Caco-2 Hücre Hattı In Vitro Epitelyal Mezenkimal Geçiş Modelinde Glutatyon S-Transferaz-π Düzeyleri

Year 2019, Volume: 39 Issue: 1, 10 - 16, 15.01.2019

Seyhan Türk , İpek Baysal Gülberk Uçar

Abstract

Modern tıptaki gelişmelere rağmen, adjuvant/neoadjuvan tedavilere direnç geli- şimi tedaviyi karmaşıklaştırabilecek en önemli sorunlardan biridir. Son yıllarda, kanser hücrelerinin epitelyal mezenkimal geçiş (EMT) aktivasyonunun, metastaz eğilimi ve kemoterapiye direncinde önemli bir rol oynadığını göstermiştir. Bu ça- lışmada CaCo-2 hücre hattında in vitro EMT modeli oluşturularak direnç meka- nizmalarının belirlenmesi hedeflenmiştir. Bu hücreler dönüşüm yetenekleri açı- sından iyi bilinen hücre hatlarıdır ve farklılaşma potansiyelleri nedeniyle yapısal ve fonksiyonel çalışmalar için önemli araçlardır. Farklılaşma 0., 15. ve 30. gün- lerde takip edilmiştir ve model validasyonu için epitelyal ve mezenkimal belir- teçler (E-kadherin ve Vimentin) kullanılmıştır. EMT’nin iki formunda da direnç mekanizmalarını aydınlatmak üzere ELISA ile Glutatyon- S-Transferaz-π (GST-π) düzeyleri analiz edilmiştir. Mezen- kimal formda beklendiği şekilde GST-π düzeyinin önemli derecede arttığı gösterilmiştir. Ayrıca mezenkimal formda direnci ortadan kaldırmaya yönelik GST-π inhibitörü hi- perisin kullanılmıştır. Hiperisinin farklı konsantrasyonları (2μM ve 20μM) ile muamele sonucu GST-π düzeylerin- de anlamlı derecede azalma tespit edilmiştir. Bu sonuçlar, adjuvan terapiye ek olarak direncin ortadan kaldırılmasını sağlayacak mekanizmaların aktifleştirilmesi ile kullanılan tedavinin etkinliğinin arttırabileceğini ortaya koymuştur.

Keywords

Kolorektal kanser, Epitelyal Mezenkimal Geçiş, Glutatyon S-Transferaz-π

Glutathione S-Transferase-π Levels of In Vitro Generated Epithelial Mesenchymal Transition Model in Caco-2 Cell Line

Abstract

Despite the developments in the modern medicine, the resistancy to adjuvant/ne- oadjuvant therapies is one of the most important problem that may complicate the treatment. In recent years, studies have shown that activation of epithelial-mes- enchymal transition (EMT) may plays an important role in cancer cells tendency to metastasis and resistance to chemotherapy. In this work, in vitro EMT model was generated with Caco-2 cell line to determine the mechanisms of response and resistancy. This cells are well-known for their ability to transform and are important tools for structural and functional studies due to their differentiation potential. Differentiation was monitored on days 0th, 15th and 30th and epithe- lial and mesenchymal markers (E-cadherin and Vimentin) were used for model validation with qRT-PCR. Glutathione S-Transferase-π (GST-π) levels were de- termined to elucidate the mechanisms of resistancy in both forms of EMT with ELISA. As expected, it was shown that GST-π levels increased significantly in mesenchymal form. Also, GST-π inhibitor hypericin was used to eliminate resist- ancy in mesenchymal form. Significant decrease in GST-π levels was determined with the different concentrations of hypericin treatment (2μM and 20μM). These results showed that, activation of mechanisms to eliminate resistancy in addition to adjuvant therapy may increase the effectiveness of treatment.

Keywords

Colorectal cancer, Epithelial Mesenchymal Transition, Glutathione S-Transferase-π

References

• 1.Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA: Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease. The Journal of clinical investigation 2009, 119(6):1438-1449.
• 2.Boyer B, Valles AM, Edme N: Induction and regulation of epithelial-mesenchymal transitions. Biochemical Pharmacology 2000, 60(8):1091-1099.
• 3.Micalizzi D, Farabaugh S, Ford H: Epithelial-Mesenchymal Transition in Cancer: Parallels Between Normal Development and Tumor Progression. Journal of mammary gland biology and neoplasia 2010, 15:117-134.
• 4. Popov Z, Gil-Diez de Medina S, Lefrere-Belda MA, Hoznek A, Bastuji-Garin S, Abbou CC, et al.: Low E-cadherin expression in bladder cancer at the transcriptional and protein level provides prognostic information. British journal of cancer 2000, 83(2):209-214.
• 5. Radisky DC: Epithelial-mesenchymal transition. Journal of cell science 2005, 118(Pt 19):4325-4326.
• 6. Casas E, Kim J, Bendesky A, Ohno-Machado L, Wolfe CJ, Yang J: Snail2 is an essential mediator of Twist1-induced epithelial mesenchymal transition and metastasis. Cancer research 2011, 71(1):245-254.
• 7. Nieto MA: The snail superfamily of zinc-finger transcription factors. Nature reviews Molecular cell biology 2002, 3(3):155- 166.
• 8. Zhao XJ, Li H, Chen H, Liu YX, Zhang LH, Liu SX, et al.: Expression of e-cadherin and beta-catenin in human esopha- geal squamous cell carcinoma: relationships with prognosis. World journal of gastroenterology 2003, 9(2):225-232.
• 9. Kang Y, Massague J: Epithelial-mesenchymal transitions: twist in development and metastasis. Cell 2004, 118(3):277- 279.
• 10. Sayan AE: Tumour-promoting role of EMT-inducing trans- cription factor ZEB1 in mantle cell lymphoma. Cell death and differentiation 2014, 21(2):194-195.
• 11. Yao D, Dai C, Peng S: Mechanism of the mesenchymal-epit- helial transition and its relationship with metastatic tumor for- mation. Molecular cancer research : MCR 2011, 9(12):1608-1620.
• 12. ArmstrongRN:Structure,catalyticmechanism,andevolution of the glutathione transferases. Chemical research in toxico- logy 1997, 10(1):2-18.
• 13. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al.: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008, 133(4):704-715.
• 14. Hayes JD, Flanagan JU, Jowsey IR: Glutathione transferases. Annual review of pharmacology and toxicology 2005, 45:51- 88.
• 15. LabordeE:Glutathionetransferasesasmediatorsofsignaling pathways involved in cell proliferation and cell death. Cell de- ath and differentiation 2010, 17(9):1373-1380.
• 16. Aliya S, Reddanna P, Thyagaraju K: Does glutathione S-transferase Pi (GST-Pi) a marker protein for cancer? Mole- cular and cellular biochemistry 2003, 253(1-2):319-327.
• 17. MannervikB,BoardPG,HayesJD,ListowskyI,PearsonWR: Nomenclature for mammalian soluble glutathione transferases. Methods in enzymology 2005, 401:1-8.
• 18. Noguti J, Barbisan LF, Cesar A, Dias Seabra C, Choueri RB, Ribeiro DA: Review: In vivo models for measuring placental glutatione-S-transferase (GST-P 7-7) levels: a suitable biomar- ker for understanding cancer pathogenesis. In vivo (Athens, Greece) 2012, 26(4):647-650.
• 19. Adler V,YinZ,Fuchs SY,Benezra M,Rosario L,Tew KD,et al.: Regulation of JNK signaling by GSTp. The EMBO journal 1999, 18(5):1321-1334.
• 20. Lu WD, Atkins WM: A novel antioxidant role for ligan- din behavior of glutathione S-transferases: attenuation of the photodynamic effects of hypericin. Biochemistry 2004, 43(40):12761-12769.
• 21. Zhao L, Li C, Zhou B, Luo C, Wang Y, Che L, et al.: Crucial role of serum response factor in renal tubular epithelial cell epithelial-mesenchymal transition in hyperuricemic nephro- pathy. Aging 2019, 11.
• 22. Zhong X, Tang J, Li H, Shi X, Wu Y, Xia D, et al.: MiR-3175 promotes epithelial mesenchymal transition by targeting Smad7 in human conjunctiva and pterygium. FEBS letters 2019.
• 23. Chen T, You Y, Jiang H, Wang ZZ: Epithelial-mesenchymal transition (EMT): A biological process in the development, stem cell differentiation, and tumorigenesis. J Cell Physiol 2017, 232(12):3261-3272.
• 24. Tania M, Khan MA, Fu J: Epithelial to mesenchymal transiti- on inducing transcription factors and metastatic cancer. Tumo- ur biology : the journal of the International Society for Onco- developmental Biology and Medicine 2014, 35(8):7335-7342.
• 25. Giannoni E, Parri M, Chiarugi P: EMT and oxidative stress: a bidirectional interplay affecting tumor malignancy. Antioxi- dants & redox signaling 2012, 16(11):1248-1263.
• 26. Basu S, Chaudhary A, Chowdhury P, Karmakar D, Basu K, Karmakar D, et al.: Evaluating the role of hsa-miR-200c in reversing the epithelial to mesenchymal transition in prostate cancer. Gene 2019, 144264.
• 27. Turk S, Kulaksiz Erkmen G, Dalmizrak O, Ogus IH, Ozer N: Purification of Glutathione S-Transferase pi from Erythrocy- tes and Evaluation of the Inhibitory Effect of Hypericin. The protein journal 2015, 34(6):434-443.
• 28. Dabrowski MJ, Maeda D, Zebala J, Lu WD, Mahajan S, Ka- vanagh TJ, et al.: Glutathione S-transferase P1-1 expression modulates sensitivity of human kidney 293 cells to photody- namic therapy with hypericin. Archives of biochemistry and biophysics 2006, 449(1-2):94-103.
• 29. Dalmizrak O, Kulaksiz-Erkmen G, Ozer N: Evaluation of the in vitro inhibitory impact of hypericin on placental glutathione S-transferase pi. The protein journal 2012, 31(7):544-549.
• 30. Halder M, Chowdhury PK, Das R, Mukherjee P, Atkins WM, Petrich JW: Interaction of glutathione S-transferase with hype- ricin: A photophysical study. The journal of physical chemistry B 2005, 109(41):19484-19489.