A549 Akciğer Kanseri Hücre Hattında TIGAR'ın Susturulması, NF-κB ve HO-1 Ekspresyonlarının Modülasyonu ile Apoptozis ve Otofaji İndükler

Year 2019, , 310 - 320, 01.03.2019

Can Ali Ağca , Osama Hamid Shareef

https://doi.org/10.21597/jist.446068

Abstract

TP53 kaynaklı
glikoliz ve apoptozis düzenleyici (TIGAR) protein, glikoz metabolizması
sırasında Fru-2, 6-P2 seviyelerini glukoz metabolizması sırasında kontrol eder
ve nikotinamid adenin dinükleotit fosfat (NADPH) düzeyini devam ettirerek hücre
içi anahtar bir antioksidan olan glutatyonun (GSH) geri dönüştürmesine yardımcı
olur. Bu çalışma, A549 hücre hattında TIGAR'ın susturulmasının altında yatan,
reaktif oksijen türleri (ROS) aracılı apoptotik ve otofajik mekanizmaları
araştırmak için tasarlanmıştır. siRNA-TIGAR'ın A549 akciğer kanseri hücreleri
üzerindeki etkisini saptamak için hücre canlılığı, koloni oluşumu, ROS ve NADPH
analizlerini gerçekleştirdik. Ek olarak, protein ve mRNA ekspresyon seviyeleri
sırası ile Western blot ve Real-time PCR yöntemleri ile belirlendi. TIGAR’ın
A549 hücre hattında susturulmasının ardından, çeşitli parametreler analiz
edildi ve TIGAR'ın down regülasyonunun hücre canlılığını inhibe ettiği ve
koloni oluşumunu azalttığı gösterildi. TIGAR’ın susturulmasının apoptozis ve
otofajiyi tetiklediğini ve bunu Nükleer faktör-kappa B (NF-κB) ve Hem
oksijenaz-1 (HO-1)’in indüksiyonun izlediği belirledik. Dahası, artmış ROS
düzeyi ve azalmış NADPH seviyeleri gözlemlendik. Bu çalışma, akciğer kanseri
hücrelerinde, NF-κB ve HO-1 ekspresyonları ile apoptozis ve otofajiyi arttırmak
için TIGAR susturulmasının kullanılmasını desteklemekte ve akciğer kanserinin
tedavisi için potansiyel bir hedef olarak TIGAR önermektedir.

Keywords

TIGAR, Otofaji, NF-κB, Akciğer kanseri, Apoptozis, HO-1

Knockdown of TIGAR Induces Apoptosis and Autophagy with Modulates NF-κB and HO-1 Expression in A549 Lung Cancer Cells

Abstract

The tp53-induced
glycolysis and apoptosis regulator (TIGAR) protein controls fructose-2, 6-
bisphosphate (Fru-2, 6-P2) levels during glucose metabolism and helps maintain
nicotinamide adenine dinucleotide phosphate (NADPH) levels to recycle
glutathione (GSH), a key intracellular antioxidant. The present study was
designed to investigate the apoptosis and autophagy mechanisms via reactive
oxygen species (ROS) that underlie TIGAR knockdown in the A549 cell line. To
detect the influence of siRNA-TIGAR on A549 lung cancer cells, we performed
cell viabilty, colony formation, ROS, and NADPH assays. In addition, Western
blotting and real-time polymerase chain reaction (PCR) assays were used to
measure protein and mRNA expression levels, respectively. After TIGAR knockdown
in A549 cell lines, various assay parameters were analyzed and showed that
down-regulation of TIGAR inhibited viability and decreased colony formation. We
also demonstrated that TIGAR knockdown induced apoptosis and autophagy,
followed by an induction of nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-κB) and heme oxygenase-1 (HO-1) expression. Furthermore,
increased ROS levels and decreased NADPH levels were observed. This study
supports our understanding of the possibility of employing TIGAR knockdown in
lung cancer cells to enhance apoptosis and autophagy with NF-κB and HO-1
expression and then suggest TIGAR as a potential target for the treatment of
lung cancer.

Keywords

TIGAR, NF-κB, Lung cancer, Apoptosis, Autophagy, HO-1

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