Rheum ribes L. bitki ekstrelerinin kolorektal kanser hücre hatlarında antikanserojenik etkisinin araştırılması
Yükleniyor...
Dosyalar
Tarih
2018
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Necmettin Erbakan Üniversitesi Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Kanserin tedavisinde kullanılan kemoterapötik ilaçların ciddi yan etkilere neden olmaları ve tedavinin ileri dozlarında kemodirenç gelişimi nedeniyle alternatif yeni sitotoksik ajanların belirlenmesine yönelik çalışmalar devam etmektedir. Bizim çalışmamızda ise ülkemizin doğusunda geniş yayılış gösteren ve halk arasında sıklılıkla tüketilen Rheum ribes L. (Polygonaceae) bitki türünün CRC hücre hatları üzerinde antikanserojenik etkiye sahip olup olmadığı araştırılacaktır. Bu bitki türü ile ilgili yapılan çalışmalar daha çok fitokimyasal analiz ve antioksidan ve antibakteriyel etkisinin değerlendirilmesine yönelik olup antikanserojenik etkilerinin moleküler düzeyde araştırıldığı herhangi bir çalışmaya rastlanmamıştır. Çalışmamızda R. ribes'e ait kök ve gövde kısımlarından 6 farklı ekstre elde edilmiştir. Tüm ekstrelerin toplam fenolik, flavonoit madde miktarları, antioksidan aktivitelerive HCT 116 ve HT-29 kolorektal kanser hücreleri üzerindeki sitotoksik etkileri belirlenmiştir. Bu analizler sonucunda en düşük dozla en yüksek sitotoksik aktivite gösteren ekstrenin kök metanol (KM) ekstresi olduğu belirlenmiş ve çalışmaya KM ekstresi ile devam edilmiştir. Sonrasında KM ekstresinin kromatografik analizle biyoaktif madde içeriği tespit edilmiştir. KM ekstresinin, CRC'nin tedavisinde yaygın olarak kullanılan kemoterapi ajanı 5-FU ile kombinasyon dozları izobologram analizi için kanser hücrelerine uygulanmış ve kombinasyonun IC50 dozunun her iki hücre içinde hafif sinerjizm gösterdiği belirlenmiştir. KM, 5-FU ve KM+5-FU'nun IC50 dozlarının uygulandığı hücrelerde miR-200 ailesi (miR-200a/b/c ve miR-141) ve hedef genlerin (EMT ve invazyonla ilişkili ZEB1, CDH1 (E-kaderin), CDH2 (N-kaderin); apoptoz ile ilişkili BCL-2, BAX, Kaspaz-3,7,8,9, CYCS (Sitokrom C), PPARG, XIAP, FAS/CD95, FAP-1; hücre döngüsü ile ilişkili p53, p21, CDK4, CDK6, CCND1, CCND2, CCND3, CDNK1B, RND3; ve TGFβ yolağıyla ilişkili TGFβ1, TGFβR1, TGFβR2, SMAD2,3,4,7) ekspresyon seviyelerindeki farklılıklar gerçek zamanlı qPCR ile değerlendirilmiştir. Morfolojik düzeyde apoptoz, TUNEL ve Anneksin V analizi ile; hücre döngüsü evrelerinin belirlenmesi, hücre döngüsü analizi ile; hücrelerin invaziv karakterleri; invazyon testi ile BCL-2, ZEB1, GATA4 ve FAS/CD95 proteinlerinin ekspresyon düzeyindeki farklılıkları ise ELISA yöntemi ile belirlenmiştir. miRNA ekspresyon analizi sonuçlarına göre, her iki hücre hattında tümör baskılayıcı fonksiyon gösteren 4 miRNA'nın ekspresyon seviyesinde de anlamlı (p<0,05) artışın olduğu tek doz grubu yalnız KM'dir. Doz gruplarına göre; apoptoz, hücre döngüsü, EMT ve TGFβ yolağıyla ilişkili hedef genlerdeki anlamlı artış ve azalışlar birlikte değerlendirildiğinde, antiproliferatif ve apoptotik etkinin tek başına uygulanan 5-FU'ya göre KM ve KM+5-FU kombine gruplarında daha yüksek olduğu belirlenmiştir. Elde ettiğimiz sonuçlar, KM'nin miR-200a/b/c ve miR-141'in ekspresyonlarında anlamlı artışa neden olduğunu;, BCL-2, ZEB1, GATA4 ekspresyonlarını baskıladığını göstermişir. KM'nin tek başına ya da 5-FU ile kombine uygulamasının, kanser hücrelerinin tek başına 5-FU ile tedavisine göre daha iyi yanıt oluşturduğunu düşündürmüştür.
Studies are continuing to identify alternative new cytotoxic agents as chemotherapeutic drugs used in the treatment of cancer due to the serious side effects and chemo-dimer resistance in further doses of the treatment. In our study, we will investigate whether Rheum ribes L. (Polygonaceae), widespread in the eastern part of our country and a frequently consumed plant species by the population, has anti-carcinogenic effect on CRC cell lines. Studies related to this plant species mostly focuse on phytochemical analysis and estimation of antioxidant and antibacterial effects, and no studies have been reported that investigated the anti-cancerogenic effects of this plant species at the molecular level. In our study, 6 different extracts were obtained from the root and stem parts of R.ribes. Total phenolic and flavonoid contents, antioxidant activities, and cytotoxic effects of all extracts on HCT 116 and HT-29 colorectal cancer cells were determined. According to the results of these analyses, it was found that, the extract which showed the highest cytotoxic activity at the lowest dose was the root methanol (KM) extract, and hence, the study proceeded with the KM extract. Afterwards, the bioactive content of KM extract was determined by chromatographic analysis. The combination of KM extract with 5-FU, a widely used chemotherapeutic agent in the treatment of CRC, was a dministered to cancer cells for isobologram analysis and it was determined that, the IC50 dose of the combination shows slight synergism in both cells. Differences in expression levels of the miR-200 family (miR-200a/b/c and miR-141) and target genes (EMT and invasion related ZEB1, CDH1 (E-cadherin), CDH2 (N-cadherin); apoptosis related BCL-2, BAX, Caspase-3,7,8,9, CYCS (Cytochrome C), PPARG, XIAP, FAS/CD95, FAP-1; cell cycle related p53, p21, CDK4, CDK6, CCND1, CCND2, CCND3, CDNK1B, RND3; and TGFβ pathway related TGFβ1, TGFβR1, TGFβR2, SMAD2,3,4,7) were assessed by real-time qPCR in cells in which IC50 doses of KM, 5-FU and KM+5-FU were administered. Apoptosis at morphological level was determined by TUNEL and Annexin V analysis; detection of cell cycle stages was determined by cell cycle analysis; invasive character of the cells was determined by invasion test; and finally, the differences in the expression levels of BCL-2, ZEB1, GATA4 and FAS/CD95 proteins were determined by ELISA method. According to the results of miRNA expression analysis, the only dose group with a significant increase (p <0,05) in the expression level of 4 miRNAs that showed tumor suppressor function in both cell lines was KM alone. According to dose groups, when the significant increases and decreases in the target genes associated with apoptosis, cell cycle, EMT and TGFβ pathway were evaluated together, anti-proliferative and apoptotic effects were found to be higher in the combined groups of KM and KM+5-FU than the 5-FU alone. Our results showed that, KM caused a significant increase in the expressions of miR-200a/b/c and miR-141, and it suppressed BCL-2, ZEB1, GATA4 expressions, with this way. Thus, it has been suggested that the administration of KM alone, or combined administration of KM with 5-FU result in a better response than cancer cells treated with 5-FU alone.
Studies are continuing to identify alternative new cytotoxic agents as chemotherapeutic drugs used in the treatment of cancer due to the serious side effects and chemo-dimer resistance in further doses of the treatment. In our study, we will investigate whether Rheum ribes L. (Polygonaceae), widespread in the eastern part of our country and a frequently consumed plant species by the population, has anti-carcinogenic effect on CRC cell lines. Studies related to this plant species mostly focuse on phytochemical analysis and estimation of antioxidant and antibacterial effects, and no studies have been reported that investigated the anti-cancerogenic effects of this plant species at the molecular level. In our study, 6 different extracts were obtained from the root and stem parts of R.ribes. Total phenolic and flavonoid contents, antioxidant activities, and cytotoxic effects of all extracts on HCT 116 and HT-29 colorectal cancer cells were determined. According to the results of these analyses, it was found that, the extract which showed the highest cytotoxic activity at the lowest dose was the root methanol (KM) extract, and hence, the study proceeded with the KM extract. Afterwards, the bioactive content of KM extract was determined by chromatographic analysis. The combination of KM extract with 5-FU, a widely used chemotherapeutic agent in the treatment of CRC, was a dministered to cancer cells for isobologram analysis and it was determined that, the IC50 dose of the combination shows slight synergism in both cells. Differences in expression levels of the miR-200 family (miR-200a/b/c and miR-141) and target genes (EMT and invasion related ZEB1, CDH1 (E-cadherin), CDH2 (N-cadherin); apoptosis related BCL-2, BAX, Caspase-3,7,8,9, CYCS (Cytochrome C), PPARG, XIAP, FAS/CD95, FAP-1; cell cycle related p53, p21, CDK4, CDK6, CCND1, CCND2, CCND3, CDNK1B, RND3; and TGFβ pathway related TGFβ1, TGFβR1, TGFβR2, SMAD2,3,4,7) were assessed by real-time qPCR in cells in which IC50 doses of KM, 5-FU and KM+5-FU were administered. Apoptosis at morphological level was determined by TUNEL and Annexin V analysis; detection of cell cycle stages was determined by cell cycle analysis; invasive character of the cells was determined by invasion test; and finally, the differences in the expression levels of BCL-2, ZEB1, GATA4 and FAS/CD95 proteins were determined by ELISA method. According to the results of miRNA expression analysis, the only dose group with a significant increase (p <0,05) in the expression level of 4 miRNAs that showed tumor suppressor function in both cell lines was KM alone. According to dose groups, when the significant increases and decreases in the target genes associated with apoptosis, cell cycle, EMT and TGFβ pathway were evaluated together, anti-proliferative and apoptotic effects were found to be higher in the combined groups of KM and KM+5-FU than the 5-FU alone. Our results showed that, KM caused a significant increase in the expressions of miR-200a/b/c and miR-141, and it suppressed BCL-2, ZEB1, GATA4 expressions, with this way. Thus, it has been suggested that the administration of KM alone, or combined administration of KM with 5-FU result in a better response than cancer cells treated with 5-FU alone.
Açıklama
Doktora Tezi
Anahtar Kelimeler
Antikanser, HCT 116, HT-29, İnvazyon, miRNA, Polygonaceae, Rheum ribes, Sitotoksite, Anticancer, Invasion, Polygonaceae, Cytotoxicity
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Çınar, İ. (2018). Rheum ribes L. bitki ekstrelerinin kolorektal kanser hücre hatlarında antikanserojenik etkisinin araştırılması. (Yayımlanmamış doktora tezi). Necmettin Erbakan Üniversitesi, Sağlık Bilimleri Enstitüsü Tıbbi Biyoloji Anabilim Dalı, Konya.
