Browsing by Author "Napieralski, Rudolf"
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Item Changes in gene methylation following chemotherapy in breast cancer cell lines(Walter De Gruyter, 2013) Napieralski, Rudolf; Arı, Ferda; Ulukaya, Engin; Uludağ Üniversitesi/Fen-Edebiyat Fakültesi/Biyoloji Anabilim Dalı.; Uludağ Üniversitesi/Tıp Fakültesi/Biyokimya Anabilim Dalı.; 0000-0002-6729-7908; K-5792-2018; AAG-7012-2021; 24376085300; 6602927353Objective: Epigenetic modulation of gene expression by DNA promoter methylation may contribute to acquired resistance to chemotherapy in cancer cells. Decitabine (5-aza-2'-deoxycytidine), a demethylating agent, may act synergistically with standard chemotherapy regimens to activate epigenetically silenced genes. In the present in vitro study, it was investigated the effect of gene methylation level after treatment with decitabine and combination of decitabine with anthracycline-based therapeutics (5-fluorouracil plus epirubicine plus cyclophosphamide; FEC) on breast cancer cells (MCF-7 and MDA-MB-231). Methods: The effect of decitabine and its combination with FEC on different genes methylation level has been tested in MDA-MB-231 and MCF-7 human breast cancer cell lines. The effect of decitabine on the cell viability was assayed by MTT assay. Methylight real-time PCR and methylation specific PCR were carried out to determine the methylation status of certain genes: DAPK, TMS1, MGMT and the global methylation marker LINE-1. Results: The LINE-1 methylation status significantly decreased in both cell lines after treatment with the combination of decitabine with FEC. In MDA-MB-231 cells, methylation of the TMS1 and the MGMT gene promoter was significantly reduced by FEC plus decitabine while no effect was observed in MCF-7 cells. Conclusion: Anthracycline-based therapy regimens in combination with demethylating agents such as decitabine may affect chemotherapy outcome by modulation of apoptosis-relevant genes by methylation. More importantly, this modulation seems to be dependent on the cell type.Publication Epigenetic modulators combination with chemotherapy in breast cancer cells(Wiley, 2021-02-20) Arı, Ferda; Napieralski, Rudolf; Akgün, Oğuzhan; Magdolen, Viktor; Ulukaya, Engin; ARI, FERDA; Akgün, Oğuzhan; Bursa Uludağ Üniversitesi/Fen-Edebiyat Fakültesi/Biyoloji Bölümü; 0000-0002-6729-7908; 0000-0002-8410-1786; A-5608-2019; AAG-7012-2021Despite the concerning adverse effects on tumour development, epigenetic drugs are very promising in cancer treatment. The aim of this study was to compare the differential effects of standard chemotherapy regimens (FEC: 5-fluorouracil plus epirubicine plus cyclophosphamide) in combination with epigenetic modulators (decitabine, valproic acid): (a) on gene methylation levels of selected tumour biomarkers (LINE-1, uPA, PAI-1, DAPK); (b) their expression status (uPA and PAI-1); (c) differentiation status (5meC and H3K27me3). Furthermore, cell survival as well as changes concerning the invasion capacity were monitored in cell culture models of breast cancer (MCF-7, MDA-MB-231). A significant overall decrease of cell survival was observed in the FEC-containing combination therapies for both cell lines. Methylation results showed a general tendency towards increased demethylation of the uPA and PAI-1 gene promoters for the MCF-7 cells, as well as the proapoptotic DAPK gene in the treatment regimens for both cell lines. The uPA and PAI-1 antigen levels were mainly increased in the supernatant of FEC-only treated MDA-MB-231 cells. DAC-only treatment induced an increase of secreted uPA protein in MCF-7 cell culture, while most of the VPA-containing regimens also induced uPA and PAI-1 expression in MCF-7 cell fractions. Epigenetically active substances can also induce a re-differentiation in tumour cells, as shown by 5meC, H3K27me3 applying ICC.Significance of the study Epigenetic modulators especially in the highly undifferentiated and highly malignant MDA-MB-231 tumour cells significantly reduced tumour malignancy thus; further clinical studies applying specific combination therapies with epigenetic modulators may be warranted.Item Modulation of protein expression levels and DNA methylation status of breast cancer metastasis genes by anthracycline-based chemotherapy and the demethylating agent decitabine(Wiley, 2011-12) Napieralski, Rudolf; Colling, Christoph; Honert, Katja; Krueger, Achim; Schmitt, Manfred; Kiechle, Marion; Arı, Ferda; Ulukaya, Engin; Dere, Egemen; Uludağ Üniversitesi/Fen-Edebiyat Fakültesi/Biyoloji Anabilim Dalı.; Uludağ Üniversitesi/Tıp Fakültesi/Biyokimya Anabilim Dalı.; 0000-0002-6729-7908; AAG-7012-2021; AAH-5068-2021; K-5792-2018; 24376085300; 6602927353; 6603627015Epigenetic drugs are promising add-ons to cancer treatment; still, adverse effects concerning tumour promotion have been reported occasionally. In this in vitro study, we investigated the effect of combination treatment of decitabine with anthracycline-based chemotherapy [5-fluorouracil plus epirubicine plus cyclophosphamide (FEC)] on viability and metastatic activity of breast cancer cell lines, MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). The effect of decitabine and its combined treatment with FEC on viability of both cancer cell lines was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide and adenosine triphosphate (ATP) cell survival assays. DNA methylation specific real-time polymerase chain reaction (PCR) (Methylight (R)) was employed to document the methylation status of the metastasis-relevant urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-I (PAI-1) genes. Additionally, protein expression levels of uPA and PAI-1 were determined using enzyme-linked immunosorbent assays. Invasion capacity of cells was assayed using Matrigel (R) invasion assay. Decitabine lowered the viability of MCF-7 cells, although MDA-MB-231 cells were not affected. Decitabine did not augment FEC-mediated cytotoxicity in both cell lines. In MCF-7 cells, methylation of the uPA and PAI-1 gene promoter was significantly reduced by decitabine or decitabine plus FEC. Protein levels of uPA and PAI-1 were induced by all treatments. Decitabine significantly induced the invasion capacity of MCF-7 cells, whereas all of the drugs resulted in decreased invasion capacity of MDA-MB-231. Our results suggest differential effects of single-dose decitabine and its combination with FEC on the metastatic capacity and survival of breast cancer cell lines endowed with different metastatic behaviour.