Honokiol traverses the blood-brain barrier and induces apoptosis of neuroblastoma cells via an intrinsic bax-mitochondrion-cytochrome c-caspase protease pathway.

Neuroblastomas, an embryonic cancer of the sympathetic nervous system, often occur in young children. Honokiol, a small-molecule polyphenol, has multiple therapeutic effects and pharmacological activities. This study was designed to evaluate whether honokiol could pass through the blood-brain barrier (BBB) and induce death of neuroblastoma cells and its possible mechanisms. Primary cerebral endothelial cells (CECs) prepared from mouse brain capillaries were cultured at a high density for 4 days, and these cells formed compact morphologies and expressed the ZO-1 tight-junction protein. A permeability assay showed that the CEC-constructed barrier obstructed the passing of FITC-dextran. Analyses by high-performance liquid chromatography and the UV spectrum revealed that honokiol could traverse the CEC-built junction barrier and the BBB of ICR mice. Exposure of neuroblastoma neuro-2a cells and NB41A3 cells to honokiolinduced cell shrinkage and decreased cell viability. In parallel, honokiol selectively induced DNA fragmentation and cell apoptosis rather than cell necrosis. Sequential treatment of neuro-2a cells with honokiol increased the expression of the proapoptotic Bax protein and its translocation from the cytoplasm to mitochondria. Honokiol successively decreased the mitochondrial membrane potential but increased the release of cytochrome c from mitochondria. Consequently, honokiol induced cascade activation of caspases-9, -3, and -6. In comparison, reducing caspase-6 activity by Z-VEID-FMK, an inhibitor of caspase-6, simultaneously attenuated honokiol-induced DNA fragmentation and cell apoptosis. Taken together, this study showed that honokiol can pass through the BBB and induce apoptotic insults to neuroblastoma cells through a Bax-mitochondrion-cytochrome c-caspase protease pathway. Therefore, honokiol may be a potential candidate drug for treating brain tumors.

Lipoteichoic acid-induced TNF-α and IL-6 gene expressions and oxidative stress production in macrophages are suppressed by ketamine through downregulating Toll-like receptor 2-mediated activation oF ERK1/2 and NFκB.

Lipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that ketamine has anti-inflammatory and antioxidant effects on gram-negative LPS-induced macrophage activation. In this study, we further evaluated the effects of ketamine on the regulation of LTA-induced TNF-alpha and IL-6 gene expressions and oxidative stress production in macrophages and its possible mechanisms. Exposure of macrophages to a therapeutic concentration of ketamine (100 microM) inhibited LTA-induced TNF-alpha and IL-6 expressions at protein or mRNA levels. In parallel, ketamine at 100 microM reduced LTA-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Sequentially, ketamine reduced the LTA-triggered translocation of nuclear factor-kappaB (NFkappaB) from the cytoplasm to nuclei and its transactivation activity. Pretreatment with PD98059, an inhibitor of ERK, decreased LTA-enhanced NFkappaB activation and TNF-alpha and IL-6 mRNA syntheses. Cotreatment with ketamine and PD98059 synergistically suppressed the LTA-induced translocation and transactivation of NFkappaB and biosyntheses of TNF-alpha and IL-6 mRNA. Application of Toll-like receptor 2 (TLR2) small interfering RNA (si)RNA into macrophages decreased the levels of this receptor, and simultaneously ameliorated LTA-augmented NFkappaB transactivation and consequent production of TNF-alpha and IL-6 mRNA. Cotreatment with ketamine and TLR2 siRNA synergistically lowered TNF-alpha and IL-6 mRNA syntheses in LTA-activated macrophages. Ketamine and TLR2 siRNA could reduce the LTA-induced increases in production of nitrite and intracellular reactive oxygen species in macrophages, and their combination had better effects than a single exposure. Thus, this study shows that one possible mechanism involved in ketamine-induced inhibition of LTA-induced TNF-alpha and IL-6 gene expressions and oxidative stress production is through downregulating TLR2-mediated phosphorylation of ERK1/2 and the subsequent translocation and transactivation of NFkappaB.