ABSTRACT
Repigmentation of vitiliginous lesions relies on the proliferation and migration of melanoblasts from hair follicles to the epidermis. Pulsed ultrasound has been demonstrated to have stimulatory effects on cell proliferation and migration and has been applied clinically to enhance tissue repair. To clarify the biologic effects and signaling mechanisms of pulsed ultrasound on melanoblast proliferation and migration, two melanoblast cell lines, the undifferentiated NCCmelb4 cells and the differentiated NCCmelan5 cells, were examined. We demonstrated that pulsed ultrasound increased cell migration in a dose-dependent manner without altering cell proliferation. Pulsed ultrasound enhanced autocrine secretion of macrophage colony-stimulating factor (M-CSF), which subsequently activated the focal adhesion kinase (FAK) pathway to promote melanoblast migration. Furthermore, conditioned medium from mouse embryonic fibroblasts NIH 3T3 and primary human keratinocytes treated with pulsed ultrasound could stimulate melanoblast migration through a paracrine effect. Our results provide a novel mechanism to promote migration of melanoblasts by pulsed ultrasound stimulation.
Subject(s)
Autocrine Communication , Cell Movement , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Macrophage Colony-Stimulating Factor/metabolism , Melanocytes/cytology , Paracrine Communication , Ultrasonics , Up-Regulation , Adult , Animals , Autocrine Communication/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Culture Media, Conditioned/pharmacology , Embryo, Mammalian/cytology , Enzyme Activation/drug effects , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Keratinocytes/cytology , Keratinocytes/drug effects , Keratinocytes/metabolism , Male , Melanins/metabolism , Melanocytes/enzymology , Melanocytes/metabolism , Mice , NIH 3T3 Cells , Phosphorylation/drug effects , Signal Transduction/drug effects , Up-Regulation/drug effectsABSTRACT
Dehydroeburicoic acid (DeEA) is a triterpene purified from medicinal fungi such as Antrodia camphorate, the crude extract of which is known to exert cytotoxic effects against several types of cancer cells. We aim to test the hypothesis that DeEA possesses significant cytotoxic effects against glioblastomas, one of the most frequent and malignant brain tumors in adults. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release assays indicated that DeEA inhibited the proliferation of the human glioblastoma cell U87MG. In addition, Annexin V and propidium iodide staining showed that DeEA treatment led to a rapid increase of glioblastomas in the necrotic/late apoptotic fraction, whereas cell cycle analysis revealed that DeEA failed to significantly enhance the population of U87MG cells in the hypodiploid (sub-G1) fraction. Using electron microscopy, we found that DeEA induced significant cell enlargements, massive cytoplasmic vacuolization, and loss of mitochondrial membrane integrity. DeEA treatment triggered an intracellular Ca(2+) increase, and DeEA-induced cell death was significantly attenuated by BAPTA-AM but not ethylenediaminetetraacetic acid or ethylene glycol tetraacetic acid. DeEA instigated a reduction of both mitochondrial transmembrane potential and intracellular ATP level. Moreover, DeEA induced proteolysis of alpha-spectrin by calpain, and DeEA cytotoxicity in U87MG cells was caspase-independent but was effectively blocked by calpain inhibitor. Interestingly, DeEA also caused autophagic response that was prevented by calpain inhibitor. Taken together, these results suggest that in human glioblastomas, DeEA induces necrotic cell death that involves Ca(2+) overload, mitochondrial dysfunction, and calpain activation.
