Endoplazmik retikulum stres belirteçlerinden ATF5 ve fosforile eIF2α düzeylerinin nöroblastoma hücrelerinde kainik asit muamelesi sonrası incelenmesi

Amaç: Bu çalışmada, amacımız, kainik asite bağlı eksitotoksisite ve endoplazmik retikulum (ER) stres arasındaki ilişkiyi iki majör endoplazmik retikulum stres markırı olan ATF5 ve fosforile olmuş eIF2a analiz ederek incelemektir.Gereç ve Yöntem: Neuroblastoma hücrelerini 1 mM kainik asit ile 24 saat muamele ettik. ATP ölçümü, kainik asit muamelesi yapılan hücrelerde ya da kontrol hücrelerinde bioluminesans bir yöntem ile yapılmıştır. Total protein, kainik asit muamelesi yapılmış ya da kontrol hücrelerinden izole edilmiş ve ATF5 ve fosforile olmuş eIF2 markırları western blot ile incelenmiştir.Bulgular: Kainik asit ile muamele edilmiş neuroblastoma hücrelerinde ATF5 ve fosforile olmuş eIF2a seviyelerinin kontrole göre değişmediğini ilk kez gösterdik. Kainik asit ile muamele edilmiş hücrelerde ATP seviyesinin düştüğünü gösterdik.Sonuç: 1 mM ve 24 saat süren kainik asit muamelesi, ATF5 ve fosforile olmuş eIF2 ile gösterilebilecek endoplazmik retikulum stresi yaratmak için yeterli olmayabilir. Süre ve konsantrasyon olarak arttırılmış kainik asit muamelesi ya da farklı markırlar gerekmektedir. Eksitotoksisiteye bağlı beyin hastalıklarında ER stres yolaklarını araştırmak, yeni tedavi yolları bulmak ve birden fazla hastalığı aynı anda engellemek adına önemli olacaktır.

Anahtar Kelimeler:

endoplasmic reticulum, stress, ATF5, eIF2a

The investigation of endoplasmic reticulum stress markers ATF5 and phosphorylated eIF2α after kainic acid treatment in neuroblastoma cells

Purpose: The aim of this study was to investigate the relationship between kainic acid induced excitotoxicity and endoplasmic resticulum (ER) stress by analyzing two major ER stress markers such as ATF5 and phosphorylated eIF2 in neuroblastoma cells. Materials and Methods: Neuroblastoma cells were treated with 1 mM kainic acid for 24 hours. ATP measurement was performed in kainic acid-treated and vehicle-treated neuroblastoma cells via ATP bioluminescence assay. Total protein was isolated from kainic acid-treated and control cells. Via western blotting, the expression levels of ATF5 and phosphorylated eIF2α were analyzed.Results: We showed for the first time that as a result of kainic acid treatment in neuroblastoma cells, the protein expression levels of ER stress markers ATF5 and phosphorylated eIF2 did not display any change when compared to control cells. We also showed that ATP levels were decreased in kainic acid-treated cells.Conclusion: This study may show that the level of stress that kainic acid causes at 1 mM for 24 hours in neuroblastoma cells was not adequate to lead to ER stress which is measurable by ATF5 and phosphorylated eIF2. Either an increased level of treatment of kainic acid via increased duration or concentration is necessary or different markers should be tried. The investigation of the ER stress pathways in the excitotoxicity-related brain diseases will pave the way for new therapies based on ER stress and combat more than one disease simultaneously.

Keywords:

endoplasmic reticulum, stress, ATF5,

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