Poliamin biyosentezi ve glikoliz yolaklarının inhibisyonunun pankreatik adenokarsinoma tedavisindeki etkinliğinin değerlendirilmesi
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Date
2024-01-10
Authors
Bozkurt, Aybike Sarıoğlu
Journal Title
Journal ISSN
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Publisher
Bursa Uludağ Üniversitesi
Abstract
Pankreas kanserlerinde 5-yıllık sağ kalım oranı yaklaşık %6-8 dolaylarında olup yeni tedavi yaklaşımlarına ihtiyaç duyulmaktadır. Pankreas kanserlerinin büyük çoğunluğunu oluşturan pankreatik duktal adenokarsinom (PDAK)'ların %90'ından fazlasında KRAS geninin aktive edici mutasyonlarına rastlanmaktadır. KRAS aktivasyonu PDAK hücrelerinin özgün metabolik bağımlılıklar geliştirmesine neden olur. Yapılan çalışmalar KRAS'ın glikoliz ve poliamin biyosentezi yolaklarının aktivitesini arttırdığını göstermektedir. Bu tez çalışmasında; KRAS mutasyonuna sahip olan ve metabolik olarak agresif oldukları bilinen PDAK hücre modellerinde glikolitik regülatör 6-fosforukto-2-kinaz/fruktoz-2,6-bisfosfataz-3 (PFKFB3) ve poliamin biyosentez yolağı hız sınırlayıcı enzimi ornitin dekarboksilaz (ODC1)'ın genetik ve farmakolojik yöntemler kullanılarak tekli ve kombine inhibisyonlarının hücrelerin in vitro metabolik, proliferatif ve onkojenik potansiyelleri üzerindeki etkisi araştırıldı. Çalışmada hücre modelleri olarak PANC1 ve MIA PaCa-2; genetik baskılama yöntemi olarak siRNA ve farmakolojik hedefleme olarak PFKFB3 için AZ PFKFB3 26 (AZP3-26) ve ODC1 için diflurometilornitin (DFMO) kullanıldı. qPCR, Western blot, F2,6BP ve metabolik analizler PDAK hücrelerinde PFKFB3 ve ODC1'nin birbirlerinin aktivitelerini düzenleyebildiğini gösterdi. PFKFB3 ve ODC1 aktivitelerinin siRNA ve özgün kimyasal inhibitörlerle birlikte baskılanması PDAK hücrelerinin proliferasyonunu tekli baskılamalara göre daha yüksek oranda azalttı. PFKFB3 ve ODC1 aktivitelerinin AZP3-26 ve DFMO ile inhibisyonu tekli kullanımlara göre her iki hücre hattının yumuşak agarda büyüme ve koloni oluşturma kapasitelerini önemli oranda düşürdü. Ayrıca PANC1 hücrelerinde, AZP3-26 ve DFMO'un birlikte kullanımı, tekli kullanımlara göre, hücrelerin Matrijel invazyon kapasitesini daha anlamlı olarak azalttı. Bu tez çalışması kapsamında elde edilen veriler; metabolik agresifliği ve plastisitesi ile öne çıkan PDAK tümörlerinde PFKFB3 ve ODC1 aktivitelerinin baskılanmasının in vitro olarak tekli inhibisyonlara göre daha kuvvetli anti-onkojenik etkiye sahip olabileceğini göstermektedir.
The 5-year survival rate for pancreatic cancer is approximately 6-8%, and new treatment approaches are needed. Activating mutations of the KRAS gene are found in more than 90% of pancreatic ductal adenocarcinomas (PDAC), which constitute the majority of pancreatic cancers. KRAS activation causes PDAC cells to develop unique metabolic dependencies. Studies show that KRAS increases the activity of glycolysis and polyamine biosynthesis pathways. In this thesis study; the effect of single and combined targeting the glycolytic glycolytic regulator 6- phosphoructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) and the polyamine biosynthesis pathway rate-limiting enzyme ornithine decarboxylase (ODC1) using genetic and pharmacological approaches on the metabolic, proliferative and oncogenic potential of mutant KRAS bearing PDAC cell models. PANC1 and MIA PaCa-2 were used as cell models; siRNA was used as a genetic suppression method and AZ PFKFB3 26 (AZP3-26) for PFKFB3 and difluromethylornithine (DFMO) for ODC1 were used as pharmacological targeting. qPCR, Western blot, F2,6BP and metabolic analyzes showed that PFKFB3 and ODC1 could regulate each other's activities in PDAK cells. Co-suppression of PFKFB3 and ODC1 activities with siRNA and specific chemical inhibitors reduced the proliferation of PDAK cells more than single suppressions. Inhibition of PFKFB3 and ODC1 activities with AZP3-26 and DFMO significantly reduced the growth and colony formation capacities of both cell lines in soft agar compared to single use. Additionally, in PANC1 cells, the combined use of AZP3-26 and DFMO reduced the Matrigel invasion capacity of the cells more significantly than single use. Data obtained within the scope of this thesis study suggest that combined targeting of PFKFB3 and ODC1 activities in PDAC tumors, which are known to exhibit elevated metabolic aggressiveness and plasticity, may have a stronger anti-oncogenic effect than single inhibitions in vitro.
The 5-year survival rate for pancreatic cancer is approximately 6-8%, and new treatment approaches are needed. Activating mutations of the KRAS gene are found in more than 90% of pancreatic ductal adenocarcinomas (PDAC), which constitute the majority of pancreatic cancers. KRAS activation causes PDAC cells to develop unique metabolic dependencies. Studies show that KRAS increases the activity of glycolysis and polyamine biosynthesis pathways. In this thesis study; the effect of single and combined targeting the glycolytic glycolytic regulator 6- phosphoructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) and the polyamine biosynthesis pathway rate-limiting enzyme ornithine decarboxylase (ODC1) using genetic and pharmacological approaches on the metabolic, proliferative and oncogenic potential of mutant KRAS bearing PDAC cell models. PANC1 and MIA PaCa-2 were used as cell models; siRNA was used as a genetic suppression method and AZ PFKFB3 26 (AZP3-26) for PFKFB3 and difluromethylornithine (DFMO) for ODC1 were used as pharmacological targeting. qPCR, Western blot, F2,6BP and metabolic analyzes showed that PFKFB3 and ODC1 could regulate each other's activities in PDAK cells. Co-suppression of PFKFB3 and ODC1 activities with siRNA and specific chemical inhibitors reduced the proliferation of PDAK cells more than single suppressions. Inhibition of PFKFB3 and ODC1 activities with AZP3-26 and DFMO significantly reduced the growth and colony formation capacities of both cell lines in soft agar compared to single use. Additionally, in PANC1 cells, the combined use of AZP3-26 and DFMO reduced the Matrigel invasion capacity of the cells more significantly than single use. Data obtained within the scope of this thesis study suggest that combined targeting of PFKFB3 and ODC1 activities in PDAC tumors, which are known to exhibit elevated metabolic aggressiveness and plasticity, may have a stronger anti-oncogenic effect than single inhibitions in vitro.
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Keywords
Pankreas duktal adenokarsinomu, ODC1, PFKFB3, DFMO, AZ PFKFB3 26, Pancreatic ductal adenocarcinoma
Citation
Bozkurt, A. S. (2024). Poliamin biyosentezi ve glikoliz yolaklarının inhibisyonunun pankreatik adenokarsinoma tedavisindeki etkinliğinin değerlendirilmesi. Yayınlanmamış doktora tezi. Bursa Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.