Rusalatide Acetate (TP508) Mitigation of Genotoxic Radiation Damage in Human Lens Epithelial Cells

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Cancer patients benefit from radiation therapy but can incur side effects to non-targeted tissues including cataracts. Although not directly life threatening, cataract disease has major medical, economic, and social impacts on individuals, families, and society as a whole. Radiation-induced lens opacification is a complex event and has been attributed to DNA double strand breaks (DSB) in the germinative epithelium, leading to defective differentiation of lens fiber cells and subsequent abnormal folding of lens proteins. Rusalatide acetate (TP508) is a radio-modulating peptide that has been shown to increase survival of irradiated animals via activation of signal transduction pathways in endothelial cells, initiating repair of DSB, increasing NO levels and reversing of endothelial cell dysfunction. This investigation will determine if, in the absence of endothelial cells, TP508 will have a similar effect on human lens epithelial cells (HLEC) and mitigate radiation induced pathophysiological pathways that lead to DSB. The hypothesis is that through the direct activation of molecular pathways in irradiated HLEC, TP508 treatment will mitigate or repair DSB. In contrast to other investigative approaches that focus on a single downstream mechanism, this investigation will examine molecular activity of TP508 across multiple pathophysiological pathways associated with the health of HLEC. Study aims are to establish the molecular activity and optimum dosage thresholds, and timing of treatments of TP508 in mitigating X-ray or proton radiation damage with single fraction exposures of 0.5, 1.0, 2.0, or 4 Gy in HLEC (CRL-11421 [B3] and SRA01/04 lines). Aim (1) is to determine the most optimized concentration and administration schedule for TP508 effects on radiation induced HLEC viability using a clonogenic survival assay, MTT assay, and cell doubling time to assess the effects of various concentrations of TP508 on the sensitivity of HLECs applied before and after irradiation. Aim (2) is to further identify the effects of TP508 on specific HLEC molecular responses, using the most optimized concentration and administration schedule of TP508 comparing the single fraction radiation exposures applied with or without TP508, and analyzed with assays for apoptosis, necrosis, senescence, mitotic catastrophe and protein expression; 53BP1 foci staining for dynamics of DSB; and western blot assays for related signaling pathways and protein profiles including amyloid beta, an early marker of cataract formation. Studies are expected to provide the following: (i) determine if TP508 provides a survival effect on irradiated HLEC at different doses applied before and after radiation; (ii) identify if the molecular mechanisms and protein profiles underlying the protective effect of TP508 in HLEC at different doses of radiation are attributed to DSB repair; and (iii) determine the most effective dosage and timing (before or after radiation) of TP508 application. Successfully developed, future investigations of TP508 could expand its application to mitigate additional radiation side effects including dry eye, retinopathy, and xerostomia. .