Gen metilasyonu inhibitörü desitabin'in meme kanseri tedavisindeki yerinin in vitro araştırılması
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Date
2010
Authors
Arı, Ferda
Journal Title
Journal ISSN
Volume Title
Publisher
Uludağ Üniversitesi
Abstract
Demetile edici bir ajan olan desitabin'in epigenetik olarak susturulmuş genlerin tekrar aktifleştirilmesini sağlayarak apoptozisi artırdığı gösterilmiştir. Bu nedenle, desitabinin standart kemoterapiyle sinerjistik etkileşimi yeni bir tedavi modalitesi olabilir. Bu çalışmada, klasik FEC tedavisi (5-florourasil+Epirubisin+4-Hidroksisiklofosfamid) ile desitabin kombinasyonunun meme kanserinde yeni bir tedavi yaklaşımı olup olamayacağı araştırılmıştır.Desitabin ve FEC ile kombinasyonu, östrojen reseptörü negatif (MDA-MB-231) ve pozitif (MCF-7) meme kanseri hücre soyları üzerinde test edildi. uPA, PAI-1 (metastaz genleri), DAPK, TMS1 (apoptozis-indükleyici genler), MGMT (DNA onarım geni) genleri ve LINE-1 (genel metilasyon belirteci) dizisinde metilasyon seviyelerindeki değişiklikler metilasyon spesifik ?real-time PCR (methylight)? ile analiz edildi. Tüm tedavilerin sitotoksik etkileri MTT ve ATP canlılık testleri, apoptozis indükleyici etki ise M30-antijen testi ile belirlendi.Desitabinin, MCF-7 hücrelerinde apoptozisi arttırarak büyüme baskılayıcı etki göstermesine rağmen, MDA-MB-231 hücrelerini etkilemediği gözlenmiştir. Bununla birlikte kombinasyon tedavisinden (%100 TDC FEC+10µM desitabin) sonra her iki hücre soyunda LINE-1 metilasyon seviyesinde belirlenen anlamlı azalma sonucu genomdaki genel metilasyon seviyesinin azaldığı belirlendi (p<0.05). Her iki hücre soyunda desitabin (10µM) ve kombinasyon tedavisinden sonra DAPK gen promotör metilasyon seviyesinde azalma belirlendi. Ayrıca MDA-MB-231 hücrelerinde kombinasyon tedavisi sonucunda TMS1 gen promotör metilasyonunda anlamlı azalmalar gözlendi (p<0.05). Bu sonuçlara rağmen bu tedavilerin sadece MCF-7 hücrelerinde apoptozisi indüklediği M30 antijen testiyle belirlendi. Ayrıca MCF-7 hücrelerinde desitabin ve kombinasyon tedavisi sonucunda uPA ve PAI-1 gen promotör metilasyon seviyelerinde anlamlı azalma belirlendi. Bu da bu hücrelerde metastaz yapabilme yeteneğinin değişebileceğini göstermektedir.Sonuç olarak, standart kemoterapi ile demetile edici ajan kombinasyonlarının apoptozis ile ilişkili genlerin aktifleşmesini sağlayarak meme kanseri tedavisinin etkinliğini arttırabileceği düşünülmüştür. Fakat diğer taraftan metastaz genlerindeki aktifleşme, bu tedavilerin tümör tipine göre ve dikkatle uygulanması gerektiğini göstermektedir.
Decitabine as a demethylating agent may act synergistically with standard chemotherapy regimens to restore apoptosis by upregulation of epigenetically silenced genes. In this study, we investigated whether or not the combination of classical FEC treatment (5-FU+Epirubicine+4-Hydroxyclophosphamide) with decitabine might be a novel therapy option in breast cancer.The effect of decitabine and its combination with FEC has been tested on estrogen receptor negative (MDA-MB-231) and estrogen receptor positive (MCF-7) breast cancer cell lines. The changes in the methylation status of uPA, PAI-1 (metastasis promoter), DAPK, TMS1 (apoptosis-inducer) MGMT (DNA repair) genes and LINE-1 (global methylation marker) have been analyzed by methylation specific realtime PCR (methylight) after the application of decitabine, FEC and their combination treatment. Anti-growth effects of treatments were assayed by the MTT assay and the ATP assay; induction of apoptosis by the M30 antigen assay.It is shown that decitabine had anti-growth effect only on MCF-7 cells by induction of apoptosis but had no effect on MDA-MB-231 cells. However, LINE-1 methylation status significantly decreased after combination treatment (%100 TDC FEC+10µM decitabine) (p<0.05) which show that whole genom methylation decreased in both cell lines. The methylation ratio of DAPK promoter was significantly decreased after decitabine (10µM) and combination regimens in both cell line. In MDA-MB-231 cells, methylation of the TMS1 gene promoter was significantly reduced by combination treatment (p<0.05). Although these results, it?s found that apoptosis only induced in MCF-7 cell lines by M30 antigen test. In addition methylation of the uPA and PAI-1 promoter was significantly reduced by treatment with decitabine or the combination treatment in MCF-7 (p<0.05). This shows that the ability of metastasis may change in these cells.In conclusion, combination of standard chemotherapy with the combination of demethylating agents may influence therapy outcome by upregulation of apoptosis relevant genes in breast cancer therapy. But reactivation of metastasis genes shows that these therapies have to be used carefully according to tumor types.
Decitabine as a demethylating agent may act synergistically with standard chemotherapy regimens to restore apoptosis by upregulation of epigenetically silenced genes. In this study, we investigated whether or not the combination of classical FEC treatment (5-FU+Epirubicine+4-Hydroxyclophosphamide) with decitabine might be a novel therapy option in breast cancer.The effect of decitabine and its combination with FEC has been tested on estrogen receptor negative (MDA-MB-231) and estrogen receptor positive (MCF-7) breast cancer cell lines. The changes in the methylation status of uPA, PAI-1 (metastasis promoter), DAPK, TMS1 (apoptosis-inducer) MGMT (DNA repair) genes and LINE-1 (global methylation marker) have been analyzed by methylation specific realtime PCR (methylight) after the application of decitabine, FEC and their combination treatment. Anti-growth effects of treatments were assayed by the MTT assay and the ATP assay; induction of apoptosis by the M30 antigen assay.It is shown that decitabine had anti-growth effect only on MCF-7 cells by induction of apoptosis but had no effect on MDA-MB-231 cells. However, LINE-1 methylation status significantly decreased after combination treatment (%100 TDC FEC+10µM decitabine) (p<0.05) which show that whole genom methylation decreased in both cell lines. The methylation ratio of DAPK promoter was significantly decreased after decitabine (10µM) and combination regimens in both cell line. In MDA-MB-231 cells, methylation of the TMS1 gene promoter was significantly reduced by combination treatment (p<0.05). Although these results, it?s found that apoptosis only induced in MCF-7 cell lines by M30 antigen test. In addition methylation of the uPA and PAI-1 promoter was significantly reduced by treatment with decitabine or the combination treatment in MCF-7 (p<0.05). This shows that the ability of metastasis may change in these cells.In conclusion, combination of standard chemotherapy with the combination of demethylating agents may influence therapy outcome by upregulation of apoptosis relevant genes in breast cancer therapy. But reactivation of metastasis genes shows that these therapies have to be used carefully according to tumor types.
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Keywords
Metilasyon, Meme kanseri, Apoptozis, Kemoterapi, LINE-1, DAPK, TMS1, uPA, PAI-1, Methylation, Breast cancer, Apoptosis, Chemotherapy
Citation
Arı, F. (2010). Gen metilasyonu inhibitörü desitabin'in meme kanseri tedavisindeki yerinin in vitro araştırılması. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.