Miyazoe Yuri, Miuma Satoshi, Miyaaki Hisamitsu, Kanda Yasuko, Nakashiki Suguru, Sasaki Ryu, Haraguchi Masafumi, Shibata Hidetaka, Honda Takuya, Taura Naota, Nakao Kazuhiko
Department of Gastroenterology and Hepatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8501, Japan.
Biomed Rep. 2020 Apr;12(4):163-170. doi: 10.3892/br.2020.1279. Epub 2020 Feb 14.
Since the discovery of the senescence-associated secretory phenotype, the role of senescent hepatic stellate cells (HSCs) in hepatocellular carcinoma (HCC) development has gained increasing attention. Similar to cytokines, extracellular vesicles (EVs) are essential for intercellular communication. However, the function of EVs derived from senescent HSCs in HCC progression has not been extensively studied. The aims of the present study were to characterize the EVs derived from senescent HSCs and determine their role in the tumor microenvironment. Cellular senescence was induced in human hepatic stellate cells (HHSteCs) with various concentrations of etoposide. Induction was confirmed using EdU staining and 53BP1 and p21 immunostaining. EVs were isolated by ultracentrifugation and analyzed by nanoparticle tracking analysis. Multiplex immunoassays were used to compare the levels of growth factors secreted from hepatoma cell lines and macrophage cells pretreated with EVs derived from senescent HHSteCs (senescent EVs) with those pretreated with EVs derived from normal cultured HHSteCs (normal EVs). Treatment with 25 µM etoposide for 3 days was the most effective at inducing senescence in HHSteCs. This finding was confirmed by induction of irreversible cell-cycle arrest, upregulation of 53BP1 and p21 expression, and increased SA-β-gal staining. Senescent HHSteCs released increased quantities of EV particles compared with normally cultured HHSteCs. Multiplex analysis revealed that there was no difference between hepatoma cell lines treated with normal EVs and those treated with senescent EVs in growth factor secretion. In contrast, the secretion of epidermal growth factor (EGF) was increased by macrophage cells treated with senescent EVs compared with those treated with normal EVs. Furthermore, senescent EVs did not affect the viability of hepatoma cells but increased the viability of hepatoma cells co-cultured with macrophage cells. In conclusion, the release of EVs from senescent HSCs was higher compared with normal HSCs. Furthermore, senescent EVs promoted HCC development by upregulating EGF secretion from macrophages.
自从衰老相关分泌表型被发现以来，衰老的肝星状细胞（HSC）在肝细胞癌（HCC）发展中的作用日益受到关注。与细胞因子类似，细胞外囊泡（EV）对于细胞间通讯至关重要。然而，源自衰老HSC的EV在HCC进展中的功能尚未得到广泛研究。本研究的目的是表征源自衰老HSC的EV，并确定它们在肿瘤微环境中的作用。用不同浓度的依托泊苷诱导人肝星状细胞（HHSteC）发生细胞衰老。通过EdU染色以及53BP1和p21免疫染色确认诱导情况。通过超速离心分离EV，并通过纳米颗粒跟踪分析进行分析。采用多重免疫测定法比较用源自衰老HHSteC的EV（衰老EV）预处理的肝癌细胞系和巨噬细胞分泌的生长因子水平与用源自正常培养的HHSteC的EV（正常EV）预处理的细胞系分泌的生长因子水平。用浓度为25µM的依托泊苷处理3天对诱导HHSteC衰老最为有效。不可逆的细胞周期停滞、53BP1和p21表达上调以及衰老相关β-半乳糖苷酶（SA-β-gal）染色增加证实了这一发现。与正常培养的HHSteC相比，衰老的HHSteC释放的EV颗粒数量增加。多重分析显示，用正常EV处理的肝癌细胞系和用衰老EV处理的肝癌细胞系在生长因子分泌方面没有差异。相比之下，与用正常EV处理的巨噬细胞相比，用衰老EV处理的巨噬细胞分泌的表皮生长因子（EGF）增加。此外，衰老EV不影响肝癌细胞的活力，但增加了与巨噬细胞共培养的肝癌细胞的活力。总之，与正常HSC相比，衰老HSC释放的EV更多。此外，衰老EV通过上调巨噬细胞的EGF分泌促进HCC发展。
