Erythropoietin Attenuates Neuronal Cell Death Following Radiation

Article Outline

• Purpose/Objective(s)
• Materials/Methods
• Results
• Conclusions
• Copyright

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Purpose/Objective(s) 

Erythropoietin (EPO) is a cytokine hormone clinically exploited for stimulation of erythropoiesis under hypoxic conditions. EPO receptors (EPO-R) are abundantly expressed in nerve tissue. Furthermore, EPO and the EPO-R are upregulated in vivo following ischemic nerve injury. EPO treatment preceding nerve transection in rats has been shown to promote the regeneration and function of insulted nerves. The relationship between radiation-induced neuronal injury and EPO-R has not been characterized. Furthermore, it is not known whether neuronal injury from radiation can be modulated by EPO-R. To study this, we investigated EPO-R expression as a function of radiation exposure. Additionally, we used survival assays to determine the protective effects of EPO, in both an in vitro mammalian neuronal model as well as in human prostate cancer cell lines.

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Materials/Methods 

PC-12 (rat pheochromocytoma cell line) cells were differentiated with low serum media and nerve growth factor to produce previously well characterized neuronal morphology and functional phenotype. Ionizing radiation was applied at 0, 3, or 6 Gy and protein extracts were made immediately after radiation exposure as well as 8 hours, and 24 hours post treatment. EPO-R protein levels were determined by SDS-PAGE and immunoblotting. PC-12 cells were then differentiated, pretreated with either vehicle, 1 ng/ml EPO, or 10 ng/ml EPO and then irradiated at 0, 3 or 6 Gy. Survival analysis was determined with clonogenic assays. Clonogenic survival was also performed using irradiated PC-3 and DU-145 prostate cancer cell lines to determine if treatment with exogenous EPO treatment had similar effects on radiation-induced cell death.

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Results 

EPO protein expression was increased three fold with radiation exposure compared to unirradiated controls in differentiated PC-12 cells. Maximal expression of protein EPO-R was seen within 30 min. following radiation exposure with increased protein expression enduring 24 hours post radiation. PC-12 cells treated with 1 ng/ml EPO and then irradiated exhibited significantly less cell death than PC-12 cells treated with vehicle only followed by radiation at both 3 and 6 Gy (p = 0.002 and p < .001 respectively). Protective effects were also seen by 10 ng/ml EPO treatment preceding radiation at 3 and 6 Gy (p < 0.001 and p = 0.004 respectively). Protective effects of EPO treatment were not seen in irradiated human prostate cancer cell lines (p = .67).

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Conclusions 

These data are the first to demonstrate a relationship between radiation exposure and EPO-R expression in a time and radiation dose dependent fashion. These are also the first data to document that pretreatment with EPO significantly decreases radiation-induced neuronal cell death but does not exert a similarly protective effect in irradiated human prostate cancer cell lines. Taken together, these data suggest that radiation associated neuronal injury may in part, be modulated through pharmologic targeting of EPO-R providing a therapeutic target for clinical-translation of this work.