Optimized mixture of As, Cd and Pb induce mitochondria-mediated apoptosis in C6-glioma via astroglial activation, inflammation and P38-MAPK

Arsenic (As), cadmium (Cd), and lead (Pb) in select combinations are proved to affect the viability of astrocyte. However, their role in glioma, an aggressive astroglial tumor, is unexplored. We analyzed the effect of As+Cd+Pb on C6-glioma cells derived from rat glioma. We determined the lethal concentration (LC) of individual metal, and then treated C6-glioma cells with As+Cd+Pb at LC-5 (As: 5 mM, Cd: 2.5 mM and Pb: 15 mM), and concentrations that were double or triple of LC5. As+Cd+Pb induced dose-dependent reduction in C6-glioma viability. Cell death was due to apoptotic DNA fragmentation, detected through terminal deoxynucleotidyl transferase-mediated dUTP-nick-end labeling. An enhanced cleavage of caspase-9 indicated the apoptosis to be mitochondria-mediated. An increase in pro-apoptotic Bcl-2-associated-X protein (Bax) and decrease in anti-apoptotic Bcl2 resulting in a Bax/Bcl2 ratio > 1.0 validated mitochondrial apoptosis. Exploring apoptotic regulatory mechanism revealed an alteration in glial cell morphology and augmentation of astroglial marker, glial fibrillary acidic protein (GFAP), that demonstrated co-localization with cleaved caspase-9. The glial activation was accompanied by inflammation, involving the up-regulation of interleukin-1 (IL-1) and IL-1-receptor. IL-1 also contributed to apoptosis, as evident from the attenuation of cleaved caspase-9 upon treatment with IL-1receptor antagonist. Investigating the involvement of Mitogen-activated protein kinases (MAPKs) revealed the activation of P38 as indicated by an increased phospho-p38 expression. p38-MAPK inhibitor, SB203580, prevented caspase-9 activation, which further suppoted the involvement of p38-MAPK in C6-glioma apoptosis. Overall our data demonstrate the toxic effect of As+Cd+Pb on C6-glioma, which is mediated by mitochondria-dependent apoptosis that requires astroglial activation, inflammation and p38-MAPK signaling. As+Cd+Pb combination treatment may have a potential therapeutic usage against glial tumors.