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

Abstract

Objective: Autophagy (AP) is a well-preserved cell survival mechanism that plays a critical role by degrading dysfunctional organelles and misfolded proteins. There are a limited number of studies examining 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 APrelated genes Beclin-1, Ulk1, Ulk2 and Uvrag in HT-29 colorectal adenocarcinoma cells. Material and Methods: HT-29 human colorectal adenocarcinoma cells were plated into growth medium. The next day, 25 µM DZNep, an inhibitor of S-Adenosylmethionine-dependent methyltransferase, was added to the cells. DZNep were removed from the cells after 4 days by refreshing the media. The cells were maintained in a tissue culture incubator. Total RNA isolation was 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 by quantitative 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 Ulk1 and Uvrag expression levels were higher than that of Beclin-1, and Ulk2 had the highest expression level in untreated HT-29 colorectal cancer lines. Conclusion: The autophagy related genes studied here consisting of Beclin-1, Ulk1, Ulk2, and Uvrag 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.