Synergistic effect of RAD50 downregulation on combination of rucaparib and doxorubicin

Yıl 2023, Cilt: 62 Sayı: 2, 289 - 300, 12.06.2023

Ramazan Gundogdu Mehmet Kadir Erdoğan Aydın Sever Yusuf Toy

Öz

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.

Anahtar Kelimeler

RAD50, MRN complex, Rucaparib, doxorubicin

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

RAD50, MRN kompleksi, Rucaparib, doksorubisin

Kaynakça

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