Synergistic effect of RAD50 downregulation on combination of rucaparib and doxorubicin

Ramazan Gundogdu; Mehmet Kadir Erdoğan; Aydın Sever; Yusuf Toy

doi:10.19161/etd.1160550

TR EN

RAD50'nin downregülasyonunun rucaparib ve doksorubisin kombinasyonuna sinerjik etkisi

Öz

Amaç: MRN (MRE11-RAD50-NBS1) protein kompleksi bir DNA hasar sensörü olarak işlev görür ve homolog rekombinasyon onarım mekanizması ile DNA çift sarmal kopmalarının onarımının koordine edilmesinde önemli roller oynar. Fonksiyonel olmayan MRN aktivitesinin kanser hücrelerini DNA'ya zarar veren ajanlara veya PARP inhibitörlerine karşı duyarlı hale getirdiği gösterilmiş olsa da, RAD50'nin rucaparib ve doksorubisin tedavileri üzerindeki fonksiyonel önemi henüz araştırılmamıştır. Bu araştırmanın amacı, RAD50 defektif kanser hücrelerinin rucaparib ve doksorubisin kombinasyonuna yanıtının araştırılmasıdır. Gereç ve Yöntem: Bu çalışmada RAD50 ekspresyon seviyelerinin terapötik potansiyelini değerlendirmek için insan kemiği osteosarkoma epitel hücreleri (U2OS) kullanıldı. RAD50 mRNA aktivitesinin ifadesini susturmak için RNA interferans teknolojisi uygulandı. İlgili genlerin mRNA ekspresyon seviyelerini araştırmak için qRT-PCR tekniği kullanıldı. İlgili protein ekspresyon seviyelerini değerlendirmek için Western blot analizi yapıldı. RAD50 kaybının rucaparib ve doksorubisin kombinasyon tedavisi üzerindeki etkisini incelemek için klonojenik sağkalım analizi gerçekleştirildi. Bulgular: Azalan RAD50 ifadesinin, MRE11 ve NBS1 protein seviyelerinde önemli bir düşüşe neden olduğu gözlenirken, p53 ve p21’in mRNA ve protein seviyelerini etkilemediği görüldü. Ayrıca, RAD50 kaybı olan hücrelerin akut doksorubisin tedavisi ile DNA hasar yanıt aktivasyonunu kaybettiği belirlendi. Son olarak, RAD50 susturulmasının PARP inhibitörü olan rucaparib ile birleştirildiğinde doksorubisinin sitotoksisitesini arttırdığı gözlendi. Sonuç: Tüm bu sonuçlar birlikte ele alındığında, klinik öncesi bulgularımız, PARP inhibitörlerinin kanser tedavisinde kullanılmasında RAD50 ekspresyon seviyelerinin prediktif bir biyobelirteç olarak araştırılabileceğini göstermektedir.

Anahtar Kelimeler

Synergistic effect of RAD50 downregulation on combination of rucaparib and doxorubicin

Abstract

Aim: The MRN (MRE11-RAD50-NBS1) protein complex functions as a DNA damage sensor and plays essential roles to coordinate the repair of DNA double-strand breaks. Although dysfunctional MRN activity has been shown to sensitize cancer cells to certain DNA-damaging agents or PARP inhibitors, the functional significance of RAD50 upon rucaparib and doxorubicin treatments has yet to be studied. The aim of this research was to investigate the response of RAD50-defective cancer cells toward the combination of rucaparib and doxorubicin. Materials and Methods: Human bone osteosarcoma epithelial cells (U2OS) were used in this study to assess the therapeutic potential of RAD50 expression levels. The RNA interference technology was applied to silence the expression of the RAD50 mRNA activity. The qRT-PCR technique was used to investigate the mRNA expression levels of the relevant genes. Western blotting analysis was conducted to assess the relevant protein expression levels. Clonogenic survival assay was performed to dissect the effect of RAD50-loss on the rucaparib and doxorubicin combination treatment. Results: RAD50 knockdown resulted in a significant decrease in MRE11 and NBS1 protein levels, whereas it did not affect p53 and p21 expressions at mRNA and protein levels. Furthermore, the cells with RAD50-loss had impaired DNA damage response activation against acute doxorubicin treatment. We finally showed that RAD50 depletion increased the cytotoxicity of doxorubicin when combined with the PARP inhibitor rucaparib. Conclusion: Taken together, our preclinical findings suggest that RAD50 expression levels can be explored as a predictive biomarker in the evaluation for precision cancer treatments involving PARP inhibitors.

Keywords

Kaynakça

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Ayrıntılar

Birincil Dil

İngilizce

Konular

Sağlık Kurumları Yönetimi

Bölüm

Araştırma Makalesi

Yazarlar

Ramazan Gundogdu ^*
0000-0001-5230-2121
Türkiye

Mehmet Kadir Erdoğan
0000-0002-1579-5737
Türkiye

Aydın Sever
0000-0002-6727-1556
Türkiye

Yusuf Toy
0000-0003-1901-9994
Türkiye

Yayımlanma Tarihi

12 Haziran 2023

Gönderilme Tarihi

11 Ağustos 2022

Kabul Tarihi

7 Aralık 2022

Yayımlandığı Sayı

Yıl 2023 Cilt: 62 Sayı: 2

DOI

https://doi.org/10.19161/etd.1160550

IZ

https://izlik.org/JA97ZF29YT

Kaynak Göster

RIS / Bibtex

Vancouver

1.Ramazan Gundogdu, Mehmet Kadir Erdoğan, Aydın Sever, Yusuf Toy. Synergistic effect of RAD50 downregulation on combination of rucaparib and doxorubicin. ETD. 01 Haziran 2023;62(2):289-300. doi:10.19161/etd.1160550