HEK293 Hücreleri Üzerine Endoplazmik Retikulum Stresi Uyaranı Tunikamisinin Etkisi

Abstract

DOI: 10.26650/experimed.2018. 462982

Amaç: Endoplazmik Retikulum (ER), protein katlanması ve işlenmesi yanı sıra Ca+2 ve glikojen deposu ve hücre membran lipidlerinin biyogenezi gibi fonksiyonlara sahiptir. Hipoksi, viral enfeksiyonlar, yanlış protein katlanmaları, protein birikimleri ve bazı kimyasallar hücrede, ER stresine sebep olarak katlanmamış protein yanıtı (UPR) sistemlerini devreye sokmaktadır. Bu kimyasallardan biri olan Tunikamisin, asparajin-bağlı glikoproteinlerin sentezinin ilk adımında görev alan GPT (GlcNAc fosfotransferaz) enzimini inhibe ederek hücrede ER stresine sebep olan bir antibiyotiktir. Bu çalışmada, ER stresini in vitro araştırmak için en uygun deney şartlarının belirlenmesi amaçlanmıştır.

Gereç ve Yöntem: HEK293 hücre hattı uygun kültür koşullarında çoğaltılarak 5 farklı dozda (0,5-1-2-5 ve 10 µg/mL), 12 saatlik Tunikamisin uygulanmıştır. 1-2-4-8 ve 12 saatlik sürelerle ışık mikroskobunda morfolojik değerlendirilmeleri yapılmıştır. Normal HEK293 hücreleri ile 0,5 ve 1 µg/mL Tunikamisin uygulanan hücrelerden total RNA izolasyonu ve cDNA sentezi yapılmıştır. Tunikamisin uygulamasının ER stres açısından değerlendirilmesi için UPR sisteminden seçilen CHOP, BiP, GADD34, ATF4, EDEM1, ASK1, XBP1 (spliced, unspliced ve total), TRAF2, HERP, PDIA4 genlerinin qRT-PCR yöntemi ile gen ekspresyonları normal hücrelere göre kıyaslanmıştır.

Bulgular: Tunikamisin uygulamasından 1-2-4-8 ve 12 saatlik ışık mikroskobunda HEK293 hücrelerinin morfolojilerinin sadece 0,5 ve 1 µg/mL konsantrasyonlarda korunduğu gözlenmiştir. Diğer dozlarda ise daha ilk saatten hücrelerin morfolojik yapılarının bozulduğu gözlenmiştir. qRT-PCR yöntemi ile UPR-ilişkili genlerden BiP, ASK1, PDIA4, s-xBP1, us-XBP1, t-XBP1, HERP ve CHOP rölatif kantitasyon değerlerinde Tunikamisin uygulanmış HEK293 hücrelerin normal hücrelere göre anlamlı bir artış saptanmıştır. ASK1 ile aynı yolaktan olmasına rağmen TRAF2 geni rölatif kantitasyon değerinde azalma gözlenmiştir.

Sonuç: HEK293 hücrelerinde ER stresinin oluşturulabilmesinde 0.5 ve 1 µg/mL’lik  Tunikamisin uygulaması uygun bulunmuştur. Daha yüksek dozların HEK293 hücrelerini apoptoza yönlendirdiği düşünülmektedir. 

Keywords

• Endoplazmik retikulum stresi,tunikamisin,HEK293 hücreleri,katlanmamış protein yanıtı

The Effect of Endoplasmic Reticulum Stress Activator-Tunicamycin on HEK293 Cells

Abstract

DOI: 10.26650/experimed.2018. 462982

Objective: Among the functions of the Endoplasmic Reticulum (ER) are many different reactions that include the regulation of protein folding and modifications in the lumen, as well as the use of Ca+2 and glycogen storage, the biogenesis of cell membrane lipids. ER homeostasis becomes unbalanced and is recognized as ER stress by the cell. It triggers Unfolded Protein Responce (UPR) systems. Hypoxia, viral infections, unfolded protein accumulation, and some chemicals cause ER stress. Among the chemicals, tunicamycin is an antibiotic known to induce ER stress in the cell by inhibiting the enzyme GlcNAc phosphotransferase (GPT), which is involved in the first step of synthesis of asparagine-linked glycoproteins. The aim of this study is to determine the optimal experimental conditions for investigating ER stress in vitro.

Material and Method: The HEK293 cells were cultured at the appropriate culture conditions and treated with five different doses (0.5, 1, 2, 5, and 10 μg/mL) of tunicamycin for 12 hours. The morphology of the cells was evaluated with a light microscope for 1, 2, 4, 8, and 12 hours. The total RNA isolation and cDNA synthesis were performed from normal and treated HEK293 cells with 0.5 and 1 μg/mL of tunicamycin. Expressions of CHOP, BiP, GADD34, ATF4, EDEM1, ASK1, XBP1 (spliced, unspliced and total), TRAF2, HERP, and PDIA4 selected from the UPR system were compared to normal cells for the evaluation of ER stress via tunicamycin application by qRT-PCR

Results: After 1, 2, 4, 8, and 12 hour observations using light microscopy after treatment of tunicamycin, the morphology of HEK293 cells were preserved at concentrations of only 0.5 and 1 μg/mL. In all other doses, the morphological structures of the cells were observed to be impaired within the first hour. Relative quantitation values of BiP, ASK1, PDIA4, s-xBP1, us-XBP1, t-XBP1, HERP, and CHOP were increased significantly on HEK293 cells treated with tunicamycin as commpared to normal cells. Despite ASK1 being in the same pathway, a decrease of TRAF2 relative quantitation was observed.

Conclusion: It was found that the appropriate doses to induce ER stress with tunicamycin utilizing an HEK293 cell culture were 0.5 and 1 μg/mL. Higher doses are thought to lead to cell apoptosis.

Keywords

• Endoplasmic reticulum stress,tunicamycin,HEK293 cells,unfolded protein response

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