Sorafenib ve doxorubicin'in lösemi hücrelerinde URG4/URGCP üzerindeki apoptotik ve hücre döngüsü etkilerinin incelenmesi

Year 2024, Volume: 17 Issue: 3, 498 - 508, 05.07.2024

Yavuz Dodurga Levent Elmas Mücahit Seçme Nazlı Demirkıran Çığır Biray Avcı Gülseren Bağcı Sevda Sağ Lale Şatıroğlu Tufan

https://doi.org/10.31362/patd.1476105

Abstract

Amaç: Çalışmanın amacı, Sorafenib (SOR) ve Doxorubicin (DOX) gibi antikanser ilaçlarının K562 ve HL-60 lösemi hücrelerinde URG4/URGCP mRNA düzeyleri üzerindeki etkilerini inceleyerek, apoptoz ve hücre döngüsü üzerindeki etkilerini açıklığa kavuşturmaktır. Bu ilaçların lösemi hücrelerinde apoptoz ve hücre döngüsü üzerindeki etkileri araştırılmıştır. Bu araştırma, lösemi tedavisinde kullanılan ilaçların hücresel etkilerini anlamak ve lösemi tedavisinde ilaç geliştirme süreçlerine değerli bilgilerle katkıda bulunmayı amaçlamaktadır.
Gereç ve yöntem: DOX ve SOR’in K562 ve HL-60 hücre hatlarında IC50 ATP ölçümüne dayanan CellTiter-Glo (Promega, ABD) tarafından değerlendirildi. Uygulama yapılan her iki hücre hattının kontrol ve doz gruplarında Trizol kimyasalı kullanılarak total RNA izolasyonu yapıldı. RNA izolasyonunun ardından “Transcriptor High Fidelity cDNA Sentez Kiti” ile cDNA'lar sentezlendi. Daha sonra URG4/URGCP, Casp-3, Casp-8, Casp-9, FADD, DR4, TRADD, CCDN1, CDK4, CDK6, PTEN, P53, Rel-A genlerine özgü primerler kullanılarak mRNA düzeyindeki ekspresyon değişiklikleri incelendi.
Bulgular: Çalışmada Sorafenib uygulanan gruplarda IC50 dozu HL-60 hücre hattı için 24’üncü saatte 40 μM, K562 hücre hattı için ise 48’inci saatte 40 μM olarak hesaplandı. Doksorubisin uygulanan gruplarda ise IC50 dozları, HL-60 hücre hattı için 48’inci saatte 50 μM, K562 hücre hattı için ise 72’inci saatte 50 μM olarak hesaplanmıştır. SOR uygulanan gruplarda Casp-8, Casp-9, TRADD, DR4, Rel A ve FADD genlerin mRNA ekspresyon düzeylerindeki kat değişimlerinde önemli derecede artış gözlemlenirken, URG4/URGCP, CCDN1, CDK4 ve CDK6 genlerinin mRNA ekspresyon düzeylerinde azalma olduğu gözlemlendi. DOX uygulanan gruplarda, Casp-3, Casp-8, P53 ve PTEN genlerinin kat değişimlerinde önemli derecede bir artış olduğu gözlemlenmiştir. Ancak, URG4/URGCP, CCDN1 ve CDK4 genleri üzerinde mRNA ekspresyon düzeylerinde önemli bir azalma meydana gelmiştir.
Sonuç: Sonuç olarak hem SOR hem de DOX’ in HL-60 ve K562 hücrelerinde URG4/URGCP, Casp-3, Casp-8, Casp-9, CDK6, CDK4, CCND1, P53, PTEN, TRADD, DR4, Rel A ve FADD genlerin mRNA ekspresyonları düzenlenmesinde rol alabilecekleri gösterilmiştir.

Keywords

Doksorubisin, sorafenib, hücre döngüsü, apoptoz, URG4/URGCP

Investigation of the apoptotic and cell cycle effects of sorafenib and doxorubicin on URG4/URGCP in leukemia cells

Abstract

Purpose: The aim of this study is to investigate the effects of anticancer drugs such as Sorafenib (SOR) and Doxorubicin (DOX) on URG4/URGCP mRNA levels in K562 and HL-60 leukemia cells, elucidating their effects on apoptosis and cell cycle. The effects of these drugs on apoptosis and the cell cycle in leukemia cells have been explored. This research aims to understand the cellular effects of drugs used in leukemia treatment and contribute valuable insights to the drug development processes in leukemia therapy.
Materials and methods: DOX and SOR were evaluated for their IC50 values in K562 and HL-60 cell lines using the CellTiter-Glo assay (Promega, USA), based on ATP measurement. Total RNA isolation was performed using Trizol reagent in both control and dose groups of each treated cell line. Following RNA isolation, cDNAs were synthesized using the "Transcriptor High Fidelity cDNA Synthesis Kit". Subsequently, changes in mRNA expression levels were examined using specific primers for URG4/URGCP, Casp-3, Casp-8, Casp-9, FADD, DR4, TRADD, CCDN1, CDK4, CDK6, PTEN, P53, and Rel-A genes.
Results: In the groups treated with Sorafenib, the IC50 dose for HL-60 cell line was calculated as 40 μM at the 24th hour, and for K562 cell line, it was calculated as 40 μM at the 48th hour. In the groups treated with Doxorubicin, the IC50 doses were calculated as 50 μM at the 48th hour for HL-60 cell line, and as 50 μM at the 72nd hour for K562 cell line. Significant increases were observed in the mRNA expression levels of Casp-8, Casp-9, TRADD, DR4, Rel A, and FADD genes in the groups treated with SOR, while a decrease was observed in the mRNA expression levels of URG4/URGCP, CCDN1, CDK4, and CDK6 genes. In the groups treated with DOX, significant increases were observed in the fold changes of Casp-3, Casp-8, P53, and PTEN genes. However, a significant decrease in mRNA expression levels was observed in URG4/URGCP, CCDN1, and CDK4 genes.
Conclusion: As a result, it has been demonstrated that both SOR and DOX may play a role in regulating the mRNA expressions of URG4/URGCP, Casp-3, Casp-8, Casp-9, CDK6, CDK4, CCND1, P53, PTEN, TRADD, DR4, Rel A, and FADD genes in HL-60 and K562 cells.

Keywords

Doxorubicin, sorafenib, cell cycle, apoptosis, URG4/URGCP

References

• 1. Davis AS, Viera AJ, Mead MD. Leukemia: an overview for primary care. Am Fam Physician 2014;89:731-738.
• 2. Terwilliger T, Abdul Hay M. Acute lymphoblastic leukemia: a comprehensive review and 2017 update. Blood Cancer J 2017;7:577. https://doi.org/10.1038/bcj.2017.53
• 3. Chu E, Sartorelli AC. Cancer chemotherapy. Lange’s Basic and Clinical Pharmacology 2018;948-976.
• 4. Nygren P. What is cancer chemotherapy? Acta Oncol 2001;40:166-174. https://doi.org/10.1080/02841860151116204
• 5. Sun Y, Liu Y, Ma X, Hu H. The influence of cell cycle regulation on chemotherapy. Int J Mol Sci 2021;22:6923. https://doi.org/10.3390/ijms22136923
• 6. Rini BI. Sorafenib. Expert Opin Pharmacother 2006;7:453-461. https://doi.org/10.1517/14656566.7.4.453
• 7. Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004;64:7099-7109. https://doi.org/10.1158/0008-5472.CAN-04-1443
• 8. Escudier B, Eisen T, Stadler WM, et al. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med 2007;356:125-134. https://doi.org/10.1056/NEJMoa060655
• 9. Mori S, Cortes J, Kantarjian H, Zhang W, Andreef M, Ravandi F. Potential role of sorafenib in the treatment of acute myeloid leukemia. Leuk Lymphoma 2008;49:2246-2255. https://doi.org/10.1080/10428190802510349
• 10. Iyer R, Fetterly G, Lugade A, Thanavala Y. Sorafenib: a clinical and pharmacologic review. Expert Opin Pharmacother 2010;11:1943-1955. https://doi.org/10.1517/14656566.2010.496453
• 11. Johnson Arbor K, Dubey R. Doxorubicin. In: StatPearls. StatPearls Publishing, Treasure Island (FL) 2024.
• 12. Carvalho C, Santos RX, Cardoso S, et al. Doxorubicin: the good, the bad and the ugly effect. Curr Med Chem 2009;16:3267-3285. https://doi.org/10.2174/092986709788803312
• 13. Thorn CF, Oshiro C, Marsh S, et al. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genomics 2011;21:440-446. https://doi.org/10.1097/FPC.0b013e32833ffb56
• 14. Rivankar S. An overview of doxorubicin formulations in cancer therapy. J Cancer Res Ther 2014;10:853-858. https://doi.org/10.4103/0973-1482.139267
• 15. Dodurga Y, Seçme M, Şatıroğlu Tufan NL. A novel oncogene URG4/URGCP and its role in cancer. Genes (Basel) 2018;668:12-17. https://doi.org/10.1016/j.gene.2018.05.047
• 16. Dodurga Y, Gundogdu G, Koc T, Yonguc GN, Kucukatay V, Satiroglu Tufan NL. Expressions of URG4/URGCP, Cyclin D1, Bcl-2, and Bax genes in retinoic acid treated SH-SY5Y human neuroblastoma cells. Contemp Oncol (Pozn) 2013;17:346-349. https://doi.org/10.5114/wo.2013.34634
• 17. Dodurga Y, Seçme M, Eroğlu C, et al. Investigation of the effects of a sulfite molecule on human neuroblastoma cells via a novel oncogene URG4/URGCP. Life Sci 2015;143:27-34. https://doi.org/10.1016/j.lfs.2015.10.005
• 18. Arslan G, Önkol T, Özçelik AB. Siklin bağımlı kinaz 4/6 ve inhibitörleri. Ankara Ecz Fak Derg 2022;46:193-208. https://doi.org/10.33483/jfpau.978763
• 19. Cabadak H. Hücre siklusu ve kanser. ADÜ Tıp Fakültesi Dergisi 2008;9:51-61.
• 20. Cai W, Shu LZ, Liu DJ, Zhou L, Wang MM, Deng H. Targeting cyclin D1 as a therapeutic approach for papillary thyroid carcinoma. Front Oncol 2023;13:1145082. https://doi.org/10.3389/fonc.2023.1145082
• 21. Jung YS, Qian Y, Chen X. Examination of the expanding pathways for the regulation of p21 expression and activity. Cell Signal 2010;22:1003-1012. https://doi.org/10.1016/j.cellsig.2010.01.013
• 22. Bourdon JC. p53 and its isoforms in cancer. Br J Cancer 2007;97:277-282. https://doi.org/10.1038/sj.bjc.6603886
• 23. Akşit H, Bildik A. Apoptozis. Yüzüncü Yıl Üniversitesi Vet Fak Derg 2008;19:55-63.
• 24. Coşkun G, Özgür H. Apoptoz ve nekrozun moleküler mekanizması. Arşiv Kaynak Tarama Dergisi 2011;20:145-158.
• 25. Kaya C, Çalışkan Y, Yönden Z. Apoptozis. Mustafa Kemal Üniv Tıp Derg 2012;3:26-37.
• 26. Celepli S, Bigat İ, Celepli P, Karagin PH. Apoptoz ve apoptotik yolların gözden geçirilmesi. Güncel Gastroentoloji 2020;24:103-111.
• 27. Wilson NS, Dixit V, Ashkenazi A. Death receptor signal transducers: nodes of coordination in immune signaling networks. Nat Immunol 2009;10:348-355. https://doi.org/10.1038/ni.1714
• 28. Dağdeviren T. Programlı hücre ölümü; apoptoz. Gaziosmanpaşa Üniversitesi Tıp Fak Derg 2021;13:120-135.
• 29. Dodurga Y, Eroğlu C, Seçme M, Elmas L, Avcı ÇB, Şatıroğlu Tufan NL. Anti-proliferative and anti-invasive effects of ferulic acid in TT medullary thyroid cancer cells interacting with URG4/URGCP. Tumor Biol 2016;37:1933-1940. https://doi.org/10.1007/s13277-015-3984-z
• 30. Novilla A, Astuti I, Suwito H. Molecular mechanism of synthesized potential anticancer agent chalcone in leukemia cell line K562. J Med Sci 2017;49:23-28.
• 31. Gilliland DG, Jordan CT, Felix CA. The molecular basis of leukemia. Hematology Am Soc Hematol Educ Program 2004;2004:80-97. https://doi.org/10.1182/asheducation-2004.1.80
• 32. Kaymaz BT, Çetintaş VB, Kosova B. Determination of the gene expression profiles of JAK/STAT cascade components for the potential role of capsaicin induced apoptosis of acute T-cell lymphoblastic leukemia cells. Kafkas J Med Sci 2013;3:129-135. https://doi.org/10.5505/kjms.2013.39200
• 33. Gökbulut AA, Yaşar M, Baran Y. A novel natural product, KL-21, inhibits proliferation and induces apoptosis in chronic lymphocytic leukemia cells. Turk J Haematol 2015;32:118-126. https://doi.org/10.4274/tjh.2013.0381
• 34. Erkurt MA, Kuku İ, Kaya E, Aydoğdu İ. Kanser kemoterapisi ve böbrek. İnönü Üniversitesi Tıp Fakültesi Dergisi 2009;16:63-68.
• 35. Ravandi F, Arana YiC, Cortes JE, et al. Final report of phase II study of sorafenib, cytarabine and idarubicin for initial therapy in younger patients with acute myeloid leukemia. Leukemia 2014;28:1543-1545. https://doi.org/10.1038/leu.2014.54
• 36. Liu L, Cao Y, Chen C, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 2006;66:11851-11858. https://doi.org/10.1158/0008-5472.CAN-06-1377
• 37. Zhao X, Tian C, Puszyk WM. OPA1 downregulation is involved in sorafenib-induced apoptosis in hepatocellular carcinoma. Lab Invest 2013;93:8-19. https://doi.org/10.1038/labinvest.2012.144
• 38. Mizutani H, Tada Oikawa S, Hiraku Y, Kojima M, Kawanishi S. Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide. Life Sci 2005;76:1439-1453. https://doi.org/10.1016/j.lfs.2004.05.040
• 39. Lee T, Lau T, Ng I. Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines. Cancer Chemother Pharmacol 2002;49:78-86. https://doi.org/10.1007/s00280-001-0376-4
• 40. Suzuki F, Hashimoto K, Kikuchi H, et al. Induction of tumor-specific cytotoxicity and apoptosis by doxorubicin. Anticancer Res 2005;25:887-893.
• 41. Zhao W, Zhang T, Qu B, et al. Sorafenib induces apoptosis in HL60 cells by inhibiting Src kinase-mediated STAT3 phosphorylation. Anticancer Drugs 2011;22:79-88. https://doi.org/10.1097/CAD.0b013e32833f44fd
• 42. Huang S, Sinicrope FA. Sorafenib inhibits STAT3 activation to enhance TRAIL-mediated apoptosis in human pancreatic cancer cells. Mol Cancer Ther 2010;9:742-750. https://doi.org/10.1158/1535-7163.MCT-09-1004
• 43. Şirin N, Elmas L, Seçme M, Dodurga Y. Investigation of possible effects of apigenin, sorafenib and combined applications on apoptosis and cell cycle in hepatocellular cancer cells. Genes (Basel) 2020;737:144428. https://doi.org/10.1016/j.gene.2020.144428
• 44. Zhang Y, Li G, Liu X, et al. Sorafenib inhibited cell growth through the MEK/ERK signaling pathway in acute promyelocytic leukemia cells. Oncol Lett 2018;15:5620-5626. https://doi.org/10.3892/ol.2018.8010
• 45. Wang S, Konorev EA, Kotamraju S, Joseph J, Kalivendi S, Kalyanaraman B. Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms: intermediacy of H2O2-and p53-dependent pathways. J Biol Chem 2004;279:25535-25543. https://doi.org/10.1074/jbc.M400944200
• 46. Pilco Ferreto N, Calaf GM. Influence of doxorubicin on apoptosis and oxidative stress in breast cancer cell lines. Int J Oncol 2016;49:753-762. https://doi.org/10.3892/ijo.2016.3558
• 47. Kim HS, Lee YS, Kim DK. Doxorubicin exerts cytotoxic effects through cell cycle arrest and Fas-mediated cell death. Pharmacol 2009;84:300-309. https://doi.org/10.1159/000245937
• 48. El Readi MZ, Abdulkarim MA, Abdellatif AAH, et al. Doxorubicin-sanguinarine nanoparticles: formulation and evaluation of breast cancer cell apoptosis and cell cycle. Drug Dev Ind Pharm 2024;1-15. https://doi.org/10.1080/03639045.2024.2302557
• 49. Żuryń A, Litwiniec A, Klimaszewska Wiśniewska A, et al. Expression of cyclin D1 after treatment with doxorubicin in the HL‐60 cell line. Cell Biol Int 2014;38:857-867. https://doi.org/10.1002/cbin.10290