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İnsan yumurtalık kanseri hücre hattında (Ovcar-3) zoledronik asit ve Gossypol’un sitotoksik ve apoptotik etkilerinin araştırılması Skip to content Tez Künye Durumu İnsan yumurtalık kanseri hücre hattında (Ovcar-3) zoledronik asit ve Gossypol’un sitotoksik ve apoptotik etkilerinin araştırılması / An investigation of cytotoxic and apoptotic effect of zoledronic acid and Gossypol in ovarian cancer cell line (OVCAR-3) Yazar:HARİKA ATMACA Danışman: YRD. DOÇ. DR. SELİM UZUNOĞLU Yer Bilgisi: Celal Bayar Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı / Moleküler Biyoloji Bilim Dalı Konu:Biyoloji = Biology Dizin:Sitotoksisite = Cytotoxicity Onaylandı Yüksek Lisans Türkçe 2008 75 s. Yumurtalık kanseri jinekolojik malign kanserler içinde en yaygın ölüm sebebidir. Epitelyal yumurtalık kanseri tüm yumurtalık kanserlerinin %90’ını oluşturmaktadır. Teşhis konulduğunda hastaların çoğu III ya da IV. evrede olup, kötü prognoza sahiptir. Yumurtalık kanserli hastalarda optimal kemoterapik cevap almada en iyi yaklaşım, kombinasyon veya ardışık kemoterapi uygulamalarıdır. Nükseden yumurtalık kanseri tedavisinde ise en büyük problem çoklu ilaç direncidir. Bundan dolayı, OVCAR-3 gibi yumurtalık kanseri hücre modelleri üzerinde yeni ilaç kombinasyon tedavilerinin araştırılması büyük önem taşır. Gossypol (GP), pamuk bitkisinden (Gossypium species) izole edilen antitümör özellikleri olan yeni bir ilaç adayıdır. Zoledronik asit (ZA), üçüncü nesil bisfosfonatlardan biri olup, çoğalmayı engelleyici ve apoptozu uyarıcı özellikleri birkaç kanser hücre hattında gösterilmiştir.Birincil amaç, ZA ve GP’nin OVCAR-3 hücreleri üzerindeki sitotoksik ve apoptotik etkilerini belirlemektir. İkinci amaç; ZA ve GP’yi sabit ve değişen oranlarda kombine ederek, kombinasyonlarının sitotoksik ve apoptotik etkilerini saptamaktır. Üçüncü amaç ise, nükseden yumurtalık kanserinin tedavi seçeneklerine dolaylı olarak katkıda bulunmaktır.İnsan yumurtalık kanseri hücre hattı OVCAR-3, RPMI 1640 besiyerinde çoğaltıldı ve canlılıkları tripan mavisinin dışa atılımı yöntemiyle belli aralıklarla izlendi. ZA ve GP’nin stok solüsyonları hazırlanarak her deneyde yeni aliquat kullanıldı. Kuyucuk başına 104 hücre ZA ve GP’nin artan konsantrasyonlarıyla 24, 48 ve 72 saat muamele edildi. IC50 dozları 72 saatlik inkübasyon sonunda doz-cevap eğrileri çizilerek belirlendi. Sitotoksisiste XTT kolorimetrik testiyle ve apoptoz ELISA tabanlı apoptoz dedeksiyon kitiyle saptandı. apoptotik veriler kaspaz 3/7 aktivitesi ölçülerek doğrulandı. Tüm deneyler üç kez tekrarlandı ve sonuçlar ortalama olarak ve standart sapmalarıyla birlikte verildi. İlaç kombinasyonlarındaki sinerji, medyan etki denklemini ve kombinasyon indeks (CI) yöntemlerini kullanan CalcuSyn yazılımıyla hesaplandı.ZA ve GP için IC50 değerleri sırasıyla 14 ve 25 µM olarak belirlendi. Gossypolün 72. saatteki en etkili sitotoksik dozu 80 µM olarak saptanırken, ZA’nın 50 µM idi. Hem ZA hem de GP hücre çoğalmasını zamana ve doza bağlı olarak inhibe ettiği gösterildi. ZA ve GP kombinasyonu aşağıdaki oran ve kombinasyonlarda sinerjistik sitotoksik etki göstermiştir; 1:104 (2,5 nM ZA+25 µM GP), 1:3 (5 µM ZA+15 µM GP). ZA-GP kombinasyonunun CI değerleri yukarıdaki konsantrasyonlarda 0,285, 0,163 olarak tespit edildi. Düşük CI değerleri güçlü sinerjizm göstergesi olarak yorumlandı. Aynı CI değerleri, ZA ve GP’nin sinerjistik bulunan dozları, sabit oran ve azalan katlı konsantrasyonlarda uygulandığında ise korunamadığı tespit edildi. İlaçların tek tek uygulanmasıyla elde edilen 72. saatteki apoptoz yüzdeleri kombine uygulamalarıyla karşılaştırıldı. Elde edilen apoptotik veriler kaspaz 3/7 enzim aktivitesindeki artışla doğrulandı.Bu çalışma gossypolün ve ZA-GP kombinasyonunun OVCAR-3 yumurtalık kanseri hücrelerine sitotoksik ve apoptotik etkilerinin belirlendiği ilk araştırmadır. ZA-GP kombinasyonunun OVCAR-3 hücreleri üzerinde önemli sitotoksik ve apoptotik etkileri olduğu gösterildi. Bu ön çalışma ZA-GP kombinasyonunun nükseden yumurtalık kanserinin tedavisinde umut verici bir kombinasyon tedavisi olabilir. Bu nedenle, ZA-GP kombinasyonunun belirlenen etkilerinin diğer yumurtalık kanseri hücre hatlarında da araştırılması gerekmektedir.Anahtar kelimeler: Apoptotik etki, Gossypol, İnsan Yumurtalık Kanseri Hücre Hattı OVCAR-3, Sinerjizm, Sitotoksisite, Zoledronik asit. Ovarian cancer remains the most common cause of death from a gynecologic malignancy. Epithelial ovarian cancer comprises 90% of all ovarian cancers. Most patients with this disease have advanced stage disease (ie, stage III?IV) at the time of diagnosis, which is associated with a substantially poorer prognosis than its early-stage counterparts. The `gold standard? for optimal chemotherapeutic response in women with ovarian cancer is combinational or sequential chemoterapy treatment. One of the biggest problem in the treatment of recurrent ovarian cancer is multidrug resistance. Therefore, investigation of new combinational drug treatments is of vital importance in ovarian cancer cell models, such as OVCAR-3. Gossypol (GP) extracted from cotton plant (Gossypium species), is a new drug candidate with antitumor properties. Zoledronic acid (ZA) is a third generation bisphosphonate and it?s antiproliferative and apoptotic properties were studied in several cancer cell lines.Our first objective is to determine the cytotoxic and apoptotic effects of gossypol and zoledronic acid in OVCAR-3 cells. Secondly, to find out the synergistic cytotoxic and apoptotic effect of gossypol and zoledronic acid by combining them in non-constant and constant ratios. Thirdly, to make contribution indirectly to treatment options of recurrent ovarian cancer.The human ovarian cancer cell line OVCAR-3 was cultured in RPMI 1640 and their viability monitored in certain intervals by trypan blue dye exclusion assay. Stock solutions of gossypol and zoledronic acid were prepared and new aliquots were used in each experiment. 104 cells in per well were treated with increasing concentrations of gossypol and zoledronic acid alone and in combination for 24, 48, 72 hours. The IC50 doses were determined by drawing dose-response curves after 72 h drug treatment. Drug-mediated cytotoxicity was determined by XTT colorimetric assay and apoptosis was detected by using ELISA based Cell Death Detection Kit. Apoptotic data were confirmed by using Caspase-Glo 3/7 Assay. All assays were set up in triplicate and the results were expressed as the mean + SD. The synergism in drug combinations was assessed by CalcuSyn software based on median effect equation and combination index methods.IC50 values for ZA and GP were determined as 14 and 25 µM, respectively. The most cytotoxic effect of gossypol was obtained in 80 µM dose at 72 hours, while the most cytotoxic effect of zoledronic acid was in 50 µM at 72 hours. Both gossypol and zoledronic acid inhibits cell proliferation in dose and time dependent manner.ZA and GP in combination was synergistic in following ratios and concentrations; 1:104 (2,5 nM ZA+25 µM GP), 1:3 (5 µM ZA+15 µM GP). CI values for ZA-GP combination were calculated as 0,285, 0,163 respectively in above concentrations. These lowest CI values were indicator of strong synergism. The same CI values were not maintained when the ZA-GP in synergistic constant ratio were tested in fold decreasing concentrations. The percentage of relative apoptosis at 72 hours was compared between single drugs and combination treatment. These apoptotic data were also confirmed by significant increase in caspase 3/7 enzyme activity in combined treatments.This study is the first investigation of cytotoxic and apoptotic effects of gossypol and ZA-GP combination on human OVCAR-3 ovarian cancer cells. For the first time, it is shown that ZA-GP combination treatment has significant cytotoxic and apoptotic effects on OVCAR-3 cells. Based on these preliminary data, ZA-GP combination is a new promising combination therapy for recurrent ovarian cancer. So, new studies are needed to explore the effects of ZA-GP combination on several ovarian cancer cells.Key words: Apoptotic effect, Cytotoxicity, Gossypol, Human OVCAR-3 Ovarian Cancer Cell, Synergism, Zoledronic Acid.
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