Publication: Apoptotik hücre ölümüne dirençli glioblastome multiforme hücreleri üzerine mezenkimal kök
hücre ve çeşitli inhibitörlerin etkisinin in vitro incelenmesi
Abstract
Erişkin beyin tümörleri arasında en sık görülen, oldukça agresif seyreden ve çoğunlukla tedaviye dirençli bir tümör olan Gliobastoma Multiforme (GBM)’ nin tedavisinde cerrahi, kemoterapi ve radyoterapiye ilaveten yeni ve daha etkili tedavi yöntemlerinin araştırılıp bulunması gerekli hale gelmiştir. Bu doğrultuda her dokuda bulunan ve kendisini yenileyip farklılaşan, ayrıca tümör hücrelerine migrasyon göstererek genetik olarak tümör hücresini öldüren ajanlarla yüklenebilen kök hücreler anti-tümöral tedavide yeni bir yaklaşım yöntemi olarak ortaya çıkmaktadır. Anti-tümöral tedavi amaçlı deneysel çalışmalarda kanser hücrelerinde apoptozu indükleyen ve normal hücreleri etkilemeyen tümör nekroz faktör (TNF) ailesinin bir üyesi olan TNF aracılı apoptoz indükleyici ligand (TRAIL) önemli bir avantaj sağlamaktadır. Ancak çoğu kanser türünde TRAIL’ e karşı apoptoz inhibitör protein (IAP) ailesi aracılı direnç gelişmektedir. Bu çalışmada, U-251GBM hücreleri ile adipoz doku kökenli insan mezenkimal kök hücreleri (AK- MKH) kullanıldı. Tümör hücrelerinde apoptozu indükleyen TRAIL kök hücrelere transfekte edildi. Bunun yanı sıra tümör hücrelerinin TRAIL’ e karşı direncini kırmak için, histon deasetilaz inhibitörlerinden Suberoylanilid hydroksamik asit (SAHA) ve apoptoz inhibitör protein ailesinin bir üyesi olan X’ e bağlı apoptozu inhibe eden protein (XIAP) inhibitörü olarak bilinen Embelin kullanıldı. Glioblastom hücre hattına uygulanacak olan Embelin ve SAHA’ nın IC50 değerini tespit etmek amacıyla WST-1 canlılık tayini yapıldı. Belirlenen dozlar glioblastom hücrelerine uygulandı ve 24 saat inkübe edildi. TRAIL yüklü kök hücreler ile Embelin ve SAHA’ nın belirlenen dozları uygulanmış glioblastom hücreleri 24 saat birlikte kültüre edildiler. İki grup için de flow sitometri cihazında Annexin V/ PI yöntemiyle hücre canlılığı, ‘cell cycle’ yöntemiyle hücre döngüsündeki değişimler kantitatif olarak belirlendi. Çalışmaların sonucunda, genetik olarak TRAIL ekspresyonu artırılmış mezenkimal kök hücrelerin glioblastoma hücreleri üzerinde apoptozu indükleyerek hücre canlılığını etkilediğini ve glioblastom hücrelerinin proliferasyonunu durdurduğu gösterilmiştir. oblastom, Embelin, SAHA, Mezenkimal kök hücre, TRAIL III
Glioblastoma Multiforme is the most frequent primary brain tumor in adults. It is very aggressive and resistant to treatment. Therefore, development of new and more effective therapies besides surgery, chemotherapy and radiotherapy is necessary. In that way, stem cells become significant because stem cells can be found in every tissue, can regenerate on their own and can be transfected with the agents which genetically kill the tumor cells. Stem cells are known to have affinity to tumor cells via molecules that are secreted from tumor cells. Thus a new anti-tumoral treatment method have been developed by using stem cells that transfected with specific genes and proteins which induce apoptosis in tumor cells. TRAIL, the member of the TNF family, induces the apoptosis in cancer cells where as it does not affect healthy cells. But most kind of cancer cells have resistance to TRAIL and inhibitor of apoptosis proteins (IAP) play a significant role in this resistance. In this study, glioblastoma cells and human adipose derived mesenchymal stem cells (AD- MSC) were used. Stem cells were transfected with TRAIL, which induces apoptosis in tumor cells. But it is known that tumor cells develop resistance to TRAIL easily. To overcome this resistance of tumor cells, Suberoylanilide hydroxamic acid (SAHA) as histone deacetylase inhibitor and Embelin as X-linked inhibitor of apoptosis (XIAP) inhibitor were used. Firstly WST-1 viability assay was performed to find out the IC50 level of Embelin and SAHA in glioblastoma cells. Following the determination of these doses, glioblastoma cells were incubated for 24 hours. These glioblastoma cells were cultured with TRAIL transfected stem cells for 24 hours together with the agent. Viability and cell cycle of both groups were determined by Annexin V/ PI and cell cycle method via flow cytometry. As a result genetically engineered AD-MSCs those express increased amount of TRAIL induced apoptosis in GBM cells and decreased their proliferation. oblastoma, Embelin, SAHA, Mesenchymal stem cells, TRAIL
Glioblastoma Multiforme is the most frequent primary brain tumor in adults. It is very aggressive and resistant to treatment. Therefore, development of new and more effective therapies besides surgery, chemotherapy and radiotherapy is necessary. In that way, stem cells become significant because stem cells can be found in every tissue, can regenerate on their own and can be transfected with the agents which genetically kill the tumor cells. Stem cells are known to have affinity to tumor cells via molecules that are secreted from tumor cells. Thus a new anti-tumoral treatment method have been developed by using stem cells that transfected with specific genes and proteins which induce apoptosis in tumor cells. TRAIL, the member of the TNF family, induces the apoptosis in cancer cells where as it does not affect healthy cells. But most kind of cancer cells have resistance to TRAIL and inhibitor of apoptosis proteins (IAP) play a significant role in this resistance. In this study, glioblastoma cells and human adipose derived mesenchymal stem cells (AD- MSC) were used. Stem cells were transfected with TRAIL, which induces apoptosis in tumor cells. But it is known that tumor cells develop resistance to TRAIL easily. To overcome this resistance of tumor cells, Suberoylanilide hydroxamic acid (SAHA) as histone deacetylase inhibitor and Embelin as X-linked inhibitor of apoptosis (XIAP) inhibitor were used. Firstly WST-1 viability assay was performed to find out the IC50 level of Embelin and SAHA in glioblastoma cells. Following the determination of these doses, glioblastoma cells were incubated for 24 hours. These glioblastoma cells were cultured with TRAIL transfected stem cells for 24 hours together with the agent. Viability and cell cycle of both groups were determined by Annexin V/ PI and cell cycle method via flow cytometry. As a result genetically engineered AD-MSCs those express increased amount of TRAIL induced apoptosis in GBM cells and decreased their proliferation. oblastoma, Embelin, SAHA, Mesenchymal stem cells, TRAIL