Analysis of dysregulated long non-coding RNA expressions in glioblastoma cells

WOS: 000381539800015PubMed ID: 27306825Long non coding RNAs (IcRNAs) are associated with various biological roles such as embryogenesis, stem cell biology, cellular development and present specific tissue expression profiles. Aberrant expression of IncRNAs are thought to play a critical role in the progression and development of various cancer types, including gliomas. Glioblastomas (GBM) are common and malignant primary brain tumours. Brain cancer stem cells (BCSC) are isolated from both low and high-grade tumours in adults and children, by cell fraction which express neuronal stem cell surface marker CD133. The purpose of this study was to investigate the expression profiles of IncRNAs in brain tumour cells and determine its potential biological function. For this purpose, U118MG-U87MG; GBM stem cell series were used. Human parental brain cancer cells were included as the control group; the expressions of disease related human IncRNA profiles were studied by LightCycler 480 real-time PCR. Expression profiles of 83 lncRNA genes were analyzed for a significant dysregulation, compared to the control cells. Among IncRNAs, 51 IncRNA genes down-regulated, while 8 IncRNA genes were up-regulated. PCAT-1 (2.36), MEG3 (5.34), HOTAIR (-2.48) lncRNAs showed low expression in glioblastoma compared to the human (parental) brain cancer stem cells, indicating their role as tumour suppressor genes on gliomas. As a result, significant changes for anti-cancer gene expressions were detected with disease-related human IncRNA array plates. Identification of novel target genes may lead to promising developments in human brain cancer treatment. (C) 2016 Elsevier B.V. All rights reserved.Research Foundation of Ege UniversityEge University [2.101.2014.0014]This study was supported by the Research Foundation of Ege University (Grant Number 2.101.2014.0014)

The effects of PKI-402 on breast tumor models' radiosensitivity via dual inhibition of PI3K/mTOR

PurposePI3K/Akt/mTOR pathway activation causes relapse and resistance after radiotherapy in breast cancer (BC). We aimed to radiosensitize BC cell lines to irradiation (IR) by PKI-402, a dual PI3K/mTOR inhibitor.MethodsWe performed cytotoxicity, clonogenicity, hanging drop, apoptosis and double-strand break detection, and phosphorylation of 16 essential proteins involved in the PI3K/mTOR pathway.ResultsOur findings showed that PKI-402 has cytotoxic efficiency in all cell lines. Clonogenic assay results showed that PKI-402 plus IR inhibited the colony formation ability of MCF-7 and breast cancer stem cell lines. Results showed that PKI-402 plus IR causes more apoptotic cell death than IR alone in the MCF-7 cells but did not cause significant changes in the MDA-MB-231. ; gamma;-H2AX levels were increased in MDA-MB-231 in PKI-402 plus IR groups, whereas we did not observe any apoptotic and ; gamma;-H2AX induction in BCSCs and MCF-10A cells in all treatment groups. Some pivotal phosphorylated proteins of the PI3K/AKT pathway decreased, several proteins increased and others did not change.ConclusionIn conclusion, if the combined use of PKI-402 with radiation is supported by in vivo studies, it can contribute to the treatment options and the course of the disease.This study was supported by Ege University Scientific Research Projects (BAP) Department (Grant No. 2017-TIP-022, 18-KSUAM-001).Ege University Scientific Research Projects (BAP) Department [2017-TIP-022, 18-KSUAM-001

The Evaluation of Effect of Aurora Kinase Inhibitor CCT137690 in Melanoma and Melanoma Cancer Stem Cell

Background: Dysregulation of the cell cycle is one of the main causes of melanomagenesis. Genomewide studies showed that the expression of Aurora-A and-B significantly has been upregulated in melanoma. However, there is no FDA approved drug targeting aurora kinases in the treatment of melanoma. In addition, the development of resistance to chemotherapeutic agents in the treatment of melanoma and, as a result, the relapse due to heterogeneous cell groups in patients is a second phenomenon that causes treatment failure. Therefore, there is an urgent need for therapeutic alternatives targeting both melanoma and melanoma cancer stem cells (MCSCs) in treatments. At this stage, cell cycle regulators become promising targets. Objective: In this study, we aimed to identify the effects of Aurora kinase inhibitor CCT137690 on the cytotoxicity, apoptosis, cell cycle, migration, and colony formation and expression changes of genes related to proliferation, cell death and cell cycle in melanoma and melanoma cancer stem cell. In addition, we investigated the apoptotic and cytostatic effects of CCT137690 in normal fibroblast cells. Methods: We evaluated the cytotoxic effect of CCT137690 in MCSCs, NM2C5 referring as melanoma model cells and WI-38 cells by using the WST-1 test. The effect of CCT137690 on apoptosis was detected via Annexin V and JC-1 method; on cell cycle progression by cell cycle test; on gene expression by using RT-PCR, on migration activity by wound healing assay and clonal growth by clonogenic assay in NM2C5 cells and MCSCs. The effects of CCT137690 in WI-38, referring as healthy fibroblast cell, were assessed through Annexin V and cell cycle method. Results: CCT137690 was determined to have a cytotoxic and apoptotic effect in MCSCs and melanoma. It caused polyploidy and cell cycle arrest at the G2/M phase in MCSCs and melanoma cells. The significant decrease in the expression of MMP2, MMP7, MMP10, CCNB1, IRAK1, PLK2 genes, and the increase in the expression of PTEN, CASP7, p53 genes were detected. Conclusion: Aurora kinases inhibitor CCT137690 displays promising anticancer activity in melanoma and especially melanoma cancer stem cells. The effect of CCT137690 on melanoma and MCSC may provide a new approach to treatment protocols

PI3K/mTOR dual-inhibition with VS-5584 enhances anti-leukemic efficacy of ponatinib in blasts and Ph-negative LSCs of chronic myeloid leukemia*

Ponatinib is used for advanced treatment of chronic myeloid leukemia (CML), although low doses to prevent side effects do not suppress survival pathways and eradicate leukemia stem cells (LSCs). We evaluated the potential of ponatinib and PI3K/mTOR dual-inhibitor VS-5584 combination (PoVS) therapy to increase the anti-leukemic effects of ponatinib and investigated the underlying mechanisms at the molecular level. We measured the cytotoxicities of ponatinib, VS-5584, and PoVS (CCK-8 assay), and used the median-effect equation for combination analyses. We investigated the effects of inhibitory concentrations on apoptosis, cell viability and cell-cycle regulation (flow cytometry), protein levels (ELISA, Western blot), transcriptional activities (dual-luciferase reporter assay), gene expressions (qRT-PCR). VS-5584 exerted selective cytotoxic effects against CML and LSC cell lines. VS-5584 inhibited the PI3K/Akt/mTOR pathway, resulting in reduced cell viability, slightly induced caspase-independent apoptosis, prominent G0/G1 cell-cycle blockade that is not a consequence of quiescence. Normal hematopoietic stem cell line was the least affected. Moreover, ponatinib and VS-5584 mediated synergistic anti-leukemic effects on leukemic cells. VS-5584 reduced the ponatinib dose required to target leukemic cells. PoVS treatment inhibited PI3K/Akt/mTOR pathway more consistently than either of the two agents alone through reducing p-Akt, p-mTOR, p-S6K, p-PRAS40, p-S6. The subsequent downstream effects were an increase in C/EBP transcriptional activity and decreases in activities of E2F/DP1, Myc/Max, CREB, STAT3, NF kappa B, AP-1, Elk-1/SRF. Transcriptional regulation resulted in alterations in the expression levels of target mRNAs. Our results highlight PoVS can be a promising treatment strategy for eliminating CML cells and LSCs selectively, with the reduced ponatinib doses.This work was supported by Ege University Scientific Research Projects (BAP) Department (Grand no. 15-TIP-019, 2015-TIP-063).Ege University Scientific Research Projects (BAP) Department [15-TIP-019, 2015-TIP-063

Comparative expression analysis of dasatinib and ponatinib-regulated lncRNAs in chronic myeloid leukemia and their network analysis

LncRNAs are associated with malignancies with their tumor suppressor/oncogenic properties. Although many studies are conducted related to the mechanism of action for dasatinib and ponatinib in chronic myeloid leukemia (CML), their comparative effects on lncRNA expressions are largely unknown. Hence, we aimed to define the lncRNAs involved in the treatment of CML with dasatinib and ponatinib. We measured the cytotoxicities of dasatinib/ponatinib with CCK-8 assay and identified differentially expressed lncRNAs (DEL) by qRT-PCR. We determined the principal functions of DELs by Ingenuity Pathway Analysis (IPA) and performed gene ontology (GO) analysis for apoptosis and anti-proliferation-related lncRNAs. Apoptotic and anti-proliferative activities of dasatinib/ponatinib were confirmed by flow-cytometry. In K562 cells, dasatinib/ponatinib re-regulated lncRNAs which were dysregulated in leukemia. DELs after treatment (forty with dasatinib, thirty-seven with ponatinib) were related to increased cell death; decreased cell viability, proliferation, tumor growth, invasion, migration. Dasatinib-mediated network was related to cancer, hematological disease while ponatinib-mediated network was associated with cancer, cell death/survival, cell-to-cell signaling/interaction. Both treatments predicted activation of IFN gamma, IL1 beta, TNF as upstream regulators, specially this effect was higher in dasatinib. Comparison analysis showed that ponatinib was predicted more effective in cell death of tumor cell line than dasatinib. We confirmed that ponatinib was more potent than dasatinib to induce apoptosis and inhibit proliferation of CML cells, in consensus with IPA and GO analysis results. LncRNAs are specifically involved in anti-leukemic activities of dasatinib and ponatinib. Our findings will contribute to understanding signalization occurring in CML cells after standard treatments

Structural characterization, antioxidant and cytotoxic effects of iron nanoparticles synthesized using Asphodelus aestivus Brot. aqueous extract

ASP was used to synthesize FeNPA. They were characterized by UV-vis spectroscopy, FT-IR, TEM, SEM, XRD and ZP. The aim of this study was to evaluate in vitro cytotoxic activity and antioxidant acitivities of FeNPA and ASP. The antioxidant properties were evaluated using DPPH, ABTS+ and H2O2 assays. FeNPA had higher antioxidant activity comparing to ASP according to DPPH (IC50: 3.48 μg/mL) and ABTS+ (60.52%) assays. Anti-cancer activities of FeNPA and ASP were investigated in breast cancer, melanoma and control cell lines. FeNPA was more cytotoxic than ASP in MCF-7, MeWo, CHL-1, and HEL 299 cells. FeNPA had shown that mitochondria induce apoptosis through stress in MDA-MB-231, and cells MeWo. ASP also induced apoptosis 2.23-fold in MCF-7 cells. Progesterone receptor gene expression showed a 10-fold increase in a hormone-dependent MCF-7 cell line in ASP, and FeNPA treatment. Expressions of BCL6, CXCL12, DNAJC15, RB1 and TPM1 in melanoma cancer cell lines were significantly increased in ASP and FeNPA administration. It had been shown that FeNPA regulates gene expressions that may be considered important in terms of prognosis in breast cancer and melanoma cell lines and it is suggested that gene expressions regulated by FeNPA are also evaluated in animal models in vivo

Origanum Sipyleum Methanol Extract in Combination with Ponatinib Shows Synergistic anti-Leukemic Activities on Chronic Myeloid Leukemia Cells

Origanum sipyleum is used in folk medicine due to its anti-inflammatory, antimicrobial, and antioxidant properties. Ponatinib, an effective tyrosine kinase inhibitor in the treatment of chronic myeloid leukemia (CML), has severe side effects. Thus, we aimed to determine a novel herbal combination therapy that might not only increase the anti-leukemic efficacy but also reduce the dose of ponatinib in targeting CML cells. Origanum sipyleum was extracted with methanol (OSM), and secondary metabolites were determined by phytochemical screening tests. The cytotoxic effects of OSM on K562 cells were measured by WST-1 assay. Median-effect equation was used to analyze the combination of ponatinib and OSM (p-OSM). Apoptosis, proliferation, and cell-cycle were investigated by flow-cytometry. Cell-cycle-related gene expressions were evaluated by qRT-PCR. OSM that contains terpenoids, flavonoids, tannins, and anthracenes exhibited cytotoxic effects on K562 cells. The median-effect of p-OSM was found as synergistic; OSM reduced the ponatinib dose similar to 5-fold. p-OSM elevated the apoptotic and anti-proliferative activity of ponatinib. Consistently, p-OSM blocked cell-cycle progression in G(0)/G(1), S phases accompanied by regulations in TGFB2, ATR, PP2A, p18, CCND1, CCND2, and CCNA1 expressions. OSM enhanced the anti-leukemic activity of ponatinib synergistically via inducing apoptosis, suppressing proliferation, and cell-cycle. As a result, OSM might offer a potential strategy for treating patients with CML.[110S289]The Origanum sipyleum L. methanol extract was obtained from TuBTAK project no 110S289. We thank Ege University School of Foreign Languages and Lecturer Dr. Atiyye Hilal SENGENC for providing professional English editing for this manuscript