Glikoz açlığının A549 ve beas-2B hücre hatlarında hücre canlılığı parametrelerine olan etkisi
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Date
2019
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Hatay Mustafa Kemal Üniversitesi
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info:eu-repo/semantics/openAccess
Abstract
Kanser hücreleri artan glikoz kullanımı ile karakterize, metabolik değişim geçirmiş hücrelerdir. Kanser hücrelerinin metabolizmasını anlamak, kanser tedavisinde oldukça büyük önem arz etmektedir. Çalışmamızın amacı, glikoz açlığının sağlıklı ve kanser hücrelerinde, hücre canlılığı parametreleri üzerindeki etkilerini belirleyerek, kanser tedavisini desteklemektir. Çalışmamızda; MTT testi ile 8, 16 ve 24 saatlik açlığın hücre canlılığına etkisi araştırıldı ve ileriki deneyler için 8 ve 24 saatlik açlık süreleri seçildi. Açlık süreleri sonucu; hücre canlılığı, hücre döngüsü, kuisens, senesens, apoptoz ve otofaji yolaklarında görevli mTOR, p21, kaspaz 3, beclin 1 ve ATG12 genlerinin ekspresyon seviyeleri qRT-PCR yöntemi ile belirlendi. Hücrelerde senesens olayı SA-β-Gal boyama ile görüntülendi Sonuçlarımız, açlığın kanser ve sağlıklı hücrelerde canlılığı düşürdüğünü göstermiştir. Ancak, BEAS-2B'den farklı olarak A549 kanser hücrelerinde açlık süresinin artışı hücre canlılığını önemli ölçüde düşürdü. SA-β-Gal boyama sonucu, 8 ve 24 saatlik açlıkta A549 hücresinde BEAS2B'ye göre daha fazla senesent hücre saptadık. Her iki hücre hattında artan açlık süresi ile p21 ekspresyon seviyesi artmış, hücre döngüsünü engellemiştir. MTT analizi ve SA-β-Gal boyama sonuçları göz önüne alındığında, kanser hücrelerinde açlığa bağlı hücre döngüsü inhibisyonunun sağlıklı hücrelere kıyasla daha fazla olduğu söylenebilir. Senesens profili kanser hücrelerinde 8 saat açlıkta artan mTOR ekspresyonu ile desteklenmiştir. mTOR mRNA seviyesi 8 saat açlıkta BEAS-2B hücrelerinde azalırken, 24 saat açlıkta artış göstermiştir. Otofajide, 8 saatlik açlıkta A549 hücresinde beclin 1 ekspresyonu artarken, BEAS-2B hücresinde azalmıştır. 24 saatlik açlıkta ise iki hücre hattında da ATG12 artışıyla otofaji tetiklenmiştir. Her iki hücre hattında 24 saat açlıkta apoptotik markır kaspaz 3'te artış görülmüştür. Fakat kaspaz 3'teki bu kat artışı sağlıklı hücrelere kıyasla kanser hücrelerinde daha fazladır. 8 saatlik açlıkta ise BEAS2B hücrelerinde kaspaz 3 değişmemiştir. Sonuç olarak bu çalışmamızda, artan glikoz açlığı süresi ile beraber sağlıklı ve tümör hücrelerinin hücre canlılığı parametrelerinin olumsuz yönde etkilendiğini gözlemledik. Sonuçlarımız, glikoz açlığının kanser hücrelerinde, sağlıklı hücrelere kıyasla daha olumsuz etkilere sahip olduğunu göstermektedir
Cancer cells are metabolically altered cells which are characterized by elevated rates of glucose uptake. Understanding the metabolism of cancer cells has a pivotal importance in cancer treatment. The aim of our study is to analyze the effects of glucose starvation on cell viability parameters in both healthy and cancer cells and to support cancer treatment. In our study, the effects of 8, 16 and 24 hour-long starvation on cell viability were determined with MTT assay and 8 and 24 hour-long starvation periods were selected for further experiments. After 8 and 24 hours of starvation, the gene expression levels of mTOR, p21, caspase 3, beclin 1, and ATG12, which play role in several pathways e.g. cell survival, cell cycle, quiescence, senescence, apoptosis, autophagy, were evaluated with qRT-PCR analysis. The senescence within the cells was detected by using SA-β-Gal staining assay. Our results showed that starvation significantly decreased cell viability in tumor and healthy cells. However, prolonging starvation time significantly reduced cell viability in A549 cancer cells unlike BEAS-2B cells. After SA-β-Gal staining, we detected more senescent cells in A549 than in BEAS-2B at 8- and 24-hour starvations. In both cell lines,extending starvation time also augmented p21 gene expressionlevels and halted cell cycle progression. Considering the outcomes of MTT and SA-β-Gal assays it can be stated that the cell cycle inhibition rate upon starvation in cancer cells is more than the healthy cells. The senescence profile was also supported by the increased mTOR expression in cancer cells at 8 hours starvation. While mTOR decreased in BEAS-2B cells at 8 hours, it went up at 24 hours starvation. Regarding autophagy, the beclin 1 expression at 8 hours starvation increased in A549 cells while it decreased in BEAS-2B cells. At 24 hours starvation, autophagy was trigerred in both cell lines by elevated levels of ATG12. We found an increase in caspase 3 expression at 24 hours starvation in both cell lines. However, this fold increase of caspase 3 was higher in cancer cells compared to the one in healthy cells. At 8 hours starvation, caspase 3 expression did not change in BEAS-2B cells while it significantly increased in A549 cells. As a result, we observed in this study that the cell viability parameters of both healhy and cancer cells were negatively affected with increasing glucose starvation. Our results showed that glucose starvation had more negative effects on cancer cells than healthy cells.
Cancer cells are metabolically altered cells which are characterized by elevated rates of glucose uptake. Understanding the metabolism of cancer cells has a pivotal importance in cancer treatment. The aim of our study is to analyze the effects of glucose starvation on cell viability parameters in both healthy and cancer cells and to support cancer treatment. In our study, the effects of 8, 16 and 24 hour-long starvation on cell viability were determined with MTT assay and 8 and 24 hour-long starvation periods were selected for further experiments. After 8 and 24 hours of starvation, the gene expression levels of mTOR, p21, caspase 3, beclin 1, and ATG12, which play role in several pathways e.g. cell survival, cell cycle, quiescence, senescence, apoptosis, autophagy, were evaluated with qRT-PCR analysis. The senescence within the cells was detected by using SA-β-Gal staining assay. Our results showed that starvation significantly decreased cell viability in tumor and healthy cells. However, prolonging starvation time significantly reduced cell viability in A549 cancer cells unlike BEAS-2B cells. After SA-β-Gal staining, we detected more senescent cells in A549 than in BEAS-2B at 8- and 24-hour starvations. In both cell lines,extending starvation time also augmented p21 gene expressionlevels and halted cell cycle progression. Considering the outcomes of MTT and SA-β-Gal assays it can be stated that the cell cycle inhibition rate upon starvation in cancer cells is more than the healthy cells. The senescence profile was also supported by the increased mTOR expression in cancer cells at 8 hours starvation. While mTOR decreased in BEAS-2B cells at 8 hours, it went up at 24 hours starvation. Regarding autophagy, the beclin 1 expression at 8 hours starvation increased in A549 cells while it decreased in BEAS-2B cells. At 24 hours starvation, autophagy was trigerred in both cell lines by elevated levels of ATG12. We found an increase in caspase 3 expression at 24 hours starvation in both cell lines. However, this fold increase of caspase 3 was higher in cancer cells compared to the one in healthy cells. At 8 hours starvation, caspase 3 expression did not change in BEAS-2B cells while it significantly increased in A549 cells. As a result, we observed in this study that the cell viability parameters of both healhy and cancer cells were negatively affected with increasing glucose starvation. Our results showed that glucose starvation had more negative effects on cancer cells than healthy cells.
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Keywords
Genetik, Genetics ; Tıbbi Biyoloji
