title: 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)-dimethyl-triapine (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 eIF2α 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.
authors.affiliation: Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria (R.T., L.S.F., C.R.K., M.A.J., B.K.K.); Research Platform "Translational Cancer Therapy Research" (R.T., C.R.K., P.H., U.J., M.A.J., W.B., B.K.K.), Institute of Cancer Research (P.H., U.J., W.B.), and Comprehensive Cancer Centre (P.H., U.J., W.B.), Medical University of Vienna, Vienna, Austria; Genomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, Austria (G.E.M., R.S.); Department of Inorganic and Analytical Chemistry, University of Szeged, Szeged, Hungary (E.A.E.); and Hungarian Academy of Science-USZ Bioinorganic Chemistry Research Group, Szeged, Hungary (E.A.E.).
authors.forename: Robert
authors.initials: R
authors.lastname: Trondl
authors.forename: Lea S
authors.initials: LS
authors.lastname: Flocke
authors.forename: Christian R
authors.initials: CR
authors.lastname: Kowol
authors.forename: Petra
authors.initials: P
authors.lastname: Heffeter
authors.forename: Ute
authors.initials: U
authors.lastname: Jungwirth
authors.forename: Georg E
authors.initials: GE
authors.lastname: Mair
authors.forename: Ralf
authors.initials: R
authors.lastname: Steinborn
authors.forename: Éva A
authors.initials: ÉA
authors.lastname: Enyedy
authors.forename: Michael A
authors.initials: MA
authors.lastname: Jakupec
authors.forename: Walter
authors.initials: W
authors.lastname: Berger
authors.forename: Bernhard K
authors.initials: BK
authors.lastname: Keppler
mesh_terms: D051701:Activating Transcription Factor 4 / Q000235:genetics; D051702:Activating Transcription Factor 6 / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000235:genetics; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects* / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics; D045325:HCT116 Cells; D018922:HL-60 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics; D051336:Mitochondrial Membranes / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology*; D000071701:Regulatory Factor X Transcription Factors; D013882:Thiosemicarbazones / Q000494:pharmacology*; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000235:genetics; D056811:Unfolded Protein Response / Q000187:drug effects / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D000071717:X-Box Binding Protein 1; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics
chemical_list: C497109:ATF4 protein, human; C497111:ATF6 protein, human; D051702:Activating Transcription Factor 6; D004268:DNA-Binding Proteins; D015852:Eukaryotic Initiation Factor-2; D011725:Pyridines; D000071701:Regulatory Factor X Transcription Factors; D013882:Thiosemicarbazones; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; C078157:3-aminopyridine-2-carboxaldehyde thiosemicarbazone; D051701:Activating Transcription Factor 4; D051742:Transcription Factor CHOP; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases