Glioblastoma multiforme ve glioblastoma multiforme kanser kök hücreleri ile astrosit ko-kültürlerinde, siklopamin inhibisyonu ile sonic hedgehog sinyal yolağı ve ilişkili mekanizmalarda karşılıklı bağlantıların moleküler yöntemlerle araştırılması
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Dosyalar
Tarih
2019
Yazarlar
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Yayıncı
Ege Üniversitesi, Tıp Fakültesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Kanser, bir grup hücrenin kontrolsüz bölünme, invazyon ve metastaz gibi özellikler sergileyerek oluşturduğu bir hastalıktır. Embriyogenez sırasında da kontrollü hücre bölünmesi, hücre farklılaşması, hücre göçü ve programlanmış hücre ölümü görülür. Embriyo gelişimi ve tümör oluşumu, hücre göçü, invazyon, hücre farklılaşması ve immün cevaptan kaçabilme gibi benzer hücre davranışları sergileyebildikleri gibi bu iki tip hücrenin gen ekspresyonu, protein profilleri, sinyal yolakları da benzerdir. Hedgehog gen ailesi üyelerinden Sonic Hedgehog (Shh) sinyal yolağı, hem embriyogenez hem de karsinogenezde aktif olabilen önemli sinyal yolaklarından biridir. Bu yolaktaki aktivasyon artışları ile medulloblastoma, rabdomyosarkoma, kolorektal karsinoma gibi birçok kanser tipi ortaya çıkabilir. Glioblastoma multiforme (GBM), erişkin insanlarda en sık görülen primer kötü huylu beyin tümörü olup uzak organ yayılımı olmasa da oldukça hızlı büyüyen infiltratif bir tümördür. Tümörler heterojen hücrelerden oluşmakta olup; tümör kitlesi içinde kök hücreler gibi kendini yenileme ve farklılaşma özellikleri bulunan, radyoterapi, kemoterapiye dirençli olan, uzak organ yayılımları yapabilen ve kanser rekürrensinde etkisi olduğu düşünülen kanser kök hücreleri (KaKH) de bulunmaktadır. GBM'li olguların tümör kitleleri içinde de kanser kök hücreleri tanımlanmıştır ve bu hücreler prognozun kötüleşmesine neden olabilirler. Son yıllarda GBM tedavisi için Hedgehog sinyal yolağı önemli bir hedef haline gelmeye başlamıştır. Siklopamin, Smoothened (Smo) reseptörüne bağlanarak Shh sinyal yolağının inhibe edilmesinde kullanılan ve hayvan deneyi çalışmalarında gliomalar, pankreatik ve kolon karsinomlarında tümör büyümesini de inhibe ettiği gösterilen önemli bir ajandır. GBM ile ilgili çeşitli ko-kültür ve mikro-çevre araştırmaları ise henüz yeterli değildir. Literatür bilgilerine dayanarak, Sonic Hedgehog sinyal yolağının embriyonik dönemde önemli bir rol oynadığı bilinmekte, kanser ve kanser kök hücrelerinde de aktif olduğu ve tedavi direncinde de etkili olabileceği düşünülmektedir. Literatür bilgileri ışığında, çalışmamız ile GBM ve GBM kanser kök hücrelerinin astrositler ile ko-kültüre edilmesi ve bu ortamda Shh sinyal yolağının etkisi ve bu yolağın inhibe edilerek sonuçların değerlendirilmesi planlanmıştır. Ko-kültür ortamında hücre-hücre etkileşimleri değerlendirilip GBM ve GBM kanser kök hücrelerinde aktive olan Shh sinyal yolağını astrosit varlığında siklopaminin inhibisyon etkisinde bir değişim olup olmadığı aynı zamanda hücrelerin parakrin olarah Shh protein salgılayabilme özellikleri araştırılarak hücre-hücre etkileşimlerinin de Shh sinyal yolağının siklopamin ile inhibisyonu üzerine olumlu ya da olumsuz etkisi olup olmadığı sorusuna cevap aranmıştır. Sonuç olarak GBM ve GBM kanser kök hücrelerinde Shh sinyal yolağı inhibe edildiğinde beraberinde etkilenebilecek apoptoz ve hücre siklusu gibi hücre içi mekanizmalarındaki değişimler PCR, immünofloresan, ELISA, TUNEL, hücre siklusu analizi gibi yöntemlerle araştırılmıştır. Siklopamin, Shh yolağını baskılamada etkili bir ajandır; tek başına yeterli derecede hücre ölümüne neden olamasa da; GBM ve GBM KaKH'lerinde Shh yolağı aktivasyonunun görülmesi, kombine kanser terapilerinde Shh yolağının baskılanması gerekliliği ortaya koymuştur. GBM ve GBM KaKH kontrol gruplarının sonuçları ve siklopaminden etkilenmeleri RNA ve protein düzeyinde birbirlerinden farklıdır. Bu hücreler ile ko-kültür uygulandığında çoğu zaman farklı sonuçlar elde edilmiştir. Yapılan çalışma sonucunda özellikle tekli hücre kültürü ve ko-kültür sonuçlarının farklılığı ele alındığında, hastalıklarda ve tedavilerinde mikro-çevre etkisinin önemi ortaya koyulmuştur.
Cancer is a disease caused by uncontrolled cell division, in which cells have the ability to invade and metastase. During embryogenesis, controlled cell division, differentiation, migration, and programmed cell death occur, as well. Embryo development and tumor formation comprimise similar behaviours of cells like cell migration, invasion, differentiation, and also escaping from immune system. Moreover, these two cell types are similar in gene expression, protein profiling and signalling pathways. Among the members of the Hedgehog gene family, Sonic Hedgehog (Shh) has the most prominent effects on the embryonic development at the brain and extremities. The Sonic Hedgehog signaling pathway is one of the important signaling pathways that active in both embryogenesis and carcinogenesis. Many cancer types, such as medulloblastoma, rhabdomyosarcoma, colorectal carcinoma, have increased activity of this pathway. Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adult humans and is a rapidly growing, infiltrating tumor, even if it does not metastase to the distant organs. Tumors are composed of a heterogeneous group of cells. Tumor stem cells, like stem cells, are characterized by self-renewal and differentiation and resistant to treatment regimens such as radiotherapy and chemotherapy besides, they can spread distant organs and are thought to be affected by cancer recurrence also found in tumor mass. Cancer stem cells have also been identified in tumor masses of patients with Glioblastoma multiforme, and these cells may cause worsening prognosis. In recent years, Hedgehog signaling pathway has become an important target for Glioblastoma multiforme treatment. Cyclopamine is an important agent used in the inhibition of the Shh signaling pathway by binding to the Smoothened (Smo) receptor and has shown to inhibit tumor growth in gliomas, pancreatic and colon carcinomas in animal studies. Various co-culture and microenvironmental researches related to GBM are not yet sufficient. Based on the literature, it is thought that the Sonic Hedgehog signaling pathway plays an important role in the embryonic period, is also active in cancer and cancer stem cells, and may also be effective in treatment resistance. In the context of literature information, we prospectively investigated the effect of Shh signaling pathway and its inhibition on GBM and GBM cancer stem cells cocultured with astrocytes. Cell-cell interactions in the co-culture conditions were evaluated to determine whether the Shh signaling pathway active in GBM and GBM cancer stem cells, will undergo a change in the inhibition effect of cyclopamine in the presence of astrocytes. At the same time, the ability of the cells to secrete Shh protein as paracrine signalling, and cell-cell interactions were expected to answer the question whether Shh signaling pathway has a positive or negative effect on cyclopamine inhibition. As a result, when the Shh signaling pathway was inhibited in GBM and GBM cancer stem cells, the differential changes in intracellular mechanisms such as apoptosis and cell cycle were examined by PCR, immunofluorescence, ELISA, TUNEL and cell cycle analysis. Although cyclopamine is an effective agent to suppress the Shh pathway, it does not cause sufficient cell death alone. Even so the presence of Shh pathway activation in GBM and GBM KaKH revealed the need to suppress Shh pathway in combination cancer therapies. Results of GBM and GBM KaKH control groups and cyclopamine effect on them are different at RNA and protein levels. Different results were often obtained when co-culture with these cells was obtained. As a result of this study, especially when the differences in single cell culture and co-culture results are considered, the importance of micro-environment effect in diseases and treatments has been revealed.
Cancer is a disease caused by uncontrolled cell division, in which cells have the ability to invade and metastase. During embryogenesis, controlled cell division, differentiation, migration, and programmed cell death occur, as well. Embryo development and tumor formation comprimise similar behaviours of cells like cell migration, invasion, differentiation, and also escaping from immune system. Moreover, these two cell types are similar in gene expression, protein profiling and signalling pathways. Among the members of the Hedgehog gene family, Sonic Hedgehog (Shh) has the most prominent effects on the embryonic development at the brain and extremities. The Sonic Hedgehog signaling pathway is one of the important signaling pathways that active in both embryogenesis and carcinogenesis. Many cancer types, such as medulloblastoma, rhabdomyosarcoma, colorectal carcinoma, have increased activity of this pathway. Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adult humans and is a rapidly growing, infiltrating tumor, even if it does not metastase to the distant organs. Tumors are composed of a heterogeneous group of cells. Tumor stem cells, like stem cells, are characterized by self-renewal and differentiation and resistant to treatment regimens such as radiotherapy and chemotherapy besides, they can spread distant organs and are thought to be affected by cancer recurrence also found in tumor mass. Cancer stem cells have also been identified in tumor masses of patients with Glioblastoma multiforme, and these cells may cause worsening prognosis. In recent years, Hedgehog signaling pathway has become an important target for Glioblastoma multiforme treatment. Cyclopamine is an important agent used in the inhibition of the Shh signaling pathway by binding to the Smoothened (Smo) receptor and has shown to inhibit tumor growth in gliomas, pancreatic and colon carcinomas in animal studies. Various co-culture and microenvironmental researches related to GBM are not yet sufficient. Based on the literature, it is thought that the Sonic Hedgehog signaling pathway plays an important role in the embryonic period, is also active in cancer and cancer stem cells, and may also be effective in treatment resistance. In the context of literature information, we prospectively investigated the effect of Shh signaling pathway and its inhibition on GBM and GBM cancer stem cells cocultured with astrocytes. Cell-cell interactions in the co-culture conditions were evaluated to determine whether the Shh signaling pathway active in GBM and GBM cancer stem cells, will undergo a change in the inhibition effect of cyclopamine in the presence of astrocytes. At the same time, the ability of the cells to secrete Shh protein as paracrine signalling, and cell-cell interactions were expected to answer the question whether Shh signaling pathway has a positive or negative effect on cyclopamine inhibition. As a result, when the Shh signaling pathway was inhibited in GBM and GBM cancer stem cells, the differential changes in intracellular mechanisms such as apoptosis and cell cycle were examined by PCR, immunofluorescence, ELISA, TUNEL and cell cycle analysis. Although cyclopamine is an effective agent to suppress the Shh pathway, it does not cause sufficient cell death alone. Even so the presence of Shh pathway activation in GBM and GBM KaKH revealed the need to suppress Shh pathway in combination cancer therapies. Results of GBM and GBM KaKH control groups and cyclopamine effect on them are different at RNA and protein levels. Different results were often obtained when co-culture with these cells was obtained. As a result of this study, especially when the differences in single cell culture and co-culture results are considered, the importance of micro-environment effect in diseases and treatments has been revealed.
Açıklama
Anahtar Kelimeler
Glioblastoma Multiforme, Sonic Hedgehog, Astrosit, Ko-Kültür, Siklopamin, Glioblastoma Multiforme, Sonic Hedgehog, Astrocyte, Co-Culture, Cyclopamine
