Molecular mechanisms underlying the α-tomatine-directed apoptosis in human malignant glioblastoma cell lines A172 and U-118 MG

Abstract

In the present study, the molecular mechanisms involved in the α-tomatine-induced apoptosis in human glioblastoma cell lines A172 and U-118 MG were investigated. Wright staining and ApopTag assays were conducted to confirm the apoptosis induced by α-tomatine treatment. Fura-2 assay determined an enhancement in free Ca2+ intracellularly, indicating the occurrence of Ca2+-dependent apoptosis induction. Western blot experiments were also performed to predict the apoptosis by measuring the changes in the Bax:Bcl-2 ratio. Increase of calpain activity triggered caspase-12 expression, which in turn further activated caspase-9. In addition, an increase in the ratio of Bax:Bcl-2 accounted for the mitochondrial release of cytochrome c into the cytosol for caspase-3 and caspase-9 activation. Elevated activity of calpain and caspase-3 yielded spectrin breakdown products with 145 and 120 kDa, respectively. Caspase-3 activation further cleaved the inhibitor of caspase activated DNase, while the apoptosis-inducing factor detected in the cytosol suggested that apoptosis was independent of caspase. The apoptosis induction was further supported by decreased expression levels of nuclear factor-κB and increased expression of the inhibitor of nuclear factor, IκBα. In conclusion, the presented experimental results revealed the stimulation of different molecular mechanisms for α-tomatine-mediated apoptosis in A172 and U-118 MG human glioblastoma cell lines.