Bufalin is the primary component of the traditional Chinese herb “Chan Su”. Evidence suggests that this compound possesses potent anti-tumor activities, although the exact molecular mechanism(s) is unknown. Our previous study showed that bufalin inhibited growth of human osteosarcoma cell lines U2OS and U2OS/MTX300 in culture. Therefore, this study aims to further clarify the in vitro and in vivo anti-osteosarcoma effects of bufalin and its molecular mechanism of action. We found bufalin inhibited both methotrexate (MTX) sensitive and resistant human osteosarcoma cell growth and induced G2/M arrest and apoptosis. Using a comparative proteomics approach, 24 differentially expressed proteins following bufalin treatment were identified. In particular, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27), decreased remarkably. The down-regulation of Hsp27 and alterations of its partner signaling molecules (the decrease in p-Akt, nuclear NF-κB p65, and co-immunoprecipitated cytochrome c/Hsp27) were validated. Hsp27 over-expression protected against bufalin-induced apoptosis, reversed the dephosphorylation of Akt and preserved the level of nuclear NF-κB p65 and co-immunoprecipitated Hsp27/cytochrome c. Moreover, bufalin inhibited MTX-resistant osteosarcoma xenograft growth, and a down-regulation of Hsp27 in vivo was observed. Taken together, bufalin exerted potent anti-osteosarcoma effects in vitro and in vivo, even in MTX resistant osteosarcoma cells. The down-regulation of Hsp27 played a critical role in bufalin-induced apoptosis in osteosarcoma cells. Bufalin may have merit to be a potential chemotherapeutic agent for osteosarcoma, particularly in MTX-resistant groups.
Background: Chronic neuropathic pain is a debilitating condition that remains difficult to treat. The Na þ-Ca 2þ exchanger (NCX) is a transporter that can exchange Ca 2þ with Na þ in either direction to maintain intracellular Ca 2þ homeostasis. However, the effect of NCX on neuropathic pain remains unclear. Therefore, in this study, we aimed to clarify whether neuropathic pain is altered by NCX. Methods: Adult Sprague-Dawley rats and mice (NCX2 knockout and wild type) were randomized to receive spinal nerve ligation surgery or intrathecal injection. Using behavioral testing to analyze the withdrawal thresholds and thermal withdrawal latency of rats after surgery or intrathecal injection. Immunohistochemistry and Western blotting were used to analyze the changes of NCX protein and downstream signaling pathways in rats dorsal root ganglion. We isolated the dorsal root ganglion neurons of adult rats using Fluo-4AM to detect the Ca 2þ imaging in neurons after drug treatment. Results: NCX was expressed in the sensory neurons of rodent dorsal root ganglia. NCX expression was altered in ipsilateral L4-6 dorsal root ganglion neurons in spinal nerve ligation rats. Intrathecal injection of an inhibitor of reversemode NCX activity (KB-R7943 5$20 mg) had an antinociceptive effect in spinal nerve ligation rats, and the effect lasted for 3 h. We measured the expression of signaling pathway molecules in dorsal root ganglion neurons, and only the p-extracellular signal-regulated kinase (ERK) 1/2 level was reduced after intrathecal injection in the spinal nerve ligation group compared to the control group. In cultured dorsal root ganglion neurons, inhibitors of reverse-mode NCX activity (KB-R7943 and ORM-10103) restrained Ca 2þ overload after tumor necrosis factor alpha (TNF-a) or lipopolysaccharide (LPS) treatment. NCX2 knockout mice presented an antinociceptive effect that lasted for more than 28 days after spinal nerve ligation surgery. The p-ERK1/2 level in NCX2 knockout mice ipsilateral L4-6 dorsal root ganglion neurons was lower than that in wild-type mice. Conclusions: NCX proteins may mediate neuropathic pain progression via the Ca 2þ and ERK pathways. NCX represents a potential target for the treatment of neuropathic pain.
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