Uvabain Aracılıklı Apoptotik Hücre Ölümleri ve Rho-Kinaz: Bir Proteomik Çalışma

Abstract

Abstract Ouabain-Induced Apoptotic Cell Death and Rho Kinase: A Proteomics Study Objective: In the current work, we aimed to evaluate the possible involvement of Rho kinase in the ouabain-induced apoptotic cell death, using a proteomic approach. Method: Human umbilical vein endothelial cells were exposed to 10 μM ouabain, 10 μM Y27632 (30 min before the ouabain treatment)+10 μM ouabain, and 10 μM Y27632 alone. Cell homogenates were separated by 2D gel electrophoresis and stained with Coomassie blue. Results: It was detected that 54 spots were changed with the ouabain treatment. Seven spots, changed with ouabain plus Y27632 treatment, were excised and subjected to MALDI-TOF MS to identify the proteins of interest. The levels of caspase-7 and protein unc-119 homolog B proteins increased with ouabain treatment (p<0.05) and this increase has decreased by the pretreatment with Y27632 (p<0.05) . In addition, the levels of neurensin-2, C-type lectin domain family-2 member D and putative FERM domain-containing protein FKSG43 proteins were decreased with ouabain treatment (p<0.05). This decrease has increased by the pretreatment with Y27632 (p<0.05). Conclusion: These results indicate that Rho kinase pathway may involve in the changes of expression of these proteins. Caspase-7, protein unc-119 homolog B, neurensin-2 and C-type lectin domain family-2 member D proteins are involved in apoptosis, neurotransmitter release, tumor suppression and the inhibition of osteoclast formation, respectively. However, further studies are needed to confirm our results and to determine the importance of these proteins.

Keywords

• ouabain, Rho-kinase, proteomics, apoptosis, sodium pump.

Uvabain Aracılıklı Apoptotik Hücre Ölümleri ve Rho-Kinaz: Bir Proteomik Çalışma

Abstract

Türkçe Özet: Amaç: Bu çalışmada, uvabain ile indüklenmiş apoptotik hücre ölümlerinde, Rho-kinazın olası rolünün proteomik yaklaşımlar kullanılarak değerlendirilmesi amaçlandı. Yöntem: İnsan umbilikal ven endotel hücreleri, 10 μM uvabain, 10 μM Y27632 (uvabain uygulamasından 30 dk önce)+10 μM uvabain ve yalnız 10 μM Y27632\'ye maruz bırakılmıştır. Hücre homojenatları, iki boyutlu jel elektroforezi ile ayrılmış ve Coomassie mavisi ile boyanmıştır . Bulgular: Uvabain uygulaması sonucunda, 54 spotun değiştiği tespit edilmiştir. Uvabain + Y27632 uygulamasında değiştiği gözlenmiş olan 7 protein, MALDI-TOF MS yöntemi ile tanımlanmak üzere seçilmiştir. Kaspaz-7 ve protein-unc-119 homolog B proteinlerinin miktarları uvabain uygulaması ile artmış (p<0.05) ve bu artış Y27632 ön uygulaması ile azalmıştır(p<0.05). Buna ek olarak nörensin-2, C-type lectin domain family-2 member D ve putative FERM domain-containing protein FKSG-43 proteinleri uvabain uygulaması ile azalmış (p<0.05) ve bu azalış Y27632 ön uygulaması ile artmıştır (p<0.05). Sonuç: Bu sonuçlar, bu proteinlerin ekspresyonlarındaki değişiklikler üzerinde Rho-kinaz yolağının önemini göstermektedir. Kaspaz-7, protein unc-119 homolog B, nörensin-2 ve C-tipi lektin bölgesi ailesi-2 üyesi D proteinleri sırasıyla, apoptoz, nörotransmitter salınımı, tümör süpresyonu ve osteoklast oluşumunun inhibisyonunda yer almaktadırlar. Bununla birlikte sonuçlarımızın doğrulanması ve bu proteinlerin öneminin belirlenmesi için daha başka çalışmalara ihtiyaç vardır.

Keywords

• uvabain, Rho-kinaz, proteomik, apoptoz, sodyum pompası.

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