Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic manipulation.
Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic manipulation.
复制标题
通过药理学和遗传操作确定骨肉瘤细胞系对自噬抑制的敏感性。
DOI：
10.1111/vco.12937
复制
发表时间：
2023
影响因子：
2.1
通讯作者：
VanEaton,KristenM
中科院分区：
文献类型：
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作者：
Gustafson,DanielL;Viola,LindseyO;Towers,ChristinaG;Das,Sunetra;Duval,DawnL;VanEaton,KristenM
自噬的药理学抑制可以使用亲溶酶体剂如羟氯喹（HCQ）来实现，其干扰自噬体与溶酶体的融合，从而阻止再循环过程的完成。本研究的目的是确定8个犬（cOSA）和4个人（hOSA）骨肉瘤肿瘤细胞系对溶酶体自噬抑制剂的抗增殖和细胞毒性作用的敏感性，并将这些结果与使用CRISPR/Cas9活细胞成像测定法在OSA和其他肿瘤细胞系中测量的自噬依赖性进行比较。使用活细胞成像和YOYO-1染色确定对HCQ和Lys 05的抗增殖和细胞毒性应答。CRISPR/Cas9活细胞成像筛选使用物种特异性引导RNA和将试剂转染到细胞中来完成。将对自噬核心基因的反应与对必需（PCNA）和非必需（FOXO 3A）基因的反应进行比较。cOSA和hOSA细胞系对HCQ和Lys 05显示出相似的抗增殖和细胞毒性反应，抗增殖反应的测量值的半数致死剂量（Dm）值分别为4.6-15.8 μM和2.1-5.1 μM。观察到对HCQ和Lys 05的抗增殖反应与VPS 34 CRISPR评分之间的关系，其中Dm值以物种独立的方式与VPS 34反应相关（r= 0.968和0.887）。结果表明，cOSA和hOSA细胞系的一个子集是自噬依赖性的，并且在细胞相关暴露下对HCQ敏感。
Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy‐dependence measured using a CRISPR/Cas9 live‐cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO‐1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non‐essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (Dm) values ranging from 4.6–15.8 μM and 2.1–5.1 μM for measures of anti‐proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score withDmvalues correlating with VPS34 response (r= 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy‐dependent and sensitive to HCQ at pharmacologically‐relevant exposures.