Diosmetinin kolorektal kanser hücrelerinde (HCT 116) antikarsinojenik etkinliğinin araştırılması
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Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Necmettin Erbakan Üniversitesi Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Kolon ve rektumda ortaya çıkan tümörlerle karakterize olan kolorektal kanser (KRK),
erkeklerde tüm kanserler arasında insidans ve mortalite açısından 3., kadınlarda ise insidans açısından
2. ve mortalite açısından 3. sırada yer almaktadır. KRK prognozu hastalığın evresine bağlıdır. KRK
tanısı hastaların yaklaşık üçte birinde metastatik süreç başladıktan sonra konulabilmektedir. İlerlemiş
KRK’da 5 yıllık sağkalım oranları da düşüktür. Erken evrede tanı alabilen ve tedavisine başlanan
hastalarda da nüks oranının yüksek olması, bir süre sonra kemo-rezistans gelişmesi ve tedavide
kullanılan terapötiklerin sitotoksisitelerinin yüksek olması tedavi başarısını olumsuz etkilemektedir.
Daha başarılı ve daha az yan etkili terapötik ajan adayları olarak yıllardır fitokimyasallar üzerinde
çalışılmaktadır. Bu fitokimyasallardan biri de Diosmetindir.
Baklagiller ve turunçgiller gibi birçok bitkide bulunan diosmetinin anti-bakteriyel, antiinflamatuvar,
anti-mutajenik ve anti-karsinojenik etkilere sahip olduğu bilinmektedir. Bu çalışmada
diosmetinin KRK hücre hattı HCT 116 hücrelerindeki anti-karsinojenik etki mekanizmasının ortaya
konulması amaçlanmıştır. Bu amaca yönelik olarak; diosmetinin sitotoksisitesinin ve proliferasyon
üzerindeki etkisinin belirlenmesi için XTT testi ve koloni oluşum testi yapılmış, antikarsinojenik
etkisini apoptoz üzerinden gösterip göstermediğinin anlaşılması için de apoptoz ve hücre döngüsü ile
ilişkili genlerin ekspresyonları Real time-PCR yöntemi ile değerlendirilmiştir. Ayrıca apoptozu
doğrulamak üzere ELISA yöntemi ile DNA fragmentasyon analizi gerçekleştirilmiştir.
XTT testi sonucunda diosmetinin IC50 değeri 7,5 μM olarak bulunmuş ve çalışmanın sonraki
kısımlarına IC50 değerinin bir alt ve bir üst dozu da dahil edilerek toplam üç doz (5 μM, 7,5 μM ve 10
μM) ile devam edilmiştir. Koloni oluşum testinde diosmetinin doz-bağımlı şekilde koloni oluşumunu
inhibe ettiği görülmüştür (Kontrol, 5 μM, 7,5 μM ve 10 μM dozlar için koloni sayısı sırasıyla 476±15,1;
248,33±3,51; 201±30,44 ve 106,33±8,14). Gen ekspresyon analizlerinde apoptoz-ilişkili genlerden
TP53, BAX, KASPAZ 3, KASPAZ 7, KASPAZ 8, KASPAZ 9, KASPAZ 10, FAS, FADD, CYCS,
TNF, TNFR2’nin ekspresyon seviyelerinde anlamlı düzeyde artış (p<0.05) ve BCL-2 gen ekspresyon
seviyesinde ise anlamlı düzeyde azalış (p<0,05) gözlenmiştir. Hücre döngüsü-ilişkili genlerinden RB1
gen ekspresyon seviyesinde anlamlı düzeyde bir artış (p<0,05) ve E2F4, CCND3, CDK4, CDK6 gen
ekspresyon seviyelerinde ise anlamlı düzeyde azalış (p<0,05) belirlenmiştir. DNA fragmentasyon
analizinde de apoptozun belirteçlerinden olan DNA fragmentasyonunun doz gruplarında kontrole göre
doza-bağlı şekilde artış gösterdiği tespit edilmiştir.
Sonuç olarak, diosmetin KRK hücre hattı HCT 116 hücrelerinde proliferasyonu baskılayarak
ve apoptozu uyararak anti-karsinojenik etki göstermiştir. Hücre döngüsü-ilişkili genlerdeki ekspresyon
değişiklikleri diosmetinin anti-proliferatif etkisini hücre döngüsünü durdurarak gerçekleştirdiğini
düşündürmektedir. Hem iç (intrinsik) yolak-ilişkili genlerdeki hem de dış (ekstrinsik) yolak-ilişkili
genlerdeki ekspresyon değişiklikleri, diosmetinin her iki apoptoz yolağı üzerinden de etkili olduğunu
ortaya koymaktadır.
Colorectal cancer (CRC), which is characterized by tumors arising in the colon and rectum, ranks 3rd in terms of incidence and mortality among all cancers in men, 2nd in incidence and 3rd in terms of mortality in women. The prognosis of CRC depends on the stage of the disease. The diagnosis of CRC can be made after the metastatic process has started in approximately one third of the patients. 5-year survival rates are also low in advanced CRC. The high recurrence rate, development of chemoresistance after a while, and high cytotoxicity of the therapeutics used in the treatment negatively affect the success of the treatment in patients who can be diagnosed at an early stage and whose treatment is started. Phytochemicals have been studied for years as candidates for more successful and less sideeffect therapeutic agents. One of these phytochemicals is Diosmetin. It is known that diosmetin, which is found in many plants such as legumes and citrus fruits, has anti-bacterial, anti-inflammatory, anti-mutagenic and anti-carcinogenic effects. In this study, it was aimed to reveal the anti-carcinogenic mechanism of action of diosmetin in CRC cell line HCT 116 cells. For this purpose; XTT test and colony formation test were performed to determine the cytotoxicity of diosmetin and its effect on proliferation, and the expressions of genes related to apoptosis and cell cycle were evaluated by Real time-PCR method to understand whether it shows its anticarcinogenic effect through apoptosis. In addition, DNA fragmentation analysis was performed by ELISA method to confirm apoptosis. As a result of the XTT test, the IC50 value of diosmetin was found to be 7,5 μM, and three doses (5 μM, 7,5 μM and 10 μM) were included in the next parts of the study, including one lower and one higher IC50 value. In the colony formation test, diosmetin was found to inhibit colony formation in a dose-dependent manner (Colony count for Control, 5 μM, 7,5 μM, and 10 μM doses, respectively 476±15.1; 248.33±3.51; 201±30.44 and 106.33±8.14). In gene expression analyzes, the expression levels of apoptosis-related genes TP53, BAX, CASPASE 3, CASPASE 7, CASPASE 8, CASPASE 9, CASPASE 10, FAS, FADD, CYCS, TNF, TNFR2 were significantly increased (p<0.05) and BCL- A significant decrease (p<0.05) was observed in 2 gene expression levels. A significant increase (p<0.05) in RB1 gene expression levels and a significant decrease (p<0.05) in E2F4, CCND3, CDK4, CDK6 gene expression levels were determined. In DNA fragmentation analysis, it was determined that DNA fragmentation, which is one of the markers of apoptosis, increased in a dose-dependent manner in the dose groups compared to the control. In conclusion, diosmetin showed anti-carcinogenic effect by suppressing proliferation and stimulating apoptosis in CRC cell line HCT 116 cells. Expression changes in cell cycle-related genes suggest that diosmetin exerts its anti-proliferative effect by arresting the cell cycle. Expression changes in both intrinsic pathway-related genes and extrinsic pathway-related genes suggest that diosmetin acts on both apoptosis pathways.
Colorectal cancer (CRC), which is characterized by tumors arising in the colon and rectum, ranks 3rd in terms of incidence and mortality among all cancers in men, 2nd in incidence and 3rd in terms of mortality in women. The prognosis of CRC depends on the stage of the disease. The diagnosis of CRC can be made after the metastatic process has started in approximately one third of the patients. 5-year survival rates are also low in advanced CRC. The high recurrence rate, development of chemoresistance after a while, and high cytotoxicity of the therapeutics used in the treatment negatively affect the success of the treatment in patients who can be diagnosed at an early stage and whose treatment is started. Phytochemicals have been studied for years as candidates for more successful and less sideeffect therapeutic agents. One of these phytochemicals is Diosmetin. It is known that diosmetin, which is found in many plants such as legumes and citrus fruits, has anti-bacterial, anti-inflammatory, anti-mutagenic and anti-carcinogenic effects. In this study, it was aimed to reveal the anti-carcinogenic mechanism of action of diosmetin in CRC cell line HCT 116 cells. For this purpose; XTT test and colony formation test were performed to determine the cytotoxicity of diosmetin and its effect on proliferation, and the expressions of genes related to apoptosis and cell cycle were evaluated by Real time-PCR method to understand whether it shows its anticarcinogenic effect through apoptosis. In addition, DNA fragmentation analysis was performed by ELISA method to confirm apoptosis. As a result of the XTT test, the IC50 value of diosmetin was found to be 7,5 μM, and three doses (5 μM, 7,5 μM and 10 μM) were included in the next parts of the study, including one lower and one higher IC50 value. In the colony formation test, diosmetin was found to inhibit colony formation in a dose-dependent manner (Colony count for Control, 5 μM, 7,5 μM, and 10 μM doses, respectively 476±15.1; 248.33±3.51; 201±30.44 and 106.33±8.14). In gene expression analyzes, the expression levels of apoptosis-related genes TP53, BAX, CASPASE 3, CASPASE 7, CASPASE 8, CASPASE 9, CASPASE 10, FAS, FADD, CYCS, TNF, TNFR2 were significantly increased (p<0.05) and BCL- A significant decrease (p<0.05) was observed in 2 gene expression levels. A significant increase (p<0.05) in RB1 gene expression levels and a significant decrease (p<0.05) in E2F4, CCND3, CDK4, CDK6 gene expression levels were determined. In DNA fragmentation analysis, it was determined that DNA fragmentation, which is one of the markers of apoptosis, increased in a dose-dependent manner in the dose groups compared to the control. In conclusion, diosmetin showed anti-carcinogenic effect by suppressing proliferation and stimulating apoptosis in CRC cell line HCT 116 cells. Expression changes in cell cycle-related genes suggest that diosmetin exerts its anti-proliferative effect by arresting the cell cycle. Expression changes in both intrinsic pathway-related genes and extrinsic pathway-related genes suggest that diosmetin acts on both apoptosis pathways.
Açıklama
Yüksek Lisans Tezi
Anahtar Kelimeler
Anti-kanserojen, Apoptoz, Diosmetin, HCT 116, Anti-carcinogene, Apoptosis
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Yörük, M. (2021). Diosmetinin kolorektal kanser hücrelerinde (HCT 116) antikarsinojenik etkinliğinin araştırılması. (Yayımlanmamış yüksek lisans tezi). Necmettin Erbakan Üniversitesi, Sağlık Bilimleri Enstitüsü Tıbbi Biyoloji Anabilim Dalı, Konya.
