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PRIMA1 ile lösemi hücrelerinde Bruton tirozin kinaz inhibisyonu aracılığı apoptoz uyarımı

Amaç: Bruton’s tirozin kinaz (Btk) , B lenfositlerinin gelişimi, proliferasyonu ve B hücre serilerinin farklılaşmasında, B hücre reseptörü (BCR) aracılı sinyal iletiminde kritik rol oynadığı bilinmektedir. Bu çalışmada PRIMA-1’in Btk aktivitesi üzerindeki antitumor etkisi ve altta yatan apopitoz mekanizması araştırılmıştır. Gereç ve Yöntem: KBM3 ve Namalwa hücrelerinde Btk ekpresyon seviyesi üzerine PRIMA 1’in etkisini kontrol etmek için kantitatif real time PCR ve Western Blot analizi yapılmıştır. FACS ve flouresan mikroskobi kullanılarak apoptozda Btk ‘nın rolünü belirlemek için, wild tip Btk ve Btk-NLS ( Nükleus hedefli Btk) ekpresyon plazmidleri COS-7 hücrelerine transfekte edildi.Bulgular: Bu çalışmada, akut myelomonositik lösemi hücrelerine anti lösemik ilaç uygulanması sonucunda, Btk ekspresyonun mRNA ve protein seviyesinde baskılanmasını gözlemledik. Böylece Nrf2 ve HO-1 proteinlerinin ekpresyonlarında bir azalma olduğunu da farkettik. Dikkat çekici bir şekilde Btk nükleer lokalizasyonu, düşük dozda PRIMA-1 uygulaması ile artıyorken, yüksek dozda PRIMA -1 uygulaması ile Btk ekspresyonu baskılanıyor. Buna ek olarak, wild tip Btk ile kıyaslandığında, nükleer hedefli aşırı ekspresyonda, apoptozisde azalma ve hücre canlılık oranında artış ile sonuçlanıyor. Sonuç: Bulgularımıza göre, PRIMA-1 uygulaması ile Nrf2 ve HO-1 oksidatif cevap aracılığı ile nükleer Btk, hücre apoptozunu azalmaktadır.

Anahtar Kelimeler:

Lösemi, apoptoz, nükleer Btk, Nrf2

PRIMA-1 induces apoptosis of leukemic cells via inhibiting Bruton's tyrosine kinase

Purpose: Bruton's tyrosine kinase (Btk) is known to be critical for B-lymphocyte development, proliferation and differentiation of B-cell lineages, convey signal transduction through B cell receptor (BCR). The present study examined the anti-tumor effects of PRIMA-1 on Btk activity and explored the underlying mechanism, such as apoptosis.Materials and Methods: Western blot analysis and Quantitative real-time polymerase chain reaction were performed to check the effects of PRIMA-1 on Btk expression level in KBM3 and Namalwa cells. Wild-type Btk and Btk-NLS (nuclear targeted Btk) expression plasmids were transfected in COS-7 cells to establish and characterize the role of Btk in apoptosis using fluorescent microscopy and FACS assay. Results: İn this study, we observed that exposure of acute myelomonocytic leukemia cells to anti-leukemic drug (PRIMA-1) suppressed Btk expression at mRNA and protein level. Consequently, we also noticed a reduction in the expression of Nrf2 and HO-1 proteins. Remarkably, Btk nuclear localization was increased in response to low PRIMA-1 exposure, while higher concentrations of PRIMA-1 suppressed Btk expression. Furthermore, overexpression of nuclear targeted decreased apoptosis and increased cell viability compared to the wild-type Btk.Conclusion: Our findings suggest that nuclear Btk reduces the cell apoptosis in response to PRIMA-1 exposure through oxidative response via Nrf2 and HO-1.

Keywords:

Leukemia, apoptosis, nuclear Btk, Nrf2,

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