Establishment of drug-resistant cell lines in non-small cell lung cancer
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Tarih
2019
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Adana Alparslan Türkeş Bilim ve Teknoloji Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Küçük hücreli dışı akciğer kanseri dünyada kansere bağlı ölümlerin önemli sebeplerinden birisidir. Bunun temel nedenlerinden birisi tedavi sırasında ya da hasta tümörlerinin genetik yapısına bağlı olarak terapi ilaçlarına karşı gelişen kimyasal dirençtir. Her ne kadar ilaç direncini aşmak için çeşitli moleküler ajanlar geliştirilmiş olsa da hastalığın dirence bağlı olarak tekrarlaması, günümüzde halen etkili tedavi için önemli bir engel oluşturmaktadır. Bu nedenle, kemorezistans mekanizmalarını anlamak, hastalığın başarılı bir şekilde tedavisi için gereklidir. Bu çalışmada ileride ilaç direnç mekanizmalarının aydınlatılması ve yeni direnç karşıtı ajanların tespiti için kullanılacak hücre hatlarının oluşturulması için in vitro da hasta kemoterapi koşulları taklit edilerek ilaç dirençli hücre hatları oluşturmak amaçlanmıştır. Bu amaç doğrultusunda Epitelyal Büyüme Faktörü Reseptörü'ne hedeflemeli bir ilaç olan Erlotinib'e dirençli hücre alt hatları, A549 küçük hücreli dışı akciğer kanseri hattının Erlotinib'in artan konsantrasyonlarına maruz bırakılmasıyla oluşturulmuştur. Direnç gelişimi sırasında oluşabilecek morfolojik ve moleküler değişimlerin karşılaştırılması amacıyla anaç A549 alt hattının parelel kültürü sürdürülmüştür. Her iki hücre alt hattında meydana gelen canlılık değişimleri hücre canlılık testi ile gösterilmiştir. Her iki hücre grubu, metastatik gelişmelerdeki farklılıkların gözlenmesi için yara kapama testine maruz bırakılmış, öte yandan tümörijenik kapasiteleri yumuşak agar koloni oluşum testi ile ölçülmüştür. Buna ek olarak, Erlotinib'in kimyasal direncin önemli bir özelliği olan epitelyal hücre tipinden ve mezenkimal tipe geçiş işaretleyicilerinin gen ifadeleri üzerindeki etkisi, kantitatif ters transkriptaz polimeraz zincir reaksiyon analizi ile belirlenmiştir. İlaç koşullarında artan hücre canlılığının yanı sıra, dirençli alt hattın anaç hatta göre daha tümörijenik ve daha yüksek göç kapasitesine sahip olduğu gözlenmiştir. Son olarak Erlotinib dirençli alt hatta mezenkimal hücre tipi işaretleyicisi N-Cadherin ifadesinin önemli derecede arttığı görülmüştür.
Non-small cell lung cancer is one of the leading causes of cancer-related deaths worldwide. One of the major reasons for this is the development of resistance against theurapatic drugs during treatment or due to genetic make up of tumors in patients. Although several molecular agents have been developed to overcome drug resistance, risk of recurrence is still being a challenge for efficient therapy at the present time. Thus, understanding chemoresistance mechanisms is crucial for successful treatment of this disease. In this study, it was aimed to establish drug-resistant sub-cell lines by mimicking treatment conditions of non-small cell lung cancer patients in vitro to study drug resistance mechanisms and to generate cell lines for future purposes to discover novel anti-chemoresistance agents. In accordance with this purpose, human non-small cell lung cancer cell line A549 was exposed to increasing concentrations of Epithelial Growth Factor Receptor targeting drug Erlotinib to create an Erlotinib resistant subline. To compare possible morphological and molecular changes during resistance development, a parental A549 subline was maintained in a parallel culture. The cell viability changes in both sublines were demonstrated by cell viability assays. Both groups of sublines were subjected to wound healing assay to monitor the differences in metastatic progression; on the other hand, tumorigenic capacities were measured by soft agar colony formation assays. Furthermore, the effects of Erlotinib on the gene expression changes of epithelial to mesenchymal transition markers, a key feature of chemoresistance, were detected by quantitative reverse transcription polymerase chain reaction analysis. It was found that besides increased cell viability under drug conditions, Erlotinib resistant subline was more tumorigenic with a greater migration capacity than the parental subline. Finally mesenchymal biomarker N-Cadherin gene expression was significantly increased in Erlotinib resistant subline.
Non-small cell lung cancer is one of the leading causes of cancer-related deaths worldwide. One of the major reasons for this is the development of resistance against theurapatic drugs during treatment or due to genetic make up of tumors in patients. Although several molecular agents have been developed to overcome drug resistance, risk of recurrence is still being a challenge for efficient therapy at the present time. Thus, understanding chemoresistance mechanisms is crucial for successful treatment of this disease. In this study, it was aimed to establish drug-resistant sub-cell lines by mimicking treatment conditions of non-small cell lung cancer patients in vitro to study drug resistance mechanisms and to generate cell lines for future purposes to discover novel anti-chemoresistance agents. In accordance with this purpose, human non-small cell lung cancer cell line A549 was exposed to increasing concentrations of Epithelial Growth Factor Receptor targeting drug Erlotinib to create an Erlotinib resistant subline. To compare possible morphological and molecular changes during resistance development, a parental A549 subline was maintained in a parallel culture. The cell viability changes in both sublines were demonstrated by cell viability assays. Both groups of sublines were subjected to wound healing assay to monitor the differences in metastatic progression; on the other hand, tumorigenic capacities were measured by soft agar colony formation assays. Furthermore, the effects of Erlotinib on the gene expression changes of epithelial to mesenchymal transition markers, a key feature of chemoresistance, were detected by quantitative reverse transcription polymerase chain reaction analysis. It was found that besides increased cell viability under drug conditions, Erlotinib resistant subline was more tumorigenic with a greater migration capacity than the parental subline. Finally mesenchymal biomarker N-Cadherin gene expression was significantly increased in Erlotinib resistant subline.
Açıklama
Fen Bilimleri Enstitüsü, Nanoteknoloji ve Mühendislik Bilimleri Ana Bilim Dalı
Anahtar Kelimeler
Moleküler Tıp, Molecular Medicine
