A p38MAPK/MK2 signaling pathway leading to redox stress, cell death and ischemia/reperfusion injury

Background Many diseases and pathological conditions are characterized by transient or constitutive overproduction of reactive oxygen species (ROS). ROS are causal for ischemia/reperfusion (IR)-associated tissue injury (IRI), a major contributor to organ dysfunction or failure. Preventing IRI with antioxidants failed in the clinic, most likely due to the difficulty to timely and efficiently target them to the site of ROS production and action. IR is also characterized by changes in the activity of intracellular signaling molecules including the stress kinase p38MAPK. While ROS can cause the activation of p38MAPK, we recently obtained in vitro evidence that p38MAPK activation is responsible for elevated mitochondrial ROS levels, thus suggesting a role for p38MAPK upstream of ROS and their damaging effects.<p></p> Results Here we identified p38MAPKα as the predominantly expressed isoform in HL-1 cardiomyocytes and siRNA-mediated knockdown demonstrated the pro-oxidant role of p38MAPKα signaling. Moreover, the knockout of the p38MAPK effector MAPKAP kinase 2 (MK2) reproduced the effect of inhibiting or knocking down p38MAPK. To translate these findings into a setting closer to the clinic a stringent kidney clamping model was used. p38MAPK activity increased upon reperfusion and p38MAPK inhibition by the inhibitor BIRB796 almost completely prevented severe functional impairment caused by IR. Histological and molecular analyses showed that protection resulted from decreased redox stress and apoptotic cell death.<p></p> Conclusions These data highlight a novel and important mechanism for p38MAPK to cause IRI and suggest it as a potential therapeutic target for prevention of tissue injury.<p></p&gt

Isolation of a novel Thioflavin S-derived compound that inhibits BAG-1-mediated protein interactions and targets BRAF inhibitor-resistant cell lines

Protein-protein interactions mediated through the C-terminal BAG domain of BAG-1 are critical for cell survival and proliferation. Thioflavin S (NSC71948) - a mixture of compounds resulting from the methylation and sulfonation of primulin base - has been shown to dose-dependently inhibit the interaction between BAG-1 and Hsc70 in vitro. In human breast cancer cell lines, with high BAG-1 expression levels, Thioflavin S reduces the binding of BAG-1 to Hsc70, Hsp70 or CRAF and decreases proliferation and viability. Here we report the development of a protocol for the purification and isolation of biologically active constituents of Thioflavin S and the characterization of the novel compound Thio-2. Thio-2 blocked the growth of several transformed cell lines but had much weaker effects on untransformed cells. Thio-2 also inhibited the proliferation of melanoma cell lines that had become resistant to treatment with PLX4032, an inhibitor of mutant BRAF. In transformed cells Thio-2 interfered with intracellular signaling at the level of RAF but had no effect on the activation of AKT. Thio-2 decreased binding of BAG-1 to Hsc70 and to a lesser extent BRAF in vitro and in vivo, suggesting a possible mechanism of action. Given that tumors frequently develop resistance to kinase inhibitors during treatment, Thio-2 and related compounds may offer promising alternative strategies to currently available therapies