miTuner - a kit for microRNA based gene expression tuning in mammalian cells

The purpose of this RFC is to introduce a modular expression tuning kit for use in mammalian cells. The kit enables the regulation of the gene expression of any gene of interest (GOI) based on synthetic microRNAs, endogenous microRNAs or a combination of both

miMeasure – a standard for miRNA binding site characterization in mammalian cells

This RFC proposes a standard for the quantitative characterization of miRNA binding sites (miRNA-BS) in mammalian cells. The miMeasure standard introduces a ready-to-use standard measurement plasmid (pSMB_miMeasure, BBa_K337049) enabling rapid experimental characterization of any miRNA-BS of choice. We recommend a new standard unit, RKDU (relative knock-down unit) to describe the knock-down efficiency of a miRNA-BS in a specific cell type. pSMB_miMeasure allows for an easy and fast measurement of RKDU while providing effective normalization against variance stemming from differences in transfection efficiency and from other sources

Introducing the 4‑Phenyl-1,2,3-Triazole Moiety as a Versatile Scaffold for the Development of Cytotoxic Ruthenium(II) and Osmium(II) Arene Cyclometalates

Herein we report the synthesis, anticancer potency <i>in vitro</i>, biomolecule interaction, and preliminary mode of action studies of a series of cyclometalated 1,2,3-triazole-derived ruthenium­(II) (<b>2a</b>–<b>e</b>) and osmium­(II) (<b>3a</b>–<b>e</b>) organometallics of the general form [(η⁶-<i>p</i>-cym)­RuCl­(κ²-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 IC₅₀ 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 IC₅₀ 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 <b>2b</b> and <b>3b</b> showed pronounced effects on cell cycle distribution

Introducing the 4‑Phenyl-1,2,3-Triazole Moiety as a Versatile Scaffold for the Development of Cytotoxic Ruthenium(II) and Osmium(II) Arene Cyclometalates

Herein we report the synthesis, anticancer potency <i>in vitro</i>, biomolecule interaction, and preliminary mode of action studies of a series of cyclometalated 1,2,3-triazole-derived ruthenium­(II) (<b>2a</b>–<b>e</b>) and osmium­(II) (<b>3a</b>–<b>e</b>) organometallics of the general form [(η⁶-<i>p</i>-cym)­RuCl­(κ²-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 IC₅₀ 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 IC₅₀ 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 <b>2b</b> and <b>3b</b> showed pronounced effects on cell cycle distribution

Molecular mode of action of NKP-1339 – a clinically investigated ruthenium-based drug – involves ER- and ROS-related effects in colon carcinoma cell lines

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