Genistein-induced mir-23b expression inhibits the growth of breast cancer cells

Aim of the study: Genistein, an isoflavonoid, plays roles in the inhibition of protein tyrosine kinase phosphorylation, induction of apoptosis, and cell differentiation in breast cancer. This study aims to induce cellular stress by exposing genistein to determine alterations of miRNA expression profiles in MCF-7 cells. Material and methods: XTT assay and trypan blue dye exclusion assays were performed to examine the cytotoxic effects of genistein treatment. Expressions of miRNAs were quantified using Real-Time Online RT-PCR. Results: The IC50 dose of genistein was 175 μM in MCF-7 cell, line and the cytotoxic effect of genistein was detected after 48 hours. miR-23b was found to be up-regulated 56.69 fold following the treatment of genistein. It was found that miR-23b was up-regulated for MCF-7 breast cancer cells after genistein treatment. Conclusions: Up-regulated ex-expression of miR-23b might be a putative biomarker for use in the therapy of breast cancer patients. miR-23b up-regulation might be important in terms of response to genistein. © 2015, Termedia Publishing House Ltd. All rights reserved

Antidiabetic exendin-4 activates apoptotic pathway and inhibits growth of breast cancer cells

Exendin-4 is a GLP-1 analog used for the treatment of type 2 diabetes mellitus in its synthetic form. As women with diabetes have higher breast cancer incidence and mortality, we examined the effect of the incretin drug exendin-4 on breast cancer cells. The aim of the study is to investigate anticancer mechanism of exendin-4 in MCF-7 breast cancer cells. Cytotoxic effects of exendin-4 were determined by XTT assay. IC50 dose in MCF-7 cells were detected as 5 μM at 48th hour. Gene messenger RNA (mRNA) expressions were evaluated by real-time PCR. According to results, caspase-9, Akt, and MMP2 expression was reduced in dose group cells, compared with the control group cells. p53, caspase-3, caspase-8, caspase-10, BID, DR4, DR5, FADD, TRADD, PARP, PTEN, PUMA, NOXA, APAF, TIMP1, and TIMP2 expression was increased in dose group cells, compared with the control group cells. Effects of exendin-4 on cell invasion, colony formation, and cell migration were detected by Matrigel chamber, colony formation assay, and wound-healing assay, respectively. To conclude, it is thought that exendin-4 demonstrates anticarcinogenesis activity by effecting apoptosis, invasion, migration, and colony formation in MCF-7 cells. Exendin-4 may be a therapeutic agent for treatment of breast cancer as single or in combination with other agents. More detailed researches are required to define the pathways of GLP-1 effect on breast cancer cells because of the molecular biology of breast cancer that involves a complex network of interconnected signaling pathways that have role in cell growth, survival, and cell invasion. © 2015, International Society of Oncology and BioMarkers (ISOBM)

Extract of Calvatia gigantea inhibits proliferation of A549 human lung cancer cells

In this study, in order to investigate the anticancer mechanism of Calvatia gigantea extract, edible mushroom species, which belong to Lycoperdaceae family, changes of CCND1, CCND2, CDK4, p21, Akt, Bax, Bcl-2, p53, caspase-3 and caspase-9 were evaluated in A549 lung cancer cells. Cytotoxic effect of C.gigantea extract was evaluated by using XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5 carboxanilide). The C. gigantea extract was treated in a time and dose dependent manner within the range 25 μg/ml–2 mg/ml to determine the IC50 dose. IC50 dose for C. gigantea extract was detected as 500 μg/ml for 72 h. According to expression results, while CCND1, CCND2, CDK4, Akt and Bcl-2 expression clearly decreased, Bax, p53, caspase-3 and caspase-9 expression clearly increased in the dose group cells (A549 cells treated with 500 μg/ml dose of C. gigantea extract for 72 h). However, there was no change in p21 expression. C. gigantea extract induced cell cycle arrest and apoptosis by decreasing the CCND1, CCND2, CDK4, Akt and Bcl-2 expression and by increasing Bax, p53, caspase-3 and caspase-9 expression in A549 cells. Mushrooms are eukaryotic organisms heavily used because of their supposedly anticancer effect. Many mushroom species have been used for medical purposes, as a result of also having many effects such as antibiotic, antiviral and anticancer effects. It is thought that the C. gigantea extract may be a significant agent for treatment of lung cancer as a single agent or in combination with other drugs. © 2016, Springer Science+Business Media Dordrecht

Zoledronic acid induces apoptosis via stimulating the expressions of ERN1, TLR2, and IRF5 genes in glioma cells

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor that affects older people. Although the current therapeutic approaches for GBM include surgical resection, radiotherapy, and chemotherapeutic agent temozolomide, the median survival of patients is 14.6 months because of its aggressiveness. Zoledronic acid (ZA) is a nitrogen-containing bisphosphonate that exhibited anticancer activity in different cancers. The purpose of this study was to assess the potential effect of ZA in distinct signal transduction pathways in U87-MG cells. In this study, experiments performed on U87-MG cell line (Human glioblastoma-astrocytoma, epithelial-like cell line) which is an in vitro model of human glioblastoma cells to examine the cytotoxic and apoptotic effects of ZA. IC50 dose of ZA, 25 μM, applied on U87-MG cells during 72 h. ApoDIRECT In Situ DNA Fragmentation Assay was used to investigate apoptosis of U87MG cells. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) (LightCycler480 System) was carried out for 48 gene expression like NF-κB, Toll-like receptors, cytokines, and inteferons. Our results indicated that ZA (IC50 dose) increased apoptosis 1.27-fold in U87MG cells according to control cells. According to qRT-PCR data, expression levels of the endoplasmic reticulum-nuclei-1 (ERN1), Toll-like receptor 2 (TLR2), and human IFN regulatory factor 5 (IRF5) tumor suppressor genes elevated 2.05-, 2.08-, and 2.3-fold by ZA, respectively, in U87MG cells. Our recent results indicated that ZA have a key role in GBM progression and might be considered as a potential agent in glioma treatment. © 2015, International Society of Oncology and BioMarkers (ISOBM)

Anti-proliferative and anti-invasive effects of ferulic acid in TT medullary thyroid cancer cells interacting with URG4/URGCP

Ferulic acid (4-hydroxy-3-methoxycinnamic acid; FA), a common dietary plant phenolic compound, is abundant in fruits and vegetables. The aim of present study is to investigate the effects of FA on cell cycle, apoptosis, invasion, migration, and colony formation in the TT medullary thyroid cancer cell line. The effect of FA on cell viability was determined by using CellTiter-Glo assay. IC50 dose in the TT cells was detected as 150 μM. URG4/URGCP (upregulated gene-4/upregulator of cell proliferation) is a novel gene in full-length mRNA of 3.607 kb located on 7p13. It was determined that FA caused a decrease in the expression of novel gene URG4/URGCP, CCND1, CDK4, CDK6, BCL2, MMP2, and MMP9, a significant increase in the expression of p53, PARP, PUMA, NOXA, BAX, BID, CASP3, CASP9, and TIMP1 genes in TT human thyroid cancer cell line by using real-time PCR. It was found that FA in TT cells suppressed invasion, migration, and colony formation by using matrigel invasion chamber, wound healing, and colony formation assay, respectively. In conclusion, it is thought that FA indicates anticarcinogenesis activity by affecting cell cycle arrest, apoptosis, invasion, migration, and colony formation on TT cells. © 2015, International Society of Oncology and BioMarkers (ISOBM)

Expression of URG4/URGCP, Cyclin D1, Bcl-2, and Bax genes in retinoic acid treated SH-SY5Y human neuroblastoma cells

Retinoic acid (RA) plays important roles in development, growth, and differentiation by regulating the expression of its target genes. The pro-apoptotic Bax gene may form channels through oligomerization in the mitochondrial membrane and facilitate the cytosolic release of cytochrome c. The anti-apoptotic Bcl-2 gene can inhibit this process. Up-regulated gene 4/Upregulator of cell proliferation (URG4/URGCP) is a novel gene located on 7p13. URG4/ URGCP also stimulates cyclin D1 (CCND1) mRNA expression, and RNAi-mediated URG4/URGCP silencing diminishes CCND1mRNA expression in HepG2 cells. In this study, the effects of RA treatment on URG4/URGCP, CCND1, Bcl-2 and Bax gene expression changes in undifferentiated and differentiated SHSY5Y neuroblastoma cells was analyzed. SHSY5Y cells were cultured in the appropriate conditions. To induce differentiation, the cells were treated with 10micromolar RA in the dark for 3-10 days. SHSY5Y cells possess small processes in an undifferentiated state, and after treatment with RA, the cells developed long neurites, resembling a neuronal phenotype. Total RNA was isolated with Tri-Reagent. Expression profiles of the target genes were determined by semi-quantitative RT-PCR. According to the results, Bcl-2 and CCND1 gene expression levels were increased, while URG4/URGCP and Bax gene expression was decreased in RA treated cells compared to the control cells. Our preliminary results suggest that RA may induce cell proliferation and escape apoptosis using a novel pathway by the URG4/URGCP gene. Further investigations are needed to clarifymore direct transcriptional targets of RA signaling and the interaction of RA pathways with other pro-regenerative signals

Regulation of URG4/URGCP and PPARα gene expressions after retinoic acid treatment in neuroblastoma cells

Neuroblastoma (NB), originating from neural crest cells, is the most common extracranial tumor of childhood. Retinoic acid (RA) which is the biological active form of vitamin A regulates differentiation of NB cells, and RA derivatives have been used for NB treatment. PPARα (peroxisome proliferator-activated receptor) plays an important role in the oxidation of fatty acids, carcinogenesis, and differentiation. URG4/URGCP gene is a proto-oncogene and that overexpression of URG4/URGCP is associated with metastasis and tumor recurrence in osteosarcoma. It has been known that URG4/URGCP gene is an overexpressed gene in hepatocellular carcinoma and gastric cancers. This study aims to detect gene expression patterns of PPARα and URG4/URGCP genes in SH-SY5Y NB cell line after RA treatment. Expressions levels of PPARα and URG4/URGCP genes were analyzed after RA treatment for reducing differentiation in SH-SY5Y NB cell line. To induce differentiation, the cells were treated with 10 μM RA in the dark for 3-10 days. Gene expression of URG4/URGCP and PPARα genes were presented as the yield of polymerase chain reaction (PCR) products from target genes compared with the yield of PCR products from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. SH-SY5Y cells possess small processes in an undifferentiated state, and after treatment with RA, the cells developed long neurites, resembling a neuronal phenotype. PPARα gene expression increased in RA-treated groups; URG4/URGCP gene expression decreased in SH-SY5Y cells after RA treatment compared with that in the control cells. NB cell differentiation might associate with PPARα and URG4/URGCP gene expression profile after RA treatment. © 2013 International Society of Oncology and BioMarkers (ISOBM)

Effects of quercetin induced cell death on a novel gene "URG4/URGCP" expression in leukemia cells

The present study aimed to investigate anti-proliferative and apoptotic effects of quercetin on human leukemia cells and effects of quercetin-induced cell death on a novel gene Up-regulated gene 4/upregulator of cell proliferation (URG4/URGCP), in leukemia cells. URG4/URGCP expression is determined by using RT-PCR. IC 50 of quercetin was determined as 25 microM in CCRF-CEM, HL-60 and K562 cells. In IC 50 dose group, URG4/URGCP expression was decreased 99% in HL-60 cells, 90% in CCRF-CEM cells, and 52% (24 hour) - 99% (72 hour) in K-562 cells. URG4/URGCP may play important roles in the development of leukemia, and might be a useful molecular marker for predicting the prognosis of leukemia via quercetin treatment. © 2012 Dodurga Y, et al

Fibroblast growth factor receptor 3 (fgfr3) a248c, s249c, g372c, t375c mutations incidence in bladder cancer

Üriner sistemde oluşan kanserlerinin büyük kısmı mesaneden gelişir ve yaklaşık %90’ı transisyonel hücre karsinomdur. Fibroblast büyüme faktörü reseptörleri (FGFR) yapısal olarak reseptör tirozin kinaz ailesindendir ve hücre farklılaşmasında, proliferasyonunda, sinyal yollarının düzenlenmesinde ve embriyonik gelişmede önemli roller oynamaktadır. FGFR3 geni kromozom 4p16.3 bölgesine lokalizedir ve çeşitli yanlış anlamlı mutasyonlar sonucunda otozomal kalıtımlı çeşitli iskelet sistemi hastalıkları ortaya çıkmaktadır ve ayrıca onkogenik etkisi olduğu da düşünülmektedir. Bu yüksek lisans tezinde, FGFR3 tanatoforik displazi mutasyonları olarak gruplandırılan ekzon 7 A248C, S249C ve ekzon 10 G372C, T375C kodonlardaki olası yanlış anlamlı mutasyonların mesane kanseri hasta popülasyonundaki varlığı ve klinikopatolojik parametreler üzerindeki etkisi araştırılmıştır. Denizli Devlet Hastanesi Patoloji bölümüne tanı alma amacıyla başvuran 56 olguya ait mesane transizyonel hücre karsinomu doku örneği çalışma kapsamına alınmıştır. Parafin kesitlerden DNA izolasyonunun sonrasında, FGFR3 geni ekzon 7 A248C, S249C ve ekzon 10 G372C ve T375C kodonlarını içeren DNA parçaları polimeraz zincir reaksiyonu ile çoğaltılıp, A248C ve S249C kodonlarındaki mutasyonlar Restriksiyon Parça Uzunluk Polimorfizmi yöntemi ile ve ekzon 10 G372C ve T375C kodonlarındaki mutasyonlar DNA dizi analizi ile araştırılmıştır. Bu çalışma kapsamına alınan 56 adet mesane transizyonel hücre karsinomunun 14 tanesi (%25) pTa, 23 tanesi (%41,1) pT1, 19 tanesi (%33,9) pT2 olarak evrelendirilmiştir. Ayrıca 56 olgunun 5 tanesi (% 8.9) G1, 25 tanesi (% 44.6) G2, 26 tanesi (% 46.4) G3 olarak derecelendirilmiştir. FGFR3 genindeki tanatoforik displazi mutasyonları olarak gruplandırılan ekzon 7 A248C, S249C ve ekzon 10 G372C, T375C kodonlardaki tespit edilen toplam mutasyon varlığı 56 olgunun 23 tanesi (%41.1) normal, 33 tanesi (%58.9) heterozigot mutant olarak değerlendirilmiştir. 56 olgunun 7’sinde (%12.5) A248C ve 28’inde (%50) S249C, 3’ünde (%5.4) T375C kodonlarında yanlış anlamlı mutasyon saptanmış olup bu oran literatürdeki diğer çalışmaları desteklemektedir. FGFR3 genindeki ekzon 7 A248C, S249C ve ekzon 10 G372C ve T375C mutasyon sonuçları tek tek ve toplu olarak değerlendirildiğinde, daha önce yapılmış çalışmalardan farklı olarak tümör evresine ve derecesine göre homojen dağılımlı olduğu saptanmıştır. Ancak daha fazla sayıda erken evre ve düşük derece tümör örneğinin araştırma kapsamında değerlendirilmesi gerektiği düşünülmektedir.Bladder cancer is the most frequent cancer derived from urinary system and %90 of them is transitional cell carcinomas. Fibroblast growth factor receptors (FGFR) belong to the tyrosine kinase family and have important roles in cellular differentiation, proliferation, cell signaling and embryonic development. FGFR3 is localized chromosome 4p16.3. Missense mutations of fibroblast growth factor receptor 3 (FGFR3) are associated with autosomal dominant human skeletal disorders and some oncogenic effects. In this thesis study, we investigated the incidence of FGFR3 thanatophoric dysplasia mutations located at exon 7 A248C, S249C and exon 10 G372C, T375C in a bladder carcinoma patient population and its correlation to clinicopathological parameters. Fifty six paraffin embedded specimens of transitional cell carcinoma of the urinary bladder were obtained from Denizli State Hospital Pathology department and included in this study. After the DNA isolation from the paraffin sections, PCR amplification of DNA segments incuding exon 7 A248C, S249C and exon 10 G372C, T375C was performed for Restriction Fragment Length Polymorphism (RFLP) and DNA sequencing analysis. Among the 56 transitional cell carcinomas, 14 of them (%25) were classified as pTa, 23 of them (%41,1) were classified as pT1 and 19 of them (%33,9) were classified as pT2. In addition, 5 of them (% 8.9) were graded as G1, 25 of them (% 44.6) were graded as G2, 26 of them (% 46.4) were graded as G3. FGFR3 thanatophoric dysplasia mutations located at exon 7 A248C, S249C and exon 10 G372C, T375C were detected in 33 of the patients (%58.9) (heterozygous mutant) and 23 of the patients (%41.1) were found to be normal. Among the 56 transitional cell carcinomas, missense point mutations were detected in 7 of them (%12.5) at codon A248C, 28 of them (%50) at codon S249C, and 2 of them (%5.4) at codon T375C and these results are in agreement with the previous research reports in the literature. When the results of the FGFR3 thanatophoric dysplasia mutations located at exon 7 A248C, S249C and exon 10 G372C, T375C were analyzed one by one or as a group, despite the findings of previous research reports, it has been suggested that these mutations are detected homogenously regardless of the tumor classification and tumor grade in our study. Hence, it is our intention to include more early stage and low grade tumor samples in this research project

Lökomogenez'de yeni bir yaklaşım olarak ifadesinde artış gözlenen gen 4 (URG4)’ün sinyal iletim yolaklarındaki gen ekspresyonları ile korelasyonunun araştırılması

Bu çalışmada, hücre döngüsünde fonksiyonel olan genlerin ve yeni onkogen adayı URG4’ün işlevsel rollerini, ekspresyon düzeyinde kantitatif olarak tanımlayarak, elde edilecek bulgulardan geliştirilecek olan gen ekspresyon profil modeli ile lökomogenezdeki değişimlerin araştırılması amaçlandı. Çalışmamıza 0-18 yaş aralığında 40 yeni tanı ALL-AML olguları ve 6 kontrol grubuna ait kemik iliği örnekleri çalışmamıza dahil edildi. Toplam RNA’ları elde edildikten sonra, komplementer DNA sentezleri gerçekleştirildi. Elde edilen cDNA’lar 96 kuyucuklu plate’lere uygun karışımlar hazırlanarak yüklendi ve LightCycler® 480 Gerçek Zamanlı RT-PZR’da kantitatif olarak değerlendirildi. T-ALL grubunda, CHEK1, URG4, CCNG1, CCNC, CDC16, KRAS, CDKN2D; Prekürsör B-ALL grubunda CCND2, ATM, CDK8, CHEK1, TP53, CHEK2, CCNG2, CDK4, CDKN2A, E2F4, CCNC, KRAS; AML grubunda CCND2, CDK6 genlerinde ekspresyon artışlarının anlamlı oldukları saptandı. Lökomogenezde sorumlu sinyal yolakları kısmen tanımlanmış olsa da, akut lösemi alt gruplarında anlamlı olarak saptanan yeni genetik belirteçler, akut lösemilerin erken tanı ve tedavisinde yeni protokollerinin geliştirilmesine olanak sağlayabilecektir