MTOR Pathway Is Restrained At Downstream Of CypD-mediated Mitochondrial Permeability Transition In Honokiol Triggered Regulated Necrosis

Honokiol is a pharmacologically active small molecule isolated from Chinese traditional medicinal herb-Houpu. It may induce diversified kinds of regulated cell deaths dependent on different cell types and drug concentrations. We previously reported that honokiol may trigger a cyclophilin D (CypD)-mediated regulated necrosis in several cell lines (MCF-7, HL-60and HEK-293) at certain concentrations (twofold higher than its IC50). Our subsequent work revealed that honokiol induced cell death transition from early-apoptosis to regulated necrosis with the increase of honokiol dose. In the current study, we found honokiol at a lower concentration (30μg/ml) than previously reported also induced simplex CypD-mediated mitochondrial permeability transition (MPT) associated regulated necrosis in human embryonic kidney cell line HEK-293. Honokiol at concentration of30μg/ml induced a necrotic cell death in HEK-293cells which manifested as propidium iodide (Pl)-stained positive. Neither DNA ladder pattern nor apoptotie bodies were detected in cells undergoing this necrotic cell death. Both caspase-8and-3were not activated during the process of honokiol induced necrosis. Both pan-caspase inhibitor z-VAD-fmk and RIP1inhibitor Nec-1failed to inhibit honokiol induced necrosis. However, cyclophilin D inhibitor cyclosporin A (CsA) totally blocked honokiol induced necrosis. These results suggest honokiol at30μg/ml may also induce CypD-mediated MPT associated regulated necrosis in HEK-293cells. Further, we found mTOR pathway was restrained accompanying with honokiol triggered regulated necrosis. And pretreatment with CsA not only prominently inhibited honokiol triggered regulated necrosis, but also apparently reversed dephosphorylation of Akt on the Ser473/Thr308,4E-BP1and S6K. This indicates mTOR pathway takes effects at downstream of the CypD-mediated mitochondrial permeability transition and before the onset of plasma membrane breakdown in honokiol induced regulated necrosis process. In conclusion, we demonstrate for the first time that mTOR pathway is restrained at downstream of the CypD-mediated MPT in honokiol induced regulated necrosis process.