Palladyum (II) bileşiği ve canertinibin kolon kanser hücrelerinde sitotoksisite, anjiyogenez, ilaç dirençliliği ve otofaji mekanizmaları üzerine etkileri
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Date
2017-12-29
Authors
Aydınlık, Şeyma
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Publisher
Uludağ Üniversitesi
Abstract
Metastatik kolon kanseri agresif kemoterapi rejimlerinin kullanılmasına rağmen, % 5'den az bir hayatta kalma oranına sahiptir. Kolon kanserinde epidermal büyüme faktörü reseptörünün (EGFR) prognostik ve prediktif önemi hala tartışmalıdır. Bu nedenle EGFR reseptörünü bloke etmek amacıyla bu çalışmada tirozin kinaz inhibitörü canertinib kullanılmıştır. Bu amaçla Uludağ üniversitesi kimya bölümü tarafından sentezlenenen Palladyum(Pd) (II) bileşiği [PdCl(terpy)](sac).2H2O] ve bu bileşiğin canertinib ile kombinasyonunun etkileri araştırılmıştır. Ayrıca kemoterapötik ilaç 5-Fluorourasil (5-FU) ve bu ajanın canertinib ile kombinasyonu ise pozitif kontrol olarak kullanılmıştır. Dolayısıyla bu tez çalışmasında, Palladyum (II) bileşiği ile canertinib tek başına ve kombinasyon tedavileri ve aynı şekilde 5- Fluorourasil ve bu ajanın canertinib ile kombinasyonunun insan kolon kanseri hücre soyları (HCT-15, HT-29) üzerine sitotoksik etkileri SRB canlılık testi ile belirlenmiştir. Kombinasyon tedavisinin sitotoksik etkilerinden sorumlu hücre ölümü (apoptozis/otofaji) akım sitometrisi, hücrelerde apoptoz varlığı Anneksin-V-Cy3/SYTOX ikili boyama yöntemi ile asidik veziküler organellerin varlığı akridin boyaması ile mitokondri depolarizasyonu tetra etil benzimidazol karboksinin iyodür (JC-1) ile floresan mikroskopta görüntülenerek desteklenmiştir. EGFR ilişkili yaşam yolaklarında, bu reseptörün inhibe edilmesi ile gerçekleşen değişimler, epidermal mezenkimal dönüşüm (EMT) ile ilişkili proteinlerdeki değişimler ve apoptoz/otofaji ilişkili proteinlerdeki değişimler ise western blot yöntemi ile değerlendirildi. Bunun dışında ilaç taşıyıcı proteinler üzerine etkisi MDCKII polarize hücre soyları kullanılarak önce SRB yöntemi ile daha sonra ilişkili proteinlerdeki değişimler western blot ile belirlendi. Ayrıca bu yaşam yolağının inhibisyonunun kombinasyon tedavileri ile birlikte invazyon, yara iyileşmesi ve koloni oluşturma yetenekleri üzerine etkileri belirlendi. Son olarak HUVEC endotel hücrelerinde EGFR inhibisyonun kombinasyon tedavileri ile birlikte tüp oluşturma yeteneği in vitro matrigel testi ile ve damar oluşumu üzerine etkileri ayrıca in vivo Chorio-allantoik membran (CAM) testi ile değerlendirildi. Sonuç olarak, Pd (II) bileşiği ve canertinib kombinasyonunun kolon kanseri hücre soylarında sitotoksik aktivite ve apoptoz artışına, invazyon, yara iyileşmesi yeteneklerinde ve damar oluşumunda azalmaya neden olduğu bulunmuştur.
Metastatic colon cancer has a survival rate less than %5 despite the use of effective chemotherapeutic regimen. Prognostic and predictive importance of epidermal growth factor receptor (EGFR) is still contradictive in colon cancer. For this reason, the tyrosine kinase inhibitor canertinib was used in this study to block the EGFR receptor. The effect of Palladium (Pd) (II) compound [PdCl(terpy)](sac).2H2O] which was synthesized by Uludağ University chemistry department and canertinib alone and the effect of Pd(II) compound and canertinib combination treatment was investigated in this study. 5-fluorouracil (5-FU), a chemotherapeutic drug commonly used in the treatment of cancer and its combination with canertinib was used as positive control. Therefore, the cytotoxic effects of the combination of Palladium (II) compound and EGFR inhibitor canertinib and the chemotherapeutic agent 5-fluorouracil and canertinib combination on human colon cancer cell line (HCT-15, HT-29) were also determined by the SRB viability test. Cell death (apoptosis/autophagy) responsible for the cytotoxic effects of the combination therapy, presence of apoptosis in cells and the presence of acidic vesiculations, mitochondrial depolarization were determined by flow cytometry, Annexin-V-Cy3/SYTOX dual staining method, acridine staining and tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining respectively. Alterations in proteins related with EGFR-associated survival pathway and alterations in proteins associated with epidermal mesenchymal transformation (EMT) and transformation in apoptosis/autophagy-related proteins were evaluated by western blot. In addition, effects on drug carrier proteins were first determined by SRB method using MDCKII polarize cell lines and then changes in proteins were determined by western blot. Also, effects of inhibition of this life pathway with combination therapy together on invasion, wound healing and colony forming ability were determined. Finally, effects of EGFR inhibition with combination treatments together on the ability to form tubes in HUVEC endothelial cells was assessed by in vitro matrigel test and the effects on vessel formation were also assessed by in vivo Chorio Allantoic Membrane (CAM) test. In conclusion, the combination of Pd (II) compound and canertinib has been shown to increase cytotoxic activity, induced apoptosis, decreased invasion, wound healing abilities and vessel formation in colon cancer cell lines.
Metastatic colon cancer has a survival rate less than %5 despite the use of effective chemotherapeutic regimen. Prognostic and predictive importance of epidermal growth factor receptor (EGFR) is still contradictive in colon cancer. For this reason, the tyrosine kinase inhibitor canertinib was used in this study to block the EGFR receptor. The effect of Palladium (Pd) (II) compound [PdCl(terpy)](sac).2H2O] which was synthesized by Uludağ University chemistry department and canertinib alone and the effect of Pd(II) compound and canertinib combination treatment was investigated in this study. 5-fluorouracil (5-FU), a chemotherapeutic drug commonly used in the treatment of cancer and its combination with canertinib was used as positive control. Therefore, the cytotoxic effects of the combination of Palladium (II) compound and EGFR inhibitor canertinib and the chemotherapeutic agent 5-fluorouracil and canertinib combination on human colon cancer cell line (HCT-15, HT-29) were also determined by the SRB viability test. Cell death (apoptosis/autophagy) responsible for the cytotoxic effects of the combination therapy, presence of apoptosis in cells and the presence of acidic vesiculations, mitochondrial depolarization were determined by flow cytometry, Annexin-V-Cy3/SYTOX dual staining method, acridine staining and tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining respectively. Alterations in proteins related with EGFR-associated survival pathway and alterations in proteins associated with epidermal mesenchymal transformation (EMT) and transformation in apoptosis/autophagy-related proteins were evaluated by western blot. In addition, effects on drug carrier proteins were first determined by SRB method using MDCKII polarize cell lines and then changes in proteins were determined by western blot. Also, effects of inhibition of this life pathway with combination therapy together on invasion, wound healing and colony forming ability were determined. Finally, effects of EGFR inhibition with combination treatments together on the ability to form tubes in HUVEC endothelial cells was assessed by in vitro matrigel test and the effects on vessel formation were also assessed by in vivo Chorio Allantoic Membrane (CAM) test. In conclusion, the combination of Pd (II) compound and canertinib has been shown to increase cytotoxic activity, induced apoptosis, decreased invasion, wound healing abilities and vessel formation in colon cancer cell lines.
Description
Keywords
Trozin kinaz inhibitörleri, Anjiyogenez, Otofaji, EMT, İlaç dirençliliği, Tyrosine kinase inhibitors, Angiogenesis, Autophagy, Drug resistance
Citation
Aydınlık, Ş. (2017). Palladyum (II) bileşiği ve canertinibin kolon kanser hücrelerinde sitotoksisite, anjiyogenez, ilaç dirençliliği ve otofaji mekanizmaları üzerine etkileri. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.