Glisirretinik asit türevi olan soloksolon metil'in meme kanseri hücreleri üzerindeki sitotoksik ve apoptotik etkilerinin araştırılması
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Date
2016-06-03
Authors
Alper, Pınar
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Publisher
Uludağ Üniversitesi
Abstract
Kadınlarda kansere bağlı ölümlerin en önemli nedenlerinden olan meme kanserinin tedavisinde doğal bileşiklerden elde edilen çeşitli kemoterapötik ajanlar kullanılmaktadır ve yeni ilaç geliştirme çalışmalarına devam edilmektedir. Glycyrrhiza glabra (meyan kökü) bitkisinin sekonder metaboliti olan Glisiretinik asitin potansiyel bir antikanser ajan olduğu daha önce yapılan çalışmalarda gösterilmiştir. Bu tez çalışmasında, Rusya Bilim Akademisi, Kimyasal Biyoloji ve Temel Bilimler Enstitüsü'nde sentezlenen, Glisiretinik asitin yarı sentetik bir türevi olan Soloksolon metil bileşiğinin sitotoksik ve apoptotik etkileri insan meme kanseri hücre soylarında (MCF-7, MDA-MB-231 ve T-47D) araştırılmıştır. Soloksolon metil'in hücre canlılığı üzerine olan etkisi (24 ve 48 saat) MTT ve ATP canlılık testleriyle belirlenmiştir. Hücrelerde ölüm modunu (apoptoz ve nekroz) belirlemek amacıyla floresan boyama (Hoechst 33342 ve propidyum iyodür) ve kaspazla kırılmış sitokeratin 18 (M30) metodları kullanılmıştır. Buna ilaveten apoptozis, gen (PCR yöntemi) ve protein (Western Blot) düzeyinde araştırılmıştır. Soloksolon metil'in her üç hücrede doza ve zamana bağlı olarak hücre canlılığını azalttığı, 48 saat sonrasında IC50 değerleri MCF-7, MDA-MB-231 ve T-47D hücrelerinde sırayla 4,22 µM, 5,97 µM ve 6,18 µM olarak belirlenmiştir. Hücrelerde Soloksolon metil'in 10 µM dozunun, 24 ve 48 saat sonrasında erken apoptoza (piknotik nükeus ve propidyum iyodür negatifliği); 72 saat sonrasında geç apoptotik/sekonder nekroza (piknotik nükleus ve propidyum iyodür pozitifliği) neden olduğu gözlenmiştir. MCF-7 ve MDA-MB-231 hücrelerinde Soloksolon metil etkisiyle apoptotik gen ekspresyonlarının arttığı tespit edilmiştir. Ayrıca MDA-MB-231 hücre soyunda apoptotik proteinlerin yanı sıra endoplazmik retikulum stresiyle ilişkili proteinlerin (IRE1-α, Bip, CHOP) artışı da belirlenmiştir. Sonuç olarak, Soloksolon metil bileşiğinin MCF-7 ve MDA-MB-231 hücre soyları üzerine olan etkisinin sitotoksik etkisini apoptozis üzerinden gerçekleştirdiği, özellikle MDA-MB-231 hücre soyunda endoplazmik retikulum aracılı apoptozis yolağından ilerlediği belirlenmiştir. Alınan sonuçlar Soloksolon metil'in meme kanseri tedavisinde etkili bir ajan olabileceğini ileri analizlerin (in vitro ve in vivo) yapılması gerektiği sonucuna varılmıştır.
Being one of the leading causes of cancer death among women, various chemotherapeutic agents isolated from natural compounds are used in breast cancer treatment and studies to develop new drugs still continues. There are several studies on glycyrrhetinic acid, a secondary metabolite which is found in Glycyrrhiza glabra (Liquorice), as a potential anticancer agent. In this thesis, the cytotoxic and apoptotic effects of soloksolon methyl compound, a semi-synthetic derivative of glycyrrhtenic acid synthesized by the Russian Academy of Sciences, Chemical Biology and Basic Sciences Institute in Siberia were investigated on breast cancer cell lines (MCF-7, MDA-MBA-231 and T-47D). The antigrowth effects (24 and 48 hours) of Soloksolon methyl were determined using MTT and ATP assays. To identify the mode of cell death (apoptosis/necrosis), fluorescent staining (Hoechst 33342 and Propidium iodide) and caspase-cleaved cytokeratin 18 (M30) test were used. In addition, apoptotic genes and protein levels were investigated by PCR method and Western Blotting. Soloksolon methyl decreased cell viability in all three cell lines in a dose and time-dependent manner. The IC50 values in MCF-7, MDA-MBA-231, and T-47D cells after 48 hours of treatment are 4,22 µM, 5,97 µM and 6,18 µM respectively. Soloksolon methyl induces early apoptosis (pyknotic nuclei and negative Propidium iodide) at 10 µM after 24 and 48 hours, and late apoptosis or secondary necrosis (pyknotic nuclei and positive Propidium iodide) after 72 hours in all cell lines. An increase of apoptotic gene expressions was determined in MCF-7 and MDA-MB-231 cells treated with Soloksolon methyl. Moreover, an increase of apoptotic proteins related to endoplasmic reticulum (IRE1-α, Bip, CHOP) stress was also determined in MDA-MB-231 cell line. In conclusion, Soloksolon methyl is found to be cytotoxic on MCF-7 and MDA-MBA-231 cells by inducing apoptosis, especially in MDA-MB-231 cell line where ER stress-induced apoptosis is triggered. Advance analyses (in vitro and in vivo) should be performed to elucidate the potential of Soloksolon methyl as anticancer agent in breast cancer treatment.
Being one of the leading causes of cancer death among women, various chemotherapeutic agents isolated from natural compounds are used in breast cancer treatment and studies to develop new drugs still continues. There are several studies on glycyrrhetinic acid, a secondary metabolite which is found in Glycyrrhiza glabra (Liquorice), as a potential anticancer agent. In this thesis, the cytotoxic and apoptotic effects of soloksolon methyl compound, a semi-synthetic derivative of glycyrrhtenic acid synthesized by the Russian Academy of Sciences, Chemical Biology and Basic Sciences Institute in Siberia were investigated on breast cancer cell lines (MCF-7, MDA-MBA-231 and T-47D). The antigrowth effects (24 and 48 hours) of Soloksolon methyl were determined using MTT and ATP assays. To identify the mode of cell death (apoptosis/necrosis), fluorescent staining (Hoechst 33342 and Propidium iodide) and caspase-cleaved cytokeratin 18 (M30) test were used. In addition, apoptotic genes and protein levels were investigated by PCR method and Western Blotting. Soloksolon methyl decreased cell viability in all three cell lines in a dose and time-dependent manner. The IC50 values in MCF-7, MDA-MBA-231, and T-47D cells after 48 hours of treatment are 4,22 µM, 5,97 µM and 6,18 µM respectively. Soloksolon methyl induces early apoptosis (pyknotic nuclei and negative Propidium iodide) at 10 µM after 24 and 48 hours, and late apoptosis or secondary necrosis (pyknotic nuclei and positive Propidium iodide) after 72 hours in all cell lines. An increase of apoptotic gene expressions was determined in MCF-7 and MDA-MB-231 cells treated with Soloksolon methyl. Moreover, an increase of apoptotic proteins related to endoplasmic reticulum (IRE1-α, Bip, CHOP) stress was also determined in MDA-MB-231 cell line. In conclusion, Soloksolon methyl is found to be cytotoxic on MCF-7 and MDA-MBA-231 cells by inducing apoptosis, especially in MDA-MB-231 cell line where ER stress-induced apoptosis is triggered. Advance analyses (in vitro and in vivo) should be performed to elucidate the potential of Soloksolon methyl as anticancer agent in breast cancer treatment.
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Keywords
Glisirotinik asit, Soloksolon metil, Meme kanseri, Apoptoz, Sitotoksite, Glycrrotinic acide, Soloksolon methyl, Breast cancer, Apoptosis, Cytotoxicity
Citation
Alper, P. (2016). Glisirretinik asit türevi olan soloksolon metil'in meme kanseri hücreleri üzerindeki sitotoksik ve apoptotik etkilerinin araştırılması. Yayınlanmamış yüksek lisans tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.