PANC-1 ve BxPC3 pankreas kanser hücre hatlarında juglonun mitokondrial fisyon ve füzyon mekanizmaları üzerine etkileri
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Tarih
2025
Yazarlar
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Cilt Başlığı
Yayıncı
Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Pankreas kanseri, dünya genelinde en ölümcül kanser türlerinden biri olup, erken evrede tanı konulamaması ve agresif biyolojik davranışı nedeniyle kötü bir prognoza sahiptir. Dünya genelinde kansere bağlı ölümler arasında 7. sırada yer almakta olan pankreas kanserinde 5 yıllık sağkalım oranı %10'dur. Ülkemizde de pankreas kanseri insidansı dünya genelindeki oranlara paralel olarak artış göstermektedir. Ekzokrin bezlerde ortaya çıkan pankreas duktal adenokarsinomu (PDAK), en yaygın pankreas kanseri türüdür ve vakaların yaklaşık %90'ını oluşturur. Kanser Genom Atlas verilerine göre, PDAK pankreas kanserlerinin yaklaşık %90'ında KRAS sürücü mutasyonu bulunmaktadır. Pankreas kanseri, erken evrelerinde asemptomatik olduğundan çoğu vaka ileri evreye erişinceye kadar teşhis edilememektedir. Tanının genellikle metastaz aşamasında konulması cerrahi tedaviyi sınırlamakta ve sıklıkla mevcut kemoterapi/radyoterapiye karşı direnç gelişiminin olması sağkalım oranını düşürmektedir. Bu nedenlerle oldukça agresif seyreden pankreas kanseriyle mücadelede kemoterapötik ajanlara ihtiyaç vardır. Juglon, ceviz ağaçlarının taze olgun meyve kabuğunda, köklerinde ve yapraklarında bulunan bir tür naftokinondur. Juglonun çeşitli kanser hücre hatlarında sitotoksik ve apoptotik etkisi gösterilmiş, daha önceki araştırmalarda PANC-1 ve BxPC3 insan pankreas kanseri hücre hatlarında bu etkilerinin yanı sıra anti-invaziv ve anti-metastatik etki gösterdiği ortaya konulmuştur. Son çalışmalarımızda Juglonun mitokondrial biyogenezini düzenleyen sinyal yolakları üzerine etki gösterdiğine dair elde ettiğimiz bulgular ışığında bu tez çalışmasında Juglonun mitokondrial biyogenez süreci üzerine etkilerini araştırmayı hedefledik. Mitokondrial biyogenez, füzyon-fisyon döngüleri arasında devam eden yaşamsal bir mekanizmadır. Kanser hücrelerinde bu dengenin genellikle fisyon yönünde kayma gösterdiği ve fragmente mitokondrilerin hücre proliferasyonunu teşvik ettiği düşünülmektedir. Bu nedenle fisyon inhibisyonunun önemli bir terapötik yaklaşım olduğu kanısına varılmıştır. Çalışmamızda Juglonun PANC-1 ve BxPC3 pankreas kanseri hücrelerinde, biyogenezin anahtar düzenleyicisi olan PGC1α ,füzyon ve fisyonun temel düzenleyicileri MFN1, MFN2 ile DRP1 üzerine etkilerinin araştırılması amaçlandı. Hücreler uygun besiyerlerinde 370C'de %5CO2 ortamında kültüre edildi. Sitotoksisite analiz sonuçları dikkate alınarak Juglon uygulama dozları 5µM, 10µM, 15µM ve 20µM olacak şekilde belirlendi. Hedef genlerin ifadeleri qPCR yöntemi ile analiz edildi ve sonuçlar 2-ΔΔCt metodu ile değerlendirildi. Çalışmamızda PCG1α gen ifadesinin 5µM, 10µM, 15µM ve 20µM Juglon uygulamalarında PANC-1 hücre hattında sırasıyla 3,34, 5,51, 3,31 ve 7,12 kat artış, BxPC3 hücrelerinde 5µM, 15µM ve 20µM Juglon uygulamasında sırasıyla 4,41, 4,41 ve 3,65 kat artış gösterdiği tespit edildi. PANC-1 hücrelerinde MFN1 gen ifadesi bakımından 5µM, 10µM, 15µM ve 20µM Juglon uygulaması sonrasında sırasıyla 3,34, 5,51, 3,31 ve 7,12 kat artış görülürken, BxPC3 hücrelerinde önemli düzeyde bir değişiklik tespit edilmedi. MFN2 gen ifadesi bakımından değerlendirildiğinde, PANC-1 hücrelerinde 15µM ve 20µM Juglon uygulamasında 3,64 ve 3,08 kat artış, 10 µM'da 4,46 kat azalış gözlendi. BxPC3 hücrelerinde de MFN2 gen ifadesinde artışlar gözlenmekle beraber anlamlı düzeyde değildi. PANC-1 hücrelerinde DRP1 gen ifade seviyeleri 10µM, 15µM ve 20µM Juglon uygulamalarında sırasıyla 8,82, 4,57 ve 2,35 kat azalış gösterirken, BxPC3 hücrelerinde gen ifade düzeylerinde değişiklik yoktu. Çalışma bulgularımız Juglonun PANC-1 hücrelerinde füzyonu güçlü bir şekilde teşvik ettiğini fisyonu ise baskıladığını göstermektedir. PDAK pankreas kanserinde yaygın olarak KRAS sürücü mutasyonunun varlığı düşünüldüğünde, Juglonun KRAS sürücü mutasyonuna sahip PANC-1 hücre hattında füzyonu aktive edici, fisyonu baskılayıcı etki göstermesi PDAK tedavisinde etkin olarak kullanılabilme potansiyelinin olduğunu göstermektedir. Ayrıca her iki hücre hattında da PGC1α gen ifadesinin Juglon uygulaması sonucu önemli düzeyde artması, Juglonun KRAS bağımsız BxPC3 hücrelerinde mitokondri üzerinde fisyon ve füzyon dışında farklı yollarla etki gösterdiğini düşündürmektedir. Sonuç olarak Juglon pankreas kanserinde mitokondri hedefli terapötik potansiyele sahip görünmekle birlikte bu önemli bulguların daha kapsamlı çalışmalarla teyit edilmesine ihtiyaç vardır.
Pancreatic cancer is one of the deadliest cancers worldwide, with a poor prognosis due to its inability to be diagnosed at early stages and its aggressive biological behavior. Pancreatic cancer ranks as the 7th leading cause of cancer-related deaths worldwide, with a 5-year survival rate of only 10%. The incidence of pancreatic cancer in our country is also increasing in parallel with the rates worldwide. Pancreatic ductal adenocarcinoma (PDAC), which originates from the exocrine glands, is the most common type of pancreatic cancer, accounting for approximately 90% of cases. According to The Cancer Genome Atlas data, around 90% of PDACs harbor KRAS driver mutations. Pancreatic cancer is often asymptomatic in its early stages, which means most cases are diagnosed only when the disease is in an advanced stage. Diagnosis is typically made at the metastatic stage, which limits the possibility of surgical treatment and often leads to the development of resistance to existing chemotherapy/radiotherapy, thereby reducing survival rates. Therefore, there is a need for chemotherapeutic agents in the fight against the highly aggressive pancreatic cancer. Juglone (5-hydroxy-1,4-naphthoquinone) is a type of naphthoquinone found in the fresh mature fruit peel, roots, and leaves of walnut trees. Juglone has been shown to have cytotoxic and apoptotic effects in various cancer cell lines, and previous studies have demonstrated that, in addition to these effects, it also exhibits anti-invasive and anti-metastatic properties in PANC-1 and BxPC3 human pancreatic cancer cell lines. Based on our recent findings, which suggest that Juglone affects signaling pathways regulating mitochondrial biogenesis, this thesis aims to investigate the effects of Juglone on the mitochondrial biogenesis process. Mitochondrial biogenesis is a vital mechanism involving continuous fusion-fission cycles. In cancer cells, this balance often shifts towards fission, and fragmented mitochondria are thought to promote cell proliferation. Therefore, inhibition of fission has been considered an important therapeutic strategy. Our study aimed to investigate the effects of Juglone on the key regulator of biogenesis, PGC1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha), and the main regulators of fusion and fission, MFN1, MFN2, and DRP1, in PANC-1 and BxPC3 pancreatic cancer cells. The cells were cultured in appropriate media at 37°C in a 5%CO2 atmosphere. Based on cytotoxicity assay results, Juglone concentrations of 5µM, 10µM, 15µM, and 20µM were determined. The expression of target genes was analyzed using qPCR, and the results were evaluated by the 2-ΔΔCt method. In our study, PGC1α gene expression increased by 3,34, 5,51, 3,31 and 7,12 fold in PANC-1 cells following treatment with 5µM, 10µM, 15µM, and 20µM Juglone, respectively. In BxPC3 cells, PGC1α expression increased by 4,41, 4,41, and 3,65 fold after treatment with 5µM, 15µM, and 20µM Juglone, respectively. In PANC-1 cells, MFN1 gene expression increased by 3,34, 5,51, 3,31, and 7,12 fold following treatment with 5µM, 10µM, 15µM, and 20µM Juglone, respectively, while no significant change was observed in BxPC3 cells. Regarding MFN2 gene expression, PANC-1 cells showed an increase of 3,64 and 3,08 fold after treatment with 15µM and 20µM Juglone, and a decrease of 4,46 fold at 10µM. In BxPC-3 cells, although increases in MFN2 expression were observed, they were not statistically significant. In PANC-1 cells, DRP1 gene expression decreased by 8,82, 4,57, and 2,35 times following 10µM, 15µM, and 20µM Juglone treatment, respectively, while no changes were detected in BxPC3 cells. Our findings indicate that Juglone strongly promotes fusion in PANC-1 cells while inhibiting fission. Considering the widespread presence of KRAS driver mutations in PDAC, Juglone's ability to activate fusion and inhibit fission in the KRAS-mutant PANC-1 cell line suggests its potential for effective use in PDAC treatment. Additionally, the significant increase in PGC1α gene expression in both cell lines following Juglone treatment suggests that Juglone may exert effects on mitochondria through mechanisms beyond fusion and fission in KRAS-independent BxPC3 cells. In conclusion, Juglone appears to have therapeutic potential targeting mitochondria in pancreatic cancer, but these important findings need to be confirmed through more comprehensive studies.
Pancreatic cancer is one of the deadliest cancers worldwide, with a poor prognosis due to its inability to be diagnosed at early stages and its aggressive biological behavior. Pancreatic cancer ranks as the 7th leading cause of cancer-related deaths worldwide, with a 5-year survival rate of only 10%. The incidence of pancreatic cancer in our country is also increasing in parallel with the rates worldwide. Pancreatic ductal adenocarcinoma (PDAC), which originates from the exocrine glands, is the most common type of pancreatic cancer, accounting for approximately 90% of cases. According to The Cancer Genome Atlas data, around 90% of PDACs harbor KRAS driver mutations. Pancreatic cancer is often asymptomatic in its early stages, which means most cases are diagnosed only when the disease is in an advanced stage. Diagnosis is typically made at the metastatic stage, which limits the possibility of surgical treatment and often leads to the development of resistance to existing chemotherapy/radiotherapy, thereby reducing survival rates. Therefore, there is a need for chemotherapeutic agents in the fight against the highly aggressive pancreatic cancer. Juglone (5-hydroxy-1,4-naphthoquinone) is a type of naphthoquinone found in the fresh mature fruit peel, roots, and leaves of walnut trees. Juglone has been shown to have cytotoxic and apoptotic effects in various cancer cell lines, and previous studies have demonstrated that, in addition to these effects, it also exhibits anti-invasive and anti-metastatic properties in PANC-1 and BxPC3 human pancreatic cancer cell lines. Based on our recent findings, which suggest that Juglone affects signaling pathways regulating mitochondrial biogenesis, this thesis aims to investigate the effects of Juglone on the mitochondrial biogenesis process. Mitochondrial biogenesis is a vital mechanism involving continuous fusion-fission cycles. In cancer cells, this balance often shifts towards fission, and fragmented mitochondria are thought to promote cell proliferation. Therefore, inhibition of fission has been considered an important therapeutic strategy. Our study aimed to investigate the effects of Juglone on the key regulator of biogenesis, PGC1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha), and the main regulators of fusion and fission, MFN1, MFN2, and DRP1, in PANC-1 and BxPC3 pancreatic cancer cells. The cells were cultured in appropriate media at 37°C in a 5%CO2 atmosphere. Based on cytotoxicity assay results, Juglone concentrations of 5µM, 10µM, 15µM, and 20µM were determined. The expression of target genes was analyzed using qPCR, and the results were evaluated by the 2-ΔΔCt method. In our study, PGC1α gene expression increased by 3,34, 5,51, 3,31 and 7,12 fold in PANC-1 cells following treatment with 5µM, 10µM, 15µM, and 20µM Juglone, respectively. In BxPC3 cells, PGC1α expression increased by 4,41, 4,41, and 3,65 fold after treatment with 5µM, 15µM, and 20µM Juglone, respectively. In PANC-1 cells, MFN1 gene expression increased by 3,34, 5,51, 3,31, and 7,12 fold following treatment with 5µM, 10µM, 15µM, and 20µM Juglone, respectively, while no significant change was observed in BxPC3 cells. Regarding MFN2 gene expression, PANC-1 cells showed an increase of 3,64 and 3,08 fold after treatment with 15µM and 20µM Juglone, and a decrease of 4,46 fold at 10µM. In BxPC-3 cells, although increases in MFN2 expression were observed, they were not statistically significant. In PANC-1 cells, DRP1 gene expression decreased by 8,82, 4,57, and 2,35 times following 10µM, 15µM, and 20µM Juglone treatment, respectively, while no changes were detected in BxPC3 cells. Our findings indicate that Juglone strongly promotes fusion in PANC-1 cells while inhibiting fission. Considering the widespread presence of KRAS driver mutations in PDAC, Juglone's ability to activate fusion and inhibit fission in the KRAS-mutant PANC-1 cell line suggests its potential for effective use in PDAC treatment. Additionally, the significant increase in PGC1α gene expression in both cell lines following Juglone treatment suggests that Juglone may exert effects on mitochondria through mechanisms beyond fusion and fission in KRAS-independent BxPC3 cells. In conclusion, Juglone appears to have therapeutic potential targeting mitochondria in pancreatic cancer, but these important findings need to be confirmed through more comprehensive studies.
Açıklama
Anahtar Kelimeler
Juglon, Mitokondrial Biyogenez, Mitokondrial Fisyon, Mitokondrial Füzyon, Pankreas Kanseri, Juglone, Mitochondrial Biogenesis, Mitochondrial Fision, Mitochondrial Fusion
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Ayhan, E. (2025). PANC-1 ve BxPC3 pankreas kanser hücre hatlarında juglonun mitokondrial fisyon ve füzyon mekanizmaları üzerine etkileri. (Yüksek Lisans Tezi). Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü, Konya.
