Comparative EPR Studies on the Influence of Genistein on Free Radicals in Non-Irradiated and UV-Irradiated MCF7, T47D and MDA-MB-231 Breast Cancer Cells.

The antioxidant activity and the association of genistein with carcinogenesis are widely documented. Few studies directly measure the number of free radicals generated in cells, either during the action of factors stimulating their formation, e.g., ultraviolet (UV), or after exposure to antioxidants. The most suitable method for analysing free radicals is electron paramagnetic resonance (EPR) spectroscopy. The EPR method detects a paramagnetic centre with a single electron. Antioxidants neutralize free radicals, therefore, EPR analysis of antioxidant efficacy is as valuable and important as studying the paramagnetic centres of radicals. The aim of the study was to determine the influence of genistein on free radicals basal level and after UV exposure in breast cancer cell lines MCF7, T47D and MDA-MB-231 cell lines. The impact of genistein on cell viability was investigated at concentrations of 0.37 µM, 3.7 µM, 37 µM and 370 µM. Genistein at a concentration of 370 µM revealed a cytotoxic effect on the cells of all three tested breast cancer lines. Genistein at a concentration of 0.37 µM showed no significant effect on the cell viability of all tested breast cancer lines. Therefore, cell proliferation and antioxidant properties were examined using genistein at a concentration of 0.37 µM and 37 µM. X-band (9.3 GHz) EPR spectra of three different types of breast cancer cells (ER-positive, PR-positive and HER-2 negative: MCF7 and T47D and triple-negative MDA-MB-231) were compared. UV irradiation was used as a factor to generate free radicals in cells. The effect of free radical interactions with the antioxidant genistein was tested for non-UV-irradiated (corresponding to the basal level of free radicals in cells) and UV-irradiated cells. The levels of free radicals in the non-irradiated cells studied increased in the following order in breast cancer cells: T47D < MDA-MB-231 < MCF7 and UV-irradiated breast cancer cells: MDA-MB-231 < MCF7 < T47D. UV-irradiation altered free radical levels in all control and genistein-cultured cells tested. UV irradiation caused a slight decrease in the amount of free radicals in MCF7 cells. A strong decrease in the amount of free radicals was observed in UV-irradiated MDA-MB-231 breast cancer cells. The amount of free radicals in T47D cancer cells increased after UV irradiation. Genistein decreased the amount of free radicals in non-irradiated and UV-irradiated MCF7 cells, and only a weak effect of genistein concentrations was reported. Genistein greatly decreased the amount of free radicals in UV-irradiated T47D cancer cells cultured with genistein at a concentration of 3.7 µM. The effect of genistein was negligible in the other samples. Genistein at a concentration of 3.7 µM decreased the amount of free radicals in non-irradiated MDA-MB-231 cancer cells, but genistein at a concentration of 37 µM did not change the amount of free radicals in these cells. An increase in the amount of free radicals in UV-irradiated MDA-MB-231 cancer cells was observed with increasing genistein concentration. The antioxidant efficacy of genistein as a potential plant-derived agent supporting the treatment of various cancers may be determined by differences in signalling pathways that are characteristic of breast cancer cell line subtypes and differences in activation of oxidative stress response pathways.

A Statistical Approach to Neutron Stars' Crust-Core Transition Density and Pressure.

In this paper, a regression model between neutron star crust-core pressure and the symmetry energy characteristics was estimated using the Akaike information criterion and the adjusted coefficient of determination Radj2. The most probable value of the transition density, which should characterize the crust-core environment of the sought physical neutron star model, was determined based on the obtained regression function. An anti-correlation was found between this transition density and the main characteristic of the symmetry energy, i.e., its slope L.

Impact of local delivery of allogeneic chondrocytes on the biological response and healing of the sternum bones after sternotomy.

Median sternotomy is the surgical method of choice for many procedures where one of the main problems is the long post-operative wound healing process leading to sternal dehiscence and the development of infection. This leads to prolonged hospital stay and increased mortality due to post-operative complications. A promising solution seems to be the use of allogeneic chondrocytes for wound treatment, whose properties in the field of cartilage reconstruction are widely used in medicine, mainly in orthopedics. In the present study, we investigated the effect of local delivery of allogeneic chondrocytes on the biological response and healing of the sternum after sternotomy. We optimized the culture conditions for the isolated chondrocytes, which were then applied to the sternal incision wound. Chondrocytes in the culture were assessed on the basis of the presence of chondrocyte-specific genes: Sox9, Aggrecan and Collagen II. In turn, the histopathological and immunohistochemical evaluation was used to assess the safety of implantation. In our work, we demonstrated the possibility of obtaining a viable culture of chondrocytes, which were successfully introduced into the sternal wound after sternotomy. Importantly, implantation of allogeneic chondrocytes showed no significant side effects. The obtained results open new possibilities for research on the use of allogeneic chondrocytes in the process of accelerating wound healing after median sternotomy.

Cisplatin in Ovarian Cancer Treatment-Known Limitations in Therapy Force New Solutions.

Cisplatin is one of the most commonly used anticancer drugs worldwide. It is mainly used in the treatment of ovarian cancer, but also used in testicular, bladder and lung cancers. The significant advantage of this drug is the multidirectional mechanism of its anticancer action, with the most important direction being damaging the DNA of cancer cells. Unfortunately, cisplatin displays a number of serious disadvantages, including toxicity to the most important organs, such as kidneys, heart, liver and inner ear. Moreover, a significant problem among patients with ovarian cancer, treated with cisplatin, is the development of numerous resistance mechanisms during therapy, including changes in the processes of cellular drug import and export, changes in the DNA damage repair mechanisms, as well as numerous changes in the processes of apoptosis and autophagy. Due to all of the mentioned problems, strategies to increase the effectiveness of cisplatin in the treatment of ovarian cancer are intensively sought. The most important strategy includes the development of less toxic cisplatin analogs. Another important direction is combination therapy, involving the simultaneous use of cisplatin with different anticancer drugs, substances derived from plants, temperature or radiotherapy. Many years of observations accompanying the presence of cisplatin in the therapy made it possible to provide a series of verifiable, statistically significant data, but also to show how, over time, with the new information and scientific discoveries, it is possible to describe and understand the therapeutic problems observed in practice, such as the acquisition of drug resistance by tumor cells or induction of changes in the tumor microenvironment. According to the authors, confronting what we knew so far with what new trends offer has a profound meaning. This paper presents information on the history of cisplatin and describes the molecular mechanisms of its action and the development of resistance by cancer cells. In addition, our goal was to highlight a number of therapeutic strategies to increase the effectiveness of cisplatin in the treatment of ovarian cancer, as well as to identify methods to eliminate problems associated with the use of cisplatin.

Alpha Mangostin and Cisplatin as Modulators of Exosomal Interaction of Ovarian Cancer Cell with Fibroblasts.

The diversity of exosomes and their role in the microenvironment make them an important point of interest in the development of cancer. In our study, we evaluated the effect of exosomes derived from ovarian cancer cells on gene expression in fibroblasts, including genes involved in metastasis. We also attempted to evaluate the indirect effect of cisplatin and/or α-mangostin on metastasis. In this aspect, we verified the changes induced by the drugs we tested on vesicular transfer associated with the release of exosomes by cells. We isolated exosomes from ovarian cancer cells treated and untreated with drugs, and then normal human fibroblasts were treated with the isolated exosomes. Changes in the expression of genes involved in the metastasis process were then examined. In our study, we observed altered expression of genes involved in various steps of the metastasis process (including genes related to cell adhesion, genes related to the interaction with the extracellular matrix, the cell cycle, cell growth and proliferation, and apoptosis). We have shown that α-mangostin and/or cisplatin, as chemotherapeutic agents, not only directly affect tumor cells but may also indirectly (via exosomes) contribute to delaying metastasis development.

Selective human cells in vitro transduction with porcine endogenous retrovirus (PERV).

Porcine xenograft transplantation raises concerns in humans about the risk of infection with porcine endogenous retroviruses (PERV) as they are an integral part of the pig genome and are therefore very difficult to exclude. In this study, for the first time, a relationship between the provirus genes sequences and released virions from pig cell line and the embedded sequence of this retrovirus in infected human cells was analyzed. PERV infection of human cells HEK-293 and HeLa and detection of PERV in pig PK-15 cells and supernatant were assessed by QPCR or RT-QPCR using primers specific for envA, envB, gag, pol genes and LTR region. Sequence analysis was performed at the DNA level and changes in the amino acid sequence were deduced in silico. Fifty nucleotide substitutions (45 in pol, 3 in gag and one each in envA and envB) were detected and most of these were heterozygous (42), which were present mainly in PK-15 cells. Our results show that sequence of the pol gene and the Pol protein is less conserved compared to the other PERV genes and PERV with some polymorphisms were not released from pig cells or/and do not infect human cells. PERV virions with a homozygous allele system were released from PK-15 cells, although their sequence replicated on the basis of the heterozygous PERV provirus sequence in PK-15. The newly discovered selective transduction of human cells with PERV will be helpful in studying the characteristics and genetic variability of the retrovirus genes to ensure safe xenotransplantation. Keywords: PERV; porcine endogenous retroviruses; infection; genetic polymorphism; xenotransplantation.

The Effect of α-Mangostin and Cisplatin on Ovarian Cancer Cells and the Microenvironment.

Ovarian cancer is one of the cancers that, unfortunately, is detected at a late stage of development. The current use of treatment has many side effects. Notably, up to 20% of patients show cisplatin resistance. We assess the effects of cisplatin and/or α-mangostin, a natural plant derivative, on ovarian cancer cells and on the cancer cell microenvironment. The effect of cisplatin and/or α-mangostin on the following cells of ovarian cancer lines: A2780, TOV-21G, and SKOV-3 was verified using the XTT cytotoxicity assay. The separate and combined effects of tested drugs on ovarian cancer cell viability were assessed. We assessed the influence of chemotherapeutic agents on the possibility of modulating the microenvironment. For this purpose, we isolated exosomes from drug-treated and untreated ovarian cancer cells. We estimated the differences in the amounts of exosomes released from cancer cells (NTA technique). We also examined the effects of isolated exosome fractions on normal human cells (NHDF human fibroblast line). In the present study, we demonstrate that treatment of A2780, SKOV-3, and TOV-21G cells with α-mangostin in combination with cisplatin can allow a reduction in cisplatin concentration while maintaining the same cytotoxic effect. Ovarian cancer cells release a variable number of exosomes into the microenvironment when exposed to α-mangostin and/or cisplatin. However, it is important to note that the cargo carried by exosomes released from drug-treated cells may be significantly different.

Aspects of the Epigenetic Regulation of EMT Related to Cancer Metastasis.

Epithelial to mesenchymal transition (EMT) occurs during the pathological process associated with tumor progression and is considered to influence and promote the metastatic cascade. Characterized by loss of cell adhesion and apex base polarity, EMT enhances cell motility and metastasis. The key markers of the epithelial to mesenchymal transition are proteins characteristic of the epithelial phenotype, e.g., E-cadherin, cytokeratins, occludin, or desmoplakin, the concentration and activity of which are reduced during this process. On the other hand, as a result of acquiring the characteristics of mesenchymal cells, an increased amount of N-cadherin, vimentin, fibronectin, or vitronectin is observed. Importantly, epithelial cells undergo partial EMT where some of the cells show both epithelial and mesenchymal characteristics. The significant influence of epigenetic regulatory mechanisms is observed in the gene expression involved in EMT. Among the epigenetic modifications accompanying incorrect genetic reprogramming in cancer are changes in the level of DNA methylation within the CpG islands and posttranslational covalent changes of histone proteins. All observed modifications, which are stable but reversible changes, affect the level of gene expression leading to the development and progression of the disease, and consequently affect the uncontrolled growth of the population of cancer cells.

Novel Xanthone Derivatives Impair Growth and Invasiveness of Colon Cancer Cells In Vitro.

Natural xanthones are a large group of compounds from which promising anticancer properties could be further developed by chemical modifications. This study aimed to investigate the influence of four novel xanthone derivatives based on a naturally occurring xanthone skeleton on the invasiveness of colon cancer cells in vitro. First, the concentrations required to inhibit growth of three colorectal cancer cell lines to 50% (GI50) of all the studied compounds, as well as the natural xanthones used as a reference (gambogic acid and α-mangostin), have been established (MTS reduction test). Next, the assays determining several aspects of the GI25 xanthones influence on colorectal cancer cells, including cytotoxicity, migration and invasion potential, interaction with extracellular matrix and endothelial cells, as well as expression of selected invasiveness related genes have been performed. Our results demonstrate that these novel xanthone derivatives impair colorectal cancer proliferation, motility, adhesion to extracellular matrix and to endothelial cells, and also induce apoptosis and cell death. Moreover, their activity is comparable to cisplatin and 5-fluorouracil, used as reference compounds. Conducted research indicates our compounds for further research and development as novel drugs in colorectal cancer treatment.

Morin exerts anti-metastatic, anti-proliferative and anti-adhesive effect in ovarian cancer cells: an in vitro studies.

The influence of morin hydrate on changes of proliferative, metastatic, and adhesive potential of human ovarian cancer cells concerning the influence of decitabine, and decitabine with trichostatin A, and in comparison to untreated cells, were analyzed. The effect of morin hydrate, decitabine, and trichostatin A were examined in A2780 and SKOV-3 ovarian cancer cell lines using MTS assay, clonogenic assay, adhesion to endothelial HMEC-1 cells, transwell migration assay and cell cycle analysis. The expression level of epithelial to mesenchymal transition (EMT) markers was quantified using PCR Array in relation to the level of global methylation determined with Methylated DNA Quantification Kit. We observed statistically significant inhibition of adhesive and migratory potential of both cell lines and the accumulation of G0/G1 phase A2780 cells after treatment with morin hydrate. Our studies confirmed the influence of morin hydrate on down-regulation of genes considered as up-regulated during EMT, and up-regulation of some genes considered as down-regulated during EMT in A2780 and SKOV-3 cells. Phenotypic changes were associated with molecular changes in cells, eg. decrease of the expression level of genes associated with adhesion, and an increase of genes down-regulated during EMT, after morin hydrate treatment in comparison to untreated control cells in both cell lines, were observed.

Influence of New Synthetic Xanthones on the Proliferation and Migration Potential of Cancer Cell Lines In Vitro.

BACKGROUND: Natural plant metabolites and their semisynthetic derivatives have been used for years in cancer therapy. Xanthones are oxygenated heterocyclic compounds produced as secondary metabolites by higher plants, fungi or lichens. Xanthone core may serve as a template in the synthesis of many derivatives that have broad biological activities. OBJECTIVE: This study synthesized a series of 17 new xanthones, and their anticancer potential was also evaluated. METHODS: The anticancer potential was evaluated in vitro using a highly invasive T24 cancer cell line. Direct cytotoxic effects of the xanthones were established by IC50 estimation based on XTT assay. RESULTS: 5 compounds of the total 17 showed significant cytotoxicity toward the studied cancer cultures and were submitted to further detailed analysis, including studies examining their influence on gelatinase A and B expression, as well as on the cancer cells migration and adhesion to an extracellular matrix. These analyses were carried out on five human tumor cell lines: A2780 (ovarian cancer), A549 (lung cancer), HeLa (cervical cancer), Hep G2 (liver cancer), and T24 (urinary bladder cancer). All the compounds, especially 4, showed promising anticancer activity: they exhibited significant cytotoxicity towards all the evaluated cell lines, including MCF-7 breast cancer, and hindered migration-motility activity of cancer cells demonstrating more potent activity than α-mangostin which served as a reference xanthone. CONCLUSION: These results suggest that our xanthone derivatives may be further analyzed in order to include them in cancer treatment protocols.

MiR-424-3p suppresses galectin-3 expression and sensitizes ovarian cancer cells to cisplatin.

PURPOSE: Assessment of miR-424-3p mimic capability to sensitize SK-OV-3 and TOV-21G ovarian cancer cells to cisplatin by decreasing the expression of galectin-3, which is an anti-apoptotic protein overexpressed in ovarian cancer and associated with resistance to chemotherapy. METHODS: We performed a reverse transfection of miR-424-3p mimic into SK-OV-3 and TOV-21G ovarian cancer cells, followed by Real Time™ RT-PCR analysis of the expression of miR-424-3p and galectin-3 mRNA as well as ELISA assay for galectin-3 protein level. Next, we studied the viability (XTT assay), proliferation (EdU incorporation assay), and apoptosis (ELISA assay) of the both cell lines transfected with the mimic and treated with cisplatin. RESULTS: We demonstrated that miR-424-3p mimic effectively transfects into SK-OV-3 and TOV-21G ovarian cancer cells in which it significantly suppresses the expression of galectin-3 at the protein level, but not at the mRNA level. Reverse transfection of both cell lines with the mimic, followed by treatment with cisplatin, resulted in a reduction in cell viability and proliferation as well as an increase in the induction of apoptosis. CONCLUSIONS: MiR-424-3p mimic sensitizes SK-OV-3 and TOV-21G ovarian cancer cells to cisplatin by decreasing the expression of galectin-3.

The involvement of multifunctional TGF-ß and related cytokines in pathogenesis of endometriosis.

PURPOSE: Transforming growth factor ß (TGF-ß) is one of the major immune and inflammation factors responsible for regulating cell proliferation, differentiation, angiogenesis, and immune responses. Deregulated TGF-ß activity, especially its influence in peritoneal cytokine cross-talk, has been implicated in pathologies of endometriosis. The aim of this study was to determine whether TGF-ß could be involved in the pathogenesis of endometriosis. For this purpose, we evaluated concentrations of TGFß1, TGF-ß2, TGF-ß3 and interleukin (IL)-1ß, IL-6, IL-10, IL-17, IL-21 and IL-22 in peritoneal fluid (PF) and serum of women with endometriosis. METHODS: A total of 66 women of reproductive age were involved in the study, 51 endometriosis patients, and 15 women from the control group. PF and serum levels of all cytokines were measured with ELISA in women with or without endometriosis. RESULTS: Higher PF and serum levels of TGF-ß1, TGF-ß2, TGF-ß3, presented also as a total TGF-ß in women with endometriosis compared to control were observed. The biggest increase was measured in the case of TGF-ß1. The higher levels of IL-1ß, IL-6, IL-10, and IL-17 in PF and serum of endometriosis women than control was observed. Higher PF levels of studied parameters in comparison with serum levels were found. CONCLUSIONS: In endometriosis, TGF-ß could affect differentiation of T helper (Th) cells, hence produce more IL-17 and IL-10 to PF and might have an indirect influence on inflammation, which is associated with higher IL-1ß and IL-6 levels. In consequent, TGF-ß in peritoneal fluid may promote an environment favorable to ectopic lesion formation.

Morin decreases galectin-3 expression and sensitizes ovarian cancer cells to cisplatin.

PURPOSE: This study aimed at evaluating whether morin (a natural flavonoid and a known inhibitor of NF-κB) can sensitize ovarian cancer cells to cisplatin by decreasing the expression of galectin-3, which is an anti-apoptotic protein regulated by NF-κB transcription factor. METHODS: To assess the possibility of augmentation the activity of cisplatin by morin, we studied the separate and the combined effect of morin and cisplatin on viability, proliferation, and apoptosis of TOV-21G (cisplatin-sensitive) and SK-OV-3 (cisplatin-resistant) ovarian cancer cells. We also analysed the effect of morin and cisplatin on galectin-3 expression at the mRNA and protein levels. RESULTS: We demonstrated that morin possess antitumor activity against TOV-21G and SK-OV-3 ovarian cancer cells by reducing cell viability and proliferation as well as increasing the induction of apoptosis. Co-treatment of the cells with selected concentrations of morin and cisplatin, accordingly to specific treatment approaches, reveals a synergism, which leads to sensitization of the cells to cisplatin. During this sensitization, morin significantly reduces the expression of galectin-3 at the mRNA and protein level, regardless of the presence of cisplatin. CONCLUSIONS: Morin sensitizes TOV-21G and SK-OV-3 ovarian cancer cells to cisplatin, what is associated with a decrease of the expression of galectin-3.

Analysis of swine leukocyte antigen class I gene profiles and porcine endogenous retrovirus viremia level in a transgenic porcine herd inbred for xenotransplantation research.

Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety.

Contribution of reactive oxygen species to the anticancer activity of aminoalkanol derivatives of xanthone.

Reactive oxygen species (ROS) are critically involved in the action of anticancer agents. In this study, we investigated the role of ROS in the anticancer mechanism of new aminoalkanol derivatives of xanthone. Most xanthones used in the study displayed significant pro-oxidant effects similar to those of gambogic acid, one of the most active anticancer xanthones. The pro-oxidant activity of our xanthones was shown both directly (by determination of ROS induction, effects on the levels of intracellular antioxidants, and expression of antioxidant enzymes) and indirectly by demonstrating that the overexpression of manganese superoxide dismutase decreases ROS-mediated cell senescence. We also observed that mitochondrial dysfunction and cellular apoptosis enhancement correlated with xanthone-induced oxidative stress. Finally, we showed that the use of the antioxidant N-acetyl-L-cysteine partly reversed these effects of aminoalkanol xanthones. Our results demonstrated that novel aminoalkanol xanthones mediated their anticancer activity primarily through ROS elevation and enhanced oxidative stress, which led to mitochondrial cell death stimulation; this mechanism was similar to the activity of gambogic acid.

DNA methyltransferase inhibitors influence on the DIRAS3 and STAT3 expression and in vitro migration of ovarian and breast cancer cells.

OBJECTIVES: Downregulation of DIRAS3 (DIRAS family, GTP-binding Ras-like 3) is related to ovarian and breast cancer progression. A possible mechanism that silences this gene is the promoter region DNA methylation. The potential reversibility of this epigenetic mechanism makes it more attractive candidate for new mode of cancer treatment. DIRAS3 regulates cell cycle, tumor dormancy and inhibits cancer cell growth and motility, all of which may indirectly depend on interaction with STAT3 (Signal Transducer and Activator of Transcription 3) classified as a potential oncogene. The restoration of DIRAS3 expression could inhibit cell proliferation and invasiveness. MATERIAL AND METHODS: Human ovarian carcinoma cell line (A2780) and human breast cancer cell line (MCF7) were exposed to two DNA methyltransferase inhibitors (DNMTi): decitabine (5-aza-2'-deoxycytidine) [25 µM and 12.5 µM] and RG108 [150 µM and 100 µM]. In vitro migration changes of cancer cells were examined with wound healing assay. After 7 days of DNMTi treatment cells were harvested and DNA and RNA was isolated. The methylation status of the promoter sequences of DIRAS3 and STAT3 genes was determined using methylation specific PCR (MS-PCR). Level of target genes' expression was quantified using quantitative reverse transcription PCR (QRT-PCR). RESULTS AND CONCLUSIONS: The in vitro wound healing assay showed changes in the migration rate of both adherent cell lines after DNMTi treatment compared to the untreated cells. Relative balance between methylated and unmethylated variants of DIRAS3 after MS-PCR was shifted towards unmethylated version after DNMTi treatment in A2780 cells. Statistically significant dose dependent effect of decitabine and RG108 on DIRAS3 expression in A2780 cells was observed.

Silencing expression of the NANOG gene and changes in migration and metastasis of urinary bladder cancer cells.

INTRODUCTION: It has been proved that expression of the NANOG gene is observed not only in embryonic-derived malignancies, but also in breast cancer, ovarian cancer, cervix cancer and bladder cancer. NANOG overexpression is correlated with high activity of MMP-2 and MMP-9. The aim of the study was to evaluate the changes in the malignant phenotype of T24 bladder cancer cells with modulated expression of the NANOG gene. MATERIAL AND METHODS: Human urinary bladder cancer cells T24 (HTB-4) were cultivated under standard conditions. Transfection of the cells with silencing constructions was performed with the application of Lipofectamine 2000 (Invitrogen) reagent. Evaluation of changes in the expression level of individual genes was performed using qRTPCR. Changes in the protein level were evaluated using the Human ELISA Kit (Abcam). The invasion capability of transfected cells was tested using Matrigel Invasion Chambers (BD Biosciences). The changes in cell migration were assessed with a wound-healing assay. RESULTS: The qRTPCR evaluation showed that silencing the NANOG gene in T24 cells led to the decrease of mRNA for the MMP-2 gene to the level of 62.4% and the MMP-9 gene to the level of 76%. The cells with modulated expression of the NANOG gene migrated slower in the Matrigel invasion assay and in the wound-healing assay. The immunoenzymatic test showed a decrease in the protein level of MMP-9. CONCLUSIONS: The transcriptional activity of the NANOG gene might be connected with some aspects of bladder cancer cell metastasis in vitro and has an influence on MMP-2 and MMP-9 expression levels.

Synthesis and in vitro Evaluation of the Anticancer Potential of New Aminoalkanol Derivatives of Xanthone.

A series of 15 derivatives of xanthone were synthesized and evaluated for the anticancer activity. The structure of the tested compounds was diversified to establish structureactivity relationships. The following evaluations were carried out: cytotoxicity-proliferation tests, apoptosis detection, expression of apoptosis and proliferation-related genes, expression and activity of gelatinases A and B, wound migration assays, and cell adhesion to MatrigelTMcoated plates. Four compounds (7, 12, 13 and 15) displayed direct cytotoxicity at micromolar concentrations toward the studied cell lines. They also significantly affected the expression of proliferationapoptosis markers, and 13 demonstrated as strong influence as α-mangostin, that served as a natural standard in our study. These four compounds also decreased the expression and activity of gelatinases, and inhibited the migration-motility potential of cancer cells. The influence of compounds 7 and 12 on MMPs mRNA levels even exceeded the activity of α-mangostin and shRNA-mediated silencing; zymography revealed that 7, 13 and 15 were as equally active as α-mangostin, despite their higher IC50 values. The highest activity to inhibit motility and migration of cancer cells was demonstrated by 7, 12, 15, and by α-mangostin; and this was almost equal to shRNA-mediated silencing. Structural features predetermining compound activity were: substitution at position C4 instead of C2, and presence of a chlorine atom and allyl moiety. These results indicate that synthesis of aminoalkanol derivatives of xanthone may lead to successful establishment of new potential anticancer chemicals.

The role of NANOG transcriptional factor in the development of malignant phenotype of cancer cells.

NANOG is a transcription factor that is involved in the self-renewal of embryonic stem cells (ES) and is a critical factor for the maintenance of the undifferentiated state of pluripotent cells. Extensive data in the literature show that the NANOG gene is aberrantly expressed during the development of malignancy in cancer cells. ES and cancer stem cells (CSCs), a subpopulation of cancer cells within the tumor, are thought to share common phenotypic properties. This review describes the role of NANOG in cancer cell proliferation, epithelial-mesenchymal transition (EMT), apoptosis and metastasis. In addition, this paper illustrates a correlation between NANOG and signal transducer and activator of transcription 3 (STAT3) in the maintenance of cancer stem cell properties and multidrug resistance. Together, the available data demonstrate that NANOG is strictly involved in the process of carcinogenesis and is a potential prognostic marker of malignant tumors.