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

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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