Astroglial U87 Cells Protect Neuronal SH-SY5Y Cells from Indirect Effect of Radiation by Reducing DNA Damage and Inhibiting Fas Mediated Apoptotic Pathway in Coculture System.

Recent studies provide the evidence that indirect effects of radiation could lead to neuronal cells death but underlying mechanism is not completely understood. On the other hand astroglial cells are known to protect neuronal cells against stress conditions in vivo and invitro. Yet, the fate of neuronal cells and the neuroprotective effect of coculture system (with glial cells) in response to indirect radiation exposure remain rarely discussed. Here, we purpose that the indirect effect of radiation may induce DNA damage by cell cycle arrest and receptor mediated apoptotic cascade which lead to apoptotic death of neuronal SH-SY5Y cells. We also hypothesized that coculture (with glial U87) may relieved the neuronal SH-SY5Y cells from toxicity of indirect effects radiation by reducing DNA damage and expression of apoptotic proteins in vitro. In the present study irradiated cell conditioned medium (ICCM) was used as source of indirect effect of radiation. Neuronal SH-SY5Y cells were exposed to ICCM with and without coculture with (glial U87) in transwell coculture system respectively. Various endpoints such as, cell survival number assay, Annexin V/PI assay, cell cycle analysis by flow cytometer, mRNA level of Fas receptor by q RT-PCR, expression of key apoptotic proteins by western blot and estimation of neurotrophic factors by ELISA method were analyzed into neuronal SH-SY5Y cells with and without co culture after ICCM exposure respectively. We found that ICCM induced DNA damage in neuronal SH-SY5Y cells by significant increase in cell cycle arrest at S-phase (***P < 0.001) which was further supported by over expression of P53 protein (**P < 0.01). While coculture (with glial U87), significantly reduced the ICCM induced cell cycle arrest and expression of P53 ((###) P < 0.001) neuronal SH-SY5Y cells. Further investigation of the underlying apoptotic mechanism revealed that in coculture system; ICCM induced elevated level of FAS mRNA level was significantly reduced ((###) P < 0.001) in neuronal SH-SY5Y cells which was followed by significant reduction in expression of key apoptotic protein i.e., FADD ((###) P < 0.001), caspase-8 ((###) P < 0.001), and cleaved caspase-3 ((###) P < 0.001) as compare to neuronal SH-SY5Y cells which received ICCM without coculture. Intriguingly, concentration of neurotrophic factors such as, GDNF and BDNF were significantly increased ((###) P < 0.001) in neuronal SH-SY5Y after coculture (with glial U87) cells. Hence, these findings infer that the receptor mediated pathway could be the one way through which indirect effects of radiation cause neurotoxicity. However, in co-cultures system (with glial U87) neuronal SH-SY5Y depicts remarkable resistance against ICCM induced neurotoxicity.