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

Yıl 2019, , 230 - 239, 30.08.2019

Pınar Buket Atalay Nur Kaluç

https://doi.org/10.26559/mersinsbd.517361

Öz

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.

Anahtar Kelimeler

Oxidative stress resistance, Mitotic arrest, Yca1, Saccharomyces cerevisiae

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

Öz

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

Kaynakça

• 1. Stobbe CC, Park SJ, Chapman JD. The radiation hypersensitivity of cells at mitosis. Int J Radiat Biol 2002;78(12):1149-57.
• 2. Hughes AF. The effect of inhibitory substances on cell division; a study on living cells in tissue cultures. Q J Microsc Sci 1950;91(3):251-77.
• 3. Mukhtar E, Adhami VM, Mukhtar H. Targeting Microtubules by Natural Agents for Cancer Therapy. Mol Cancer Ther 2014;13, 275–284.
• 4. Dominguez-Brauer C, Thu KL, Mason JM, Blaser H, Bray MR, Mak TW. Targeting Mitosis in Cancer: Emerging Strategies. Mol Cell 2015;60(4):524-36.
• 5. Lara-Gonzalez P, Westhorpe FG, Taylor SS. The spindle assembly checkpoint. Curr Biol 2012; 22(22):R966-80.
• 6. Scully R. The spindle-assembly checkpoint, aneuploidy, and gastrointestinal cancer. N Eng J Med 2010;363(27), 2665-6.
• 7. Sies H, Berndt C, Jones DP. Oxidative Stress. Annu Rev Biochem 2017;86:715-748.
• 8. Pelicano H, Carney D, Huang P. ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 2004;7, 97-110.
• 9. Liou GY, Storz P. Reactive oxygen species in cancer. Free Radic. Res 2010; 44, 10.3109/ 10715761003667554.
• 10. Flattery-O'Brien JA, Dawes IW. Hydrogen peroxide causes RAD9-dependent cell cycle arrest in G2 in Saccharomyces cerevisiae whereas menadione causes G1 arrest independent of RAD9 function. J Biol Chem 1998;273(15):8564-71.
• 11. McArthur K, Kile BT. Apoptotic Caspases: Multiple or Mistaken Identities? Trends Cell Biol 2018;28(6):475-493.
• 12. Hill SM, Hao X, Liu B, Nyström T. Life-span extension by a metacaspase in the yeast Saccharomyces cerevisiae. Science 2014;344(6190):1389-92.
• 13. Uren AG, O'Rourke K, Aravind LA, Pisabarro MT, Seshagiri S, Koonin EV, Dixit VM. Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. Mol Cell 2000;6(4):961-7.
• 14. Madeo F, Herker E, Maldener C, Wissing S, Lächelt S, Herlan M, Fehr M, Lauber K, Sigrist SJ, Wesselborg S, Fröhlich KU. A caspase-related protease regulates apoptosis in yeast. Mol Cell 2002;9(4):911-7.
• 15. Lee RE, Puente LG, Kaern M, Megeney LA. A non-death role of the yeast metacaspase: Yca1p alters cell cycle dynamics. PloS One 2008;3(8), e2956.
• 16. Shrestha A, Megeney LA. The non-death role of metacaspase proteases. Front Oncol 2012;2, 78.
• 17. Hill SM, Nyström T. The dual role of a yeast metacaspase: What doesn't kill you makes you stronger. BioEssays 2015;37(5), 525-31.
• 18. Madeo F, Fröhlich, E, Ligr M, Grey M, Sigrist SJ, Wolf DH, Fröhlich KU. Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol 1999;145 (4), 757-67.
• 19. Wu D, Yotnda P. Production and Detection of Reactive Oxygen Species (ROS) in Cancers. J Vis Exp 2011;(57):3357.
• 20. Chen B, Retzlaff M, Roos T, Frydman J. Cellular strategies of protein quality control. Cold Spring Harb Perspect Biol 2011;3(8):a004374.
• 21. Hartl FU, Bracher A, Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis. Nature 2011;475(7356):324–332.