Mitoz sırasında H2O2 ile indüklenen oksidatif strese karşı dirençte Yca1’in rolünün incelenmesi

Amaç: Günümüzde başarıyla kullanılan antikanser ilaçlar antitümör etkilerini mikrotübüllerin yapısını bozup hücrelerde uzun süreli mitotik arreste yol açarak gösterirler. Uzun süreli mitotik arrestin sonuçlarından biri apoptozdur. Metakaspazlar, metazoan haricindeki canlılarda bulunan ve apoptozda önemli rol oynadıkları bilinen yapısal olarak kaspaz homologlarıdır. Bu çalışmada, Saccharomyces cerevisiae’de mikrotübülleri hedef alan antikanser ilaçlarla indüklenen mitotik arrestin oksidatif stres direncine etkisinin ve mayadaki tek metakaspaz olan Yca1’in bu etkideki olası rolünün araştırılması amaçlanmıştır. Yöntem: Bu amaç için, yabanıl tip (YT) ve Yca1 delesyon mutant (yca1Δ) suşlarında mitotik arrest nocodazole veya carbendazim ile; G1-arrest ise alfa faktör ile tetiklenmiştir. Her iki suştaki protein agregat oluşumu ise cycloheximide muamelesi ile engellenmiştir. Bu koşullardan her biri altında 6 mM H2O2 ile indüklenen oksidatif stresin YT ve yca1Δ suşlardaki sağkalım üzerine etkisi koloni oluşturan birim (cfu) ve nokta ekim yöntemleriyle; reaktif oksijen türleri (ROT) birikimi üzerine etkisi ise H2DCFDA yöntemiyle incelenerek karşılaştırılmıştır. Bulgular: Nocodazole veya carbendazim ile tetiklenen mitotik arrest YT S.cerevisiae hücrelerinde oksidatif stres direnci oluştururken, aynı koşullardaki yca1Δ suşunda ise direnç oluşturmadığı tespit edilmiştir. Diğer yandan, alfa faktörle tetiklenen G1-arrestin YT veya yca1Δ suşlarında oksidatif stres hassasiyeti üzerinde önemli bir etkisi olmadığı gözlenmiştir. Ayrıca, cycloheximide muamalesi ile protein agregat oluşumunun engellenmesinin yca1Δ suşunda YT suşa benzer şekilde mitotik arrest sonucu oksidatif stres direnci oluşmasına yol açtığı görülmüştür. Sonuç: Mikrotübülleri hedef alan antikanser ilaçlarla tetiklenen mitotik arrest YT S.cerevisiae hücrelerinde oksidatif stres direncine yol açar. Sadece mitozda gelişen bu dirençte Yca1’in protein agregat oluşumunu engelleme fonksiyonu önemli rol oynamaktadır.

Anahtar Kelimeler:

Oksidatif stres direnci, Mitotik arrest, Yca1, Saccharomyces cerevisiae

Investigating the role of Yca1 in resistance to H2O2-induced oxidative stress during mitosis

Aim: The most successfully used anticancer drugs today exhibit their antitumor effects by causing a prolonged mitotic arrest through disrupting the structure of microtubules. One of the outcomes of the prolonged mitotic arrest is apoptosis. Metacaspases are the structural homologs of caspases that are found in living organisms other than metazoan and are known to play an important role in apoptosis. The aim of this study was to investigate the effect of mitotic arrest induced by microtubule-targeting anticancer drugs on oxidative stress resistance in Saccharomyces cerevisiae and the possible role of Yca1, the only metacaspase in yeast, in this effect. Methods: For this purpose, mitotic arrest was induced by either nocodazole or carbendazim; G1-arrest was triggered by alpha factor in the wild type (WT) and Yca1 deletion mutant (yca1Δ) strains. Protein aggregation was prevented by cycloheximide treatment in both strains. Under each of these conditions, the effect of oxidative stress induced by 6 mM H2O2 on the viability of the WT and yca1Δ strains was evaluated by the colony forming unit (cfu) and spotting assays; accumulation of the reactive oxygen species (ROS) was evaluated and compared by the H2DCFDA assay. Results: It was observed that mitotic arrest induced by nocodazole or carbendazim resulted in resistance to oxidative stress in WT S.cerevisiae cells, while the same conditions did not cause resistance to oxidative stress in yca1Δ cells. On the other hand, G1-arrest induced by alpha factor did not have a dramatic effect on the sensitivities of WT or yca1Δ strains to oxidative stress. Additionally, we observed that prevention of protein aggregation by the cycloheximide treatment led to oxidative stress resistance resulting from mitosis in the yca1Δ strain, similar to WT. Conclusion: Mitotic arrest induced by microtubule targeting anticancer drugs results in resistance to oxidative stress. In this resistance, which develops only in mitosis, function of Yca1 in the prevention of protein aggregation plays an important role.

Keywords:

Oxidative stress resistance, Mitotic arrest, Yca1, Saccharomyces cerevisiae,

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