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Öğe Different doses of radiation on agar colony forming development in C6 glioma cells(Ortadogu Ad Pres & Publ Co, 2007) Ozmen, Tolga; Oktem, Gulperi; Tuna, Sevilcan; Biltekin, Burcu; Denir, Secnur; Bilir, AyhanObjective: Gliomas are relatively frequent in adults, and are among the most malignant primary brain tumors. Glioblastoma. multiforme, like many other tumors that exhibit radiation sensitivity in vitro, seems to be very resistant to radiation in vivo, thus suggesting that irradiation may not be a rate-limiting factor for malignant glioma tumor growth. In this study, we aimed to determine the optimal dose of radiation in C6 glioma colony forming assay, which is a valuable tool for antitumor treatment screening. Material and Methods: 10(5) cell/lamella colony forming cells were radiated with 200 cGy, 400 cGy, 800 cGy and 1600 cGy for 10 minutes. Radiosensitivity was measured systematically 24, 48, 72 and 96 hours after the radiation by three methods: soft-agar bilayer assay, thymidinE incorporation, and bromodeoxyuridine (BrdU) incorporation. Results: The soft-agar bilayer assay, which assessed the colony-forming units, showed that the number of colonies in the control group (609, 3 +/- 86.8) were decreased after 200 cGy (8.3 +/- 3.6) and 400 cGy (7.2 +/- 4.3). No colony was detected in 800 cGy and 1600 cGy irradiated cells [3H] Thymidine incorporation was more prominent with higher doses of radiation. BrdU incorporation revealed that even at low doses (200 cGy) of radiation there was a significant decrease of cell proliferation. On higher doses like 1600 cGy it was more prominent. Conclusion: Cell survival, doubling time, and cell phases are parameters of growth kinetics, and the results suggest that C6 glioma cells are radiosensitive and are virtually affected by all radiation doses in our experiment even 200 cGy at 24 hours. Besides, colony forming assay with thymidine labeling index, and BrdU labeling index may be used as new methods for determining radiotherapy doses in clinical applications.Öğe The flavonoid apigenin reduces prostate cancer CD44+ stem cell survival and migration through PI3K/Akt/NF-?B signaling(Pergamon-Elsevier Science Ltd, 2016) Erdogan, Suat; Doganlar, Oguzhan; Doganlar, Zeynep B.; Serttas, Riza; Turkekul, Kader; Dibirdik, Ilker; Bilir, AyhanAims: Cancer stem cells (CSCs) are involved in drug resistance, metastasis and recurrence of cancers. The efficacy of apigenin on cell survival, apoptosis, migration and stemness properties were analyzed in CSCs. Main methods: Prostate CSCs (CD44(+)) were isolated from human prostate cancer (PCa) PC3 cells using a magnetic-activated cell sorting system. PC3 and CSCs were treated with various concentrations of apigenin, docetaxel and their combinations for 48 h. Key findings: Apigenin dose dependently inhibited CSCs and PC3 cell survival, and this was accompanied with a significant increase of p21 and p27. Apigenin induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mRNA expressions of caspases-8,-3 and TNF-alpha, but failed to regulate the intrinsic pathway as determined by the Bax, cytochrome c (Cyt-c) and APAF-1 in CSCs. In contrary to CSCs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of Bax, Cyt-c and caspase-3 while caspase-8, TNF-alpha and Bcl-2 levels remained unchanged in PC3 cells. The flavonoid strongly suppressed the migration rate of CSCs compared to untreated cells. Significant downregulation of matrix metallopeptidases-2,-9, Snail and Slug exhibits the ability of apigenin treatment to suppress invasion. The expressions of NF-kappa B p105/p50, PI3K, Akt and the phosphorylation of pAkt were decreased after apigenin treatment. Moreover, apigenin treatment significantly reduced pluripotency marker Oct3/4 protein expression which might be associated with the down-regulation of PI3K/Akt/NF-kappa B signaling. Significance: Our data indicated that, apigenin could be a useful compound to prevent proliferation and migration of cancer cells as well as CSCs. (C) 2016 Elsevier Inc. All rights reserved.Öğe Midkine downregulation increases the efficacy of quercetin on prostate cancer stem cell survival and migration through PI3K/AKT and MAPK/ERK pathway(Elsevier France-Editions Scientifiques Medicales Elsevier, 2018) Erdogan, Suat; Turkekul, Kader; Dibirdik, Ilker; Doganlar, Oguzhan; Doganlar, Zeynep B.; Bilir, Ayhan; Oktem, GulperiAims: To examine the functions of growth factor midkine (MK) and a flavonoid quercetin on survival, apoptosis and migration of prostate cancer (PCa) stem cells (CSCs). Main methods: CD44(+)/CD133(+) and CD44(+) stem cells were isolated from PC3 and LNCaP cells, respectively by magnetic-activated cell sorting system. 3D cell culture was used to evaluate the ability of quercetin, MK siRNA, and the combination of both to inhibit spheroid formation, apoptosis and cell cycle arrest. Image-based cytometer, RT-qPCR, Western blotting and transwell migration assays were performed. Key findings: Quercetin treatment for 24-72 h inhibited PC3 and CD44+/CD133+ stem cell proliferation in a time-and dose-dependent manner. Knockdown of endogenous MK expression significantly suppressed proliferation of CD44(+)/CD133(+) and CD44(+) cells as well as their parent cells. Co-administration of MK siRNA and quercetin reduced the cell survival, induced apoptosis and caused G1 phase cell cycle arrest more effectively than the individual therapy. Knockdown of MK significantly enhanced the inhibitory effect of quercetin on CD44(+)/CD133(+) migration and spheroid formation. In addition, the combined therapy inhibited the phosphorylation of PI3K, AKT and ERK1/2, and reduced the protein expression of p38, ABCG2 and NF-kappa B. Significance: Quercetin alone exhibited significant cytotoxic effects on CD44(+)/CD133(+). MK plays an important role in the proliferation of CD44(+)/CD133(+) and CD44(+) cells in particular, and quercetin and MK-silencing therapy may be an important strategy in targeting CSCs that play a role in relapse, migration and drug resistance.Öğe Midkine silencing enhances the anti-prostate cancer stem cell activity of the flavone apigenin: cooperation on signaling pathways regulated by ERK, p38, PTEN, PARP, and NF-?B(Springer, 2020) Erdogan, Suat; Turkekul, Kader; Dibirdik, Ilker; Doganlar, Zeynep B.; Doganlar, Oguzhan; Bilir, AyhanProstate cancer (PCa) is the most common cancer in men worldwide. Midkine (MK) is overexpressed in PCa, as well as in tumor-initiating cells termed cancer stem cells (CSCs). Apigenin is a dietary flavone with considerable anti-tumor activities. In this study, we explored the possible therapeutic use of MK silencing, apigenin treatment, and a combination of both on human PCa and prostate cancer stem cells (PCSCs). CD44(+)CD133(+) PC3 and CD44(+) LNCaP CSCs were isolated from their parent cell lines. Both MK knockdown and apigenin treatment resulted in loss of cell viability in PCSCs, and these effects were significantly elevated when apigenin was applied with MK silencing. Combined treatment of CD44(+)CD133(+) PC3 cells with apigenin and MK siRNA was also more effective in inducing apoptotic and non-apoptotic cell death when compared with individual applications. Treatment of CD44(+) LNCaP cells with apigenin significantly decreased viability, although the combination treatment did not markedly alter the individual therapy. Molecular events underlying cell cycle arrest and inhibition of the survival, proliferation, and migration of CD44(+)CD133(+) PC3 cells were found to be associated with upregulated p21, p27, Bax, Bid, caspase-3, and caspase-8 expression, as well as downregulated p-p38, p-ERK, NF-kappa B, and PARP. In addition, the combination of apigenin treatment and MK silencing showed better outcomes on the anticancer efficacy of docetaxel in CD44(+)CD133(+) PC3 cells. In conclusion, MK-regulated events are different between PCSCs, and when combined with apigenin plus MK silencing, docetaxel treatment may be a valuable approach for the eradication of PCSCs.Öğe Prostat Kanser Kök Hücrelerinin Apoptozu, Proliferasyonu ve Anti-neoplastik Tedavilerinde Midkin Büyüme Faktörünün Etkinliğinin Araştırılması(2018) Erdoğan, Suat; Öktem, Gülperi; Doğanlar, Oğuzhan; Dıbırdık, Ilker; Bilir, Ayhan; Doğanlar, Zeynep BanuProstat kanseri (PCa), özellikle gelişmiş ülkelerde erkeklerde yaygın görülen malignitedir ve kanserle ilişkili mortalitenin ikinci en sık nedenini oluşturur. Bu çalışmada midkin (MK) büyüme faktörünün PCa ve bunların kanser kök hücrelerinin (KKH) sağkalımı ve migrasyonuna etkinliği, kemoterapi ilaç tedavisine yanıtları ve seçilmiş doğal flavonoid bileşiklerin kemoterapi ilaçları ile kombinasyonlarının anti-kanser potansiyeli analiz edildi. Kanser kök hücreleri manyetik aktive edilmiş hücre izolasyon sistemi ile androjen-dirençli PC3 ve androjen-duyarlı LNCaP hücrelerinden ayrıştırıldı. Midkin proteinin rolü MK-spesifik siRNA transfeksiyonu ile mRNA sessizleştirilen (knock down) hücrelerde belirlendi. Hücrelere tedavi amaçlı farklı konsantrasyon ve sürelerde MK siRNA, enzalutamid, dozetaksel ve flavonoidler ayrı ayrı ve bunların formülasyonları kullanıldı. Analizlerde hücrelerin sağkalımı MTT testi, apoptoz ve hücre siklusu fazları sitometrik, protein modifikasyonları Western blot, mRNA ifade analizleri RT-qPCR, migrasyon ise yara iyileşmesi ve transwel deneyleri ile incelendi. Apoptozun hücresel düzeyde analizinde Hoechst boyası kullanıldı, tedavi sonrası hücrelerin ultrastruktürel değişimleri geçirimli elektron mikroskopisi ile değerlendirildi. Midkinin sessizleştirilmesi PC3, LNCAP ve bunlardan izole edilen kanser kök hücrelerinde hücre sağkalımını % 20 ? 25 düzeyinde azalttı ve hücre migrasyonunu baskıladı. Kullanılan dört farklı flavonoid bileşikten apigenin düşük konsantrasyonları ve kuersetin hücrelerde apoptozu uyarırken hücre döngüsünü G1 fazında tutarak proliferasyonu ve hücre migrasyonunu baskıladı. Midkin protein ifadesinin inhibisyonu ile birlikte uygulanan kemoterapi, tedavi gücünü belirgin düzeyde güçlendirdi ve hücre migrasyonunu inhibe etti. Bu tedaviye flavonoid eklenmesi androjen-duyarlı hücrelerde sitotoksik etkiyi önemli düzeyde yükseltti. Birleşik tedavi PI3K, AKT ve ERK1/2'nin fosforilasyonunu inhibe etti ve p38, ABCG2 ve NF-?B'nin protein ifadesini azalttı. Elde edilen verilere göre, MK, PCa ve KKH'lerin proliferasyonunda önemli rol oynamaktadır. Kemoterapi yanısıra flavonoid kullanımı ve midkin aracılı tedavi hastalığın relapsı, hücre göçü ve ilaç direncinde rol oynayan kanser kök hücrelerinin hedeflenmesinde önemli bir stratejiyi oluşturabilir.