Pankreas kanseri hücre hatlarında NaV1.7 kanalının siRNA aracılı hedeflenmesinin anti-kanser etkileri
Küçük Resim Yok
Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Dünya genelinde ve Türkiye'de kanser vaka sayıları her geçen yıl daha da artış gösterme seyrindedir. Ortalama insan ömründeki artış ve yaşam kalitesi seviyelerindeki azalmayla birlikte kanser sebebiyle sağlık ekonomisinin ve dolayısıyla da ülkelerin karşılaşacağı ekonomik zorluklar da artış gözlenecektir. Kansere yönelik devam edilen bu savaş sürecinde terapötik amaçlı olarak kullanılabilecek, ciddi potansiyele sahip yeni nesil tedavi girişimlerinin etkinliklerinin, kullandıkları moleküler mekanizmalar ve henüz açıklanamamış özelliklerinin değerlendirilmesi hem dünyamız hem de ülkemiz adına kritik öneme sahiptir. Pankreas kanseri, son yıllarda kanser kaynaklı ölümler arasında Amerika Birleşik Devletleri'nde 4.sırada, dünya genelinde ise ilk 5 kanserin içerisinde yer alması nedeniyle en önemli kanser türlerinden birisidir. Voltaj-kapılı sodyum kanalları (VGSC) uzun zamandan beri birçok metastatik hücre hattında ve meme, akciğer, prostat kanseri dışında başka doku örneklerinde de tespit edilmiştir. Önceki çalışmalara ait ve tümör doku veritabanı üzerinden elde edilen sonuçlar baz alınarak kurgulanan çalışmada; pankreas kanseri hücrelerinde öncelikle NaV1.7 inhibisyonunun ve daha sonra da nNaV1.7'nin, pankreas kanser hücrelerinin progresif ve metastatik özelliklerinin baskılanmasında tedavi edici bir etkiye sahip olup olmadığının araştırılması amaçlanmıştır. Tezimde insan normal keratinosit hücre hattı HaCaT ve pankreas kanseri hücre hatları olan PANC-1 ve MiaPaCa-2 kullanıldı. Pankreas kanseri invazyonu, metastazı ve progresyonu üzerine NaV1.7 kanalının adult ve neonatal izoformlarının olası rolleri ve etkileri araştırıldı. Bu amaç ile her iki kanala da spesifik siRNA'lar uygulanarak terapötik olarak gen susturma gerçekleştirildi. Çalışmada in vitro olarak hücrelerdeki proliferasyon MTS testi ile değerlendirilirken, hücrelerin koloni şekillendirme kapasiteleri, gemsitabine olan direnç ve kombinasyonların etkileri, hücrelerde invazyon, migrasyon ve yara iyileşmesi düzeyleri, flow sitometrik olarak apoptoz ile hücre siklusu düzeylerindeki değişimler, bu etkilerin tümüyle ilişkili olabilecek potansiyel role sahip protein sinyal yolakları western blot yöntemi ile, her iki kanal izoformunun da mRNA ekspresyon düzeyleri Real-Time ve RT-PCR yöntemleri aracılığıyla değerlendirildi. Analizler sonucunda PANC-1 ve MiaPaCa-2 hücre hatlarında HaCaT hücrelerine kıyasla adult ve neonatal NaV1.7 mRNA ekspresyonlarının anlamlı olarak daha yüksek olduğu ve her iki spesifik siRNA ile kanal mRNA ve protein ekspresyonlarının downregüle edildiği tespit edildi. PANC-1 ve MiaPaCa-2 pankreas kanseri hücrelerinde spesifik NaV1.7 siRNA tedavilerinin kanser hücre proliferasyonu ve koloni şekillendirme kapasitelerinde anlamlı düzeyde inhibisyon meydana getirdiği gözlendi. Fakat NaV1.7 siRNA'larının sağlıklı keratinosit hücre hattı HaCaT'lar üzerine herhangi bir sitotoksik etki göstermedikleri belirlendi. PANC-1 hücre hattında gemsitabin ile spesifik NaV1.7 siRNA kombinasyonlarının ilacın etkinliğinde artış meydana getirdiği gözlendi. Bunlarla birlikte NaV1.7 siRNA'larının her iki hücre hattında da hücrelerin invazyonu, migrasyonu ve yara iyileşmesi düzeylerini anlamlı şekilde inhibe ettikleri bulundu. Yine her iki hücrede NaV1.7 siRNA'larının apoptozu ve hücre siklusunda G1 arrestini indükledikleri tespit edildi. NaV1.7 siRNA'larının gözlenen bu etkilerine dair hücre içi sinyal yolaklarında etkili olabilecek protein ekspresyonlarında da belirgin düzeyde inhibisyon yaptıkları gözlendi. Sonuç olarak bu çalışmayla NaV1.7 kanal izoformlarının pankreas kanseri gelişimi, progresyonu ve metastazında önemli rol oynadığı ayrıntılı biçimde ortaya konuldu. Metastaz sürecine odaklanarak planlanan çalışmada kullanılan diğer analiz ve belirteçler ile NaV1.7 kanalının pankreas kanserindeki olası diğer etkileri de ilk kez gösterilmiş oldu. Tüm bu sebeplerle çalışma pek çok konuda sunduğu bulgular ile literatürde bu kapsamda ilk çalışma olması nedeniyle önem arz etmektedir.
The number of cancer cases in the world and in Turkey is on the course of increasing every year. With the increase in the average human lifespan and the decrease in the quality of life, the economic difficulties that the health economy and therefore the countries will face due to cancer will also increase. Evaluating the efficacy, molecular mechanisms and yet unexplained features of new generation treatment interventions with serious potential that can be used for therapeutic purposes during this ongoing battle against cancer is of critical importance for both our world and our country. Pancreatic cancer is one of the most important cancer types because it ranks 4th in the United States of America and among the top 5 cancers worldwide in recent years among cancer-related deaths. Voltage-gated sodium channels (VGSC) have long been detected in many metastatic cell lines and in tissue samples other than breast, lung, and prostate cancer. In the study, which was based on the results of previous studies and obtained from the tumor tissue database; In this study, it was aimed to investigate whether the inhibition of NaV1.7 and then nNaV1.7 in pancreatic cancer cells has a therapeutic effect in suppressing the progressive and metastatic features of pancreatic cancer cells. Human normal keratinocyte cell line HaCaT and pancreatic cancer cell lines PANC-1 and MiaPaCa-2 were used in the study. The possible roles and effects of adult and neonatal isoforms of the NaV1.7 channel on pancreatic cancer invasion, metastasis and progression were investigated. For this purpose, therapeutic gene silencing was performed by applying specific siRNAs to both channels. In the study, cell proliferation in vitro was evaluated with the MTS test, while colony forming capacities of cells, resistance to gemcitabine and the effects of combinations, levels of invasion, migration and wound healing in cells, apoptosis in flow ctometric and changes in cell cycle levels have a potential role that may be related to all of these effects. Protein signaling pathways were evaluated by western blot method, and mRNA expression levels of both channel isoforms were evaluated by Real-Time and RT-PCR methods. In the study, it was determined that adult and neonatal NaV1.7 mRNA expressions were significantly higher in PANC-1 and MiaPaCa-2 cell lines compared to HaCaT cells, and channel mRNA and protein expressions were downregulated with both specific siRNAs. It was observed that specific NaV1.7 siRNA treatments in PANC-1 and MiaPaCa-2 pancreatic cancer cells caused significant inhibition of cancer cell proliferation and colony forming capacities. However, it was determined that NaV1.7 siRNAs did not show any cytotoxic effect on healthy keratinocyte cell line HaCaTs. It was observed that the combination of gemcitabine and specific NaV1.7 siRNA in the PANC-1 cell line increased the efficacy of the drug. In addition, NaV1.7 siRNAs were found to significantly inhibit cell invasion, migration and wound healing levels in both cell lines. Again, it was determined that NaV1.7 siRNAs induced apoptosis and G1 arrest in the cell cycle in both cells. Regarding these observed effects of NaV1.7 siRNAs, it was observed that they inhibited protein expressions that may be effective in intracellular signaling pathways. In conclusion, this study revealed in detail that NaV1.7 channel isoforms play an important role in the development, progression and metastasis of pancreatic cancer. Other analyzes and markers used in the study, which was planned by focusing on the metastasis process, and other possible effects of the NaV1.7 channel in pancreatic cancer were shown for the first time. For all these reasons, the study is important as it is the first study in this context in the literature with the findings it presents on many subjects.
The number of cancer cases in the world and in Turkey is on the course of increasing every year. With the increase in the average human lifespan and the decrease in the quality of life, the economic difficulties that the health economy and therefore the countries will face due to cancer will also increase. Evaluating the efficacy, molecular mechanisms and yet unexplained features of new generation treatment interventions with serious potential that can be used for therapeutic purposes during this ongoing battle against cancer is of critical importance for both our world and our country. Pancreatic cancer is one of the most important cancer types because it ranks 4th in the United States of America and among the top 5 cancers worldwide in recent years among cancer-related deaths. Voltage-gated sodium channels (VGSC) have long been detected in many metastatic cell lines and in tissue samples other than breast, lung, and prostate cancer. In the study, which was based on the results of previous studies and obtained from the tumor tissue database; In this study, it was aimed to investigate whether the inhibition of NaV1.7 and then nNaV1.7 in pancreatic cancer cells has a therapeutic effect in suppressing the progressive and metastatic features of pancreatic cancer cells. Human normal keratinocyte cell line HaCaT and pancreatic cancer cell lines PANC-1 and MiaPaCa-2 were used in the study. The possible roles and effects of adult and neonatal isoforms of the NaV1.7 channel on pancreatic cancer invasion, metastasis and progression were investigated. For this purpose, therapeutic gene silencing was performed by applying specific siRNAs to both channels. In the study, cell proliferation in vitro was evaluated with the MTS test, while colony forming capacities of cells, resistance to gemcitabine and the effects of combinations, levels of invasion, migration and wound healing in cells, apoptosis in flow ctometric and changes in cell cycle levels have a potential role that may be related to all of these effects. Protein signaling pathways were evaluated by western blot method, and mRNA expression levels of both channel isoforms were evaluated by Real-Time and RT-PCR methods. In the study, it was determined that adult and neonatal NaV1.7 mRNA expressions were significantly higher in PANC-1 and MiaPaCa-2 cell lines compared to HaCaT cells, and channel mRNA and protein expressions were downregulated with both specific siRNAs. It was observed that specific NaV1.7 siRNA treatments in PANC-1 and MiaPaCa-2 pancreatic cancer cells caused significant inhibition of cancer cell proliferation and colony forming capacities. However, it was determined that NaV1.7 siRNAs did not show any cytotoxic effect on healthy keratinocyte cell line HaCaTs. It was observed that the combination of gemcitabine and specific NaV1.7 siRNA in the PANC-1 cell line increased the efficacy of the drug. In addition, NaV1.7 siRNAs were found to significantly inhibit cell invasion, migration and wound healing levels in both cell lines. Again, it was determined that NaV1.7 siRNAs induced apoptosis and G1 arrest in the cell cycle in both cells. Regarding these observed effects of NaV1.7 siRNAs, it was observed that they inhibited protein expressions that may be effective in intracellular signaling pathways. In conclusion, this study revealed in detail that NaV1.7 channel isoforms play an important role in the development, progression and metastasis of pancreatic cancer. Other analyzes and markers used in the study, which was planned by focusing on the metastasis process, and other possible effects of the NaV1.7 channel in pancreatic cancer were shown for the first time. For all these reasons, the study is important as it is the first study in this context in the literature with the findings it presents on many subjects.
Açıklama
Anahtar Kelimeler
Pankreas Kanseri, Voltaj-Kapılı Sodyum Kanalları, NaV1.7, Proliferasyon, İnvazyon, siRNA, Pancreatic Cancer, Voltage-Gated Sodium Channels, NaV1.7, Proliferation, Invasion, siRNA
