Betül ÜNAL, Timur KOCA, Gülsüm Özlem ELPEK, Cumhur İbrahim BAŞSORGUN, Ersin AKINCI

DZNep Kolon Kanseri Hücre Hattı HT-29ʼda Otofaji Gen İfadelerini Etkilememektedir

Amaç: Otofaji (AP), fonksiyonel olmayan organelleri ve hatalı katlanmış proteinleri degrade ederek, hücrenin hayatta kalmasını sağlayan iyi korunmuş bir mekanizmadır. 3-Deazaneplanocin A hydrochloride (DZNep) ve otofaji arasındaki ilişkiyi inceleyen az sayıda çalışma bulunmaktadır. Bu öncül çalışmanın amacı, DZNep'in kolon kanseri hücre hattı HT-29'da otofaji ile ilişkili genler olan, Beclin-1, Ulk1, Ulk2 ve Uvrag'a olan etkilerini araştırmaktır. Gereç ve Yöntemler: HT-29 insan kolorektal adenokarsinom hücreleri kültür ortamına ekildi. Ertesi gün, S-Adenosylmethionine bağımlı metiltransferaz inhibitörü olan 25 µmol DZNep, hücre kültürüne ilave edildi. DZNep, dört gün sonra kültür ortamı değiştirilerek hücrelerden uzaklaştırıldı. Hücreler deney süresince doku kültürü inkübatöründe tutuldu. DZNep uygulamasının 4. gününde, DZNep uygulanmış ve uygulanmamış hücrelerden total RNA izolasyonu yapıldı. cDNA’lar, DNaz uygulanmış 2 µg RNA’dan ters transkriptaz ile sentezlendi. cDNA’lar daha sonra kantitatif polimeraz zincir reaksiyonu ile analiz edildi. Sonuçlar, HT-29 hücrelerinde relatif ekspresyon düzeyleri şeklinde sunuldu. Bulgular: DZNep, HT-29 kolorektal kanser hücrelerinin %50’sinden fazlasını öldürerek ekstrem hücre ölümünü indükledi. Otofaji genlerinin ifadeleri şu şekildeydi; Beclin-1 için hafif, Ulk1 ve Uvrag için orta ve Ulk2 için yüksek. Sonuç: Burada çalışılan bazı otofaji genleri; Beclin-1, Ulk1, Ulk2, Uvrag, DZNep uygulamasından etkilenmemiştir. DZNep uygulaması sonucunda, HT-29 hücrelerinde meydana gelen hücre ölümü otofaji aracılı değildir.

DZNep Does Not Influence Autophagy Gene Expression in the Colon Cancer Cell Line HT-29

Objective: Autophagy (AP) is a well-preserved cell survival mechanism that plays a critical role bydegrading dysfunctional organelles and misfolded proteins. There are a limited number of studiesexamining the relationship between 3-Deazaneplanocin A hydrochloride (DZNep) and autophagy.The purpose of this preliminary study was to assess the effect of DZNep on the expression of the APrelatedgenes Beclin-1, Ulk1, Ulk2 and Uvrag in HT-29 colorectal adenocarcinoma cells.Material and Methods: HT-29 human colorectal adenocarcinoma cells were plated intogrowth medium. The next day, 25 µM DZNep, an inhibitor of S-Adenosylmethionine-dependentmethyltransferase, was added to the cells. DZNep were removed from the cells after 4 days byrefreshing the media. The cells were maintained in a tissue culture incubator. Total RNA isolationwas performed from DZNep treated and untreated HT-29 cells 4 days after DZNep application.cDNAs were reverse transcribed from 2 µg of DNase treated RNA. cDNAs were then analyzed byquantitative PCR. The results were presented as relative expression level in HT-29 cells.Results: DZNep induced extreme cell death on HT-29 colorectal cancer cell lines; more than 50%of the colorectal cancer cells were dead. The expression level of Beclin-1 was the smallest while Ulk1and Uvrag expression levels were higher than that of Beclin-1, and Ulk2 had the highest expressionlevel in untreated HT-29 colorectal cancer lines.Conclusion: The autophagy related genes studied here consisting of Beclin-1, Ulk1, Ulk2, andUvrag were not affected by DZNep application. Based on mRNA expression levels of AP genes,administration of DZNep does not stimulate cell toxicity through autophagy in the HT-29 cells.

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