Sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (NKP-1339) is a clinically investigated ruthenium-based metal complex, which shows promising results in solid tumors, such as non-small cell lung cancer, colorectal carcinoma, and most distinctively in gastrointestinal neuroendocrine tumors. In previous studies, fast binding to albumin as well as transferrin could be shown. The enhanced permeability and retention (EPR) effect, which is diversely being exploited for tumor targeting, could therefore be applicable for NKP-1339. Here we studied the serum dependence of its biological activity in various methods, influencing its cellular accumulation, cytotoxicity as well as the generation of reactive oxygen species (ROS). ROS lead to Nrf2 activation, which is known to activate antioxidant response gene transcription. GRP78 down-regulation on the protein level suggests ER associated protein degradation (ERAD) as a mode of action, as RNA levels are only mildly affected. Another important part for the mode of action is endoplasmic reticulum (ER) stress, as different factors are highly upregulated on the protein level. For example PERK, a transmembrane receptor which is released by GRP78 when the ER is disturbed, is upregulated and phosphorylated. EIF2α is phosphorylated, which leads to an inhibition of CAP-dependent translation and other stress responses. The transcription factor CHOP (DDIT3), which promotes ER stress dependent apoptosis, is time and concentration dependently upregulated. Finally cytotoxicity tests could prove that inhibition of ER stress and ER stress-mediated apoptosis leads to decreased cytotoxic effects of NKP-1339, which highlights the involvement of this mechanism in the mode of action.Electronic supplementary materialThe online version of this article (doi:10.1007/s10637-016-0337-8) contains supplementary material, which is available to authorized users.

Herein we report the synthesis, anticancer potency in vitro, biomolecule interaction, and preliminary mode of action studies of a series of cyclometalated 1,2,3-triazole-derived ruthenium(II) (2a-e) and osmium(II) (3a-e) organometallics of the general form [(η-p-cym)RuCl(κ-C^N-L)] with varying substituents in postion 1 of the 1,2,3-triazole moiety. These cyclometalates were characterized by standard analytical methods and their structures unambiguously assigned by single crystal X-ray crystallography. The anticancer activity of these novel compounds was tested in the human tumor cell lines A549 (non-small cell lung cancer), SW480 (colon adenocarcinoma), and CH1/PA-1 (ovarian teratocarcinoma), and preliminary structure-activity relationships were derived from the obtained data sets. Various representatives exhibit promising antineoplastic effects with IC values down to the low micromolar range. The compounds readily formed stable DMSO adducts after aquation in DMSO-containing solution, but employing DMSO as solubilizer in cytotoxicity assays had no pronounced effect on the cytotoxicity, compared to analogous experiments with DMF for most compounds. We isolated and characterized selected DMSO adducts as triflate salts and found that they show activities in the same range as the parent chlorido metalacycles in MTT assays with the use of DMSO. Osmium(II) cyclometalates exhibited higher antiproliferative activities than their ruthenium(II) counterparts. The IC values within each metal series decreased with increasing lipophilicity, which was attributed to higher cellular accumulation. Investigations on their mode of action revealed that the prepared organometallics were unable to inhibit topoisomerase IIα. Still, the most cytotoxic representatives 2b and 3b showed pronounced effects on cell cycle distribution.

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.