Palladin Institute of Biochemistry, NAS of Ukraine, Kyiv 01030, Ukraine.
Oles' Honchar Dnipro National University, Dnipro 49050, Ukraine.
Exp Oncol. 2020 Dec;42(4):270-276. doi: 10.32471/exp-oncology.2312-8852.vol-42-no-4.15253.
Pericellular plasmin generation triggers apoptosis/anoikis in normal adherent cells. However, cancer cells are notoriously resistant to anoikis, enabling metastasis and new tumor growth beyond their original environment. Autophagy can be a major contributor to anoikis resistance in cancer.
To investigate if protective autophagy can be induced in lung adenocarcinoma cells in response to plasminogen treatment.
Human lung adenocarcinoma A549 cells were incubated with Glu-plasminogen (0.1-1.0 µM) for 24 h. Pericellular plasmin activity was monitored spectrophotometrically by a cleavage of the specific chromogenic- substrate S-2251. Cell survival was assessed by 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)-test. Degradation of fibronectin, levels of autophagy markers (beclin-1 and light chain 3 (LC3)) and glycolysis regulator (TIGAR) were evaluated by western blot. Intracellular localization of LC-3 was visualized by immunocytochemistry.
It was shown that plasminogen is converted into plasmin on the surface of adenocarcinoma cells in a dose-dependent manner. Plasmin disrupted cellular adhesive contacts resulting in cell detachment. A549 cells did not loss their viability after plasminogen treatment for 24 h, while 1.0 µM plasminogen was cytotoxic for non-transformed fibroblasts. Plasminogen 0.1, 0.5, and 1.0 µM induced 7.08-, 5.18-, and 3.78-fold elevation of TIGAR expression (p < 0.05), respectively. Enhanced TIGAR expression indicates switch on pentose phosphate pathway, protection against oxidative stress to prevent apoptosis, facilitation of DNA repair and the degradation of their own organelles (autophagy). Exposure of adenocarcinoma cells to plasminogen in concentrations of 0.1 and 0.5 µM caused 1.74- and 2.19-fold elevation of beclin-1 expression vs untreated cells (p < 0.05), respectively. Unlike K1-3 fragment, plasminogen treatment (0.1-0.5 µM) resulted in increased expression of LC3-I and stimulated rapid conversion of LC3-I to LC3-II. Up-regulation of beclin-1 levels and enhanced LC3-I/II conversion in plasminogen-treated A549 cells are the hallmarks of autophagy induction. According to immunocytochemistry data, increased LC3 puncta and autophagosome formation after exposure to plasminogen could reflect autophagy activation.
Therefore, we showed stimulation of prosurvival signals and induction of autophagy in plasminogen-treated adenocarcinoma cells rendering them resistant to apoptosis/anoikis. Based on the obtained data, autophagy has a great potential for novel targets that affect cancer cell death, in addition to the current cytotoxic agents.
研究纤溶酶原处理能否诱导肺腺癌细胞发生保护性自噬。
用 Glu-纤溶酶原（0.1-1.0 μM）孵育人肺腺癌细胞 A549 24 小时。通过特定显色底物 S-2251 的裂解，分光光度法监测细胞外泌体中纤溶酶的活性。通过 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑溴盐（MTT）试验评估细胞存活率。通过 Western blot 评估纤连蛋白降解、自噬标志物（beclin-1 和 LC3）和糖酵解调节剂（TIGAR）的水平。通过免疫细胞化学观察 LC-3 的细胞内定位。
结果表明纤溶酶原在腺癌细胞表面呈剂量依赖性转化为纤溶酶。纤溶酶破坏细胞黏附接触，导致细胞脱落。纤溶酶原处理 24 小时后，A549 细胞并未丧失活力，而 1.0 μM 的纤溶酶原对非转化成纤维细胞具有细胞毒性。纤溶酶原 0.1、0.5 和 1.0 μM 分别诱导 TIGAR 表达增加 7.08、5.18 和 3.78 倍（p < 0.05）。增强的 TIGAR 表达表明戊糖磷酸途径的开启，对细胞凋亡的氧化应激的保护，促进 DNA 修复和自身细胞器（自噬）的降解。用纤溶酶原 0.1 和 0.5 μM 处理腺癌细胞，beclin-1 表达分别增加 1.74 倍和 2.19 倍（p < 0.05）。与 K1-3 片段不同，纤溶酶原处理（0.1-0.5 μM）导致 LC3-I 表达增加，并刺激 LC3-I 向 LC3-II 的快速转化。纤溶酶原处理的 A549 细胞中 beclin-1 水平的上调和 LC3-I/II 转化的增强是自噬诱导的标志。根据免疫细胞化学数据，纤溶酶原处理后 LC3 斑点增加和自噬体形成可能反映了自噬的激活。
因此，我们表明纤溶酶原处理刺激了腺癌细胞中的生存信号，并诱导了自噬，使它们对细胞凋亡/失巢凋亡产生抗性。基于获得的数据，自噬除了当前的细胞毒性药物外，还有很大的潜力成为影响癌细胞死亡的新靶点。
