Triapine and a More Potent Dimethyl Derivative Induce Endoplasmic Reticulum Stress in Cancer Cells

Abstract

Triapine (3-AP; 3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a ribonucleotide reductase inhibitor, has been extensively evaluated in clinical trials in the last decade. This study addresses the role of endoplasmic reticulum (ER) stress in the anticancer activity of 3-AP and the derivative N 4 ,N 4 -dimethyltriapine (3-AP-Me), differing from 3-AP only by dimethylation of the terminal nitrogen. Treatment of colon cancer cells with 3-AP or 3-AP-Me activated all three ER stress pathways (PERK, IRE1a, ATF6) by phosphorylation of eIF2a and upregulation of gene expression of activating transcription factors ATF4 and ATF6. In particular, 3-AP-Me led to an upregulation of the alternatively spliced mRNA variant XBP1 (16-fold). Moreover, 3-AP and 3-AP-Me activated the cellular stress kinases c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases, and inhibition of JNK activity antagonized the cytotoxic effect of both compounds. Subsequent to induction of the unfolded protein response, a significant upregulation of proapoptotic proteins was detected, including the transcription factor CHOP and Bim, an essential factor for ER stress-related apoptosis. In correlation with the higher degree of ER stress after 3-AP-Me treatment, also a more potent depolarization of mitochondrial membranes was found. These data suggest that 3-AP and 3-AP-Me induce apoptosis via ER stress. This was further corroborated by showing that inhibition of protein biosynthesis with cycloheximide prior to 3-AP and 3-AP-Me treatment leads to a significant reduction of the antiproliferative properties of both compounds. Taken together, this study demonstrates that induction of ER stress contributes to the mode of action of 3-AP and that terminal dimethylation leads to an even more pronounced manifestation of this effect.

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“…ATF5 was also reported to enhance radioresistance and malignancy in cancer cells [ 33 ]. ATF6, a constitutively expressed, endoplasmic reticulum (ER) membrane-anchored transcription factor, is associated with liver cancer [ 34 ], breast cancer [ 35 ], prostate cancer [ 36 ], colonic neoplasm [ 37 ] and soft tissue sarcoma [ 38 ]. ATF7, a novel bZIP protein that interacts with PTP4A1 [ 39 ], was recently identified as a favorable factor for survival of patients with colorectal cancer [ 40 ].…”

The thiosemicarbazone Me2NNMe2 induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition

“…After 24 h recovery, cells were treated for another 24 h with the indicated drug concentrations. Then, cells were either stained with annexin V-APC (AV) and propidium iodide (PI) or with 10 µg/ml JC-1 as previously described 51 , 52 .…”

A comparative study of α- N -pyridyl thiosemicarbazones: Spectroscopic properties, solution stability and copper(II) complexation

“…TSCs often possess intrinsic fluorescence properties due to their rigid structure and the extended conjugated electron system. This advantageous feature allows the monitoring of the cellular accumulation and distribution in biological samples like cancer cells by fluorescence microscopy [50]. In order to compare the fluorescence of the investigated TSCs, their excitation, emission and UV-vis spectra were recorded under identical conditions.…”