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

癌症患者从放射治疗中受益，但可能会对包括眼睛在内的正常组织产生副作用 晶状体，导致白内障。虽然不会直接危及生命，但白内障疾病在医疗、经济、 以及对个人、家庭和整个社会的影响。辐射引起的晶状体混浊是一种 这一复杂事件被归因于萌发上皮中的DNA双链断裂(DSB)， 导致晶状体纤维细胞分化缺陷，进而导致晶状体蛋白质的异常折叠。 醋酸鲁沙拉肽(TP508)是一种放射调节肽，已被证明可提高人卵巢癌的存活率。 通过激活内皮细胞中的信号转导通路，启动DSB的修复， 提高NO水平，逆转内皮细胞功能障碍。这项调查将确定TP508 将对人类晶状体上皮细胞(HLEC)产生类似的影响，并减轻辐射诱导的 与纤维细胞异常分化、蛋白质异常相关的病理生理途径 折叠和混浊。假说是通过直接激活分子通路 照射后的HLEC、TP508可减轻或修复DSB，恢复正常细胞分化。相比之下， 对于其他侧重于单一下游机制的调查方法，这项调查将 检测TP508在与正常相关的多个病理生理通路中的分子活性 HLEC的分化。研究的目的是建立分子活性和最佳剂量阈值，以及 TP508在减轻0.5，1.0， 2.0GyHLEC(CRL-11421[B3]、SRA01/04和HLEpiC细胞)。目的(1)是确定药物的毒性 用克隆存活试验和细胞倍增时间确定TP508的最佳浓度 和(辐射前或辐射后)对辐射诱导产生防护作用的给药时间表 HLEC的生存能力。AIM(2)将确定TP508是否能够维持或恢复正常的细胞分化 正常蛋白质折叠，使用目标1中建立的最优剂量和给药程序。 调查将包括TP508对分子标记和与异常相关的蛋白质的影响 晶状体纤维细胞(CryAB[αB-晶体蛋白]、CRYBB2[βB2-晶体蛋白])及其参与蛋白的分化 细胞凋亡、坏死、衰老和有丝分裂灾难的信号通路。预计研究将 提供以下资料：(I)确定TP508剂量增加时的毒性限度和对 在辐射前后应用不同剂量的辐照HLEC；以及(Ii)识别分子是否 与晶状体混浊进展相关的机制和蛋白质标志物可通过 TP508(来自AIM 1)在不同辐射水平下在HLEC中的最佳剂量。成功开发，未来 TP508的研究可以扩大其应用范围，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.