Curcumin activated both receptor-mediated and mitochondria-mediated proteolytic pathways for apoptosis in human glioblastoma T98G cells

Abstract

The therapeutic effect of curcumin (CCM), a polyphenolic compound from the rhizome of Curcuma longa, has not yet been examined in glioblastoma. We used human glioblastoma T98G cells to explore the efficacy of CCM for inducing apoptosis and identifying proteolytic mechanisms involved in this process. Trypan blue dye exclusion test showed decrease in cell viability with increasing dose of CCM. Wright staining and ApopTag assay showed, respectively, morphological and biochemical features of apoptosis in T98G cells exposed to 25μM and 50μM of CCM for 24h. Treatment with CCM activated receptor-mediated pathway of apoptosis as Western blotting showed activation of caspase-8 and cleavage of Bid to tBid. Besides, CCM caused an increase in Bax:Bcl-2 ratio, and mitochondrial release of cytochrome c, Second mitochondrial activator of caspases/Direct IAP binding protein with low pI (Smac/Diablo), and apoptosis-inducing-factor (AIF) indicating involvement of mitochondria-mediated pathway as well. Down regulation of the nuclear factor kappa B (NFκB), increased expression of inhibitor of nuclear factor kappa B alpha (IκBα), and decreased expression of inhibitor-of-apoptosis proteins (IAPs) such as c-IAP1 and c-IAP2 in T98G cells following CCM treatment suggested suppression of survival signal. Activation of caspase-9 and caspase-3 was detected in generation of 35kD and 20kD active fragments, respectively. Calpain and caspase-3 activities cleaved 270kD α-spectrin at specific sites to generate 145kD spectrin break down product (SBDP) and 120kD SBDP, respectively. Our results strongly suggest that CCM induced both receptor-mediated and mitochondria-mediated proteolytic mechanisms for induction of apoptosis in T98G cells.