Epigallokateşin-3- gallat uygulamasına bağlı olarak kronik miyeloid lösemi hücrelerinde genetik ve epigenetik otofaji regülatörlerinin ekspresyon değişimlerinin değerlendirilmesi

Öz

Amaç: Kronik miyeloid lösemi, Bcr-Abl1 füzyon tirozin kinazının oluşumu aktivitesi ile tanımlanır. Füzyon proteinini hedefleyen inhibitörün keşfi sağ kalım oranlarında büyük artış meydana getirmiştir. Ancak bu inhibitöre karşı direnç gelişimi yeni terapi hedeflerinin belirlenmesini zorunlu kılmaktadır. Otofaji, güncel kanser araştırmalarında dikkat çekici bir iki yönlü hedeftir. Kanser hücrelerini stres koşullarına adapte etmeyi sağladığı için genellikle otofajinin inhibisyou hedeflenmektedir. Epigallokateşin-3-gallat yeşil çayda bulunan temel bir fitokimyasaldır. mikroRNA’lar gibi epigenetik regülatörlerin üzerindeki etkinliği tedavide olduğu kadar hastaların beslenme önerilerinin düzenlenmesinde de önem taşımaktadır. Bu çalışmada bir flavanoid olan epigallokateşin-3-gallatın kronik miyeloid lösemi hücrelerinde otofajiyi düzenleyen genetik ve epigenetik faktörler üzerindeki etkinliğinin araştırılması amaçlanmıştır.

Gereç ve yöntem: mikroRNA-mRNA etkileşimini değerlendiren veritabanları kullanılarak otofaji ilişkili genleri hedefleyen mikroRNA’lar belirlenmiştir. 50 µikromolar Epigallokateşin-3-gallat uygulanan K-562 hücrelerinde gerçek zamanlı kantitatif PCR yöntemi kullanılarak otofaji ilişkili genlerin ve bu genleri hedefleyen mikroRNA’ların ekspresyon seviyeleri belirlenmiştir.

Bulgular: Epigallokateşin-3-gallat uygulaması K-562 hücrelerinde otofajinin pozitif regülatörü olan genlerin ekspresyon seviyesinde anlamlı azalışa, bu genleri hedefleyen mikroRNA’ların ekspresyon seviyelerinde ise anlamlı artışa neden olmuştur.

Sonuç: Elde edilen veriler doğrultusunda epigallokateşin-3-gallatın, kronik miyeloid lösemi hücrelerinde genetik ve epigenetik regülasyonla otofaji inhibisyonuna neden olduğu belirlenmiştir. Otofajinin çift yönlü mekanizması göz önünde bulundurulduğunda bu sonuç epigallokateşin-3-gallatın ilaç potansiyeli olma konusunda olduğu kadar hastaların beslenme şeklinde de önem taşıyan bir kimyasal olduğu gerçeğini ortaya koymaktadır.

Anahtar Kelimeler

• Kronik Miyeloid Lösemi,miRNA,Otofaji

Evaluation of expression changes of genetic and epigenetic autophagy regulators in chronic myeloid leukemia cells due to epigallocatechin-3-gallate treatment

Öz

Purpose: Chronic myeloid leukemia is characterized by the formation and activation of Bcr-Abl1 fusion tyrosine kinase. Discovery of the inhibitor which targets the fusion protein results in a significant increase in survival rates.  However the development of resistance to the inhibitor necessitates determining new therapy targets. Autophagy is a remarkable dual target in recent cancer researches. It is usually aimed at inhibition of autophagy because autophagy allows cells to adapt to stress conditions. Epigallocatechin-3-gallate is a basic phytochemical found in green tea. The efficacy of epigenetic regulators, such as microRNAs, is important in the regulation of dietary recommendations as well as in treatment. In this study, it was aimed to investigate the effect of epigallocatechin-3-gallate, a flavanoid, on genetic and epigenetic factors regulating autophagy in chronic myeloid leukemia cells.

Materials and methods: microRNAs which are target autophagy-related genes have been identified using appropriate databases. Expression levels of autophagy-related genes and microRNAs targeting these genes were determined using real-time quantitative PCR method in 50 micromolar Epigallocatechin-3-gallate treated K-562 cells.

Results: Epigallocatechin-3-gallate treatment resulted in a significant decrease and increase the expression levels of autophagy-related genes and microRNAs targeting these genes in K-562 cells, respectively.

Conclusion: In the direction of the results, epigallocatechin-3-gallate was found to cause autophagy inhibition by genetic and epigenetic regulation in chronic myeloid leukemia cells. Considering the dual mechanism of autophagy, this result suggests that epigallocatechin-3-gallate is a chemical that is important for the nutrition of patients as well as its potential to be used as a drug.

Anahtar Kelimeler

• Chronic Myeloid Leukemia,miRNA,Autophagy

Kaynakça

1. Apperley JF. Chronic myeloid leukaemia. Lancet. 2015;385:1447-1459.
2. Sun Y, Peng ZL. Programmed cell death and cancer. Postgrad Med J. 2009;85:134-140.
3. Yonekawa T, Thorburn A. Autophagy and cell death. Essays Biochem. 2013;55:105-117.
4. Singh BN, Shankar S, Srivastava RK. Green tea catechin, epigallocatechin-3-gallate (EGCG): mechanisms, perspectives and clinical applications. Biochem Pharmacol. 2011;82:1807-1821.
5. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol. 2014;15:509-524.
6. Goker B, Caliskan C, Onur Caglar H. et al. Synergistic effect of ponatinib and epigallocatechin-3-gallate induces apoptosis in chronic myeloid leukemia cells through altering expressions of cell cycle regulatory genes. J BUON. 2014;19:992-998.
7. Mao Y, Li L, Liu J, Wang L, Zhou Y. MiR-495 inhibits esophageal squamous cell carcinoma progression by targeting Akt1. Oncotarget. 2016;7:51223-51236.
8. Chen PH, Cheng CH, Shih CM. et al. The inhibition of microRNA-128 on IGF-1-activating mTOR signaling involves in temozolomide-induced glioma cell apoptotic death. PLoS One. 2016;11:e0167096.

9. Mou X, Liu S. MiR-485 inhibits metastasis and EMT of lung adenocarcinoma by targeting Flot2. Biochem Biophys Res Commun. 2016;477:521-526.
10. Ma Y, Yang HZ, Dong BJ, et al. Biphasic regulation of autophagy by miR-96 in prostate cancer cells under hypoxia. Oncotarget. 2014;5:9169-9182.
11. Soni M, Patel Y, Markoutsa E, et al. Autophagy, cell viability and chemo-resistance are regulated by miR-489 in breast cancer. Mol Cancer Res. 2018.
12. Chen X, Zhang Y, Shi Y, et al. MiR-129 triggers autophagic flux by regulating a novel Notch-1/ E2F7/Beclin-1 axis to impair the viability of human malignant glioma cells. Oncotarget. 2016;7:9222-9235.
13. Cao W, Qian G, Luo W, et al. miR-125b is downregulated in systemic lupus erythematosus patients and inhibits autophagy by targeting UVRAG. Biomed Pharmacother. 2018;99:791-797.
14. Capizzi M, Strappazzon F, Cianfanelli V, Papaleo E, Cecconi F. MIR7-3HG, a MYC-dependent modulator of cell proliferation, inhibits autophagy by a regulatory loop involving AMBRA1. Autophagy. 2017;13:554-566.
15. Yang J, He Y, Zhai N, Ding S, Li J, Peng Z. MicroRNA-181a inhibits autophagy by targeting Atg5 in hepatocellular carcinoma. Front Biosci (Landmark Ed). 2018;23:388-396.
16. Wang L, Song LF, Chen XY, et al. MiR-181b inhibits P38/JNK signaling pathway to attenuate autophagy and apoptosis in juvenile rats with kainic acid-induced epilepsy via targeting TLR4. CNS Neurosci Ther. 2018.
17. Ham O, Lee SY, Lee CY, et al. let-7b suppresses apoptosis and autophagy of human mesenchymal stem cells transplanted into ischemia/reperfusion injured heart 7by targeting caspase-3.Stem Cell Res Ther. 2015;6:147.
18. Zhang J, Ma J, Long K, et al. Overexpression of exosomal cardioprotective miRNAs mitigates hypoxia-induced H9c2 cells apoptosis. Int J Mol Sci. 2017;18.
19. Pan B, Feng B, Chen Y, et al. MiR-200b regulates autophagy associated with chemoresistance in human lung adenocarcinoma. Oncotarget. 2015;6:32805-328520.
20. Liguori M, Nuzziello N, Licciulli F, et al. Combined microRNA and mRNA expression analysis in pediatric multiple sclerosis: an integrated approach to uncover novel pathogenic mechanisms of the disease.Hum Mol Genet. 2018;27:66-79.
21. Xu Y, Yang J, Li F, Lian G, Ouyang M. MiR-29a inhibited intestinal epithelial cells autophagy partly by decreasing ATG9A in ulcerative colitis. Anticancer Drugs. 2018.
22. Gan J, Cai Q, Qu Y, et al. miR-96 attenuates status epilepticus-induced brain injury by directly targeting Atg7 and Atg16L1. Sci Rep. 2017;7:10270.
23. Li X, Li Y, Fang S, Su J, et al. Downregulation of autophagy-related gene ATG5 and GABARAP expression by IFN-λ1 contributes to its anti-HCV activity in human hepatoma cells. Antiviral Res. 2017;140:83-94.
24. Etna MP, Sinigaglia A, Grassi A, et al. Mycobacterium tuberculosis-induced miR-155 subverts autophagy by targeting ATG3 in human dendritic cells. PLoS Pathog. 2018;14:e1006790.
25. Wang C, Zhang ZZ, Yang W, et al. MiR-210 facilitates ECM degradation by suppressing autophagy via silencing of ATG7 in human degenerated NP cells. Biomed Pharmacother. 2017;93:470-479.
26. Amaravadi R, Kimmelman AC, White E. Recent insights into the function of autophagy in cancer. Genes Dev. 2016;30:1913-1930.
27. Shinohara H, Taniguchi K, Kumazaki M, et al. Anti-cancer fatty-acid derivative induces autophagic cell death through modulation of PKM isoform expression profile mediated by bcr-abl in chronic myeloid leukemia. Cancer Lett. 2015;360:28-38.
28. He W, Ye X, Huang X, et al. Hsp90 inhibitor, BIIB021, induces apoptosis and autophagy by regulating mTOR-Ulk1 pathway in imatinib-sensitive and -resistant chronic myeloid leukemia cells. Int J Oncol. 2016;48:1710-1720.
29. Mitchell R, Hopcroft LEM, Baquero P, et al. Targeting BCR-ABL-independent TKI resistance in chronic myeloid leukemia by mTOR and autophagy inhibition. J Natl Cancer Inst. 2018;110:467-478.
30. Ianniciello A, Dumas PY, Drullion C, et al. Chronic myeloid leukemia progenitor cells require autophagy when leaving hypoxia-induced quiescence. Oncotarget. 2017;8:96984-96992.
31. Lu Z, Xu N, He B, et al. Inhibition of autophagy enhances the selective anti-cancer activity of tigecycline to overcome drug resistance in the treatment of chronic myeloid leukemia. J Exp Clin Cancer Res. 2017;36:43.
32. El-Khattouti A, Selimovic D, Haikel Y, Hassan M. Crosstalk between apoptosis and autophagy: molecular mechanisms and therapeutic strategies in cancer. J Cell Death. 2013;6:37-55.
33. Yuan CH, Horng CT, Lee CF, et al. Epigallocatechin gallate sensitizes cisplatin-resistant oral cancer CAR cell apoptosis and autophagy through stimulating AKT/STAT3 pathway and suppressing multidrug resistance 1 signaling. Environ Toxicol. 2017;32:845-855.
34. Irimie AI, Braicu C, Zanoaga O, et al. Epigallocatechin-3-gallate suppresses cell proliferation and promotes apoptosis and autophagy in oral cancer SSC-4 cells. Onco Targets Ther. 2015;8:461-470.
35. Holczer M, Besze B, Zámbó V, Csala M, Bánhegyi G, Kapuy O. Epigallocatechin-3-Gallate (EGCG) Promotes Autophagy-Dependent Survival via Influencing the Balance of mTOR-AMPK Pathways upon Endoplasmic Reticulum Stress. Oxid Med Cell Longev. 2018;2018:6721530.
36. Kim SW, Moon JH, Park SY. Activation of autophagic flux by epigallocatechin gallate mitigates TRAIL-induced tumor cell apoptosis via down-regulation of death receptors. Oncotarget. 2016;7:65660-65668.
37. Sethi S, Li Y, Sarkar FH. Regulating miRNA by natural agents as a new strategy for cancer treatment. Curr Drug Targets. 2013;14:1167-1174.
38. Milenkovic D, Jude B, Morand C. miRNA as molecular target of polyphenols underlying their biological effects. Free Radic Biol Med. 2013;64:40-51.
39. Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol. 2011;27:107-132.
40. Wang J, Man GCW, Chan TH, Kwong J, Wang CC. A prodrug of green tea polyphenol (-)-epigallocatechin-3-gallate (Pro-EGCG) serves as a novel angiogenesis inhibitor in endometrial cancer. Cancer Lett. 2018;412:10-20.
41. Liu S, Xu ZL, Sun L, et al. (‑)‑Epigallocatechin‑3‑gallate induces apoptosis in human pancreatic cancer cells via PTEN. Mol Med Rep. 2016;14:599-605.
42. Jang JE, Eom JI, Jeung HK, et al. AMPK-ULK1-Mediated Autophagy Confers Resistance to BET Inhibitor JQ1 in Acute Myeloid Leukemia Stem Cells. Clin Cancer Res. 2017;23:2781-2794.
43. Zachari M, Ganley G. The mammalian ULK1 complex and autophagy initiation. Essays Biochem. 2017;61:585–596.
44. Burman C, Ktistakis NT. Regulation of autophagy by phosphatidylinositol 3-phosphate. FEBS Lett. 2010;584:1302-1312.
45. Matsushita M, Suzuki NN, Obara K, Fujioka Y, Ohsumi Y, Inagaki F. Structure of Atg5.Atg16, a complex essential for autophagy. J Biol Chem. 2007;282:6763-6772.
46. Chen L, Ye HL, Zhang G, et al. Autophagy inhibition contributes to the synergistic interaction between EGCG and doxorubicin to kill the hepatoma Hep3B cells. PLoS One. 2014;9:e85771.
47. Schaaf MB, Keulers TG, Vooijs MA, Rouschop KM. LC3/GABARAP family proteins: autophagy-(un)related functions. FASEB J. 2016;30:3961-3978.
48. Tanida I, Sou YS, Minematsu-Ikeguchi N, Ueno T, Kominami E. Atg8L/Apg8L is the fourth mammalian modifier of mammalian Atg8 conjugation mediated by human Atg4B, Atg7 and Atg3. FEBS J. 2006;273:2553-2562.