Glioblastoma ve nöroblastoma tip beyin tümör hücre serileri üzerinde temozolomid ile paklitaksel, melatonin ve ergosterol ile oluşturulan kombine terapilerinin etkisi
Küçük Resim Yok
Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Trakya Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Glioblastoma multiforme (GBM) ve Nöroblastoma (NB), merkezi sinir sisteminin (MSS) en ölümcül habis tümör türleridir ve heterojenik özellikleri tedaviyi zorlaştırmaktadır. Kanserlerin heterojenliği ve karmaşıklığı nedeniyle, kombinasyon terapi rejimleri, ölümcül beyin tümörlerine karşı standart tedaviler haline gelmiştir. Temozolomid ve paklitaksel kombinasyonu, Glioblastoma için en yaygın kullanılan kemoterapi rejimidir. Ancak, tedavi edilen hastaların yaklaşık yarısında temozolomid direnci ve ciddi yan etkiler gelişir ve hastaların büyük bir kısmında sonuç olarak tedavi başarısız olur. Her ikisi de uzun süredir antitümör etkileri ile bilinen çok işlevli bir hormon melatonin ve doğal ürün olan ergosterol, tümörleri normal hücrelerden seçici şekilde etkileme yeteneği ile kombinasyon kanser tedavisinde büyük bir avantaja sahiptir. Bu tezde, kemoterapi ajanlarının tek başına IC50 dozlarını belirledikten sonra, temozolomid (TMZ) ile birlikte paklitaksel (PTX), melatonin (MLT) ve ergosterol (ERG) kombinasyonlarının, oksidatif stres, DNA hasarı, ısı şoku proteinleri, Nf-kb, Pı3k / Akt / mTOR ve apoptoz sinyal yolakları üzerinden Glioblastoma (C6) ve Nöroblastoma (N1E-115) hücre serilerinin in vitro progresyonu üzerinde inhibisyon etkileri araştırıldı. Hem C6 hem de N1E-115 hücre hatlarında sitotoksisiteyi indüklemek için tek başına ve PXT, MLT veya ERG (tümü IC50) ile kombinasyon halinde iki TMZ konsantrasyonu (IC50 ve 1/2 IC50) kullanıldı. Canlı, ölü ve apoptotik hücreler, Anneksin V:PI boyama ile belirlendi. Sinyal yollarına ait gen ifadeleri, kantitatif ters transkriptaz polimeraz zincir reaksiyonu (qRT-PCR) ile değerlendirildi ve ana proteinler western blot analizi ile belirlendi. IC50 seviyelerindeki tüm tedaviler, taşıt madde ile tedavi edilen kontrollere kıyasla hem Glioma hem de Nöroblastoma hücrelerinin canlılığını önemli ölçüde azalttı. TMZ'nin 1/2 IC50 konsantrasyonunda, özellikle melatonin kombinasyonu, kontrol ve diğer kombinasyon gruplarına kıyasla sinerjik etki ile önemli hücre ölümü gösterdi. Her iki hücre hattında da TMZ-MLT kombinasyonu oksidatif stres, DNA hasarı ve katlanmamış protein yanıtını indükledi. Nf-kb ve Pı3k / Akt ekseniyle ilişkili proteinler önemli ölçüde azaldı. TMZ (1/2 IC50) ile MLT ve ERG kombinasyon tedavileri, hücre döngüsü tutuklanması ve P53 ekspresyonlarının yanı sıra pro-apoptotik gen ve proteinleri (Bax, Kaspaz 3 ve 8) artırırken, Bcl-2 proteini önemli ölçüde azalttı. Bu moleküler sinyaller, C6 ve N1E-115 hücrelerinde hem apoptotik hem de nekrotik hücre ölümünü önemli ölçüde tetikledi. Ayrıca, bu etki seçiciydi ve tek başına TMZ tedavisi ile karşılaştırıldığında C8 sağlıklı astrosit hücre serilerinde önemli ölçüde yüksek hücre canlılığı belirlendi. Sonuçlarımız, özellikle MLT ve ikincil olarak ERG'ün, düşük doz TMZ ile kombinasyonunun, oksidatif stres ile ilişkili DNA-protein hasarı ve apoptoz arasında ortak bir hücre ölümü yukarı akış sinyalleri olarak hareket edebildiğini, bu sayede, hem Glioma hem de Nöroblastoma için TMZ-PXT tedavisine alternatif olan yeni seçici terapötik stratejilerin geliştirilmesinde kullanabileceğini önermektedir.
Glioblastoma multiforme (GBM) and Neuroblastoma (NB) is the deadliest malignant tumour types of the central nervous system (CNS) and their heterogenic properties make treatment difficult. Due to heterogeneity and complexity of cancers, combination regimens have become standard treatments against fatal brain tumours. Temozolomide and paclitaxel combination is the most widely used chemotherapy regime for Glioblastoma, however, approximately half of treated patients have temozolomide resistance and serious side effect and most of patients finally fail therapy. A multifunctional hormone, melatonin, and natural product, ergosterol, that both have long been known for its antitumor effects have a great advantage in combination cancer therapy with ability to differentially influence tumours from normal cells. In this thesis, after determining IC50 doses of chemotherapy agents alone, we investigated the effectiveness of a combination of temozolomide (TMZ) plus paclitaxel (PTX), melatonine (MLT) or ergosterol (ERG) on the inhibition of Glioma (C6) and Neuroblastoma (N1E-115) progress in vitro, via impact on oxidative stress, DNA damage, heat shock proteins, Nf-kb, Pı3k /Akt /mTOR and apoptosis signaling pathway. Two concentrations of TMZ (ie. IC50 and 1/2 IC50) alone, and in combination with PXT, MLT or ERG (all of IC50) were used to induce cytotoxicity in the both C6 and N1E-115 cell lines. Live, death and apoptotic cells were visualised by annexin V:PI staining. Relative gen expressions belong to signaling pathways were assessed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and key proteins determined by western blot assay. All treatments at IC50 levels significantly decreased the viability of both Glioma and Neuroblastoma cells compared to vehicle treated controls. At 1/2 IC50 concentration of TMZ, especially melatonin combination showed significant cell death with synergistic effect compared to control and other combination groups. In both cell lines, TMZ-MLT combination induce oxidative stress, DNA damage and unfolded protein response. Nf-kb and Pı3k / Akt axis-related proteins were significantly decreased. MLT and ERG in combination with TMZ (1/2 IC50) increased cell cycle arrest, P53 expressions as well as pro-apoptotic gene and proteins (Bax, Caspase 3 and 8), while Bcl-2 protein was significantly decreased. This molecular signals significantly triggered both apoptotic and necrotic cell death in C6 and N1E-115 cells. Furthermore, this effect was selective, and significantly high cell viability determined in C8 healthy astrocyte cell lines compared to TMZ alone. Our result suggests that, especially MLT and secondarily ERG in combination with low dose TMZ may act as a common cell death upstream signals among oxidative stress related DNA-protein damage and apoptosis, which may lead to the development of new selective therapeutic strategies that alternative for TMZ-PXT treatment for both Glioma and Neuroblastoma.
Glioblastoma multiforme (GBM) and Neuroblastoma (NB) is the deadliest malignant tumour types of the central nervous system (CNS) and their heterogenic properties make treatment difficult. Due to heterogeneity and complexity of cancers, combination regimens have become standard treatments against fatal brain tumours. Temozolomide and paclitaxel combination is the most widely used chemotherapy regime for Glioblastoma, however, approximately half of treated patients have temozolomide resistance and serious side effect and most of patients finally fail therapy. A multifunctional hormone, melatonin, and natural product, ergosterol, that both have long been known for its antitumor effects have a great advantage in combination cancer therapy with ability to differentially influence tumours from normal cells. In this thesis, after determining IC50 doses of chemotherapy agents alone, we investigated the effectiveness of a combination of temozolomide (TMZ) plus paclitaxel (PTX), melatonine (MLT) or ergosterol (ERG) on the inhibition of Glioma (C6) and Neuroblastoma (N1E-115) progress in vitro, via impact on oxidative stress, DNA damage, heat shock proteins, Nf-kb, Pı3k /Akt /mTOR and apoptosis signaling pathway. Two concentrations of TMZ (ie. IC50 and 1/2 IC50) alone, and in combination with PXT, MLT or ERG (all of IC50) were used to induce cytotoxicity in the both C6 and N1E-115 cell lines. Live, death and apoptotic cells were visualised by annexin V:PI staining. Relative gen expressions belong to signaling pathways were assessed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and key proteins determined by western blot assay. All treatments at IC50 levels significantly decreased the viability of both Glioma and Neuroblastoma cells compared to vehicle treated controls. At 1/2 IC50 concentration of TMZ, especially melatonin combination showed significant cell death with synergistic effect compared to control and other combination groups. In both cell lines, TMZ-MLT combination induce oxidative stress, DNA damage and unfolded protein response. Nf-kb and Pı3k / Akt axis-related proteins were significantly decreased. MLT and ERG in combination with TMZ (1/2 IC50) increased cell cycle arrest, P53 expressions as well as pro-apoptotic gene and proteins (Bax, Caspase 3 and 8), while Bcl-2 protein was significantly decreased. This molecular signals significantly triggered both apoptotic and necrotic cell death in C6 and N1E-115 cells. Furthermore, this effect was selective, and significantly high cell viability determined in C8 healthy astrocyte cell lines compared to TMZ alone. Our result suggests that, especially MLT and secondarily ERG in combination with low dose TMZ may act as a common cell death upstream signals among oxidative stress related DNA-protein damage and apoptosis, which may lead to the development of new selective therapeutic strategies that alternative for TMZ-PXT treatment for both Glioma and Neuroblastoma.
Açıklama
Doktora
Anahtar Kelimeler
Tıbbi Biyoloji, Medical Biology
