Pankreas kanseri ve tümör mikroçevresinin ökaryotik elongasyon faktörü 2 kinaz (EF2K) enzim inhibisyonu aracılığıyla hedeflenmesi
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Date
2017-02-24
Authors
Karakaş, Didem
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Publisher
Uludağ Üniversitesi
Abstract
Pankreatik kanser ortalama 6 aylık sağ kalım süresi ile en agresif ve ölümcül kanser türlerinden biridir. Kullanılan geleneksel tedavi yöntemleri sadece kanser hücrelerini hedeflemektedir. Ancak son yıllardaki çalışmalar ile tümör mikroçevresinin de tümör gelişimi, invazyonu, kemorezistans ve metastaz süreçlerinde aktif rol oynadığı bilinmektedir. Bu nedenle etkin bir tedavi için hem tümör hücrelerinin, hem de tümör mikroçevresinin hedeflenmesi gerekmektedir. Ökaryotik elongasyon faktörü 2 kinaz (EF2K), kanser hücrelerinde artmış aktivite gösteren ve stres koşullarında kanser hücrelerinin hayatta kalmasında rol alan bir enzimdir. EF2K pankreas kanserinde invazyon sürecinde rol almaktadır ve bu enzimin inhibisyonu kanser hücrelerinde apoptozisi tetiklemektedir. Literatürde EF2K ile tümör mikroçevresi arasındaki ilişkiyi gösteren bir çalışma bulunmamaktadır. Bu nedenle bu çalışmada tümör mikroçevresinin önemli hücre gruplarından biri olan makrofajlar ile PANC1 pankreatik kanser hücreleri arasındaki etkileşimin EF2K ekspresyonuna etkileri incelendi. Çalışmada PANC1 hücreleri ile monositik THP-1 hücreleri ve bu hücrelerden polarize edilen makrofajlar indirekt olarak kültüre edildi. Ardından PANC1 hücrelerinde EF2K ekspresyonu, hücre migrasyon ve invazyonundaki değişimler sırasıyla western blot, migrasyon ve invazyon testleri ile incelendi. PANC1 hücreleri ile monosit ve makrofajların kokültürü sonrası, tümör mikroçevresinin önemli kemokinlerinden olan monosit kemoatraktan-1 proteini (MCP-1) düzeyleri ELISA testi ile ölçüldü. MCP-1'in PANC1 hücrelerinde EF2K ekspresyonu ve migrasyon yetenekleri üzerine etkileri de incelendi. MCP-1'in monosit-makrofaj dönüşümü üzerindeki etkisi western blot ile incelendi. MCP-1 ile EF2K arasındaki etkileşimi incelemek amacıyla, EF2K overeksprese eden PANC1 hücreleri kullanılarak MCP-1 düzeyi western blot ile incelendi. Ayrıca, EF2K siRNA'sı kullanılarak PANC1 hücrelerinin koloni oluşturma, migrasyon ve invazyon yeteneklerinde ve MCP-1 ekspresyonundaki değişimler incelendi. EF2K inhibisyonunun in vivo etkilerini değerlendirmek için pankreatik ortotopik tümör modeli kullanıldı ve EF2K inhibisyonu sonrası MCP-1 düzeyi western blot ile incelendi. Tümöre infiltre olan protümörijenik makrofajlar mmünohistokimyasal boyama ile gösterildi. Sonuç olarak, makrofajlar ile PANC1 hücreleri arasındaki etkileşimin EF2K ve MCP-1 ekspresyonunu arttırdığı ve hücre migrasyon ve invazyonunda artışa neden olduğu bulundu. Ayrıca EF2K ile MCP-1 arasında iki yönlü bir etkileşim olduğu ve MCP-1'in monositlerin protümörijenik makrofajlara farklılaşmasına yol açtığı bulundu. EF2K'nin in vitro susturulması MCP-1 protein düzeyinde, hücre invazyon ve migrasyonunda azalmaya yol açtı. Ayrıca, EF2K inhibisyonu sonucu in vivo olarak tümör hacmi ve proliferasyonunda ve MCP-1 protein ekspresyonunda azalma olduğu gösterildi. EF2K inhibisyonu tümöre infiltre olan protümörijenik makrofaj sayısında da azalmaya yol açtı. Sonuç olarak, EF2K inhibisyonu ile hem kanser hücreleri hem de makrofajların hedeflenmesinin pankreatik kanser tedavisi için umut verici bir yaklaşım olabileceği sonucuna varıldı.
Pancreatic cancer is one of the most aggressive and deadliest cancer with 6 months average survival rate. Traditional therapies only target cancer cells. However, recent studies indicate that tumor microenvironment plays an important role during tumor progression, invasion, chemoresistance and metastasis. Therefore, both tumor cells and their microenvironment should be targeted for an effective treatment. Eukaryotic elongation factor 2 kinase (EF2K) is an enzyme which is overexpressed in cancer cells that plays a key role in cancer cell survival under stress conditions. EF2K participate in invasion of pancreatic cancer cells and inhibition of this enzyme triggers apoptosis in cancer cells. There is no study that have shown the relationship between EF2K and tumor microenvironment. Therefore, in this study it has been investigated that the interaction between macrophage (one of the most abundant cell type in tumor microenvironment) and PANC1 pancreatic cancer cells on EF2K expression. In this study, PANC1 cells were cultured with monocytic THP-1 cells and macrophages which were polarized from THP-1 cells. The changes in EF2K expression, cell migration and invasion were analyzed using western blot, migration and invasion assays, respectively. The effects of coculture of PANC1 cells with monocytes and macrophages on monocyte chemoattractant protein-1 (MCP-1) levels, which is one of the most important chemokines in tumor microenvironment were analyzed using ELISA. The role of MCP-1 on both EF2K and migration of PANC1 cells were investigated. The role of MCP-1 on monocyte-macrophage differentiation was also investigated. To determine the relationship between EF2K and MCP-1, EF2K was stably overexpressed in PANC1 cells and MCP-1 levels were measured using western blot. EF2K was silenced using siRNA and then the changes in MCP-1 expression levels, the ability of colony formation, migration and invasion of PANC1 cells were investigated. Pancreatic orthotopic tumor model was used to determine the in vivo effects of EF2K inhibition. Following EF2K inhibition, the changes in expression levels of MCP-1 was measured using western blot. The presence of tumor-infiltrated pro-tumorigenic macrophages were shown using immunohistochemical staining. As a result, it was found that the interaction between PANC1 cells and macrophages caused an increase in EF2K and MCP-1 proteins and this interaction accelarated cell migration and invasion. In addition, there was a bidirectional interaction between MCP-1 and EF2K. MCP-1 also caused differentiation of monocytes to pro-tumorigenic macrophages. In vitro silencing of EF2K decreased MCP-1 expression, cell invasion and migration. In vivo inhibition of EF2K also decreased MCP-1 expression, tumor volume and the number of tumor-infiltrated pro-tumorigenic macrophages. Consequently, targeting both tumor cells and macrohages through EF2K inhibition might be a promising strategy for pancreatic cancer treatment.
Pancreatic cancer is one of the most aggressive and deadliest cancer with 6 months average survival rate. Traditional therapies only target cancer cells. However, recent studies indicate that tumor microenvironment plays an important role during tumor progression, invasion, chemoresistance and metastasis. Therefore, both tumor cells and their microenvironment should be targeted for an effective treatment. Eukaryotic elongation factor 2 kinase (EF2K) is an enzyme which is overexpressed in cancer cells that plays a key role in cancer cell survival under stress conditions. EF2K participate in invasion of pancreatic cancer cells and inhibition of this enzyme triggers apoptosis in cancer cells. There is no study that have shown the relationship between EF2K and tumor microenvironment. Therefore, in this study it has been investigated that the interaction between macrophage (one of the most abundant cell type in tumor microenvironment) and PANC1 pancreatic cancer cells on EF2K expression. In this study, PANC1 cells were cultured with monocytic THP-1 cells and macrophages which were polarized from THP-1 cells. The changes in EF2K expression, cell migration and invasion were analyzed using western blot, migration and invasion assays, respectively. The effects of coculture of PANC1 cells with monocytes and macrophages on monocyte chemoattractant protein-1 (MCP-1) levels, which is one of the most important chemokines in tumor microenvironment were analyzed using ELISA. The role of MCP-1 on both EF2K and migration of PANC1 cells were investigated. The role of MCP-1 on monocyte-macrophage differentiation was also investigated. To determine the relationship between EF2K and MCP-1, EF2K was stably overexpressed in PANC1 cells and MCP-1 levels were measured using western blot. EF2K was silenced using siRNA and then the changes in MCP-1 expression levels, the ability of colony formation, migration and invasion of PANC1 cells were investigated. Pancreatic orthotopic tumor model was used to determine the in vivo effects of EF2K inhibition. Following EF2K inhibition, the changes in expression levels of MCP-1 was measured using western blot. The presence of tumor-infiltrated pro-tumorigenic macrophages were shown using immunohistochemical staining. As a result, it was found that the interaction between PANC1 cells and macrophages caused an increase in EF2K and MCP-1 proteins and this interaction accelarated cell migration and invasion. In addition, there was a bidirectional interaction between MCP-1 and EF2K. MCP-1 also caused differentiation of monocytes to pro-tumorigenic macrophages. In vitro silencing of EF2K decreased MCP-1 expression, cell invasion and migration. In vivo inhibition of EF2K also decreased MCP-1 expression, tumor volume and the number of tumor-infiltrated pro-tumorigenic macrophages. Consequently, targeting both tumor cells and macrohages through EF2K inhibition might be a promising strategy for pancreatic cancer treatment.
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Keywords
Pankreatik kanser, Tümör mikroçevresi, Makrofaj, EF2K, MCP-1, Pancreatic cancer, Tumor microenvironment, Macrophage
Citation
Karakaş, D. (2017). Pankreas kanseri ve tümör mikroçevresinin ökaryotik elongasyon faktörü 2 kinaz (EF2K) enzim inhibisyonu aracılığıyla hedeflenmesi. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.