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

Öz

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.

Anahtar Kelimeler

• 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

Öz

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.

Anahtar Kelimeler

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

Kaynakça

1. Bansal S, Biswas G, Avadhani NG, 2014. Mitochondria-targeted heme oxygenase-1 induces oxidative stress and mitochondrial dysfunction in macrophages, kidney fibroblasts and in chronic alcohol hepatotoxicity. Redox biology, 2: 273-283.
2. Bensaad K, Cheung EC, Vousden KH, 2009. Modulation of intracellular ROS levels by TIGAR controls autophagy. The EMBO journal, 28: 3015-3026.
3. Bensaad K, Tsuruta A, Selak MA, Vidal MN, Nakano K, Bartrons R, Gottlieb E, Vousden KH, 2006. TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell, 126: 107-120.
4. Bindu S, Pal C, Dey S, Goyal M, Alam A, Iqbal MS, Dutta S, Sarkar S, Kumar R, Maity P , et al., 2011. Translocation of heme oxygenase-1 to mitochondria is a novel cytoprotective mechanism against non-steroidal anti-inflammatory drug-induced mitochondrial oxidative stress, apoptosis, and gastric mucosal injury. The Journal of biological chemistry, 286: 39387-39402.
5. Chen S, Li P, Li J, Wang Y, Du Y, Chen X, Zang W, Wang H, Chu H, Zhao G , et al., 2015. MiR-144 inhibits proliferation and induces apoptosis and autophagy in lung cancer cells by targeting TIGAR. Cellular physiology and biochemistry, 35: 997-1007.
6. Cheung EC, Lee P, Ceteci F, Nixon C, Blyth K, Sansom OJ, Vousden KH, 2016. Opposing effects of TIGAR- and RAC1-derived ROS on Wnt-driven proliferation in the mouse intestine. Genes & development, 30: 52-63.
7. Gazdar AF, Girard L, Lockwood WW, Lam WL, Minna JD, 2010. Lung cancer cell lines as tools for biomedical discovery and research. Journal of the National Cancer Institute, 102: 1310-1321.
8. Gloire G, Legrand-Poels S, Piette J, 2006. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochemical pharmacology, 72: 1493-1505.

9. Han Z, Varadharaj S, Giedt RJ, Zweier JL, Szeto HH, Alevriadou BR, 2009. Mitochondria-derived reactive oxygen species mediate heme oxygenase-1 expression in sheared endothelial cells. The Journal of pharmacology and experimental therapeutics, 329: 94-101.
10. Huang S, Yang Z, Ma Y, Yang Y, Wang S, 2017. miR-101 Enhances Cisplatin-Induced DNA Damage Through Decreasing Nicotinamide Adenine Dinucleotide Phosphate Levels by Directly Repressing Tp53-Induced Glycolysis and Apoptosis Regulator Expression in Prostate Cancer Cells. DNA and cell biology, 36: 303-310.
11. Huang T, Zhang P, Li W, Zhao T, Zhang Z, Chen S, Yang Y, Feng Y, Li F, Shirley Liu X , et al., 2017. G9A promotes tumor cell growth and invasion by silencing CASP1 in non-small-cell lung cancer cells. Cell death & disease, 8: e2726.
12. Lee P, Vousden KH, Cheung EC, 2014. TIGAR, TIGAR, burning bright. Cancer & metabolism, 2: 1.
13. Leung CC, Porcel JM, Takahashi K, Restrepo MI, Lee P, Wainwright C, 2014. Year in review 2013: Lung cancer, respiratory infections, tuberculosis, cystic fibrosis, pleural diseases, bronchoscopic intervention and imaging. Respirology, 19: 448-460.
14. Li L, Tan J, Miao Y, Lei P, Zhang Q, 2015. ROS and Autophagy: Interactions and Molecular Regulatory Mechanisms. Cellular and molecular neurobiology, 35: 615-621.
15. Livak KJ, Schmittgen TD, 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods, 25: 402-408.
16. Lui VW, Lau CP, Cheung CS, Ho K, Ng MH, Cheng SH, Hong B, Tsao SW, Tsang CM, Lei KI , et al., 2010. An RNA-directed nucleoside anti-metabolite, 1-(3-C-ethynyl-beta-d-ribo-pentofuranosyl) cytosine (ECyd), elicits antitumor effect via TP53-induced Glycolysis and Apoptosis Regulator (TIGAR) downregulation. Biochemical pharmacology, 79: 1772-1780.
17. Lui VW, Wong EY, Ho K, Ng PK, Lau CP, Tsui SK, Tsang CM, Tsao SW, Cheng SH, Ng MH , et al., 2011. Inhibition of c-Met downregulates TIGAR expression and reduces NADPH production leading to cell death. Oncogene, 30: 1127-1134.
18. Mahmood T, Yang PC, 2012. Western blot: technique, theory, and trouble shooting. North American journal of medical sciences, 4: 429-434.
19. Morgan MJ, Liu ZG, 2011. Crosstalk of reactive oxygen species and NF-kappaB signaling. Cell research 21: 103-115.
20. Patra KC, Hay N, 2014. The pentose phosphate pathway and cancer. Trends in biochemical sciences 39: 347-354.
21. Pena-Rico MA, Calvo-Vidal MN, Villalonga-Planells R, Martinez-Soler F, Gimenez-Bonafe P, Navarro-Sabate A, Tortosa A, Bartrons R, Manzano A, 2011. TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown results in radiosensitization of glioma cells. Radiotherapy and oncology, 101: 132-139.
22. Qian S, Li J, Hong M, Zhu Y, Zhao H, Xie Y, Huang J, Lian Y, Li Y, Wang S , et al., 2016. TIGAR cooperated with glycolysis to inhibit the apoptosis of leukemia cells and associated with poor prognosis in patients with cytogenetically normal acute myeloid leukemia. Journal of hematology & oncology, 9: 128.
23. Rahal A, Kumar A, Singh V, Yadav B, Tiwari R, Chakraborty S, Dhama K, 2014. Oxidative stress, prooxidants, and antioxidants: the interplay. BioMed research international, 2014: 1-19.
24. Riganti C, Gazzano E, Polimeni M, Aldieri E, Ghigo D, 2012. The pentose phosphate pathway: an antioxidant defense and a crossroad in tumor cell fate. Free radical biology & medicine, 53: 421-436.
25. Scherz-Shouval R, Elazar Z, 2007. ROS, mitochondria and the regulation of autophagy. Trends in cell biology, 17: 422-427.
26. Tai G, Gu P, Zhang H, He X, Du J, Huang J, Yu J, Cai J, Liu F, 2016. TP53 induced glycolysis and apoptosis regulator knockdown radiosensitizes glioma cells through oxidative stress-induced excessive autophagy. International Journal of Clinical and Experimental Pathology, 9: 7.
27. Wang S, Yu Q, Zhang R, Liu B, 2011. Core signaling pathways of survival/death in autophagy-related cancer networks. International Journal of Biochemistry and Cell Biology, 43: 1263-1266.
28. Wang Y, Huang X, Cang H, Gao F, Yamamoto T, Osaki T, Yi J, 2007. The endogenous reactive oxygen species promote NF-kappaB activation by targeting on activation of NF-kappaB-inducing kinase in oral squamous carcinoma cells. Free radical research, 41: 963-971.
29. Wanka C, Steinbach JP, Rieger J, 2012. Tp53-induced glycolysis and apoptosis regulator (TIGAR) protects glioma cells from starvation-induced cell death by up-regulating respiration and improving cellular redox homeostasis. The Journal of biological chemistry, 287: 33436-33446.
30. Wong EY, Wong SC, Chan CM, Lam EK, Ho LY, Lau CP, Au TC, Chan AK, Tsang CM, Tsao SW , et al., 2015. TP53-induced glycolysis and apoptosis regulator promotes proliferation and invasiveness of nasopharyngeal carcinoma cells. Oncology letters, 9: 569-574.
31. Xie JM, Li B, Yu HP, Gao QG, Li W, Wu HR, Qin ZH, 2014. TIGAR has a dual role in cancer cell survival through regulating apoptosis and autophagy. Cancer research, 74: 5127-5138.
32. Ye L, Zhao X, Lu J, Qian G, Zheng JC, Ge S, 2013. Knockdown of TIGAR by RNA interference induces apoptosis and autophagy in HepG2 hepatocellular carcinoma cells. Biochemical and biophysical research communications, 437: 300-306.
33. Yin L, Kosugi M, Kufe D, 2012. Inhibition of the MUC1-C oncoprotein induces multiple myeloma cell death by down-regulating TIGAR expression and depleting NADPH. Blood, 119: 810-816.
34. Yu HP, Xie JM, Li B, Sun YH, Gao QG, Ding ZH, Wu HR, Qin ZH, 2015. TIGAR regulates DNA damage and repair through pentosephosphate pathway and Cdk5-ATM pathway. Scientific reports, 5: 9853.
35. Zhao D, Han W, Liu X, Cui D, Chen Y, 2017. MicroRNA-128 promotes apoptosis in lung cancer by directly targeting NIMA-related kinase 2. Thoracic cancer, 8: 304-311.
36. Zhuang W, Qin Z, Liang Z, 2009. The role of autophagy in sensitizing malignant glioma cells to radiation therapy. Acta biochimica et biophysica Sinica, 41: 341-351.