Department of Immunology, Medical University of Warsaw, 1A Banacha Str., F building, 02-097, Warsaw, Poland.
Postgraduate School of Molecular Medicine, Warsaw, Poland.
BMC Cancer. 2018 Feb 20;18(1):210. doi: 10.1186/s12885-018-4126-y.
Accumulating evidence suggest that autophagy plays a pivotal role in various anticancer therapies, including photodynamic therapy (PDT), acting as a pro-death or pro-survival mechanism in a context-dependent manner. Therefore, we aimed to determine the role of autophagy in Photofrin-based PDT.
In vitro cytotoxic/cytostatic effects of PDT were evaluated with crystal violet cell viability assay. Autophagy induction was analyzed by immunoblotting and immunofluorescence using anti-LC3 antibody. Autophagy was inhibited by shRNA-mediated ATG5 knockdown or CRISPR/Cas9-mediated ATG5 knockout. Apoptosis was assessed by flow cytometry analysis of propidium iodide and anexin V-positive cells as well as by detection of cleaved PARP and caspase 3 proteins using immunoblotting. Protein carbonylation was evaluated by the 2,4-dinitrophenylhydrazine (DNPH) method.
Photofrin-PDT leads to robust autophagy induction in two cancer cell lines, Hela and MCF-7. shRNA-mediated knockdown of ATG5 only partially blocks autophagic response and only marginally affects the sensitivity of Hela and MCF-7 cells to PDT. ATG5 knockout in HeLa cell line utilizing CRISPR/Cas9 genome editing results in increased PDT-mediated cytotoxicity, which is accompanied by an enhanced apoptotic response and increased accumulation of carbonylated proteins.
Altogether, these observations imply that autophagy contributes to Photofrin-PDT resistance by enabling clearance of carbonylated and other damaged proteins. Therefore, autophagy inhibition may serve as a strategy to improve PDT efficacy.
越来越多的证据表明，自噬在各种抗癌疗法中起着关键作用，包括光动力疗法（PDT），它在依赖于上下文的情况下作为一种促进死亡或促进存活的机制发挥作用。因此，我们旨在确定自噬在 Photofrin 基 PDT 中的作用。
用结晶紫细胞活力测定法评估 PDT 的体外细胞毒性/细胞抑制作用。通过免疫印迹和用抗 LC3 抗体进行免疫荧光分析来检测自噬的诱导。通过 shRNA 介导的 ATG5 敲低或 CRISPR/Cas9 介导的 ATG5 敲除来抑制自噬。通过碘化丙啶和膜联蛋白 V 阳性细胞的流式细胞术分析以及通过免疫印迹检测裂解的 PARP 和 caspase 3 蛋白来评估细胞凋亡。通过 2,4-二硝基苯肼（DNPH）法评估蛋白质羰基化。
Photofrin-PDT 在两种癌细胞系 Hela 和 MCF-7 中导致强烈的自噬诱导。shRNA 介导的 ATG5 敲低仅部分阻断自噬反应，并且仅轻微影响 Hela 和 MCF-7 细胞对 PDT 的敏感性。利用 CRISPR/Cas9 基因组编辑在 HeLa 细胞系中敲除 ATG5 会导致 PDT 介导的细胞毒性增加，这伴随着增强的凋亡反应和羰基化蛋白质的积累增加。
总的来说，这些观察结果表明，自噬通过使羰基化和其他受损蛋白质的清除来促进 Photofrin-PDT 耐药性。因此，自噬抑制可能是提高 PDT 疗效的一种策略。
