitiia pobochnykh éffektov i lekarstvennoi ustoichivosti k asparaginazam i puti ikh preodoleniia.

Asparaginase is one of the most important chemotherapeutic agents against acute lymphoblastic leukemia, the most common form of blood cancer. To date, both asparaginases from E. coli and Dickeya dadantii (formerly known as Erwinia chrysanthemi), used in hematology, induce chemoresistance in cancer cells and side effects in the form of hypersensitivity of immune reactions. Leukemic cells may be resistant to asparaginase due to the increased activity of asparagine synthetase and other mechanisms associated with resistance to asparaginase. Therefore, the search for new sources of L-asparaginases with improved pharmacological properties remains a promising and prospective study. This article discusses the mechanisms of development of resistance and drug resistance to L-asparaginase, as well as possible ways to overcome them.

[Increased suppressor activity of transformed ex vivo regulatory T-cells in comparison with unstimulated cells of the same donor]. / Povyshennaia supressornaia aktivnost' transformirovannykh ex vivo reguliatornykh T-kletok v sravnenii s nestimulirovannymi kletkami togo zhe donora.

Regulatory T-cells CD4⁺CD25⁺FoxP3⁺CD127low (Tregs) play a key role in the maintenance of tolerance to auto antigens, inhibit function of effector T and B lymphocytes, and provide a balance between effector and regulatory arms of immunity. Patients with autoimmune diseases have decreased Treg numbers and impaired suppressive activity. Transformed ex vivo autologous Tregs could restore destroyed balance of the immune system. We developed a method for Treg precursor cell cultivation. Following the method, we were able to grown up 300-400 million of Tregs cells from 50 ml of peripheral blood during a week. Transformed ex vivo Tregs are 90-95% CD4⁺CD25⁺FoxP3⁺CD127low and have increased expression of transcription genes FoxP3 and Helios. Transformed ex vivo Tregs have increased demethylation of FoxP3 promoter and activated genes of proliferation markers Cycline B1, Ki67 and LGALS 1. Transformed ex vivo Tregs have increased suppressive activity and up to 80-90% these cells secrete cytokines TNFα и IFNγ. Our data suggest transformed ex vivo autologous Tregs have genetic, immunophenotypic and functional characteristics for regulatory T-cells and further can be used for adoptive immunotherapy autoimmune diseases and inhibition of transplantation immunity.

Effect of Heterologous Expression of Chemotaxis Proteins from Genus Thermotoga on the Growth Kinetics of Escherichia coli Cells.

In the process of optimization of heterologous expression of thermostable chemotaxis proteins CheW and CheY as industrially useful polypeptides, their direct influence on the cell growth kinetics and morphology of Escherichia coli was observed. CheW and CheY of bacteria of the genus Thermotoga, being expressed in recombinant form in E. coli cells, are involved in the corresponding signal pathways of the mesophilic microorganisms. The effects of such involvement in the metabolism of "host" cells are extremely diverse: from rapid aging of the culture to elongation of the stationary growth phase. We also discuss the mechanisms of the influence of the heterologous chemotaxis proteins on cells, their positive and negative effects, as well as potential applications in industry and biomedicine.

Thermostable Recombinant Polypeptides as the Source of L-Amino Acids for Culture Media.

Mutant homologues of small chemotactic and DNA-binding proteins from thermophilic bacteria Thermotoga petrophila RKU-1 and Thermotoga naphthophila were obtained. These proteins can be expressed in the recombinant form in E. coli cells. A wide range of properties and parameters that are important for isolation of these proteins were revealed: stability in a wide temperature and pH range, high level of expression, solubility, and the possibility of using simple purification schemes with low number of successive steps. The positive effect of proteins on in vitro fibroblasts growth was demonstrated. The described properties of the target proteins indicate the possibility of their use in different biotechnology industries as an inexpensive source of L-amino acids.

Intracellular Localization of Apoptotic Endonuclease EndoG and Splice-Variants of Telomerase Catalytic Subunit hTERT.

The activity of telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) can be regulated by alternative splicing of its mRNA. The mechanism of hTERT splicing is not understood in detail. Apoptotic endonuclease EndoG is known to participate in this process. In the present work, the intracellular colocalization and mRNA levels of EndoG and hTERT splice-variants in normal and apoptotic cancer cells were studied. We found that the development of apoptosis increased the expression of EndoG and changed the ratio of hTERT splice-variants, which decreased the telomerase activity in the cells. The development of apoptosis was accompanied by changes in the amount of mRNA and in the localization of EndoG and hTERT splice-variants in the cytoplasm, nuclei, and mitochondria of the cells. The suppression of EndoG expression using RNA interference prevented induction of the α+ß- splice-variant of hTERT and inhibition of the telomerase activity. A high degree of the intracellular colocalization of EndoG and hTERT was shown. The changes in the expression and localization of EndoG corresponded with changes in the amount and localization of hTERT splice-variants. These data confirm the participation of EndoG in the alternative splicing of mRNA of the telomerase catalytic subunit and in regulation of the telomerase activity.

[Cisplatin-induced apoptotic endonuclease EndoG inhibits telomerase activity and causes malignant transformation of human CD4+ T lymphocytes]. / Indutsirovannaia tsisplatinom ékspressiia apoptoticheskoi éndonukleazy EndoG vyzyvaet ingibirovanie aktivnosti telomerazy i zlokachestvennuiu transformatsiiu CD4+ T-limfotsitov cheloveka.

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.

[Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase]. / Podavlenie aktivnosti telomerazy leikoznykh kletok mutantymi formami L-asparaginazy Rhodospirillum rubrum.

The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.

Apoptotic Endonuclease EndoG Inhibits Telomerase Activity and Induces Malignant Transformation of Human CD4+ T Cells.

Telomerase activity is regulated by an alternative splicing of mRNA of the telomerase catalytic subunit hTERT (human telomerase reverse transcriptase). Increased expression of the inactive spliced hTERT results in inhibition of telomerase activity. Little is known about the mechanism of hTERT mRNA alternative splicing. This study was aimed at determining the effect of an apoptotic endonuclease G (EndoG) on alternative splicing of hTERT and telomerase activity in CD4+ human T lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of the active full-length hTERT variant and upregulated the inactive alternatively spliced variant. Reduction of full-length hTERT levels caused downregulation of the telomerase activity, critical telomere shortening during cell division that converted cells into the replicative senescence state, activation of apoptosis, and finally cell death. Some cells survive and undergo a malignant transformation. Transformed cells feature increased telomerase activity and proliferative potential compared to the original CD4+ T cells. These cells have phenotype of T lymphoblastic leukemia cells and can form tumors and cause death in experimental mice.

[Apoptotic endonuclease EndoG induces alternative splicing of telomerase catalytic subunit hTERT and death of tumor cells]. / Éndonukleaza EndoG indutsiruet al'ternativnyi splaising kataliticheskoi sub"edinitsy telomerazy hTERT i gibel' opukholevykh kletok.

Telomerase activity is known to be regulated by alternative splicing of its catalytic subunit hTERT (human Telomerase Reverse Transcriptase) mRNA. Induction of non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. Strong correlation was found between expression of EndoG and hTERT splice-variants in 12 colon cancer cell lines. Overexpression of EndoG in СаСо-2 cells downregulated the expression of active full-length hTERT variant and upregulated non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, dramatically shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. These data indicated the participation of EndoG in alternative splicing of mRNA of telomerase catalytic subunit, regulation of telomerase activity and cell fate.

Identification of functional regions in the Rhodospirillum rubrum L-asparaginase by site-directed mutagenesis.

Site-directed mutagenesis of Rhodospirillum rubrum L-asparaginase (RrA) was performed in order to identify sites of the protein molecule important for its therapeutic and physico-chemical properties. Ten multipoint mutant genes were obtained, and five recombinant RrA variants were expressed in E. coli BL21(DE3) cells and isolated as functionally active highly purified proteins. Protein purification was performed using Q-Sepharose and DEAE-Toyopearl chromatography. Overall yield of the active enzymes was 70-80 %, their specific activity at pH 7.4 and 37 °C varied of 140-210 U/mg. L-Glutaminase activity did not exceed 0.01 % of L-asparaginase activity. All RrA mutants showed maximum enzyme activity at pH 9.3-9.5 and 53-58 °C. Km and Vmax values for L-asparagine were evaluated for all mutants. Mutations G86P, D88H, M90K (RrAH), G121L, D123A (RrАI) caused the loss of enzyme activity and confirmed the importance of these sites in the implementation of catalytic functions. Removal of four residues from C-terminal area of the enzyme (RrAK) resulted in the enzyme instability. Mutations D60K, F61L(RrАD), and R118H, G120R(RrАJ) led to the improvement of kinetic parameters and enzyme stabilization. Substitutions E149R, V150P (RrАB) improved antineoplastic and cytotoxic activity of the RrA. A64V, E67K substitutions, especially in combination with E149R, V150P (RrАE), considerably destabilized recombinant enzyme.

[Physicochemical properties and antiproliferative activity of recombinant Yersinia pseudotuberculosis L-asparaginase].

The physicochemical, catalytic, and antiproliferative activity of a recombinant L-asparaginase from Yersinia pseudotuberculosis (YpA) have been studied. The following results were obtained: the K(M) value for L-asparagine is 17 +/- 0.9 microM, the optimal temperature is 60 degrees C, pH is 8.0, pI is 5.4 +/- 0.3, the L-glutaminase activity is no more than 5-6% of the L-asparaginase activity, and the antiproliferative activity on the Fisher L5178y lymphadenosis cell line comprised T/C = 136% (p < 0.001) at a 15% recovery rate. The described characteristic allows one to regard YpA as an antitumor enzyme with biological features similar to the L-asparaginase of E. coli.

[The influence of compound aITEL1296 on telomerase activity and the growth of cancer cells].

Telomerase is a ribonucleoprotein that synthesizes telomeric repeats and identified as a promising target for anticancer therapy. Here we describe a new compound aITEL1296 as a potent telomerase inhibitor. Its inhibitory activity was a bit higher (IC50 = 0,19 +/- 0,02 ng/ml) than that of BIBR1532, one of the most potent telomerase inhibitors known to date. Besides telomerase inhibition aITEL1296 activated apoptotic mechanisms and effectively suppressed proliferation of tumor cell lines (GI50 = 5,0 +/- 0,2 ng/ml for most sensitive cell line LnCap) but not normal fibroblast cell line.