HEK293 Hücreleri Üzerine Endoplazmik Retikulum Stresi Uyaranı Tunikamisinin Etkisi

Year 2018, Volume: 8 Issue: 3, 71 - 78, 02.12.2018

Muhammed Abdulvahid Kalkan , Burçak Vural , Evrim Kömürcü Bayrak

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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