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.