THE EFFECT OF 5-FU AND RUXOLITINIB ON MITOCHONDRIAL APOPTOSIS IN GLIOBLASTOMA U87 CELL LINE

Year 2020, Volume: 7 Issue: 3, 130 - 139, 31.10.2020

Gonca Aksu Oğuzhan Doğanlar Zeynep Banu Doğanlar

Abstract

Aims: The aim of this study is to carry out the effect of 5-Fluorouracil alone or combined with Ruxolitinib on both apoptosis and JAK/STAT pathway in U87 glioblastoma cells. Methods: We used U87 glioblastoma cell lines as the human brain cancer cells. We treated the cells with 5-Fluorouracil (3.125 μM-400 μM) alone and with a combination of Ruxolitinib (100 μM or 400 μM of Ruxolitinib with 3.125-25 μM 5-Fluorouracil), and performed the MTT test for calculating IC50 value. Molecular fluorescence staining was performed with Hoechst and acridine orange/ethidium bromide probes. The alteration in mitochondrial apoptosis and JAK/STAT pathways to drug treatment was analyzed by the qRT-PCR assay. Results: Decrease in cell viability was more prominent in U87 cells treated with a combination of 5-Fluorouracil and Ruxolitinib compared to those treated with 5-Fluoro- uracil alone. In gene expression analysis, apoptosis signals were observed in cells treated with 5-Fluorouracil alone and 5-Fluo- rouracil+Ruxolitinib treatment. Conclusion: Treatment with 5-Fluorouracil alone and 5-Fluorouracil+Ruxolitinib combination increased apoptosis in U87 glioblastoma cells. However, it is difficult to mention an evident difference between treatments. Therefore, further studies are needed.

Keywords

5-Fluorouracil, Ruxolitinib, glioblastoma, apoptosis, JAK/STAT pathway

References

• 1. Bedini A, Baiula M, Vincelli G et al. Nociceptin/orphanin FQ antagonizes lipopolysaccharide-stimulated proliferation, migration and inflammatory signaling in human glioblastoma U87 cells. Bio- chem Pharmaol 2017;140:89-104.
• 2. Batash R, Asna N, Schaffer P et al. Glioblastoma multiforme, diagnosis and treatment; recent literature review. Curr Med Chem 2017;24(27):3002-9.
• 3. Alifieris C, Trafalis DT. Glioblastoma multiforme: pathogenesis and treatment. Pharmacol Ther 2015;152:63-82.
• 4. Leelakanok N, Geary S, Salem A. Fabrication and use of poly(d,l- lactide-co-glycolide)-based formulations designed for modified re- lease of 5-Fluorouracil. J Pharm Sci 2018;107(2):513-28
• 5. Diasio RB, Harris BE. Clinical pharmacology of 5-Fluorouracil. Clin Pharmacokinet 1989;16(4):215-37.
• 6. Akca H, Ozes ON. Antitumor effects of TNF-β, 5-FU and their combinations on cervical carcinoma cell lines. 2002;32:127-32.
• 7. Goker Bagca B, Avci CB. Ruxolitinib and effect mechanism. FNG & Demiroğlu Bilim Tıp Dergisi 2016;2(2):153-7.
• 8. LiverTox: Clinical and Research Information on Drug-Induced Liver Injury [Internet]. Bethesda (MD): National Institute of Dia- betes and Digestive and Kidney Diseases; 2012. Ruxolitinib. (Up- dated 2018 Jun 4). Available from: URL: https://www.ncbi.nlm.nih. gov/books/NBK548534/pdf/Bookshelf_NBK548534.pdf.
• 9. Delen E, Doganlar O, Doganlar ZB et al. Inhibition of the inva- sion of human glioblastoma U87 cell line by ruxolitinib: a molec- ular player of miR-17 and miR-20a regulating JAK/STAT pathway. Turk Neurosurg 2020;30(2):182-9.
• 10. Xu J, JiL D, Xu LH. Lead-induced apoptosis in PC 12 cells: Involvement of p53, Bcl-2 family and caspase-3. Toxicol Lett 2006;166(2):160-7.
• 11. Erdogan MK, Agca CA, Askin H. Enhanced antiproliferative and apoptotic effects of 5-fluorouracil by combined with Pistacia eurycarpa extracts on human colorectal cancer cells. BioDiCon 2019;12(1):27-38.
• 12. Winter RN, Kramer A, Borkowski A et al. Loss of caspase-1 and caspase-3 protein expression in human prostate cancer. Cancer Res 2001;61(3):1227-32.
• 13. Teitz T, Wei T, Valentine MB et al. Caspase 8 is deleted or si- lenced preferentially in childhood neuroblastomas with amplifica- tion of MYCN. Nat Med 2000;6(5):529-35.
• 14. Shivapurkar N, Toyooka S, Eby MT, et al. Differential inactiva- tion of caspase-8 in lung cancers. Cancer Biol Ther 2002;1(1):65–9.
• 15. Kasibhatla, S. Acridine Orange/Ethidium Bromide (AO/EB) Staining to Detect Apoptosis. Cold Spring Harbor Protocols, 2006 (21). doi:10.1101/pdb.prot4493
• 16. Jatiani SS, Baker SJ, Silverman LR et al. Jak/STAT pathways in cytokine signaling and myeloproliferative disorders: approaches for targeted therapies. Genes Cancer 2010;1(10):979-93.
• 17. Owen KL, Brockwell NK, Parker BS. JAK-STAT signaling: a double-edged sword of immune regulation and cancer progression. Cancers (Basel) 2019;11(12):2002.
• 18. Imada K, Leonard WJ. The JAK-STAT pathway. Molecular Im- munology 2000;37:1-11.
• 19. Schindler C, Levy DE, Decker T. JAK-STAT signaling: from interferons to cytokines. Journal of Biological Chemistry 2007;282(28):20059-63.
• 20. Masjedi A, Hashemi V, Hojjat-Farsangi M et al. The significant role of interleukin-6 and its signaling pathway in the immuno- pathogenesis and treatment of breast cancer. Biomedicine & Phar- macotherapy 2018;108:1415-24.
• 21. Rose-John S. IL-6 trans-signaling via the soluble IL-6 receptor: importance for the pro-inflammatory activities of IL-6. Int J Biol Sci 2012;8(9):1237–47.
• 22. Yu H, Lee H, Herrmann A et al. Revisiting STAT3 signalling in cancer: new and unexpected biological functions. Nat Rev Cancer 2014;14(11):736-46.