Abstract 2637: Terminal dimethyl substitution of Triapine leads to activation of CHOP and induction of apoptosis via ER stress in human colon cancer cells
Authors: Trondl Robert , Jungwirth Ute , Heffeter Petra , Kowol Christian R. , Arion Vladimir B. , Jakupec Michael A. , Keppler Bernhard K. 
Published: June 2012

Abstract
The α-N-heterocyclic thiosemicarbazone Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) has been studied in several phase I & II clinical trials and it is known as a potent ribonucleotide reductase inhibitor. We synthesized Triapine (3-AP) and the corresponding terminally N4-dimethylated derivative (3-AP-Me2). Previously, we have shown that dimethylation of the terminal amino group leads to significant enhancement of cytotoxicity. In this study we utilized the intrinsic fluorescence properties of these compounds to investigate their intracellular distribution and studied the ability of both compounds to induce endoplasmic reticulum (ER) stress in SW480 colon carcinoma cells.
Fluorescence microscopy was performed on viable SW480 cells (colon carcinoma) treated with 3-AP and 3-AP-Me2, showing a strong affinity of the compounds to the nuclear membrane and to cytosolic structures. Co-localization studies with organelle-specific staining revealed that both agents are associated with structures of ER and mitochondria. Therefore, we hypothesized an interference with ER functions. Immunoblotting showed that treatment with the dimethylated compound (3-AP-Me2) results in increased expression of BIP/GRP78 and induction of CHOP. Both are relevant stress markers for unfolded protein response (UPR), and CHOP indicates induction of apoptosis via the mitochondrial pathway. In contrast, treatment with 3-AP leads to an inhibition of BIP/GRP78 expression and does not induce CHOP expression. However, both compounds cause splicing of XBP-1 mRNA indicating activation of the IRE1-α pathway, which triggers apoptosis by activation of the ASK1-JNK pathway. In accordance with these findings 3-AP as well as 3-AP-Me2 activates c-Jun N-terminal kinase (JNK), which is commonly associated with cellular stress such as UPR. A dichlorofluorescein assay showed no generation of reactive oxygen species (ROS) neither by 3-AP nor by 3-AP-Me2, suggesting that the initiation of the ER stress pathway is ROS independent.
In conclusion, dimethylation of the terminal amino group leads to up regulation of ER stress mechanism by increase of BIP/GRP79 expression and induction of CHOP. Activation of CHOP indicates induction of apoptosis and may be crucial for the higher cytotoxicity of dimethylated derivates of thiosemicarbazones. XBP-1 splicing and phosphorylation of JNK suggest an activation of the IRE1α-ASK1-JNK signaling pathway, which is connected to cell death involving proteins of the BCL-2 family. The induction of ER stress in a ROS independent manner is a novel mechanism of action relevant for the anti-neoplastic activity of this class of compounds.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2637. doi:10.1158/1538-7445.AM2011-2637