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

Year 2024, , 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

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