Gudoitytė, Greta

Background: Mutations within genes encoding components of the PI3K/AKT/mTOR (phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin) signaling axis frequently activate the pathway in breast cancer, making it an attractive therapeutic target. Inhibition of mTORC1 (mechanistic target of rapamycin complex 1) activity upon aspirin treatment has been reported in breast cancer cells harboring PI3KCA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) mutation and is considered to account for anticancer action. Methods: MDA-MB-468 (harbors mutated PTEN (phosphatase and TENsin homolog)), MCF-7 (PI3KCA-mutated), MDA-MB-231 (no PI3K pathway mutations) cancer cell lines and MCF10A non-cancerous breast epithelial cells were employed for the assessment of modulation of mTORC1 signaling by aspirin. Targeted amplicon-based next-generation sequencing using the Ion Torrent technology was carried out to determine gene expression changes following drug treatment. Western blot was performed to analyze the expression and phosphorylation of proteins. Knockdown by siRNA approach was applied to assess the role of REDD1/DDIT4 (DNA damage-inducible transcript 4) in mTORC1 inhibition by aspirin. Results: We show a decline in phosphorylation of mTORC1 downstream substrate 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) in response to treatment with aspirin and its metabolite salicylic acid in MDA-MB-468, MCF-7, MDA-MB-231, and MCF10A cell lines. We further demonstrate a novel molecular response to aspirin in breast cancer cells. Specifically, we found that aspirin and salicylic acid increase the expression of REDD1 protein, that is known for its suppressive function towards mTORC1. Unexpectedly, we observed that siRNA knockdown of REDD1 expression facilitated aspirin-mediated suppression of mTORC1 downstream substrate 4E-BP1 phosphorylation in the MDA-MB-468 cell line. REDD1 downregulation slightly encouraged [...].

Introduction: Deregulated miRNA profiles and their contribution to carcinogenesis have been observed in virtually all types of cancer. However, their contribution to the pathogenesis of rare gastrointestinal stromal tumors (GISTs) is not well defined yet. In this study, we aimed to investigate the role of miR-375 - a well-known tumor-suppressive miRNA - in the pathogenesis of GIST. Materials and Methods: MirVana miRNA mimic and negative control were transfected to the GIST-T1 cell line using the lipofection technique. Target gene and protein expression were detected by RT-qPCR and Western Blot, respectively. Alterations in cell viability, migration rate and apoptotic cell counts, as well as miRNA-target gene interaction, were evaluated by MTT, Wound Healing, Annexin V FITC and Dual-Light Luciferase assays, respectively. Statistical analysis was performed using the computing environment R. Results: Overexpression of miR-375 reduced levels of KIT gene mRNA (1.9-fold, p<0.05) and protein (1.4-fold, p<0.05) in GIST-T1 cell line. Further functional analysis revealed a significant effect of miR-375 on cell viability (reduced by 47%, p<0.05) and migration rates (reduced by up to 28% in different time points, p<0.05). GIST-T1 cells with KIT 3’UTR construct (pos855-861) showed decreased luciferase activity down to 76% when miR-375 was overexpressed. Conclusions: Our findings indicate that miR-375 could suppress GIST by targeting KIT and contribute to the pathogenesis of GIST.

Introduction. Breast cancer (BC) is the most common cancer among women worldwide, making this disease a leading cause of morbidity and mortality. One of the most common treatments of this cancer is radiotherapy. The therapy is based on activity of ionising radiation (IR) which directly affects malignant cells by changing DNA structure stability and repair processes. However, even though it is widely used and provides good outcome this treatment, it has some limitations. It has been observed that cancer cells might become radioresistant and therefore repopulate. Hence, it is important to find molecular mechanisms by which radioresistance occur and find new therapeutic alternatives that can inhibit the viability of the cancer cells and sensitize them to radiotherapy. For a while, investigation using phytochemicals has drawn attention of the scientists. It is known that a number of phytochemicals possess anti-cancer properties. In addition, these substances might enhance cancer cells sensitivity to IR. Therefore the aim of this study was to evaluate human BC MCF-7 and MDA-MB-231 cell line response to a single dose of IR combining it with phytochemicals. Methodology. MCF-7 and MDA-MB-231 BC cell lines were used to test Resveratrol (RSV) effect. RSV was dissolved in dimethyl sufoxide. Initially, we examined cell viability after the exposure solely to RSV. In order to assess survival we incubated cells with different RSV (0, 10, 25, 50, 80, 100, 150, 200 μM) concentrations for various incubation times (24, 48, 72 h). For cells survival analysis MTT test was performed. Afterwards, the combination of RSV and IR (0, 2, 10 Gy) on cells survival (MTT method) was analysed. Results. The study showed that chosen substances had different effect on BC cell viability. Firstly, MTT assay results indicated significant decrease in cells survival after the exposure to RSV. In addition, viability of both cell lines declined depe. [...].

Deregulated microRNA (miRNA) expression profiles and their contribution to carcinogenesis have been observed in virtually all types of human cancer. However, their role in the pathogenesis of rare mesenchymal gastrointestinal stromal tumors (GISTs) is not well defined, yet. In this study, we aimed to investigate the role of two miRNAs strongly downregulated in GIST—miR-375-3p and miR-200b-3p—in the pathogenesis of GIST. To achieve this, miRNA mimics were transfected into GIST-T1 cells and changes in the potential target gene mRNA and protein expression, as well as alterations in cell viability, migration, apoptotic cell counts and direct miRNA–target interaction, were evaluated. Results revealed that overexpression of miR-375-3p downregulated the expression of KITmRNAand protein by direct binding to KIT 30UTR, reduced GIST cell viability and migration rates. MiR-200b-3p lowered expression of ETV1 protein, directly targeted and lowered expression of EGFR mRNA and protein, and negatively a ected cell migration rates. To conclude, the present study identified that miR-375-3p and miR-200b-3p have a tumor-suppressive role in GIST. Introduction Gastrointestinal stromal tumors (GISTs), while relatively rare, are the most common mesenchymal tumors of the gastrointestinal tract. GISTs are considered to originate from interstitial cells of Cajal or their precursors, residing within the muscle layers of the gastrointestinal tract and are characterized by strong immunohistochemical staining for receptor tyrosine kinase (RTK) KIT (> 95% of the cases) [1,2]. The main initiating events in GIST are gain-of-function mutations in two oncogenes from the RTK family—KIT (70–80%) or platelet-derived growth factor receptor- (PDGFRA; 10%)—that result in constitutive activation of the receptor and downstream signaling pathways, including mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/mammal...[...].

Background and Objectives While the anticancer activity of aspirin is strongly supported by epidemiological data, the underlying molecular mechanism remains uncertain. Inhibition of mTORC1 (mechanistic target of rapamycin complex 1) signaling has been proposed to contribute to the effect. Attenuation of mTORC1 activity by aspirin has been reported to be dependent and independent of AMP-activated protein kinase (AMPK). Although REDD1 (protein regulated in development and DNA damage response 1), encoded by the DDIT4 gene, is known to function as an mTORC1 inhibitor, the involvement of REDD1 in aspirin anticancer action has not been reported in the literature. Since we have previously found that DDIT4 mRNA is elevated upon aspirin treatment in breast cancer cells, here, we seek to determine whether this increase is concomitant with the induction of REDD1 protein. While aspirin has been shown to suppress mTORC1 signaling in PIK3CA-mutant breast cancer cells, we investigate the activity of mTORC1 signaling following aspirin exposure in PTEN-mutant breast cancer cells. Material and Method MDA-MB-468 (PTEN-mutant) and MCF-7 (PIK3CA-mutant) breast cancer cell lines were used for the analysis. Cells were treated with aspirin or its metabolite salicylic acid for 24 hours before cell lysates were prepared. Protein expression and phosphorylation was assessed by western blotting. mTORC1 activity was evaluated by phosphorylation levels of 4E-BP1 and S6K1. Results We demonstrated that aspirin and salicylic acid increase REDD1 protein levels in MDA-MB-468 and MCF-7 cells. In order to investigate the effect of aspirin on mTORC1 activity in MDA-MB-468 cell line we probed for phosphorylation levels of S6K1 and 4E-BP1 which are the main known targets of mTORC1. However, we found no detectable baseline S6K1 phosphorylation and therefore assessed only phosphorylation of 4E-BP1. We observed that aspirin and salicylic acid dephopho...[...].

Background and Objectives Breast cancer is the most common women's oncologic disease and leading cause of morbidity and mortality. The main treatment of breast cancer is radiotherapy, which is based on ionizing radiation (IR) activity. IR causes DNA structure instability and distruption causing activation of DNA repair mechanisms which leads to cell cycle arrest and cell death. Although radiotherapy is widely used and provides substantial benefits it has several limitations. It has been observed, that cancer cells develop radioresistance and are able to repopulate, therefore decreasing effectiveness of the treatment. Consequently, there is a growing interest in compounds which could inhibit cancer cell viability or sensitize them, thus improving treatment efficiency. Phytochemicals have attracted attention of many researchers as potential anti-cancer agent. In addition, phytochemicals might inhance cancer cells sensitivity to IR. Therefore, the aim of this study was to evaluate human breast cancer MCF-7 and MDA-MB-231 cell line response to a single dose of IR in combination with phytochemicals. Material and Method MCF-7 and MDA-MB-231 breast cancer cell lines were used to test kaempferol (KMF) effect. KMF was dissolved in dimethyl sulfoxide. Initially, we examined cell viability after the exposure solely to KMF. In order to assess survival, we incubated cells with different KMF (0, 5, 10, 20, 30, 40 μM) concentrations for various incubation times (24, 48, 72 h). For cells survival analysis MTT test was performed. Afterwards, the combination of KMF and IR (0, 2, 10 Gy) on cells survival (MTT method) was analysed. Results This study showed that KMF had different effect on chosen breast cancer cells survival. There was a significant decline in MCF-7 cell viability which was KMF concentration-dependent. The same effect was observed following all three incubation periods. However, significant decrease of MDA-MB...[...].

Introduction Recently, aspirin has attracted attention as a chemopreventive agent, which is strongly supported by epidemiological data. However, the underlying mechanisms by which aspirin exerts its antineoplastic effects are not clearly established. Aim The aim of this study was to examine the effects of aspirin and salicylic acid on the expression of genes encoding mTOR signaling pathway components and related genes in breast cancer cell lines Methods Two different commercial breast cancer cell lines MCF-7 and MDA-MB-468 were used for the investigation. Since we previously demonstrated that aspirin metabolite salicylic acid reduces cell viability to a similar extent to aspirin in these cell lines, salicylic acid treatments were examined in parallel. Gene expression changes were tested after the cells were exposed to 0.5 and 2 mM concentrations of drugs for 1 and 24 hours. Gene expression was determined by next generation sequencing, using Ion Ampliseq technology. RNA isolation, cDNA synthesis and library contruction were carried out according to manufacturer‘s instructions. Automated template preparation and chip loading was done on Ion Chef System. Samples were sequenced on the Ion S5 Sequencer. Primary analysis of sequencing data was performed using ampliseqRNA analysis plugin in the Torrent Suite Software. Reads were subsequently analyzed for differential expression using edgeR package from open-source Bioconductor Project. Four biological replicates were used for statistical analysis. Adjusted p-values for multiple testing, using Benjamini-Hochberg to estimate the false discovery rate (FDR), were calculated for final estimation of DE significance. Genes with a fold change ≥1.5 and adjusted p ≤0.05 were considered to be differentially expressed. Results We found that expression of mTOR inhibitor DDIT4 was increased after 24-hour exposure to 2mM aspirin and salicylic acid in both cell lines. Additionally, another mTOR inhibitor DEPTOR mRNA was also

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in post-transcriptional regulation of gene expression. Deregulated miRNA profiles and their contribution to carcinogenesis has been observed in different types of cancer. However, their involvement in pathogenesis of rare gastrointestinal stromal tumors (GISTs) is not yet fully understood. Therefore, the aim of this study was to determine highly deregulated miRNAs in GIST and to investigate their possible involvement in GIST pathogenesis. MiRNA expression profile was determined using small RNA-seq approach in paired tumor and adjacent tissue samples of 15 GIST patients and validated by Taq-Man low-density array in a larger sample group of 40 patients. Potential targets of deregulated miRNAs were predicted using TargetScan Release 7.1. Further transfections with miRNA mimics and functional analysis was performed in GIST-T1 cell line. Changes in target gene expression were detected using TaqMan primers and probes, protein expression analysis was performed using Western Blot technique. Alterations in cell viability and migration rates were evaluated by MTT and Wound Healing Assays. Statistical analysis was performed using the computing environment R. Sequencing data showed distinct miRNA expression profiles between tumor and adjacent tissue samples. In a further validation step, expression levels of 19 miRNAs showed significant differential expression in the same direction as in the sequencing data (Bonf. adj. p < 0.01; FC > 2). MiRNAs, potentially targeting genes involved in cancer associated signaling pathways, were selected (hsa-miR-375, hsa-miR-200b-3p, hsa-miR-490-3p) for target gene expression and functional analysis in GIST-T1 cell line. Increased amounts of hsa-miR-375 significantly reduced expression of its predicted target gene KIT (FC = 1.8, p < 0.05), however KIT protein expression remained unchanged. Overexpression of hsa-miR-200b-3p significantly reduced EGFR and ETV1 gene expression

Introduction. Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. It is now known that the main event in GIST tumorigenesis is mutations in KIT or PDGFRA, which lead to uncontrolled cell growth and tumor formation. However, it has been determined that changes in gene expression are responsible for the gradual malignant transformation in GIST, but, little is known about the underlying mechanisms regulating these expression signatures. MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranscriptional regulation of gene expression. Various studies have shown that expression deregulation of miRNAs has a crucial role in cancer and is associated with tumorigenesis, tumor progression, metastasis and may have influence in tumor drug resistance. Biological functional determination of specific miRNAs and their target genes could contribute to understanding of cancer pathogenesis and may improve its diagnostics or even treatment. AIM The aim of the study was to assess the functional role of microRNAs (hsa-miR-141-3p and hsa-miR- 200b-3p) in gastrointestinal stromal tumor pathogenesis. Methods. GIST-T1 cell line was used as a model system for this study. miRNAs of interest were chosen from previous non-coding RNA profiling study (2). Cells have been transfected using two miRNAs mimics (at concentrations 80-100 nM): hsa-miR-141-3p and hsa-miR-200b-3p. MTT assay was used for assessment of cell viability 72 h after transfection. „Wound Healing“ assay was used to determine the migration of cells after transfection. Further, target genes of miRNAs were chosen with TargetScan 7: hsa-miR-141-3p: STAT1, STAT5A, STAT5B, EGFR, KIT and PDGFRA, for hsa-miR-200b-3p: STAT1, EGFR and ETV1. Their expression was validated using qRT-PCR (internal control - GAPDH) and analysed using 2-ΔΔCt method. Finally, protein expression of targets was analyzed using Wester [...].