Nöroblastoma kanser hücre hatlarında rottlerin ve genistein'in hücre proliferasyonu, invazyonu ve hücre ölümü/siklusu üzerine etkileri
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Tarih
2017
Yazarlar
Dergi Başlığı
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Cilt Başlığı
Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Giriş ve Amaç: Nöroblastoma (NB) tüm dünyada çocukluk döneminin en yaygın kanseri olmakla beraber yine çocukluk çağındaki en sık karşılaşılan ekstrakraniyal solid kanserlerden birisidir. Tirozin kinaz sinyalizasyonu nöroblastoma da hücre farklılaşmasında temel bir role sahiptir. Mallotus philipinensis'ten (Euphorbiaceae) köken alan doğal polifenolik bir bileşik olan Rottlerin'in proliferasyon ve apoptoz gibi çeşitli hücresel prosesler üzerine olan etkilerinden dolayı kanser tedavisi için büyük bir potansiyele sahip olduğu görülmektedir. Genistein bir fitoöstrojen olup, tirozin kinaz inhibitörü olarak işlev görebilmektedir. Genistein'in büyük olasılıkla prostat, serviks, beyin ve meme gibi çeşitli kanserlerde hücre bölünmesi ve sağ kalımını inhibe etmesi yoluyla kontrolsüz kanser büyüme ve gelişimini inhibe ettiği tespit edilmiştir. Son yıllarda, atipik bir kinaz olan Ökaryotik elongasyon faktörü-2 kinaz'ın (EF2K) birçok kanser hücresinde dramatik bir biçimde upregüle olduğu ve hücre sağ kalımı ile proliferasyonu tetiklediği, ayrıca inhibisyonunun hücre büyümesi ve koloni formasyonunu önemli düzeyde azalttığı öğrenilmiştir. Rottlerin ve Genistein pankreas kanseri gibi diğer solid tümörlerde de bu kinaz üzerine inhibitörik etkiler göstermiştir. Ancak, Rottlerin ve Genistein'in bu kanserdeki etki ve rolleri, ayrıca hücre proliferasyonu, koloni formasyonu, sağkalım, invazyon/metastaz ve hücre ölüm/hücre siklusu üzerine ve bunları düzenleyen yolaklara olan etkileri ile EF2K ilişkisi tam olarak bilinmemektedir. Tüm bu bilginin ardından, çalışmada nöroblastoma hücrelerinde Rottlerin ve Genistein'in ayrı ayrı ve kombine olarak bu hücre davranışları üzerine olan etkileri incelenmiştir. Yöntemler: Bu çalışmada insan nöroblastoma kanser hücre hatları (SH-SY5Y, Kelly) kullanılmıştır. Tedavi amacıyla Rottlerin ve Genistein uygulanmıştır. İn vitro deneyler olarak, hücre proliferasyonu, koloni formasyonu, invazon/migrasyon, yara iyileşmesi testi, flow sitometri ile apoptoz ve hücre siklusu analizleri ile western blot analizi gerçekleştirilmiştir. Bulgular: Çalışmamızdaki bulgular Rottlerin ve Genistein tedavilerinin nöroblastoma hücrelerinde sırasıyla 5 µM ve 30 µM dozlarda hücre proliferasyonu, koloni formasyonu, invazyon/migrasyon ve yara iyileşmesi kapasitelerinde önemli düzeyde azalmalara neden oldukları görüldü. Bu dozların kombinasyonları da aynı analizlerdeki inhibisyon düzeylerini artırdı. Ek olarak bu ilaçlar nöroblastoma hücrelerinde apoptoz düzeylerinde de artışa sebep oldu. Bunun yanında yine aynı ilaçlar G1 hücre siklusu arrestine de neden oldu. Ayrıca, Rottlerin ve Genistein tedavilerinin EF2K overekspresyonunu önemli düzeyde azalttığı ve bu downregülasyona bağlı olarak in vitro nöroblastoma hücre proliferasyonu, koloni formasyonu ve invazyon/migrasyonunu inhibe ettikleri gösterildi. Western blot bulguları nöroblastoma hücrelerinde EF2K'in pro-tümörojenik, metastatik protein ve yolaklardaki upregülasyon üzerinden tümörogenez ve metastazı artırabileceği ve Rottlerin ile Genistein'in anti-proliferatif, anti-metastatik ve apoptotik etkilerini olasılıkla EF2K downregülasyonu üzerinden ortaya koyabildiğini gösterdi. Sonuç: Sonuç olarak tüm bu veriler ile birlikte Rottlerin ve Genistein'in nöroblastoma hücrelerinde hücre proliferasyonu, koloni formasyonu, invazyon/migrasyonu ve hücre siklusu/apoptoz üzerine önemli etkilere sahip olduğu ortaya konulmuştur. Dahası, Rottlerin ve Genistein'in bu etkilerini bu kanserde yeni bir potansiyel terapötik hedef olarak EF2K downregülasyonu üzerinden ortaya koyabilecekleri gösterilmiştir. Özetle, kombine olarak Rottlerin ve Genistein ile tedavi, ileride yapılacak ilave çalışmaların ışığında ve yeni nano ilaç taşıyıcı sistemlerin de yardımıyla nöroblastoma hastalarında faydalı ve uygulanabilir bir girişim olabilecektir.
Objectives and Aim: Neuroblastoma (NB) is the most common extracranial solid cancer in childhood and the most common cancer in infancy in the world. Tyrosine kinase signaling networks play a major role in governing cell differentiation, including in neuroblastoma. Rottlerin, a naturally occurring polyphenolic compound derived from Mallotus philipinensis (Euphorbiaceae), appears to have great potential in cancer therapy because of its effects on several cellular processes such as proliferation and apoptosis. Genistein is a phytoestrogen and acts as tyrosine kinase inhibitor. Genistein have been found to inhibit the uncontrolled cell growth of cancer, most likely by inhibiting cell division and cell survival in several cancers such as prostate, cervix, brain and breast. Recently, we learned that Eukaryotic elongation factor-2 kinase (EF2K), an atypical kinase, is dramatically up-regulated in many cancer cells and promotes cell survival and proliferation, its inhibition significantly reduces cell growth and colony formation. Rottlerin and Genistein also showed inhibitory effects on this kinase in another solid tumours like Panc Ca. However, the role that Rottlerin and Genistein in this cancer and effects of these drugs on pathways regulating cell proliferation, colony formation, survival, invasion/metastasis and resistance and relationship between EF2K and these drugs remains are largely unknown. After all of these knowledge, we investigated the effects of Rottlerin and Genistein separately and in combination on these cell behaviours in Neuroblastoma cells. Methods: In this study, the human neuroblastoma cancer cell lines (SH-SY5Y, Kelly) were used. Rottlerin and Genistein were also employed for therapy. As in vitro experiments, cell proliferation, colony formation, invasion, wound-healing tests, western blot, cell cycle and apoptosis analysis by flow cytometry were performed. Results: Our results showed that Rottlerin and Genistein treatments caused a significant reduction in cell proliferation, colony formation, invasion/wound-healing capacity in Neuroblastoma cells at concentrations of 5 µM and 30 µM, respectively (p<0,0001). The combination of these doses also empowered the level of inhibition in these analysis (p<0,0001). Additionally, these drugs also increased the level of apoptosis in Neuroblastoma cell lines (p<0,0001). Additionally, our agents caused G1 cell cycle arrest in these cells (p<0,0001). We showed that Rottlerin and Genistein treatments markedly inhibit EF2K overexpression and due to this downregulation inhibit Neuroblastoma cell proliferation, colony formation and invasion/migration in vitro. Our western blot data suggested that EF2K may enhance tumorigenesis and metastasis through the upregulation of pro-tumorigenic, metastatic proteins and pathways in Neuroblastoma cells and our agents probably showed their anti-proliferative, anti-metastatic and apoptotic effects through EF2K downregulation. Conclusion: In conclusion, it was revealed with all these results that Rottlerin and Genistein have important effects on cell proliferation, colony formation, invasion, and cell cycle/apoptosis in Neuroblastoma cells. Moreover, it has been shown that Rottlerin and Genistein may show these effects through downregulation of EF2K as a novel potential therapeutic target in this cancer. Overall, treatment with Rottlerin and Genistein in combination may be a viable approach and beneficial to neuroblastoma patients with use in new nano-drug delivery systems.
Objectives and Aim: Neuroblastoma (NB) is the most common extracranial solid cancer in childhood and the most common cancer in infancy in the world. Tyrosine kinase signaling networks play a major role in governing cell differentiation, including in neuroblastoma. Rottlerin, a naturally occurring polyphenolic compound derived from Mallotus philipinensis (Euphorbiaceae), appears to have great potential in cancer therapy because of its effects on several cellular processes such as proliferation and apoptosis. Genistein is a phytoestrogen and acts as tyrosine kinase inhibitor. Genistein have been found to inhibit the uncontrolled cell growth of cancer, most likely by inhibiting cell division and cell survival in several cancers such as prostate, cervix, brain and breast. Recently, we learned that Eukaryotic elongation factor-2 kinase (EF2K), an atypical kinase, is dramatically up-regulated in many cancer cells and promotes cell survival and proliferation, its inhibition significantly reduces cell growth and colony formation. Rottlerin and Genistein also showed inhibitory effects on this kinase in another solid tumours like Panc Ca. However, the role that Rottlerin and Genistein in this cancer and effects of these drugs on pathways regulating cell proliferation, colony formation, survival, invasion/metastasis and resistance and relationship between EF2K and these drugs remains are largely unknown. After all of these knowledge, we investigated the effects of Rottlerin and Genistein separately and in combination on these cell behaviours in Neuroblastoma cells. Methods: In this study, the human neuroblastoma cancer cell lines (SH-SY5Y, Kelly) were used. Rottlerin and Genistein were also employed for therapy. As in vitro experiments, cell proliferation, colony formation, invasion, wound-healing tests, western blot, cell cycle and apoptosis analysis by flow cytometry were performed. Results: Our results showed that Rottlerin and Genistein treatments caused a significant reduction in cell proliferation, colony formation, invasion/wound-healing capacity in Neuroblastoma cells at concentrations of 5 µM and 30 µM, respectively (p<0,0001). The combination of these doses also empowered the level of inhibition in these analysis (p<0,0001). Additionally, these drugs also increased the level of apoptosis in Neuroblastoma cell lines (p<0,0001). Additionally, our agents caused G1 cell cycle arrest in these cells (p<0,0001). We showed that Rottlerin and Genistein treatments markedly inhibit EF2K overexpression and due to this downregulation inhibit Neuroblastoma cell proliferation, colony formation and invasion/migration in vitro. Our western blot data suggested that EF2K may enhance tumorigenesis and metastasis through the upregulation of pro-tumorigenic, metastatic proteins and pathways in Neuroblastoma cells and our agents probably showed their anti-proliferative, anti-metastatic and apoptotic effects through EF2K downregulation. Conclusion: In conclusion, it was revealed with all these results that Rottlerin and Genistein have important effects on cell proliferation, colony formation, invasion, and cell cycle/apoptosis in Neuroblastoma cells. Moreover, it has been shown that Rottlerin and Genistein may show these effects through downregulation of EF2K as a novel potential therapeutic target in this cancer. Overall, treatment with Rottlerin and Genistein in combination may be a viable approach and beneficial to neuroblastoma patients with use in new nano-drug delivery systems.
Açıklama
Anahtar Kelimeler
Apoptoz, Genistein, İnvazyon, Nöroblastoma, Proliferasyon, Rottlerin, EF2K, Apoptosis, Invasion, Neuroblastoma, Proliferation
