Caco-2 Hücre Hattı In Vitro Epitelyal Mezenkimal Geçiş Modelinde Glutatyon S-Transferaz-π Düzeyleri

Year 2019, Volume: 39 Issue: 1, 10 - 16, 15.01.2019

Seyhan Türk İpek Baysal Gülberk Uçar

Abstract

Modern tıptaki gelişmelere rağmen, adjuvant/neoadjuvan tedavilere direnç geli- şimi tedaviyi karmaşıklaştırabilecek en önemli sorunlardan biridir. Son yıllarda, kanser hücrelerinin epitelyal mezenkimal geçiş (EMT) aktivasyonunun, metastaz eğilimi ve kemoterapiye direncinde önemli bir rol oynadığını göstermiştir. Bu ça- lışmada CaCo-2 hücre hattında in vitro EMT modeli oluşturularak direnç meka- nizmalarının belirlenmesi hedeflenmiştir. Bu hücreler dönüşüm yetenekleri açı- sından iyi bilinen hücre hatlarıdır ve farklılaşma potansiyelleri nedeniyle yapısal ve fonksiyonel çalışmalar için önemli araçlardır. Farklılaşma 0., 15. ve 30. gün- lerde takip edilmiştir ve model validasyonu için epitelyal ve mezenkimal belir- teçler (E-kadherin ve Vimentin) kullanılmıştır. EMT’nin iki formunda da direnç mekanizmalarını aydınlatmak üzere ELISA ile Glutatyon- S-Transferaz-π (GST-π) düzeyleri analiz edilmiştir. Mezen- kimal formda beklendiği şekilde GST-π düzeyinin önemli derecede arttığı gösterilmiştir. Ayrıca mezenkimal formda direnci ortadan kaldırmaya yönelik GST-π inhibitörü hi- perisin kullanılmıştır. Hiperisinin farklı konsantrasyonları (2μM ve 20μM) ile muamele sonucu GST-π düzeylerin- de anlamlı derecede azalma tespit edilmiştir. Bu sonuçlar, adjuvan terapiye ek olarak direncin ortadan kaldırılmasını sağlayacak mekanizmaların aktifleştirilmesi ile kullanılan tedavinin etkinliğinin arttırabileceğini ortaya koymuştur.

Keywords

Kolorektal kanser, Epitelyal Mezenkimal Geçiş, Glutatyon S-Transferaz-π

Glutathione S-Transferase-π Levels of In Vitro Generated Epithelial Mesenchymal Transition Model in Caco-2 Cell Line

Abstract

Despite the developments in the modern medicine, the resistancy to adjuvant/ne- oadjuvant therapies is one of the most important problem that may complicate the treatment. In recent years, studies have shown that activation of epithelial-mes- enchymal transition (EMT) may plays an important role in cancer cells tendency to metastasis and resistance to chemotherapy. In this work, in vitro EMT model was generated with Caco-2 cell line to determine the mechanisms of response and resistancy. This cells are well-known for their ability to transform and are important tools for structural and functional studies due to their differentiation potential. Differentiation was monitored on days 0th, 15th and 30th and epithe- lial and mesenchymal markers (E-cadherin and Vimentin) were used for model validation with qRT-PCR. Glutathione S-Transferase-π (GST-π) levels were de- termined to elucidate the mechanisms of resistancy in both forms of EMT with ELISA. As expected, it was shown that GST-π levels increased significantly in mesenchymal form. Also, GST-π inhibitor hypericin was used to eliminate resist- ancy in mesenchymal form. Significant decrease in GST-π levels was determined with the different concentrations of hypericin treatment (2μM and 20μM). These results showed that, activation of mechanisms to eliminate resistancy in addition to adjuvant therapy may increase the effectiveness of treatment.

Keywords

Colorectal cancer, Epithelial Mesenchymal Transition, Glutathione S-Transferase-π

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