Ertan KUCUKSAYAN, Aslinur SİRCAN-KUCUKSAYAN

Melanoma hücrelerinde epoksomisinin indüklediği apoptozis sürecinde gerçekleşen erken spektral değişikliklerin belirlenmesi

Amaç: Hücrelerde apoptozis sürecinde meydana gelen spektral değişikliklerin belirlenmesi yeni tedavi ve ilaç araştırmalarına önemli bilgiler sağlayabilir. Apoptotik hücreler, hücre küçülmesi ve parçalanmasına yol açan bir dizi hücre altı değişikliğe uğrar. Apoptotik süreçte bu değişimlerin erken aşamada zamana bağlı olarak belirlenmesi hücre kültürü çalışmalarına yeni bir yön verebilir. Bu çalışmanın amacı melanoma hücrelerinin apoptozis sürecinde erken aşamada meydana gelen spektral değişikliklerin zamana bağlı olarak belirlenebileceği bir yöntem geliştirmektir. Yöntem: Bu çalışmada A375 melanoma hücre hattında apoptozisi indüklemek için epoksomisin (Epo) kullanılmıştır ve apoptotik doz MTT yöntemi ile belirlenmiştir. Erken apoptotik sürecinin zamana bağlı değerlendirilebilmesi için sabit Epo dozuyla beş farklı zaman noktasında (0.5-6 saat) ölçümler yapılmıştır. Apoptozisin en önemli uyaranı olan ROS ölçümü DCFH-DA yöntemi ile yapılmıştır. Western Blot tekniği ile Bax miktarı belirlenmiştir. Geri yansıma spektroskopisi sistemi ile ölçülen spektrumlardan spektroskopik apoptozis indeksi belirlenmiştir. Bulgular: Melanoma hücrelerinin apoptozis süresinde ölçülen spektrumlar ile erken spektral değişiklikler belirlenmiştir. ROS ölçümlerinde 2., 4. ve 6. saat ölçümlerinde kontrole göre anlamlı fark bulunmuştur. Hücre canlılığının 24 saat sonra 75 ve 100 nM Epo dozlarında kontrole göre %70 azaldığı bulunmuştur. Apoptozisin bir göstergesi olarak tüm Epo gruplarında zamana bağlı olarak Bax seviyelerinin arttığı bulunmuştur. Spektroskopik apoptozis indeks değeri tüm gruplarda ROS ve Bax sonuçları ile uyumlu bulunmuştur. Sonuç: Hücre kültüründe apoptozis sürecinde erken aşamada meydana gelen spektral değişikliklerin geri yansıma spektroskopisi ile belirlenebileceği yeni bir yaklaşım sunulmuştur. Bu yaklaşımın hücre kültürü çalışmalarında apoptozisi hücre kültürü koşullarına müdahale etmeden, zamana bağlı olarak izleyebilen bir yöntem olarak geliştirilme potansiyeli vardır.

Determination of early spectral changes in melanoma cells during epoxomicin-induced apoptotic process

Objective: Determining the changes in the apoptosis process in cells can provide important information for new treatment and drug research. Apoptotic cells undergo a series of subcellular changes that lead to cell shrinkage and fragmentation. Determining these early changes in the apoptotic process depending on time may provide a new perspective to cell culture studies. The aim of this study is to develop a method in which early spectral changes occurring in the apoptosis process of melanoma cells can be determined depending on time. Methods: In this study, epoxomicin (Epo) was used to induce apoptosis in A375 melanoma cell line and apoptotic dose was determined by MTT method. In order to evaluate the early apoptotic process in a time-dependent manner, measurements were made at five different time points (0.5-6 hours) with a fixed Epo dose. DCFH-DA method was used to measure ROS, which is the most important stimulus of apoptosis. Bax amount was determined by Western Blot technique. Spectroscopic measurements were made with a back-reflection spectroscopy experiment setup consisting of spectrometer, tungsten-halogen light source and fiber optic probe. Apoptosis index values were determined from spectra. Results: Early spectral changes were determined with the spectra measured in the apoptosis time of melanoma cells. A significant difference was found in ROS measurements at 2, 4 and 6 hours compared to control. Cell viability was found to be 70% lower than control at 75 and 100 nM Epo doses after 24 hours. Time-dependent Bax levels were found to increase in all Epo groups as an indicator of apoptosis. Spectroscopic apoptosis index value was found to be compatible with ROS and Bax results at all groups. Conclusion: A new approach has been presented in which spectral changes occurring in the early stage of the apoptosis process in cell culture can be determined by back reflection spectroscopy. This approach has the potential to be developed in cell culture studies as a method that can monitor apoptosis over time without interfering with cell culture conditions.

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