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

Abstract

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.

Keywords: ER stress; Oxidative stress; Ruthenium; Unfolded protein response.

Fig. 1

Influence of serum content on cellular accumulation of Ru (from NKP-1339) in the two colon carcinoma cell lines HCT116 and SW480 (n = 3). Cells were treated for 2 h with 100 μM NKP-1339 in media containing different serum concentrations (2 %, 5 % or 10 % as indicated). An inverse correlation between cellular accumulation and serum content could be observed

Fig. 2

Cytotoxicity of NKP-1339 in the two colon carcinoma cell lines HCT116 and SW480 treated in medium containing 2 % or 10 % FCS and co-treatment with inhibitors of ER stress or responses thereto (n = 3). Cytotoxicity is illustrated by the half maximal inhibitory concentration (IC₅₀). Cytotoxicity is increased when serum concentration is reduced in both cell lines. Inhibiting protein translation by CHX as well as inhibiting translation from ER stress to apoptosis by the JNK inhibitor SP600125 decreases cytotoxicity

Fig. 3

Generation of reactive oxygen species (ROS) in HCT116 and SW480 cells after 1 h treatment with 200 μM NKP-1339 (n = 2) in the presence of different serum concentrations. The relative fluorescence units plotted on the y-axis indicate ROS levels, which are inversely dependent on serum content of the medium

Fig. 4

Nrf2 translocation. Cells treated for 6 h in medium containing 2 % FCS. Upon treatment with NKP-1339, Nrf2 is translocating to the nucleus. Pictures depict typical results. Scale bar applies to all images

Fig. 5

Western blot analysis showing p-eIF2y α and CHOP upregulation, PERK phosphorylation as well as Grp78 regulation (n = 3). Incubation time is 24 h. ß-Actin was used as a loading control

Fig. 6

Rt qPCR of UPR key factors in two cell lines upon 4 h exposure to NKP-1339 in media containing 2 % or 10 % FCS (n = 3). GRP78, ATF4 and IRE1α show no major upregulation on the mRNA level (a). CHOP and XBP1 are slightly or even distinctly upregulated. CHOP shows the highest upregulation in HCT116 cells treated in medium containing 10 % FCS, and XBP1 in SW480 cells treated in medium containing 10 % FCS (b). Note the differently scaled x-axes