Research Unit on BioActive Molecules, Department of Biological Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), 08034 Barcelona, Spain.
Unitat de Biologia Cel·lular, Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036 Barcelona, Spain.
Int J Mol Sci. 2022 Jun 29;23(13):7257. doi: 10.3390/ijms23137257.
Methuosis is a type of programmed cell death in which the cytoplasm is occupied by fluid-filled vacuoles that originate from macropinosomes (cytoplasmic vacuolation). A few molecules have been reported to behave as methuosis inducers in cancer cell lines. Jaspine B (JB) is a natural anhydrous sphingolipid (SL) derivative reported to induce cytoplasmic vacuolation and cytotoxicity in several cancer cell lines. Here, we have investigated the mechanism and signalling pathways involved in the cytotoxicity induced by the natural sphingolipid Jaspine B (JB) in lung adenocarcinoma A549 cells, which harbor the G12S K-Ras mutant. The effect of JB on inducing cytoplasmic vacuolation and modifying cell viability was determined in A549 cells, as well as in mouse embryonic fibroblasts (MEF) lacking either the autophagy-related gene or genes. Apoptosis was analyzed by flow cytometry after annexin V/propidium iodide staining, in the presence and absence of z-VAD. Autophagy was monitored by LC3-II/GFP-LC3-II analysis, and autophagic flux experiments using protease inhibitors. Phase contrast, confocal, and transmission electron microscopy were used to monitor cytoplasmic vacuolation and the uptake of Lucifer yellow to assess macropinocyosis. We present evidence that cytoplasmic vacuolation and methuosis are involved in Jaspine B cytotoxicity over A549 cells and that activation of 5' AMP-activated protein kinase (AMPK) could be involved in Jaspine-B-induced vacuolation, independently of the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) axis.
细胞自噬性溶酶体死亡是一种程序性细胞死亡形式，其特征是细胞质被充满液体的空泡所占据，这些空泡来源于巨胞饮体（细胞质空泡化）。已经有一些分子被报道在癌细胞系中作为细胞自噬性溶酶体死亡诱导剂发挥作用。Jaspine B（JB）是一种天然的无定形神经酰胺（SL）衍生物，据报道它能诱导几种癌细胞系的细胞质空泡化和细胞毒性。在这里，我们研究了天然神经酰胺 Jaspine B（JB）在携带 G12S K-Ras 突变的肺腺癌细胞 A549 中诱导细胞毒性的机制和信号通路。在 A549 细胞以及缺乏自噬相关基因 或 基因的小鼠胚胎成纤维细胞（MEF）中，测定了 JB 诱导细胞质空泡化和改变细胞活力的作用。在存在和不存在 z-VAD 的情况下，通过 Annexin V/碘化丙啶染色后用流式细胞术分析细胞凋亡。通过 LC3-II/GFP-LC3-II 分析和使用蛋白酶抑制剂的自噬流实验监测自噬。相差、共聚焦和透射电子显微镜用于监测细胞质空泡化和 Lucifer yellow 的摄取，以评估巨胞饮作用。我们提供的证据表明，细胞质空泡化和细胞自噬性溶酶体死亡参与了 Jaspine B 对 A549 细胞的细胞毒性，5' 腺苷单磷酸激活的蛋白激酶（AMPK）的激活可能与 Jaspine-B 诱导的空泡化有关，而与磷脂酰肌醇 3-激酶/蛋白激酶 B/雷帕霉素靶蛋白复合物 1（PI3K/Akt/mTORC1）轴无关。
