Ankara Üniversitesi Tıp Fakültesi Mecmuası

0365-8104 1307-5608

Ankara University

Medical Biochemistry and Metabolomics (Other)

Tıbbi Biyokimya ve Metabolomik (Diğer)

İmatinib’e Duyarlı ve Dirençli K562 Hücrelerinde Kalneksinin Protein Ekspresyonunun Araștırılması

https://orcid.org/0000-0002-2482-0359

Karabay

Arzu Zeynep

ANKARA ÜNİVERSİTESİ, ECZACILIK FAKÜLTESİ

10 16 2018

71 1 9 15 02 21 2018 04 13 2018

1947

Ankara Üniversitesi Tıp Fakültesi Mecmuası

Aim: The aim of this study is to determine the expression levels of endoplasmic reticulum (ER) chaperon protein calnexin in K562 cell line which is the cell line model of chronic myeloid leukemia characterised with the transformation of numerous blood stem cells into abnormal granulocytes. Calnexin levels in K562 cells and its resistant counterpart (K562R) to Imatinib, the first line therapy option for CML were examined. In addition, effects of first and second generation tyrosine kinase inhibitors, Imatinib and Nilotinib on calnexin levels and cell viability parameters in K562S and K562R cells were also determined.Material and Method: In this study, K562S (sensitive to Imatinib) cell line, which is the cell model of the blast phase of CML, and K562R cell lines, which are resistant to imatinib (5uM), were used. Cells were cultured in parallel. K562S cells treated with or without 0.5 uM imatinib and 0.05 uM Nilotinib and K562R cells treated with or without 20 uM Imatinib and 0.1 uM Nilotinib were collected 48 hours after treatment and cell viability, apoptosis, cell morphology and calnexin protein expression weredetermined with MTT assay, flow cytometry, light microscopy and western blot respectively.Results and conclusion: In this study, it was shown that the total cellular expression of calnexin, a chaperone that regulates the folding of proteins in the endoplasmic reticulum, did not show any significant difference between the K562S and K562R cells, On the other hand, since Imatinib and Nilotinib significantly decreased calnexin protein expression in K562S cells and did not show any significant effect on K562R cells, this may indicate that the ER stress pathway and possibly calnexin are involved in the action mechanisms of these drugs. Therefore, even if no significant difference in calnexin levels was found between K562R and K562S cells, there may be differences in the ER stress pathway between the sensitive and resistant cells. For further studies, it is suggested to investigate the distribution of calnexin in different intracellular compartments as well as its modulation with drugs.

Amaç: Bu çalıșmada, çok sayıda kan kök hücresinin, sağlıklı beyaz kan hücrelerine dönüșemeyen anormal granülositlere dönüștüğü bir hastalık olan kronik myeloid löseminin hücre serisi modeli olan K562 hücrelerinde (K562S) ve KML tedavisinde ilk seçenek olarak uygulanan İmatinib’e karșı direnç geliștirmiș K562 hücrelerinde (K562R), endoplazmik retikulum (ER) șaperon proteini kalneksinin protein düzeylerinin belirlenmesi ve KML tedavisinde kullanılan birinci ve ikinci jenerasyon tirozin kinaz inhibitörleri İmatinib ve Nilotinib’in kalneksin protein düzeyi üzerindeki etkilerinin ve hücre canlılığı parametrelerine olan etkilerinin ortaya çıkarılması amaçlanmıștır.Gereç ve Yöntem: Bu çalıșmada, KML’nin blast fazının hücre modeli olan K562S (İmatinib’e duyarlı) hücre serisi ve İmatinib’e (5uM) karșı dirençli hale getirilmiș K562R hücre serileri kullanılmıștır. Hücreler, paralel olarak kültür edilmișlerdir. 0.5 uM İmatinib ve 0.05 uM Nilotinib ile tedavi edilen ve edilmeyen K562S ve 20 uM İmatinib ve 0.1 uM Nilotinib K562R ile tedavi edilen ve edilmeyen K562R hücreleri tedaviden 48 saat sonra toplanarak MTT testi ile hücre canlılığı, akım sitometri ile apoptoz tayini, ıșık mikroskobisi ile hücre morfolojisi ve total hücre ekstraktlarında western blot ile kalneksin protein ekspresyonu belirlenmiștir.Bulgular ve sonuç: Bu çalıșmada, endoplazmik retikulumda proteinlerin katlanmasını düzenleyen bir șaperon olan kalneksinin total hücre ekspresyonunun K562S ve K562R hücreleri arasında anlamlı değișiklik göstermediği belirlenmiștir, ancak, İmatinib ve Nilotinib’in duyarlı hücrelerde bu proteinin düzeyini anlamlı olarak düșürmesi ve dirençli hücrelerde anlamlı etki göstermemesi, bu ilaçların etki mekanizması arasında ER stres yolağı ve olasılıkla kalneksin proteininin yer aldığına ișaret edebilir ve duyarlı ve dirençli hücreler arasında her ne kadar kalneksin düzeyinde fark bulunmasa da ER stres yolağında farklılıklar bulunabileceğinin bir göstergesi olabilir. İleri çalıșmalar için, ilaca duyarlı ve dirençli hücrelerde kalneksin ve ilișkili bileșenlerin farklı hücre içi kompartmanlardaki değișiminin ve ilaçlarla modülasyonunun daha geniș olarak araștırılması önerilmektedir.

Calnexin K562 leukemia drug resistance

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