Sitrat transport protein inhibisyonunun meme kanseri hücreleri üzerine olan etkilerinin araştırılması
Küçük Resim Yok
Tarih
2015
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Çağımızın en önemli sağlık sorunlarından birisi olan kanserin karakteristik özelliklerinden birisi de hücresel metabolizmada meydana gelen değişikliklerdir. Bu değişiklikler arasında glikolitik hızda ve sentez yolaklarında artış ile yıkım yolaklarında yavaşlama yer almaktadır. Hücrenin yeni hücreler oluşturmak üzere gerekli metabolitleri sentezledikleri anabolik yolaklar arasında protein, nükleotid ve yağ asidi sentezi yer almaktadır. Son yıllarda yapılan çalışmalar bu yolakta yer alan ATP-sitrat liyaz (ACLY), asetil-KoA karboksilaz ve yağ asidi sentaz enzimlerinin kanser hücrelerinde yüksek aktiviteye sahip olduğunu ortaya koymuştur. Bununla birlikte yağ asidi sentezinin gerçekleşmesinde kritik rol oynayan ve sitoplazmik asetil-KoA'nın kaynağı olan sitoplazmik sitratın mitokondriden taşınımında görev alan sitrat transport proteininin (CTP) kanserdeki rolü iyi bilinmemektedir. Bu çalışmada siRNA ve kimyasal inhibitör aracılığıyla inhibe edilen sitrat transport proteininin meme kanseri hücre hatlarında hücresel süreçlerdeki önemi incelenmiştir. Kullanılan hücre hatlarından MCF-10A normalmeme epitel hücrelerini, MCF-7 agresif olmayan meme kanserini ve MDA-MB-231 metastatik meme kanserini temsil etmektedir. Çalışmada hücreler siRNA ile muamele edildikten sonra inhibisyonun etkinliği western blot yöntemi ile gösterilmiş ve sitoplazmik sitrat düzeylerindeki değişim spektrofotometrik yöntemler kullanılarak incelenmiştir. Hücre canlılığı spektrofotometrik kristal viyole yöntemi ile; apoptoz, nekroz ve hücre yaşam döngüsünde meydana gelen değişiklikler ise akış sitometrik yöntemlerle belirlenmiştir. Otofaji hem akış sitometri hem de florasan mikroskopi yöntemleri kullanılarak incelenmiştir. Son olarak inhibisyonun sitoplazmik asetil-KoA monomerlerine ihtiyaç duyan epigenetik bir regülasyon mekanizması olan histon asetilasyonu üzerine etkileri spektrofotometrik yöntemler kullanılarak tespit edilmiştir. Denemelerde CTP ile birlikte ACLY da inhibe edilmiş ve sonuçlar karşılaştırmalı olarak değerlendirilmiştir. Elde edilen sonuçlar bütün hücre hatlarında ilgili proteinlerin eksprese edildiğini ve siRNA kullanılarak ekspresyonun etkili bir şekilde susturulabildiğini göstermektedir. Kanser agresifliği ile bazal sitoplazmik sitrat düzeyleri arasında ilişki belirlenmiş ve sitoplazmik sitrat düzeyleri CTP siRNA ile kısmen ve kimyasal inhibitör ile etkili bir şekilde inhibe edilmiştir. Canlılık verilerine göre CTP ve ACLY inhibisyonu normal hücrelere zarar vermeden kanser hücrelerinde canlılığı inhibe etmektedir. Bu inhibisyon kimyasal inhibitör ile çok daha etkili bir biçimde gerçekleşmektedir. Hücrelerde canlılık inhibisyonunun nedenleri araştırılmış ama azalmayı açıklayabilecek düzeyde apoptoz, nekroz, hücre yaşam döngüsü değişikliği ya da otofaji belirlenememiştir. Son olarak inhibisyonun hücrelerde asetilenmiş histon düzeylerinde azalmaya neden olduğu belirlenmiştir. Bu azalma, diğer deney sonuçlarıyla uyumlu bir biçimde kimyasal inhibitör ile muamele edilen hücrelerde daha etkili bir biçimde gerçekleşmiştir. Bu tez kapsamında elde edilen veriler CTP ve ACLY proteinlerinin canlılık ve histon asetilasyonu açısından önemli olduğu ve ilgili proteinlerin inhibisyonu ile bu süreçlere girişim yapılabildiğini göstermektedir. Kimyasal inhibisyonun RNA interferansa göre daha etkili olması, sitoplazmik sitrat kaynağı olarak ekstraselüler alandan geçiş yapan sitrat metabolitlerinin önemli olduğuna ve bu yolağın kimyasal inhibitörden etkilendiklerine işaret etmektedir. Bununla birlikte her iki proteinin inhibisyonunun da normal hücrelere zarar vermeden kanser hücrelerinde etkili olması bu proteinlerin hedeflenmesinin kanser terapi stratejileri açısından avantajlı olabileceğini göstermektedir
Cellular metabolic alterations in cancer cells are among the hallmarks of the cancer which is one of the most important medical problems of our age. Increased rate of glycolysis and synthesis pathways along with decreased rate of degradation pathways are mmong these alterations. Anabolic pathways required for cells to create novel cells are protein, nucleotide and fatty acid synthesis pathways. Novel studies suggest that proteins playing a role in these pathways such as ATP-citrate lyase (ACLY), acetyl-CoA carboxilase and fatty acid synthease have high activity in cancer cells. However the role of citrate transport protein (CTP) which enables transportation of citrate from mitochondria into the cytoplasm where it plays a critical role for fatty acid synthesis as a source for cytoplasmic acetyl-CoA, is rather unknown. In this study the importance of citrate transport protein for cellular processes is examined by its inhibition in breast cancer cell lines via siRNA or chemical inhibition. Among the cell lines; MCF-10A represents normal epithelial cells, MCF-7 represents less-aggressive breast cancer and MDA-MB-231 represents metastatic breast cancer. After siRNA treatment the efficiency of the inhibition was demonstrated via western blotting and the alterations in cytoplasmic citrate levels were detected via spectrophotometry. Cell viability was assessed by spectrophotometric crystal violet assay while the alterations in apoptosis, necrosis and cell cycle regulation were evaluated via flow cytometry. Autophagy was assessed both by fluorescence microscopy and flow cytometry. Lastly the effects of the inhibition on acetylation of histones, an epigenetic regulation process which requires cytopalsmic acetyl-CoA, were detected spectrophotometrically. During the experiments ACLY was also inhibited along with CTP and the results were evaluated comparatively Obtained results suggest the proteins of interest are expressed in all cell lines and siRNA treatment can effectively silence the proteins. An association between basal cytoplasmic citrate levels and the aggressiveness of the cancer was determined and these levels were reduced partially by siRNA treatment and effectively by chemical inhibition. According to the data obtained from viability experiments inhibition of CTP and ACLY inhibit cancer cell viability without affecting normal cells. This inhibition is more evident in cells treated with chemical inhibitor. The reason of the inhibition of cell viability was also studied, however no alteration in apoptosis, necrosis, cell cycle or autophagy strong enough for to cause such inhibition was detected. Lastly inhibition was also detected to inhibit histone acetylation which was more prominent in cells treated with chemical inhibitor accordance with previous experiments. Data obtained throughout this thesis demonstrates the importance of CTP and ACLY for cell viability and histone acetylation, and there processes can be interfered with by inhibiting those proteins. High potency of chemical inhibition compared to RNA interference suggest the importance of extracellular citrate as a source of cytoplasmic citrate and that this pathway is effected by chemical inhibition. However effectiveness of the inhibition of both protein on cancer cells without affecting normal cells demonstrates that targeting these proteins is advantageous for cancer therapy strategies.
Cellular metabolic alterations in cancer cells are among the hallmarks of the cancer which is one of the most important medical problems of our age. Increased rate of glycolysis and synthesis pathways along with decreased rate of degradation pathways are mmong these alterations. Anabolic pathways required for cells to create novel cells are protein, nucleotide and fatty acid synthesis pathways. Novel studies suggest that proteins playing a role in these pathways such as ATP-citrate lyase (ACLY), acetyl-CoA carboxilase and fatty acid synthease have high activity in cancer cells. However the role of citrate transport protein (CTP) which enables transportation of citrate from mitochondria into the cytoplasm where it plays a critical role for fatty acid synthesis as a source for cytoplasmic acetyl-CoA, is rather unknown. In this study the importance of citrate transport protein for cellular processes is examined by its inhibition in breast cancer cell lines via siRNA or chemical inhibition. Among the cell lines; MCF-10A represents normal epithelial cells, MCF-7 represents less-aggressive breast cancer and MDA-MB-231 represents metastatic breast cancer. After siRNA treatment the efficiency of the inhibition was demonstrated via western blotting and the alterations in cytoplasmic citrate levels were detected via spectrophotometry. Cell viability was assessed by spectrophotometric crystal violet assay while the alterations in apoptosis, necrosis and cell cycle regulation were evaluated via flow cytometry. Autophagy was assessed both by fluorescence microscopy and flow cytometry. Lastly the effects of the inhibition on acetylation of histones, an epigenetic regulation process which requires cytopalsmic acetyl-CoA, were detected spectrophotometrically. During the experiments ACLY was also inhibited along with CTP and the results were evaluated comparatively Obtained results suggest the proteins of interest are expressed in all cell lines and siRNA treatment can effectively silence the proteins. An association between basal cytoplasmic citrate levels and the aggressiveness of the cancer was determined and these levels were reduced partially by siRNA treatment and effectively by chemical inhibition. According to the data obtained from viability experiments inhibition of CTP and ACLY inhibit cancer cell viability without affecting normal cells. This inhibition is more evident in cells treated with chemical inhibitor. The reason of the inhibition of cell viability was also studied, however no alteration in apoptosis, necrosis, cell cycle or autophagy strong enough for to cause such inhibition was detected. Lastly inhibition was also detected to inhibit histone acetylation which was more prominent in cells treated with chemical inhibitor accordance with previous experiments. Data obtained throughout this thesis demonstrates the importance of CTP and ACLY for cell viability and histone acetylation, and there processes can be interfered with by inhibiting those proteins. High potency of chemical inhibition compared to RNA interference suggest the importance of extracellular citrate as a source of cytoplasmic citrate and that this pathway is effected by chemical inhibition. However effectiveness of the inhibition of both protein on cancer cells without affecting normal cells demonstrates that targeting these proteins is advantageous for cancer therapy strategies.
Açıklama
Anahtar Kelimeler
Biyokimya, Biochemistry, Tıbbi Biyoloji, Medical Biology
