PANC1 hücre hattında SsaI inhibitör uygulaması sonrası ST3Gal II, ST3Gal III, ST6Gal I ve ST6GalNac II enzim ifadelerinin real-time PCR ile analizi
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Dosyalar
Tarih
2021
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Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Memeli hücrelerinin yüzeyi glikokaliks adı verilen bir yapıyla örtülüdür ve bu yapıyı glikoproteinler, glikolipidler ve proteoglikanlar gibi birimler oluşturur. Hücre yüzeyindeki lipidlerin, proteinlerin veya organik moleküllerin glikan yapılarıyla birleşerek glikokonjugat yapılarını oluşturmalarına glikozilasyon adı verilir. Kanser araştırmalarında gözlenen önemli hücresel değişimlerden bir tanesi, glikokonjugatlardaki oligosakkaritlerin en uç monomerinde gözlenen miktar değişimidir. Bu en uç monomerler, 9 karbonlu glikan olan sialik asitlerdir. Glikozilasyon süreci sırasında oluşan bağların farklılıkları veya oluşan zincirlerin uzunluk farklılıkları glikokonjugat yapıları için, dolayısıyla da hücrelerdeki biyolojik ve fizyolojik süreçler için çeşitlilik sağlar. Kanser hücresinin yüzeyindeki değişikliğe uğramış sialik asitlerde; yani, sialillenmiş glikokonjugatlarda ciddi bir artış gözlenmektedir. Bu artışlar genellikle sialiltransferaz (ST) enzimlerindeki miktar değişiklikleri ile ilişkilidir. Bu sebeple, sialilasyon mekanizmaları ve artan sialiltransferaz ekspresyonların düşürülmesi için inhibitör arayışları hızlanmıştır. Bu çalışmalar kapsamında keşfedilen Soyasaponin I (Ssa I), ST3Gal enzim ailesinin inhibitörü olarak ortaya çıkmaktadır. Bu inhibitörün α2,6 bağlı sialik asitleri transfer eden ve ST3Gal ile aynı enzim ailesinden gelen ST6Gal ve ST6GalNAc enzim ailelerine de etki edip etmediği henüz bilinmemektedir. Sunulan tez çalışmasında, pankreas kanser hücrelerine Ssa I inhibitörü uygulaması gerçekleştirilerek tümör hücrelerinde ST enzimlerinden dört tanesinin (ST3Gal II, ST3Gal III, ST6Gal I ve ST6GalNac II) ifade değişimlerinin kontrol ve inhibitör uygulanmış hücreler arasında karşılaştırılması amaçlanmıştır. Böylece, bu dört enzimden hangisinin ifadesinde uygulama yapılmış ve yapılmamış hücreler arasına anlamlı fark olduğu tespit edilmesi hedeflenmiştir. Değişiklik tespit edilen Sialiltransferaz(lar)ın kanser teşhis ve tedavisi için hedef molekül olarak önerilmesi nihai hedeftir. Bu amaçla; kültüre alınan pankreas kanseri (PANC1) hücrelerine 0 mM, 50 mM, 100 mM ve 150 mM Ssa I inhibitörü uygulanmış ve 48 ve 72 saat sonrası hücreler kaldırılarak total RNA izolasyonu gerçekleştirilmiştir. Elde edilen total RNA örneklerinden cDNA sentezi yapılmış ve real time PCR analizi ile de ST3Gal II, ST3Gal III, ST6Gal I ve ST6GalNac II enzim ifadelerindeki zamana ve uygulama miktarına bağlı değişiklikler analiz edilmiştir. Bu analizler için actin geni housekeeping gen olarak kullanılmıştır. Her dört gen bölgesi için de, CT değerlerine baktığımızda enzim ifadesinde Ssa I uygulaması ile birlikte azalma görülmüş ve bu azalma Ssa I uygulama miktarı arttıkça daha fazla olmuştur. CT değerleri analiz edildiğinde, housekeeping gen olarak kullanılan Actin geninin de inhibitörden etkilenmiş olabileceğine dair bir bulgu elde edilmiştir. Genel olarak, sonuçlar üzerinden 150 mM uygulamanın hücrelere toksik etkisi olabileceğine dair bir çıkarım yapılmıştır. Her dört gen bölgesi için de kontrol hücrelere (0mM Ssa I) oranla 50, 100 ve 150 mM Ssa I inhibitörü uygulanmış hücrelerde enzim ifadesi azalmıştır. Her dört gen bölgesinde de enzim ifadesinde azalma gözlenmiştir. Pankreas kanserinde sialik asit artışında bu dört genin ifadesinin katkısı olabileceği yönünde ön sonuçlar elde edilmiştir. Uygulamanın, Ssa I için sitotoksisite testi yapılarak ve farklı bir housekeeping gen (ör: GAPDH) kullanılarak tekrarlanması önerilir.
The surface of mammalian cells is covered by a structure called the glycocalyx, which consists of units such as glycoproteins, glycolipids, and proteoglycans. The binding of lipids, proteins or organic molecules on the cell surface with glycan structures to form glycoconjugate structures is called glycosylation. One of the important cellular changes observed in cancer research is the amount of variation observed in the number of end monomer units of oligosaccharides within glycoconjugates. These monomers found in sugar chains are sialic acids, which are 9-carbon glycans. The differences in the bonds formed during the glycosylation process or the differences in the length of the chains formed provide diversity for the glycoconjugate structures and thus for the biological and physiological processes in the cells. In modified sialic acids on the surface of the cancer cell; that is, a significant increase in sialylated glycoconjugates is observed. These increases are generally associated with changes in the amount of sialyltransferase (ST) enzymes. For this reason, the search for inhibitors to reduce sialylation mechanisms and increased sialyltransferase expressions has accelerated. Soyasaponin I (Ssa I), discovered within the scope of these studies, emerges as an inhibitor of the ST3Gal enzyme family. It is not yet known whether this inhibitor also acts on the ST6Gal and ST6GalNAc enzyme families, which transfer α2,6-linked sialic acids and come from the same enzyme family as ST3Gal. In the presented thesis, it was aimed to compare the expression changes of four ST enzymes (ST3Gal II, ST3Gal III, ST6Gal I and ST6GalNac II) in tumor cells between control and inhibitor-treated cells by applying Ssa I inhibitor to pancreatic cancer cells. Thus, it is aimed to determine which of these four enzymes has a significant difference in the expression of cells with and without treatment. It is the ultimate goal to propose the modified Sialyltransferase(s) as a target molecule for cancer diagnosis and treatment. For this aim; the cultured pancreatic cancer (PANC1) cells were treated with 0 mM, 50 mM, 100 mM and 150 mM Ssa I inhibitors and after 48 and 72 hours, the cells were removed and total RNA isolation was performed. cDNA synthesis was made from the total RNA samples obtained, and changes in ST3Gal II, ST3Gal III, ST6Gal I and ST6GalNac II enzyme expressions depending on time and application amount were analyzed by real time PCR analysis. The actin gene was used as a housekeeping gene for these analyses. When we look at the CT values for all four gene regions, a decrease was observed in enzyme expression with Ssa I application, and this decrease was greater as the amount of Ssa I application increased. When the CT values were analyzed, it was found that the Actin gene, which is used as a housekeeping gene, may also be affected by the inhibitor. In general, it was concluded from the results that 150 mM administration may have a toxic effect on cells. Enzyme expression was decreased in cells treated with 50, 100 and 150 mM Ssa I inhibitor compared to control cells (0mM Ssa I) for all four gene regions. A decrease in enzyme expression was observed in all four gene regions. Preliminary results have been obtained that the expression of these four genes may contribute to the increase in sialic acid in pancreatic cancer. It is recommended to repeat the application by performing a cytotoxicity test for Ssa I and using a different housekeeping gene (eg GAPDH).
The surface of mammalian cells is covered by a structure called the glycocalyx, which consists of units such as glycoproteins, glycolipids, and proteoglycans. The binding of lipids, proteins or organic molecules on the cell surface with glycan structures to form glycoconjugate structures is called glycosylation. One of the important cellular changes observed in cancer research is the amount of variation observed in the number of end monomer units of oligosaccharides within glycoconjugates. These monomers found in sugar chains are sialic acids, which are 9-carbon glycans. The differences in the bonds formed during the glycosylation process or the differences in the length of the chains formed provide diversity for the glycoconjugate structures and thus for the biological and physiological processes in the cells. In modified sialic acids on the surface of the cancer cell; that is, a significant increase in sialylated glycoconjugates is observed. These increases are generally associated with changes in the amount of sialyltransferase (ST) enzymes. For this reason, the search for inhibitors to reduce sialylation mechanisms and increased sialyltransferase expressions has accelerated. Soyasaponin I (Ssa I), discovered within the scope of these studies, emerges as an inhibitor of the ST3Gal enzyme family. It is not yet known whether this inhibitor also acts on the ST6Gal and ST6GalNAc enzyme families, which transfer α2,6-linked sialic acids and come from the same enzyme family as ST3Gal. In the presented thesis, it was aimed to compare the expression changes of four ST enzymes (ST3Gal II, ST3Gal III, ST6Gal I and ST6GalNac II) in tumor cells between control and inhibitor-treated cells by applying Ssa I inhibitor to pancreatic cancer cells. Thus, it is aimed to determine which of these four enzymes has a significant difference in the expression of cells with and without treatment. It is the ultimate goal to propose the modified Sialyltransferase(s) as a target molecule for cancer diagnosis and treatment. For this aim; the cultured pancreatic cancer (PANC1) cells were treated with 0 mM, 50 mM, 100 mM and 150 mM Ssa I inhibitors and after 48 and 72 hours, the cells were removed and total RNA isolation was performed. cDNA synthesis was made from the total RNA samples obtained, and changes in ST3Gal II, ST3Gal III, ST6Gal I and ST6GalNac II enzyme expressions depending on time and application amount were analyzed by real time PCR analysis. The actin gene was used as a housekeeping gene for these analyses. When we look at the CT values for all four gene regions, a decrease was observed in enzyme expression with Ssa I application, and this decrease was greater as the amount of Ssa I application increased. When the CT values were analyzed, it was found that the Actin gene, which is used as a housekeeping gene, may also be affected by the inhibitor. In general, it was concluded from the results that 150 mM administration may have a toxic effect on cells. Enzyme expression was decreased in cells treated with 50, 100 and 150 mM Ssa I inhibitor compared to control cells (0mM Ssa I) for all four gene regions. A decrease in enzyme expression was observed in all four gene regions. Preliminary results have been obtained that the expression of these four genes may contribute to the increase in sialic acid in pancreatic cancer. It is recommended to repeat the application by performing a cytotoxicity test for Ssa I and using a different housekeeping gene (eg GAPDH).
Açıklama
Anahtar Kelimeler
Sialiltransferaz, Sialik Asit, Pankreas Kanseri, ST3GAL, Soyasaponin I, Sialyltransferase, Sialic Acid, Pancreatic Cancer, Soyasaponin I
