Novel autophagy inhibitor Lys05 enhances impact of ionizing radiation on human lung cancer cells H1299

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increasing the efficiency of radiotherapy of oncological patients. A new inhibitor, Lys05, with potential to accumulate within lysosomes and to block autophagy has been discovered recently. Several studies have described its chemosensitizing effects, but nothing is known about its impact in the context of ionizing radiation (IR). To investigate the mechanisms underlying its role in radiosensitization we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative) in combination with multiple approaches (cell biology techniques to reveal the phenotypes, and quantitative phosphoproteomics to comprehensively describe Lys05-induced changes in irradiated cells). In the presented study, we report for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in eradication of lung cancer cells. H1299 cells were either irradiated or left unirradiated and both of these groups were either treated with Lys05 or spautin or left untreated. This results in six experimental groups per one time interval. The cells were sampled at time one, twenty-four and fourty-eight hours post treatment. In each time & treatment group were n=3 samples. There were 6 experimental groups * 3 times * 3 biological replicates = 54 samples. These samples were hybridized in two-color fashion (portion of the three biological replicates were labeled with Cy3, part with Cy5) but were analyzed as single channel (quantile normalization across all channels).

通过小分子抑制剂实现自噬抑制，是提升肿瘤患者放疗效率的可行策略之一。近期研究发现了一种新型抑制剂Lys05，其具备在溶酶体内富集并阻断自噬的潜力。已有多项研究阐述了该化合物的化学增敏作用，但目前尚无关于其在电离辐射（ionizing radiation, IR）背景下的影响的相关报道。为探究Lys05介导放射增敏的作用机制，本研究采用电离辐射抗性人非小细胞肺癌细胞（H1299，p53阴性），结合多种实验手段开展研究：包括用于表型分析的细胞生物学技术，以及用于全面解析受辐照细胞中Lys05诱导蛋白变化的定量磷酸化蛋白质组学方法。在本研究中，我们首次报道了可将Lys05与电离辐射联合使用，作为一种极具前景的肺癌细胞清除策略。实验中将H1299细胞分为受辐照组与未受辐照组，每组又分别接受Lys05处理、spautin处理或不作处理，因此每个时间区间内共设6个实验组。分别于处理后1小时、24小时及48小时收集细胞样本。每个时间点与处理组合下设置3份生物学重复样本，即6个实验组 × 3个时间点 × 3份生物学重复 = 共计54份样本。所有样本采用双色荧光标记策略（3份生物学重复中的一部分以Cy3标记，其余以Cy5标记），但最终采用单通道模式进行数据分析，并对所有通道实施分位数归一化处理。