Browsing by Subject "Breast Cancer"

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Open Access
Analysis of 45S rDNA promoter methylation and expression of rRNA transcripts in breast cancer
(2015-06) Karahan, Gurbet
Ribosome biogenesis has a central role in cell growth and proliferation that is usually disrupted in tumor cells by the inactivation of tumor suppressor genes and activation of oncogenes. Ribosomal RNA (rRNA) gene expression is one of the most important factors regulating ribosome production, which is controlled by CG rich 45S ribosomal DNA (rDNA) promoter. The effect of DNA methylation at 45S rDNA promoter on rRNA gene expression is a subject of controversy in the literature. In this thesis, a 434 bp region (-380 bp to +54 bp) spanning both upstream control element (UCE) and core promoter located in 45S rDNA promoter containing 54 CpGs was analysed in breast cancer. We also analysed the related rRNA expression levels in the same samples in order to clarify the role of 45S rDNA promoter methylation on rRNA gene expression. 45S rDNA promoter region was highly methylated (74%-96%) in all cell lines including non-tumorigenic breast cell line (MCF10A). Even though 45S rDNA promoter region of breast cancer cell lines are extensively methylated, rRNAs (18S, 28S, 5.8S and 45S ETS) were expressed independent of the heavy methylation. Expression levels of rRNAs are assessed either using housekeeping genes (ACTB, TBP, ACTB&TBP) or geometric mean of rRNAs (GM-rRNAs). We propose GM-rRNA normalization as a new method to identify relative expression differences between rRNA transcripts. Epigenetic drugs 5-Aza-2'-deoxycytidine (5-AZA) and Trichostatin A (TSA) were used to determine the effect of DNA methylation and histone acetylation on rRNA expression. Demethylation with 5-AZA resulted in an unexpected decrease in the expression of all rRNA. TSA treatment did not lead to any significant expression difference in cell lines. To better evaluate the effect of DNA methylation on the expression of rRNA transcripts we analysed the methylation status of 19 breast tumor and matched normal frozen tissue samples. The results showed that majority of the tumors (13/19) have significantly higher methylation levels than their normal pairs. Using the GM-rRNA as reference helped us to determine significant differences in the proportionate expression of rRNAs in these tissue samples. The 5.8S rRNA ratio was significantly lower whereas the 18S rRNA ratio was significantly higher in breast tumor samples. Furthermore, the 45S rDNA promoter methylation levels in normal breast tissue samples were negatively correlated with the18S rRNA ratio but this correlation was disrupted in breast tumors. Similarly, rRNA transcript levels were significantly correlated with each other in normal samples, were lost in tumor samples. It is clear that, there is a dysregulation both in rDNA methylation levels and spliced rRNA transcripts specific to breast tumor samples, which was not observed in normal breast tissues. rRNA gene expression is controlled by mechanisms other than promoter DNA methylation. Tumorigenesis may cause disruption of many control mechanisms that are required for proper rRNA expression, splicing and maturation, resulting in a dysregulation of the correlation between spliced rRNA expression levels, which should be investigated further.
Open Access
A comprehensive methodology for determining the most informative mammographic features
(2013) Wu, Y.; Alagoz O.; Ayvaci, M.U.S.; Munoz Del Rio, A.; Vanness, D.J.; Woods, R.; Burnside, E.S.
This study aims to determine the most informative mammographic features for breast cancer diagnosis using mutual information (MI) analysis. Our Health Insurance Portability and Accountability Act-approved database consists of 44,397 consecutive structured mammography reports for 20,375 patients collected from 2005 to 2008. The reports include demographic risk factors (age, family and personal history of breast cancer, and use of hormone therapy) and mammographic features from the Breast Imaging Reporting and Data System lexicon. We calculated MI using Shannon's entropy measure for each feature with respect to the outcome (benign/malignant using a cancer registry match as reference standard). In order to evaluate the validity of the MI rankings of features, we trained and tested naïve Bayes classifiers on the feature with tenfold cross-validation, and measured the predictive ability using area under the ROC curve (AUC). We used a bootstrapping approach to assess the distributional properties of our estimates, and the DeLong method to compare AUC. Based on MI, we found that mass margins and mass shape were the most informative features for breast cancer diagnosis. Calcification morphology, mass density, and calcification distribution provided predictive information for distinguishing benign and malignant breast findings. Breast composition, associated findings, and special cases provided little information in this task. We also found that the rankings of mammographic features with MI and AUC were generally consistent. MI analysis provides a framework to determine the value of different mammographic features in the pursuit of optimal (i.e., accurate and efficient) breast cancer diagnosis. © 2013 Society for Imaging Informatics in Medicine.
Open Access
Effects of biological compound Turkish propolis extract on breast cancer cells
(2013) Uğurlu, Deniz
Propolis is a resinous compound which is collected from various plants then combined with wax and bee enzymes by worker bees. There are many studies conducted on propolis or its active components aiming to find new treatment possibilities in diverse research fields such as immunology, infectious diseases, allergy, diabetes, ulcers, and oncology. Chemical analysis indicated that propolis is a multicomponent mixture of various compounds with prevalence of flavonoids and phenolic acids. Therefore it is important to investigate the propolis extract mechanisms of action in order to predict possible cytotoxic and may be therapeutic effects for cancer. The most common propolis extract is ethanol extract of propolis (EEP) whereas Turkish researchers were able to extract the propolis with dimethyl sulfoxide (DMSO) which can maximize the penetration of compounds from propolis to the cells as well as DMSO is a good solvent for flavonols (one of the most common compound in propolis). There are many studies conducted on propolis or its active components for treatment of cancer which reveals the potential of this biological compound in the development of novel anti-cancerous agents. However, anti-cancer activity of DMSO extract of Turkish propolis (DEP) on human breast cancer has not been investigated yet. The aim of this study was to investigate the anti-cancer effects of DMSO extract of Turkish propolis (DEP) on cancer cells. Inhibitory effects of propolis extracts collected from different regions of Turkey were analyzed on the growth of the human breast carcinoma cells. Two different propolis extracts were used to determine their cytotoxic effects of breast carcinoma cell lines using SRB staining and IC50 values were determined. The results showed that propolis is cytotoxic in dose-dependent manner (IC50 value of diverse from 25 ug/ml to 123 ug/ml). Real time monitoring (xCELLigence system) of propolis treated cells confirmed the cytotoxic effect of propolis, since increasing concentrations of propolis decreased the cell number in a dose- and cell line- dependent way. Furthermore, propolis treatment induces apoptosis in breast carcinoma cell lines. Propolis treated cells changed their adherent morphology to round cells and detached from the surface. Hoechst 33258 staining of propolis treated cells revealed the increasing number of cells displays DNA condensation. PARP-1, a 116 kDa nuclear enzyme, is cleaved in fragments of 89 and 24 kDa during apoptosis. Western blot analysis was performed to detect the PARP-1 cleavage in propolis treated cells. Decrease in the full-length PARP-1 protein levels supports our hypothesis that propolis shows its cytotoxic effect at least partially through induction of apoptosis. The effect of propolis on cell cycle was analyzed with flow cytometer after staining the cells with Propidium iodide (PI). Increase in the G2/M cell cycle arrest was observed in propolis treated cells compare to control DMSO treated MDA-MB-231 cells. In addition to cytotoxic effects, in vitro wound healing assay revealed that propolis treated MDA-MB-231 cells shows delayed invasion of the cells to the denuded area when compared to the DMSO control cells. In conclusion, propolis showed a cytotoxic effect on breast carcinoma cell lines by inducing apoptosis, G2/M arrest as well as delaying the invasion capacity of the cells which makes it a potent anti-tumorigenic compound that may be useful in cancer chemoprevention or therapy.
Open Access
Effects of Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi on apoptosis, DNA damage response, drug sensitivity, and HSA-MIR-495-3P overexpression in breast cancer
(2018-12) Köker, Şahika Cıngır
Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) is associated with nicotine addiction and it has an important role in the prognosis of lung cancer. Despite its important cellular functions, its role in breast cancer remains to be elucidated. In this thesis, I aimed to identify the alterations in the important cancer signaling pathways occurring upon CHRNA5 depletion. Drug resistance is one of the major obstacles in breast cancer therapy. Heterogeneous nature of breast cancer necessitates identification of more biomarkers which aid in precise diagnosis and hence development of proper treatment options. In this study, by using more than one cell line which is representative of different subtypes of breast cancer, I showed the alterations occurred in cancer signaling pathways such as cell cycle and apoptosis upon CHRNA5 depletion, which could serve as a novel biomarker in breast cancer subtyping. Depending on mutation status of TP53, which is the gatekeeper protein during G1/S checkpoint, CHRNA5 depletion mostly exerted its effects over decreasing the levels of total CHEK1 and pCHEK1 (S345) which significantly altered the response of MCF7 cells to topoisomerase inhibitors in terms of enhanced drug sensitivity. Increases in apoptotic markers, such as BAX/BCL2 ratio along with increased FAS levels, further confirmed that this sensitization of MCF7 cells upon CHRNA5 depletion might have ended with apoptosis. So far in the literature, there is no study examining the regulation of CHRNA5 by small endogenous molecules such as miRNAs. Due to the predictive binding sites in 3’UTR of CHRNA5 and the importance of participating in tamoxifen resistance in breast cancer; I also examined the interplay between miR-15a family and CHRNA5 in MCF7 cells. I showed significant decrease in CHRNA5 levels upon using miR-15a mimic while demonstrating similar activity of miR-15a family mimics with CHRNA5 depletion using RT-qPCR. Another important implication of CHRNA5 depletion in MCF7 cells was the global change in miRNA expression prolife which was verified with independent microRNA arrays. Based on these in silico results, hsa-miR-495-3p appeared as the most downregulated miRNA which is known as a tumor suppressor miRNA. As stated in the literature, the role of miR-495 differs depending on the tumor type. Therefore, I tried to restore its expression by mimicking along with CHRNA5 depletion. The transcriptomic changes observed with CHRNA5 depletion was boosted with the restoration of miR-495 levels.
Open Access
Evaluation of TAGLN as a diagnostic marker in breast cancer
(2018-08) Köseer, Ayşe Sedef
Silecing of tumor suppressor genes via CpG hypermethylation in promoter regions is one of the frequent events occurring in different types of cancers. These genes have the potential as a diagnostic or a prognostic biomarker. Liquid biopsy is a relatively less invasive technique that is used for early diagnosis, therapy response prediction, minimal residual disease detection and real-time monitoring of tumor progression. In this study, a 402 bp region (-286 bp to -80 bp for Section 1, -102 bp to +115 bp for Section 2) located in TAGLN promoter containing 22 CpGs was analyzed in breast cancer patients and healthy donors to evaluate the biomarker potential of TAGLN promoter methylation levels in breast cancer. TAGLN promoter region was significantly hypermethylated in breast cancer patients (77.3%) compared to healthy donors (68.2%). Among differentially methylated CpGs, 6 out of 22 were hypermethylated and one was hypomethylated in breast cancer patients. We also analyzed the relationship between TAGLN promoter methylation levels and the patient's clinicopathological parameters. Analyses revealed that TAGLN promoter is highly methylated in breast cancer patients over 50 years of age compared to the healthy donors in the same age group. TAGLN promoter methylation did not differ as related to various clinicopathological parameters of breast cancer patients. TAGLN promoter methylation levels diagnosed breast cancer patients with 74.45% specificity and 57.58% sensitivity. Additionally, independent of the age group breast cancer patients (131.6 ng) exhibited higher levels of total cfDNA compared to healthy donors (56.4 ng). Pre- and postmenopausal breast cancer patients possessed higher total cfDNA levels compared to pre- and postmenopausal healthy donors. Total cfDNA levels did not differ in various clinicopathological parameters of breast cancer patients; however, total cfDNA levels diagnosed breast cancer patients with 73.33% specificity and 56.72% sensitivity. In summary, breast cancer patient sera can be used to identify the tumor profile, and TAGLN promoter hypermethylation and total cfDNA levels could serve as a diagnostic biomarker in breast cancer.
Open Access
Functional analysis of transgelin in breast cancer
(2018-06) Değer, Nazlı
Transgelin (TAGLN) is an actin-binding protein. It is highly expressed in fibroblasts and smooth muscle cells. In smooth muscle cells, it takes part in processes including motility and differentiation and also it has a role in the formation of stress fibers. TAGLN gene has been found to be downregulated by promoter hypermethylation in breast and colon tissues and in these tissues, it acted as a tumor suppressor gene. However, in a study on nerve sheath tumors, TAGLN expression was found as upregulated via hypomethylation and in nerve sheath tumors, it acted as a proto-oncogene. To the best of our knowledge, the functional effect of TAGLN gene expression has not been studied in detail in breast carcinoma cell lines. The aim of this study was therefore to identify the functional role of TAGLN in breast cancer development. Hence, TAGLN gene expression was silenced or overexpressed and functional analysis was performed in selected breast cancer cell lines. Breast cancer cell lines were chosen according to their subtypes such as basal, HER2 positive or triple negative; their migratory properties; epithelial or mesenchymal characteristics and the expression level of TAGLN. Therefore, triple negative and mesenchymal MDA-MB-157 cells and MDA-MB-231 cells that express TAGLN at medium level were selected to silence TAGLN expression. The same cell lines and HER2 positive and epithelial MDA-MB-361 cells which express TAGLN at very low level were selected to overexpress TAGLN gene. Immunofluorescence and western blot analysis showed that in MDA-MB-157 and MDA-MB-231 cells mesenchymal marker Vimentin expression is correlated with TAGLN gene expression level. On the contrary, a reverse relationship exists in MDA-MB-361 cells where E-Cadherin expression increased and Vimentin expression decreased in TAGLN overexpressing MDA-MB-361 cells. TAGLN silencing in MDA-MB-157 cells increased the cell spreading potential and viability capacity while TAGLN upregulated cells did not show any significant change. TAGLN silencing in MDA-MB-231 cells decreased the cell spreading potential and cell viability of the cells, TAGLN overexpression in MDA-MB-231 cells increased these properties of cells. MDA-MB-361 cells behaved differently with TAGLN overexpression; cells were able to form less colonies and cell viability decreased in TAGLN overexpressing cells. TAGLN gene silencing affected the cell cycles of MDA-MB-157 and MDA-MB-231 cells but TAGLN overexpression had no effect on the cell cycle. In conclusion, TAGLN expression has an effect on Epithelial to Mesenchymal Transition (EMT) by altering the expression of established EMT markers E-Cadherin and Vimentin and its effect is based on the original morphology of the respective cell lines. In this study, the effect of TAGLN expression on cell proliferation was also studied and TAGLN seems to be acting as a tumor suppressor in MDA-MB-157 and MDA-MB-361 cells and as an oncogene in MDA-MB-231 cells. This might be due to the invasive character of MDA-MB-231 cells and the underlying mechanisms for this outcome should be investigated. Also, in vivo experiments can be performed to see whether changes in the expression of TAGLN gene has a role in tumor formation or metastasis capacity of cells.
Open Access
qPCR validation of in vivo diagnostic importance and regulation by estrogen for CHRNA5 isoform expression in breast cancer
(2014) Özdemir, Emine Sıla
Breast cancer has multiple molecular subtypes; normal-like, basal-like, luminal A, luminal B and HER2 positive depending on receptor status of tumor cells. Cancer therapy is tailored according to the type of cancer; hence finding new diagnostic markers is important to decide on the best treatment approach. Cholinergic nicotinic receptor alpha 5 (CHRNA5) is one of the subunits of nicotinic acetylcholine receptors with significant roles in addiction and cancer. In the present study, CHRNA5 has been validated as an estrogen and/or Estrogen receptor (ER) modulated nicotinic acetylcholine receptor by qPCR in in vitro and in vivo in breast cancer samples. CHRNA5 isoform expression was measured using in vitro cell culture studies in which ER- and ER+ cell lines treated with different doses of estradiol (E2); MCF7 cell line was exposed to long-term E2 depletion, in another experiment it was treated with tamoxifen (4-OHT), an ER antagonist, and with or without E2. We found that all CHRNA5 isoforms exhibited increased expression in response to E2 dose-dependently in the ER+ MCF7 cell line while in the ER- MDAMB-231 cell line CHRNA5 isoform expression response was variable in direction and magnitude. CHRNA5 isoform expression in general steadily decreased in ER+ cell line MCF7 after 4-OHT treatment. After six months of E2 depletion, ER+ MCF7 cell line had increased CHRNA5_v3 isoform and ESR1 (ER gene) mRNA expression. In vivo, a human breast cancer cDNA panel was scanned with specially designed primers with qPCR using a custom-written GUI in MATLAB. It was found that CHRNA5, showing a statistically significant difference between normal and tumor cDNA, was a good candidate gene in diagnosis of breast cancer. CHRNA_v3 was able to distinguish between ER+ vs ER- breast tumor samples. We also addressed whether CHRNA5 isoforms exhibited differences in distinguishing tumor stage, and HER2 status. Our findings showed that expression of CHRNA5 isoforms were correlated with each other and regulated by E2 in breast cancer depending on ER receptor status.
Open Access
A simulation optimization for breast cancer screening in Turkey
(2014) Keyf, Dilek
Breast cancer is the most common cancer type among women in the world. 6.3 million women were diagnosed with breast cancer between 2007 - 2012 and 25% of cancers in women are breast cancer. Early diagnosis and early detection has an important role in survival from breast cancer. Mammographic screening is proved to be the only screening method that can reduce breast cancer mortality. Even though mammographic screening has this significant benefit, it is expensive and it can decrease life quality and it can generate false positive results. As a consequence, recommending an effective and costefficient mammographic screening policy in terms of starting and ending ages and screening frequencies has high importance. This study aims to optimize Ada’s Breast Cancer Simulation Model using Simulated Annealing. This model was run for Turkish women born in 1980 during their lifetime. The purpose of this study is to obtain an optimal or near optimal policy in terms of life years gained and cost for Turkish women. This study also aims to demonstrate the outcomes in terms of effectiveness and cost when different combinations of policy variables are used.
Open Access
Unliganded estrogen receptor-x activates transcription of the mammary gland Na+/I-symporter gene
(Elsevier, 2006) Alotaibi, H.; Yaman, E. C.; Demirpence, E.; Tazebay, U. H.
The function of sodium iodide symporter (Na+/I symporter, or NIS) in mammary epithelial cells is essential for the accumulation of I in milk; the newborn’s first source of I for thyroid hormone synthesis. Furthermore, increased mammary gland NIS expression has previously been shown in human breast cancer. Several hormones and factors including all-trans-retinoic acid (tRA) regulate the expression of NIS. In this study, using breast cancer cell lines, we established that tRA-responsive NIS expression is confined to estrogen receptor- a (ERa) positive cells and we investigated the role of ERa in the regulation of NIS expression. We showed that the suppression of endogenous ERa by RNA interference downregulates NIS expression in ERa positive mammary cells. Besides, in an ERa negative cell line, reintroduction of ERa resulted in the expression of NIS in a ligand-independent manner. We also identified a novel estrogen-responsive element in the promoter region of NIS that specifically binds ERa and mediates ERa-dependent activation of transcription. Our results indicate that unliganded ERa (apo-ERa) contributes to the regulation of NIS gene expression. 2006 Elsevier Inc. All rights reserved.