Background: 2,4-Dimethoxyphenyl-E-4-arylidene-3-isochromanone (IK11) was previously described to induce apoptotic death of A431 tumor cells. In this report, we investigated the molecular action of IK11 in the HepG2 human hepatocellular carcinoma cell line to increase our knowledge of the role of poly (ADP-ribose)-polymerase (PARP), protein kinase B/Akt and mitogen activated protein kinase (MAPK) activation in the survival and death of tumor cells and to highlight the possible role of PARP-inhibitors in co-treatments with different cytotoxic agents in cancer therapy. Results: We found that sublethal concentrations of IK11 prevented proliferation, migration and entry of the cells into their G2 phase. At higher concentrations, IK11 induced reactive oxygen species (ROS) production, mitochondrial membrane depolarization, activation of c-Jun N-terminal kinase 2 (JNK2), and substantial loss of HepG2 cells. ROS production appeared marginal in mediating the cytotoxicity of IK11 since N-acetyl cysteine was unable to prevent it. However, the PARP inhibitor PJ34, although not a ROS scavenger, strongly inhibited both IK11-induced ROS production and cell death. JNK2 activation seemed to be a major mediator of the effect of IK11 since inhibition of JNK resulted in a substantial cytoprotection while inhibitors of the other kinases failed to do so. Inhibition of Akt slightly diminished the effect of IK11, while the JNK and Akt inhibitor and ROS scavenger trans-resveratrol completely protected against it. Conclusions: These results indicate significant involvement of PARP, a marginal role of ROS and a pro-apoptotic role of Akt in this system, and raise attention to a novel mechanism that should be considered when cancer therapy is augmented with PARP-inhibition, namely the cytoprotection by inhibition of JNK2.

In recent years, several studies aimed to investigate the metabolic effects of non-functioning or absent cyclophilin D (CypD), a crucial regulatory component of mitochondrial permeability transition pores. It has been reported that the lack of CypD affects glucose and lipid metabolism. However, the findings are controversial regarding the metabolic pathways involved, and most reports describe the effect of a high-fat diet on metabolism. We performed a lipidomic analysis of plasma and liver samples of CypD-/- and wild-type (WT) mice to reveal the lipid-specific alterations resulting from the absence of CypD. In the CypD-/- mice compared to the WT animals, we found a significant change in 52% and 47% of the measured 225 and 201 lipid species in liver and plasma samples, respectively. The higher total lipid content detected in these tissues was not accompanied by abdominal fat accumulation assessed by nuclear magnetic resonance imaging. We also documented characteristic changes in the lipid composition of the liver and plasma as a result of CypD ablation with the relative increase in polyunsaturated membrane lipid species. In addition, we did not observe remarkable differences in the lipid distribution of hepatocytes using histochemistry, but we found characteristic changes in the hepatocyte ultrastructure in CypD-/- animals using electron microscopy. Our results highlight the possible long-term effects of CypD inhibition as a novel therapeutic consideration for various diseases.

Macrophage migration inhibitory factor (MIF) has been shown to induce M1 macrophage polarization with oxidative stress and associated metabolic reprogramming. Several tautomerase inhibitors were shown to selectively inhibit either MIF’s ketonase or enolase sub-activities. In this study, we aimed to investigate the role of enolase sub-activity in M1 polarization using the selective enolase inhibitor TE-91. We performed in silico molecular docking analysis and physicochemical characterization of TE-91. LPS + IFN-γ-induced RAW264.7 cells were applied as a model for M1 macrophage activation. We performed ROS and nitrite determinations, ELISA, qPCR, and immunoblot analysis, and measured mitochondrial oxygen consumption rate and extracellular acidification rate. Here, we reveal that TE-91 might directly bind to the MIF tautomerase active site. Furthermore, TE-91 reduces M1 activation by enhancing oxidative phosphorylation and reducing the glycolytic activity in LPS + IFN-γ-induced macrophage cells. In the same model, TE-91 reduces TNF-α, IL-6, CCL2, and iNOS mRNA transcription yet fails to modulate PARP1 and SOD2 mRNA transcription. It also decreases ROS, nitrite, and IL-6 production without influencing TNF-α and CCL2 protein production. TE-91 was unable to reduce either HIF-1α mRNA transcription or its protein expression. Finally, TE-91 reduced IL-1β cleavage, without affecting IL-1β protein expression. These results may highlight the importance of tautomerase sub-activities in M1 polarization.