Optimized Mixture Of As,Cd And Pb Induces C6 Glioma Cell Apoptosis And Its Mechanism Research

Background:gliomas are the most common and aggressive type of primary brain tumors,conferring poor prognosis,where drug penetrability through the blood-brain barrier(BBB)poses a problem.glioma cells exhibit an impaired apoptosis,prompting growth and invasiveness of malignant tumor.Thus,stimulation of glioma cell apoptosis is deemed as a promising treatment strategy to reduce glioma tumor invasion.Arsenic,Cd and Pb have proven anti-proliferative and pro-apoptotic effects in cancer cells as well.We thus hypothesized that As+Cd+Pb,with the ability to penetrate BBB,may affect the viability of malignant glioma and serve as chemotherapeutic agent.We assumed a greater effect in mixture than the individual metals.We assessed the potential apoptotic effect of this heavy metal mixture on C6-glioma.We examined the possibility of altered astroglial activation and inflammation,and scanned modulation in MAPK signaling.Overall,our study identified mechanism affecting viability of malignant glial cells upon As+Cd+Pb-exposure.methods:Cell culture and MTT assay,DNA fragmentation-apoptosis assay,Combination index(CI),Western Blotting,Immunocytochemistry,Statistical analysis.Results: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?M,Cd:2.5?M and Pb:15?M),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 C6 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 P3 8 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.Conclusion:The current study reveals that a mixture of As,Cd and Pb at optimized concentration is toxic to malignant glial tumor cell line,C6.A dose-dependent up-regulation of apoptosis appears responsible for the reduction in C6 cell count.The apoptotic effect is mitochondrial with an increase in Bax/Bcl2 ratio that executes nuclear fragmentation through a caspase-dependent pathway.Factors regulating C6 apoptosis include astroglial activation,accompanied with altered glial morphology,inflammation and P38-MAPK signaling.Overall,our findings are important because they provide insight for proposing As+Cd+Pb as novel therapeutic against glioma.