Meme Kanseri ve Hepatosellüler Karsinom Hücre Dizilerinde Serum Starvasyonu ve Hipoksik Ortam Koşullarının Metabolik Yolak Protein Ekspresyonlarına Etkisinin İncelenmesi
Amaç: Adenozin monofosfat (AMP) aktive edici protein kinaz (AMPK), hücrenin çeşitli metabolik stres durumlarında aktive olarak hücredeki enerjihomeostazını sağlayan önemli bir serin/treonin protein kinazdır. Besin azlığı ve hipoksi gibi stres koşullarında organizmadaki enerji miktarının düşükolduğu veya enerji tüketiminin yüksek olduğu durumlarda hücresel AMP miktarı artmakta ve AMPK aktivasyonu ile birlikte yağ asidi oksidasyonu veglikolizis gibi katabolik reaksiyonlar indüklenmektedir. Kanser hücrelerinin tümör mikro çevresinde en çok maruz kaldığı durumların oksijen ve besinyetersizliği olduğu raporlanmıştır. Bu çalışmada, kanser hücrelerinin çeşitli stres koşullarında AMPK regülasyonu sayesinde bu stres koşullarına karşıgeliştirdiği epitelyal-mezenkimal-dönüşüm epitelial mezenkimal geçiş ve metastaz gibi adaptasyon mekanizmaları ile sağkalım avantajı sağlamasınınaraştırılması amaçlanmıştır.Gereç ve Yöntem: Meme kanseri (SKBR-3, MDA-MB-453) ve hepatosellüler karsinom (HepG2, Huh-7) hücre hatlarının serum starvasyon koşulundap-AMPK, total AMPK; hipoksik ortam koşullarında HIF-1α, E-kadherin, p-AMPK ve total AMPK ekspresyonları Western-blot yöntemi ile tespitedilmiştir. Stres koşullarının kanser hücrelerinde metabolik yolak ve invazyonla ilişkili protein regülasyonlarına etkisi incelenmiştir. Bu sayede, in vivotümör mikroçevresi stres koşullarının simülasyonu aracılığıyla bu stres faktörlerinin in vitro deneysel koşullardaki etkileri araştırılmıştır.Bulgular: Çalışmadan elde edilen bulgular, starvasyon ve hipoksi koşullarının hem kanser hücresi metabolik regülasyonu hem de tümör progresyonuüzerinde belirgin ve istatistiksel olarak anlamlı etki yaratabileceğini göstermektedir. AMPK gen ekspresyonu yüksek olan hücrelerde (Huh7, SKBR-3),hücresel stres ile birlikte AMPK’nin regüle olduğu ve bu regülasyonun hücreye adaptasyon avantajı sağladı sonucuna ulaşılmıştır.Sonuç: Bu çalışmanın sonuçları, kanser hücre stres mekanizmalarına ve AMPK’ye yönelik terapötik stratejilerin belirlenebilmesine katkı sağlamaktave ileride yapılacak in vivo araştırmalar için temel oluşturarak literatüre katkı sağlamaktadır.
Evaluation of the Effects of Serum Starvation and Hypoxic Conditions on Metabolic Pathway Protein Expressions in Breast and Hepatocellular Cancers
Objectives: Adenosine monophosphate (AMP) activating-protein-kinase (AMPK) is a crucial serine/threonine protein kinase that is activated in cellular metabolic stress conditions to maintain cellular energy-homeostasis. Under conditions of stress such as starvation and hypoxia, when the amount of energy in the organism is low or energy consumption is increased, the amount of cellular AMP increases and catabolic reactions such as fatty acid oxidation and glycolysis are induced due to AMPK activation. The most common conditions that cancer cells face in tumormicroenvironment were reported to be nutrient deficiency and lack of oxygen. This study aims to investigate whether cancer cells bear survival advantages with mechanisms of adaptation such as epithelial-mesenchymal-transition and metastasis by the regulation of AMPK under various stress conditions. Materials and Methods: p-AMPK, total-AMPK protein expression levels of breast (SKBR-3, MDA-MB-453) and hepatocellular cancer (HepG2, Huh-7) cell lines under conditions of serum starvation were determined by Western-blot method. In addition, HIF-1α, E-cadherin, p-AMPK and total-AMPK protein expression levels under conditions of hypoxia were also determined by Western-blot method. The effects of stress conditions on regulation of proteins associated with metabolic pathway and invasion in cancer cells were investigated. As a result, the in vitro effects of stress factors which are observed in tumor-microenvironment in vivo were experimentally investigated by this simulation approach. Results: Our findings demonstrate that conditions of starvation and hypoxia may show prominent and statistically significant effects on both cancer cell metabolic regulation and tumor progression. AMPK was shown to be regulated as a result of cellular stress in cancer cells (Huh7, SKBR3) with high AMPK gene expression. Such a regulation seems to provide an adaptive advantage to those cells. Conclusion: Our results will contribute to determining therapeutic strategies targeting cancer cell stress mechanisms and AMPK. In addition, the results will pave the way for future in vivo studies; thus, will contribute to the literature.
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