Hippo sinyal yolağı efektör molekülü YAP1'in inhibisyonunun in vitro beyin kanseri ve beyin kanseri kök hücrelerindeki etkisinin araştırılması
Küçük Resim Yok
Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Glioblastoma yetişkinlerde daha sık görülen ve en agresif primer beyin tümörüdür. Standart tedavi protokollerinin 12-15 ay süre ile uygulandığı hastaların sadece %3-5'inin sağ kalım süresi 5 yıla kadar uzayabilmektedir. Glioblastoma tedavisinde eşzamanlı radyokemoterapi ile yoğun klinik protokollere rağmen cerrahide hiçbir küratif tedavi mevcut değildir. Aslında nüks esas olarak lokal invazivlik, radyasyon terapisindeki terapötik başarısızlık ve yetersiz ilaç yanıtının yanı sıra kanser kök hücre popülasyonunun varlığından dolayı meydana gelmektedir. İlk olarak Drosophila'da tanımlanan ve yüksek oranda korunmuş Hippo sinyal iletim yolağı, organ boyutu, hücre büyümesi ve apoptozun güçlü bir düzenleyicisidir. Hippo yolunun ana efektörü olan Yes ile ilişkili protein (YAP), transkripsiyon koaktivatörü olarak işlev görerek organ boyutu kontrolünde önemli bir rol oynar. Glioma, hepatoselüler karsinom, prostat kanseri, kolon kanseri, yumurtalık kanseri ve meme kanseri üzerinde yapılan bir çok çalışmada YAP aşırı ifadesinin onkogenik bir rolü olduğu gösterilmiştir. Bu çalışmada agresif beyin kanserlerinden olan glioblastomalarda aşırı ifade edildiği bilinen YAP1'in alternatif tedavi hedefi olma potansiyelinin belirlenebilmesi amacıyla beyin kanseri ve beyin kanser kök hücrelerinde siRNA ile hedeflenerek susturulması sağlanmış ve yeni geliştirilen bir YAP1 inhibitörü CA3 (CIL56)' ün terapotik potansiyeli ileri moleküler analizlerle in vitro olarak araştırılmıştır. Araştırma beyin kanseri, beyin kanseri kök hücresi, sağlıklı beyin kök hücresi ve insan beyin mikrovasküler endoteliyal hücre modelleri üzerinde gerçekleştirilmiştir. CA3 (CIL56) ve YAP1 siRNA'sının hücre hatları üzerindeki apoptotik etkileri Annexin V–FITC testi, ferroptoz testi (GPX aktivitesi), hücre döngüsü analizi ile belirlenmiştir. Uygulamaların hücre hareketi üzerine olan etkileri invazyon testi, migrasyon testi, adezyon testi ve epiteliyal mezenşimal transisyon belirteçleri değerlendirilerek belirlenmiştir. Köklülük üzerine olan etkileri değerlendirmek için sferoid oluşturma deneyi yapılmıştır. Ayrıca bu süreçlerde rol alan anahtar genlerin ekspresyon düzeyindeki değişimleri qRT-PCR ile değerlendirilmiştir. YAP1 inhibisyonu protein düzeyinde western blot analizi ile gösterilmiştir. Araştırmadan elde ettiğimiz bulgular sonucunda YAP1'in siRNA ve inhibitör CA3(CIL56) aracılı inhibisyonunun canlılık, motilite ve köklülük özellikleri üzerine eşit düzeyde olmasa da kuvvetli anti-tümöral etkiler göstererek, umut vadeden terapotik bir aday olabileceği gösterilmiştir.
Glioblastoma is the most common and most aggressive primary brain tumor in adults. The survival time of only 3-5% of the patients in whom standard treatment protocols are applied for 12-15 months is extended up to 5 years. Despite intensive clinical protocols with concomitant radiochemotherapy for the treatment of glioblastoma, no curative treatment is available in surgery. In fact, recurrence mainly occurs due to local invasiveness, therapeutic failure in radiation therapy, and drug response, as well as the presence of the cancer stem cell population. The Hippo signal transduction pathway, first identified in Drosophila, is a highly conserved, potent regulator of organ size, cell growth, and apoptosis. Yes-associated protein (YAP), a major effector of the hippo pathway, plays an important role in organ size control by acting as a transcriptional coactivator. Many studies on glioma, hepatocellular carcinoma, prostate cancer, colon cancer, ovarian cancer and breast cancer have shown that YAP overexpression has an oncogenic role. In this study, targeted silencing of YAP1 with siRNA was achieved in order to determine its potential as an alternative therapy target in glioblastomas, and the therapeutic potential of a newly developed YAP1 inhibitor CA3 (CIL56) was investigated in vitro with advanced molecular analyzes. The research was carried out on brain cancer, brain cancer stem cell, healthy brain stem cell and human brain microvascular endothelial cell models. The cytotoxic effects of CA3 (CIL56) and YAP1 siRNA on cell lines were determined by Annexin V-FITC test, ferroptosis test (GPX activity), cell cycle analysis. The effects of the treatments on cell movement were determined by evaluating the invasion test, migration test, adhesion test and EMT markers. Spheroid formation experiment was conducted to evaluate the effects on stemness. In addition, the changes in the expression level of key genes involved in these processes were evaluated by qRT-PCR. Inhibition of YAP1 was demonstrated at the protein level by western blot analysis. As a result of the findings we obtained from the study, it has been shown that YAP1 may be a promising therapeutic candidate by showing strong anti-tumoral effects, although not at equal levels, on siRNA and inhibitor CA3(CIL56)-mediated inhibition on viability, motility and stemness.
Glioblastoma is the most common and most aggressive primary brain tumor in adults. The survival time of only 3-5% of the patients in whom standard treatment protocols are applied for 12-15 months is extended up to 5 years. Despite intensive clinical protocols with concomitant radiochemotherapy for the treatment of glioblastoma, no curative treatment is available in surgery. In fact, recurrence mainly occurs due to local invasiveness, therapeutic failure in radiation therapy, and drug response, as well as the presence of the cancer stem cell population. The Hippo signal transduction pathway, first identified in Drosophila, is a highly conserved, potent regulator of organ size, cell growth, and apoptosis. Yes-associated protein (YAP), a major effector of the hippo pathway, plays an important role in organ size control by acting as a transcriptional coactivator. Many studies on glioma, hepatocellular carcinoma, prostate cancer, colon cancer, ovarian cancer and breast cancer have shown that YAP overexpression has an oncogenic role. In this study, targeted silencing of YAP1 with siRNA was achieved in order to determine its potential as an alternative therapy target in glioblastomas, and the therapeutic potential of a newly developed YAP1 inhibitor CA3 (CIL56) was investigated in vitro with advanced molecular analyzes. The research was carried out on brain cancer, brain cancer stem cell, healthy brain stem cell and human brain microvascular endothelial cell models. The cytotoxic effects of CA3 (CIL56) and YAP1 siRNA on cell lines were determined by Annexin V-FITC test, ferroptosis test (GPX activity), cell cycle analysis. The effects of the treatments on cell movement were determined by evaluating the invasion test, migration test, adhesion test and EMT markers. Spheroid formation experiment was conducted to evaluate the effects on stemness. In addition, the changes in the expression level of key genes involved in these processes were evaluated by qRT-PCR. Inhibition of YAP1 was demonstrated at the protein level by western blot analysis. As a result of the findings we obtained from the study, it has been shown that YAP1 may be a promising therapeutic candidate by showing strong anti-tumoral effects, although not at equal levels, on siRNA and inhibitor CA3(CIL56)-mediated inhibition on viability, motility and stemness.
Açıklama
Anahtar Kelimeler
YAP1, CA3 (CIL56), siRNA, Beyin Kanser Kök Hücresi, GBM, YAP1, CA3 (CIL56), siRNA, Brain Cancer Stem Cell, GBM
