Shi Lei, Ito Fumiya, Wang Yue, Okazaki Yasumasa, Tanaka Hiromasa, Mizuno Masaaki, Hori Masaru, Hirayama Tasuku, Nagasawa Hideko, Richardson Des R, Toyokuni Shinya
Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
Center for Advanced Medicine and Clinical Research, Nagoya University Hospital, Nagoya 466-8550, Japan.
Free Radic Biol Med. 2017 Jul;108:904-917. doi: 10.1016/j.freeradbiomed.2017.04.368. Epub 2017 May 2.
Non-thermal plasma (NTP) is a potential new therapeutic modality for cancer. However, its mechanism of action remains unclear. Herein, we studied the effect of NTP on mesothelioma cells and fibroblasts to understand its anti-proliferative efficacy. Interestingly, NTP demonstrated greater selective anti-proliferative activity against mesothelioma cells relative to fibroblasts than cisplatin, which is used for mesothelioma treatment. The anti-proliferative effect of NTP was enhanced by pre-incubation with the cellular iron donor, ferric ammonium citrate (FAC), and inhibited by iron chelation using desferrioxamine (DFO). Three oxidative stress probes (CM-H2DCFDA, MitoSOX and C11-BODIPY) demonstrated reactive oxygen species (ROS) generation by NTP, which was inhibited by DFO. Moreover, NTP decreased transferrin receptor-1 and increased ferritin-H and -L chain expression that was correlated with decreased iron-regulatory protein expression and RNA-binding activity. This regulation was potentially due to increased intracellular iron in lysosomes, which was demonstrated via the Fe(II)-selective probe, HMRhoNox-M, and was consistent with autophagic-induction. Immunofluorescence using LysoTracker and Pepstatin A probes demonstrated increased cellular lysosome content, which was confirmed by elevated LAMP1 expression. The enhanced lysosomal biogenesis after NTP could be due to the observed increase in fluid-phase endocytosis and early endosome formation. These results suggest NTP acts as a stressor, which results in increased endocytosis, lysosome content and autophagy. In fact, NTP rapidly increased autophagosome formation, as judged by increased LC3B-II expression, which co-localized with LAMP1, indicating autophagolysosome formation. Autophagic-induction by NTP was confirmed using electron microscopy. In summary, NTP acts as a cellular stressor to rapidly induce fluid-phase endocytosis, lysosome biogenesis and autophagy.
非热等离子体（NTP）是一种潜在的新型癌症治疗方式。然而，其作用机制仍不清楚。在此，我们研究了NTP对间皮瘤细胞和成纤维细胞的影响，以了解其抗增殖功效。有趣的是，相对于成纤维细胞，NTP对间皮瘤细胞表现出比用于间皮瘤治疗的顺铂更大的选择性抗增殖活性。NTP与细胞铁供体柠檬酸铁铵（FAC）预孵育可增强其抗增殖作用，而使用去铁胺（DFO）进行铁螯合则可抑制该作用。三种氧化应激探针（CM-H2DCFDA、MitoSOX和C11-BODIPY）显示NTP可产生活性氧（ROS），而DFO可抑制这种产生。此外，NTP降低了转铁蛋白受体-1的表达，并增加了铁蛋白-H和-L链的表达，这与铁调节蛋白表达和RNA结合活性的降低相关。这种调节可能是由于溶酶体中细胞内铁的增加，这通过Fe（II）选择性探针HMRhoNox-M得以证实，并且与自噬诱导一致。使用溶酶体追踪染料和胃蛋白酶抑制剂A探针进行的免疫荧光显示细胞溶酶体含量增加，LAMP1表达升高证实了这一点。NTP后溶酶体生物合成的增强可能归因于观察到的液相内吞作用和早期内体形成的增加。这些结果表明NTP作为一种应激源，导致内吞作用、溶酶体含量和自噬增加。事实上，通过增加的LC3B-II表达判断，NTP迅速增加了自噬体的形成，LC3B-II与LAMP1共定位，表明形成了自噬溶酶体。使用电子显微镜证实了NTP诱导的自噬。总之,NTP作为一种细胞应激源，可迅速诱导液相内吞作用、溶酶体生物合成和自噬。
