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

癌症患者受益于放射治疗，但可能会对正常组织（包括眼部）产生副作用。 透镜，导致白内障。虽然不直接威胁生命，但白内障疾病具有重大的医疗、经济、 对个人、家庭和整个社会的影响。辐射诱导的透镜混浊是一种 复杂的事件，并已归因于DNA双链断裂（DSB）在萌发上皮， 导致透镜纤维细胞的分化缺陷和随后的透镜蛋白质的异常折叠。 醋酸鲁沙利铂（TP 508）是一种放射性调节肽，已显示可增加 通过激活内皮细胞中的信号转导途径，启动DSB的修复， 增加NO水平和逆转内皮细胞功能障碍。本次调查将确定TP 508是否 将对人类透镜上皮细胞（HLEC）具有类似的作用，并减轻辐射诱导的 与纤维细胞异常分化、异常蛋白质 折叠和不透明化。该假说认为，通过直接激活分子途径， 照射后的HLEC，TP 508处理将减轻或修复DSB并恢复正常的细胞分化。相比之下 与其他关注单一下游机制的调查方法相比，本调查将 检查TP 508在与正常人相关的多种病理生理学途径中的分子活性， HLEC的分化。研究目的是确定分子活性和最佳剂量阈值， TP 508在减轻X射线或质子损伤中的治疗时机，单次暴露为0.5，1.0， 2.0或4戈伊（CRL-11421 [B3]、SRA 01/04和HLEpiC细胞）。目的（1）确定药物毒性 使用克隆形成存活测定和细胞倍增时间确定最佳TP 508浓度 和给药方案（辐射前或辐射后），用于对辐射诱导的 HLEC活力。目的（2）将确定TP 508是否可以维持或恢复正常细胞分化所需的正常细胞分化， 正常蛋白质折叠，使用目标1中建立的最优化剂量和给药方案。 研究将包括TP 508对与异常代谢相关的分子标志物和蛋白质的影响。 透镜纤维细胞的分化（α B-晶状体蛋白[α B-crystallin]，βB2-晶状体蛋白[βB2-crystallin]）和参与 细胞凋亡、坏死、衰老和有丝分裂灾难的信号通路。研究预计将 （i）确定TP 508剂量增加时的毒性极限和对存活的影响， 在辐射之前和之后施加不同剂量的经辐射的HLEC;以及（ii）鉴定分子 与透镜混浊的进展相关的机制和蛋白质标记物被 TP 508（来自目标1）在不同辐射水平下在HLEC中的最佳剂量。成功开发，未来 TP 508的研究可以扩展其应用，以减轻额外的眼部放射治疗副作用 包括干眼症和视网膜病。

Cancer patients benefit from radiation therapy but can incur side effects to normal tissues including the ocular lens, leading to 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 TP508 will have a similar effect on human lens epithelial cells (HLEC) and mitigate radiation-induced pathophysiological pathways that are associated with abnormal differentiation of fiber cells, abnormal protein folding and opacification. The hypothesis is that through the direct activation of molecular pathways in irradiated HLEC, TP508 treatment will mitigate or repair DSB and restore normal cell differentiation. 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 normal differentiation 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 damage with single fraction exposures of 0.5, 1.0, 2.0, or 4 Gy in HLEC (CRL-11421 [B3], SRA01/04 and HLEpiC cells. Aim (1) is to determine drug toxicity levels using clonogenic survival assays and cell doubling times to identify the optimum TP508 concentration and administration schedule (before or after radiation) for producing protective effects on radiation induced HLEC viability. Aim (2) will determine if TP508 can maintain or restore normal cell differentiation required for normal protein folding, using the most optimized dosage and administration schedule established in Aim 1. Investigations will include the effects of TP508 on molecular markers and proteins associated with abnormal differentiation of lens fiber cells (CRYAB [αB-crystallin], CRYBB2 [βB2-crystallin]) and proteins involved in signaling pathways for apoptosis, necrosis, senescence, and mitotic catastrophe. Studies are expected to provide the following: (i) determine limits of toxicity with increasing doses of TP508 and the survival effect on irradiated HLEC at different doses applied before and after radiation; and (ii) identify if the molecular mechanisms and protein markers associated with the progression of lens opacification are mitigated by the optimum doses of TP508 (from Aim 1) in HLEC at different levels of radiation. Successfully developed, future investigations of TP508 could expand its application to mitigate additional ocular radiation therapy side effects including dry eye and retinopathy.