Search

Background/aim Tuberculosis is a public health problem that still remains significant. For prevention, diagnosis, and treatment of tuberculosis more effective novel biomarkers are needed. MicroRNAs can regulate innate and adaptive immune responses, alter host-pathogen interactions, and affect progression of diseases. The relationship between microRNA expression and active pulmonary tuberculosis (APT) has not yet been investigated in the Turkish population. We aimed to test the potential diagnostic value of some microRNAs whose levels were previously reported to be altered in APT patients. Materials and methods Using two different references (U6 and miR-93), we compared the expression levels of potentially important microRNAs in serum of APT patients with healthy individuals using quantitative polymerase chain reaction (qPCR). Results miR-144 expression level was down-regulated in APT patients when either U6 or miR-93 was used for normalization. When data was normalized with miR-93, a statistically significant decrease in miR-125b (0.8 fold) and miR-146a (0.7 fold) expression levels were observed, while no differences were detected for U6. The receiver operating characteristic suggested that miR-144 may be a candidate biomarker for discriminating APT patients and controls (p < 0.05) both for U6 and miR-93. Conclusion These findings suggest that miR-144 can have potential as a biomarker for APT. Using a single reference may be misleading in evaluation of microRNA expression. U6 and miR-93 can be used in combination as references for normalization of serum microRNA expression data.

We aimed to investigate the in vitro physiologic effects of xylene, chloroform, orange oil and eucalyptus oil solvents for dissolving gutta-percha on L929 and HOB cell lines; 2.5 and 10 mu L mL(-1) of these solvents were tested for 24, 48 and 72 h. Gutta-percha solvents inhibited the proliferation rate of fibroblasts in a dose- and time-dependent manner; however, no inhibition was detected in HOB (evaluated using MTT assay). None of the solvents induced apoptosis/necrosis in HOB cells at

In this study, we compared the effects of terpinolene against the anti-cancer agent cisplatin on proliferation, apoptotic activity and intracellular ROS (reactive oxygen species) production in cancerous (MCF-7) and non-cancerous (HEK-293) cell lines. Cisplatin presented a strong growth-inhibitory effect on both MCF-7 (IC25: 18.77 mu M) and HEK-293 (IC25: 15.03 mu M) cell lines. The growth-inhibitory effect of terpinolene was found to be weaker than cisplatin (IC25: 291.18 mu M for MCF-7 and 345.35 mu M for HEK 293 cells). The expression levels of BAX (by 1.86-fold), cleaved-PARP (by 2.23-fold) and pro-caspase-8 (by 1.74-fold) proteins increased in response to terpinolene treatment in MCF-7 cells. Curiously, the increase in the expression levels of these apoptotic markers was less pronounced in terpinolene-treated non-cancerous HEK 293 cells. Terpinolene, promoted a higher rate of apoptotic and necrotic cell death on MCF-7 cells (apoptotic cells: 25.28 %; necrotic cells: 17.70 %) when compared with HEK-293 cell line (apoptotic cells: 15.47 %; necrotic cells: 9.42 %). Interestingly, terpinolene caused a marked increase (2.05-fold) in intracellular ROS production in MCF-7 cells while HEK-293 cells appeared to be resistant to terpinolene or cisplatin treatments. In conclusion, although terpinolene showed a weaker growth-inhibitory effect on MCF-7 cancer cells, it exhibited a better selectivity than cisplatin in inducing cell death and intracellular oxidative stress in MCF7 breast cancer cells.

Objectives Betahistine is a histamine analog commonly prescribed for symptomatic treatment of vertiginous symptoms. In vitro studies have shown that betahistine was not toxic at the prescribed doses in a nasal epithelial cell line. However, the effect of betahistine on other cell types has not been studied. In this study, we aimed to investigate some of the physiological effects of betahistine on L929 fibroblast, A549 lung cancer, human umbilical vein endothelial (HUVEC), and Ishikawa endometrial cell lines. Materials and methods Cellular proliferation was assed assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, apoptosis was evaluated by acridine orange-ethidium bromide staining, and cellular migration was assed assessed by scratch assay. Results Betahistine treatment (0.1-0.5 mg/mL, 24 hours) can inhibit cell proliferation and induce apoptosis in HUVEC, A549, Ishikawa, and L929 cell lines. Betahistine (≥0.1 mg/mL) significantly increased the number of apoptotic cells (HUVEC: 26.3%, A549: 17.3%, L929: 8.6%, and Ishikawa: 2.3%). Betahistine at doses over 0.1 mg/mL significantly suppressed the cell migration rate in all of the cell lines. In contrast, exposure to a low dose of betahistine (0.025 mg/mL) induced migration rates of HUVEC and Ishikawa cells by 81% and 48%, respectively. Conclusion Betahistine may alter the processes of cellular proliferation, apoptosis, and cellular migration in a cell line- and dose-dependent manner. In this sense, proliferative and metastatic properties of certain cancer cells can potentially be altered in response to betahistine treatment.

OBJECTIVES: Dehydroepiandrosterone (DHEA) is an endogenous hormone that acts as a ligand for several cellular receptors. An age-dependent decline in circulating levels of DHEA is linked to changes in various physiological functions. In gynecological clinical practice, DHEA is commonly prescribed to induce ovulation. Some clinical studies report a positive association between high serum concentrations of DHEA and an increased risk of developing ovarian cancer. However, the in vitro physiological effects of DHEA on ovarian cancerous cells have not been explored thus far. In this study, we aimed to investigate the physiological effects of DHEA treatment (0-200 μM, 24-72 hours) on MDAH-2774 human ovarian cancer cell line and primary HuVeC human endothelial cells. MATERIALS AND METHODS: The physiological effects of DHEA treatment (0-200 μM, 24-72 hours) on MDAH-2774 human ovarian cancer cell line and primary HuVeC human endothelial cells were investigated with the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, acridine orange/ethidium bromide staining, and scratch assay. RESULTS: DHEA treatment promoted proliferation of the MDAH-2774 cancer cell line in a dose-dependent manner (r=0.6906, p

Objective: Inter-retrotransposon polymorphism Polymerase Chain Reaction (IRAP-PCR) technique allows for detecting insertional polymorphisms via amplification of the DNA fragment between two retrotransposons in plant genomes. However, this method has not been reported to be used for analyzing human samples to date. Recently, Human Endogenous Retrovirus (HERV) polymorphisms gained interest due to their potential effect on pathophysiology of certain diseases. Nevertheless, the association between HERV polymorphisms and the risk for developing nasal polyposis (NP) has not been studied. In this study, we aimed to investigate whether or not IRAP-PCR could be performed in nasal swab samples for comparing HERV polymorphisms in different nasal mucosal samples. Methods: Nasal swab samples from 16 patients were used for DNA isolation. These DNA samples were used as templates for IRAP PCR of HERV-K6, HERV-K11, HERV-L1 and HERV-L2 and PCR products were analyzed by agarose gel electrophoresis. Results: Nasal swab samples yielded enough DNA material for successfully performing IRAP-PCR. We obtained specific banding patterns the three out of four HERV sequences tested in this study. No polymorphisms was detected between samples from different patients. Similarly, polymorphic bands was not detected between the polyps or nasal mucosal swab samples obtained from the same patient. Conclusion: We have, for the first time, shown that IRAPPCR can be performed in nasal swabs. Our findings suggest that this technique can serve as an inexpensive and effective screening tool for investigating links between nasal mucosal diseases and HERV polymorphisms such as nasal polyposis.

Autophagy is an evolutionarily conserved catabolic mechanism, by which eukaryotic cells recycle or degrades internal constituents through membrane-trafficking pathway. Thus, autophagy provides the cells with a sustainable source of biomolecules and energy for the maintenance of homeostasis under stressful conditions such as tumor microenvironment. Recent findings revealed a close relationship between autophagy and malignant transformation. However, due to the complex dual role of autophagy in tumor survival or cell death, efforts to develop efficient treatment strategies targeting the autophagy/cancer relation have largely been unsuccessful. Here we review the two-faced role of autophagy in cancer as a tumor suppressor or as a pro-oncogenic mechanism. In this sense, we also review the shared regulatory pathways that play a role in autophagy and malignant transformation. Finally, anti-cancer therapeutic agents used as either inhibitors or inducers of autophagy have been discussed.

Protein kinase MELK has oncogenic properties and is highly overexpressed in some tumors. In the present study, we show that a novel MELK inhibitor causes both the inhibition and degradation of MELK, culminating in replication stress and a senescence phenotype., Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELK-T1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold.

Introduction: In this cross-sectional study, we aimed to evaluate the distribution of allergic sensitivity to the common inhalant allergens in different age groups. We also aimed to determine the factors that may be associated with sensitization in adults from the European region of Istanbul. Materials and Method: Using SPSS statistical analysis software, we retrospectively evaluated Skin Prick Test (SPT) data from 2587 patients with symptoms similar to Allergic Rhinitis (AR). Results: Analysis of data revealed that that the frequency of sensitization to the common inhalant allergens significantly decreased by age. Animal dander was found to be the most common cause of allergic reactions among adult patients (p

Maternal embryonic leucine zipper kinase (MELK) belongs to the subfamily of AMP-activated Ser/Thr protein kinases. The expression of MELK is very high in glioblastoma-type brain tumors, but it is not clear how this contributes to tumor growth. Here we show that the siRNA-mediated loss of MELK in U87 MG glioblastoma cells causes a G1/S phase cell cycle arrest accompanied by cell death or a senescence-like phenotype that can be rescued by the expression of siRNA-resistant MELK. This cell cycle arrest is mediated by an increased expression of p21WAF1/CIP1, an inhibitor of cyclin-dependent kinases, and is associated with the hypophosphorylation of the retinoblastoma protein and the down-regulation of E2F target genes. The increased expression of p21 can be explained by the consecutive activation of ATM (ataxia telangiectasia mutated), Chk2, and p53. Intriguingly, the activation of p53 in MELK-deficient cells is not due to an increased stability of p53 but stems from the loss of MDMX (mouse double minute-X), an inhibitor of p53 transactivation. The activation of the ATM-Chk2 pathway in MELK-deficient cells is associated with the accumulation of DNA double-strand breaks during replication, as demonstrated by the appearance of γH2AX foci. Replication stress in these cells is also illustrated by an increased number of stalled replication forks and a reduced fork progression speed. Our data indicate that glioblastoma cells have elevated MELK protein levels to better cope with replication stress during unperturbed S phase. Hence, MELK inhibitors hold great potential for the treatment of glioblastomas as such or in combination with DNA-damaging therapies. Background: Protein kinase MELK is expressed at very high levels in glioblastomas, but it is not understood how this benefits tumor growth. Results: A deficiency of MELK causes replication stress and is associated with cell cycle arrest and senescence. Conclusion: MELK is required for progression through unperturbed S phase. Significance: The inhibition of MELK emerges as an attractive cancer therapy.

Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. This MELK-dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA-mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK-mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM.

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double-or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways., Scientific and Technological Research Council of Turkey (TÜBİTAK); Sabanci University; Swiss National Science Foundation; Scientific and Technological Research Council of Turkey (TÜBİTAK); IKU Prof. Onder Oztunali Science Award; TGC Sedat Simavi Health Sciences Award; Elginkan Foundation Technology Award

Nitric oxide synthases (NOS) catalyze the synthesis of ubiquitous signaling molecule nitric oxide (NO) which controls numerous biological processes. Using a spectrofluorometric NOS assay, we have measured the rate of total NO production in the crude cell extracts of Schizosaccharomyces pombe. NO production was reduced in the absence of NOS cofactors calmodulin and tetrahydrobiopterin, and a competitive NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was able to cause a statistically significant inhibition on the rate of total NO production. These results, for the first time, provide evidence that an enzyme with a NOS-like activity may be present in the fission yeast. In order to assess the possible regulatory roles of NO as a signaling molecule in this yeast, using the differential display technique, we screened for NO-responsive genes whose expression decreased upon exposure to L-NAME and increased in response to an NO donor, sodium nitroprusside treatment. Differential expression patterns of byr1, pek1, sid1, and wis1 genes were confirmed by quantitative real-time PCR. The physiological experiments performed based on the functions and molecular interactions of these genes have pointed to the possibility that NO production might be required for sporulation in S. pombe. Taken together, these findings suggest that NO may function as a signaling molecule which can induce both transcriptional and physiological changes in the fission yeast. Hence, these data also imply that S. pombe can be used as a model system for investigating the mechanisms underlying NO-related complex signaling pathways.

Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELK-T1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold. ispartof: Bioscience Reports vol:35 issue:6 ispartof: location:England status: published

The invertase mutant defective in the glucose signaling pathway of Schizosaccharomyces pombe (ird11) is resistant to glucose repression. This mutant is able to consume sucrose alongside glucose and grows in glucose-containing media with a generation time close to that of the wild type. Intracellular oxidation, protein carbonyl, and reduced glutathione levels and catalase, superoxide dismutase, and glutathione peroxidase activity were investigated in ird11, to determine the relationship between oxidative stress response and glucose signaling. The expression profiles of some genes involved in regulation of glucose repression (fbp1, fructose-1,6-bis-phosphatase; hxk2, hexokinase) and stress response (atf1 and pap1 transcription factors; ctt1, catalase; sod1, Cu,Zn superoxide dismutase) were analyzed using the quantitative real-time PCR technique. Oxidative stress response in ird11 seems to be affected by glucose signaling in a manner different from that caused by glucose deprivation.

We have isolated 14 different Schizosaccharontyces pombe mutants that synthesize invertase enzyme constitutively. Analyses of invertase activities revealed that the degrees of resistance to glucose repression were not similar among different complementation groups. One of the complementation groups appeared to be associated with functional and/or regulatory defects in hexose transport. Another complementation group appeared to be specific for the regulation of the inv1 gene alone, implying that these mutations might be associated with different genes acting on the glucose sensing and signaling pathway. In addition, we found that the wild-type level glucose uptake is essential for the full-level repression of inv1 expression.

Maternal embryonic leucine zipper kinase (MELK, hMP38, pEG3), a Ser/Thr protein kinase, is highly overexpressed in stem cells and cancer cells. The oncogenic role of MELK is attributed to disabling critical cell cycle check points as well as enhancing replication. Most functional studies have relied on the use of siRNA/shRNA mediated gene silencing, but this often can be associated with off-target effects. Here we want to present a novel, potent and selective small molecule inhibitor JNJ-47117096 that has enabled us to validate the biological function of MELK kinase. Outcome: MELK inhibition in cancer cells with an intact p53 signaling pathway is linked to a replicative senescence phenotype. This phenotype correlates with a rapid ATM activation and phosphorylation of CHK2 without any effects on the alternative ATR/ CHK1 DNA damage pathways. Furthermore, JNJ-47117096 induces strong phosphorylation of p53 and prolonged up-regulation of p21 without the induction of apoptosis. Strikingly, MELK inhibition in several p53 disabled cancer cells showed induction of a mitotic catastrophe phenotype followed by prominent cell killing. Finally, JNJ-47117096 triggers a rapid degradation of cellular MELK protein, independent of the cell cycle phase and its regulation by the E2F pathway. This observation can clearly be linked to a direct binding effect of JNJ-47117096 to cellular MELK protein confirmed by chemical proteomics. Conclusion: Our data generated with JNJ-47117096, confirmed by selective siRNAs, indicate MELK as a key stimulator of proliferation and replication by its ability to increase the threshold for DNA damage tolerance. Targeting MELK function by selective small molecule inhibitors might sensitise tumors to DNA-damaging agents or radiation therapy. Citation Format: Lijs Beke, Joannes T.m. Linders, Cenk Kig, An Boeckx, Erika van Heerde, Dirk Wuyts, Marc Parade, Lieven Meerpoel, Chris Johnson, Monique Beullens, Mathieu Bollen, Dirk Brehmer. JNJ-47117096, a selective small molecule inhibitor of the MELK oncogene decreases DNA damage tolerance in highly proliferating cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2936. doi:10.1158/1538-7445.AM2014-2936