目的:由于空泡质子泵在癌症中的重要性,我们研究了质子泵抑制对乳腺癌细胞迁移和增殖的影响,F-肌动蛋白聚合,laminA/C,异染色质,和ETV7表达式,核的大小和形状,和AKT/mTOR信令。
方法:用120nM质子泵抑制剂巴弗洛霉素A1处理低转移性MCF7和高转移性MDA-MB-231乳腺癌细胞24小时。用伤口划痕试验研究了细胞迁移,用化学发光测定法测定ATP水平;通过细胞面积扩增测定法定量细胞增殖。使用DAPI核染色和荧光显微镜确定核大小和形状。F-肌动蛋白的水平,laminA/C,异染色质,和ETV7使用免疫细胞化学和蛋白质印迹进行定量;p-mTORC1,p-mTORC2,mTOR,p-AKT,和AKT通过蛋白质印迹测量。
结果:我们发现质子泵抑制可降低F-肌动蛋白聚合,细胞迁移,扩散,并增加低转移和高转移细胞中的异染色质。令人惊讶的是,巴弗洛霉素可降低两种细胞系中的层粘连蛋白A/C。抑制对低转移和高转移细胞中ETV7的表达有不同的影响,以及核领域,周边,和循环性。巴弗洛霉素也显著降低p-mTORC1、p-MTORC2和MTOR在两种细胞系中的表达,而显着降低低转移细胞中的p-AKT,并令人惊讶地显着增加高转移细胞中的p-AKT。我们的质子泵抑制方案在三小时内降低了V-ATPase水平(~25%)。V-ATPase水平随时间变化的对照和抑制细胞,和抑制减少细胞ATP。
结论:质子泵促进F-肌动蛋白聚合并减少异染色质,促进入侵。这些泵还上调mTORC1和mTORC2,从而突出了空泡质子泵作为转移性癌症靶标的相关性。
OBJECTIVE: Motivated by the vacuolar proton pump\'s importance in cancer, we investigate the effects of proton pump inhibition on breast cancer cell migration and proliferation, F-actin polymerization, lamin A/C, heterochromatin, and ETV7 expressions, nuclear size and shape, and AKT/mTOR signaling.
METHODS: Lowly metastatic MCF7 and highly metastatic MDA-MB-231 breast cancer cells were treated with 120 nM of proton pump inhibitor Bafilomycin A1 for 24 hours. Cell migration was studied with wound- scratch assays, ATP levels with a chemiluminescent assay; cell proliferation was quantified by a cell area expansion assay. Nuclear size and shape were determined using DAPI nuclear stain and fluorescence microscopy. The levels of F-actin, lamin A/C, heterochromatin, and ETV7 were quantified using both immunocytochemistry and western blots; p-mTORC1, p-mTORC2, mTOR, p-AKT, and AKT were measured by western blots.
RESULTS: We reveal that proton pump inhibition reduces F-actin polymerization, cell migration, proliferation, and increases heterochromatin in both lowly and highly metastatic cells. Surprisingly, Bafilomycin decreases lamin A/C in both cell lines. Inhibition has different effects on ETV7 expression in lowly and highly metastatic cells, as well as nuclear area, perimeter, and circularity. Bafilomycin also significantly decreases p-mTORC1, p-MTORC2, and MTOR expression in both cell lines, whereas it significantly decreases p-AKT in lowly metastatic cells and surprisingly significantly increases p-AKT in highly metastatic cells. Our proton pump inhibition protocol reduces V-ATPase levels (~25%) within three hours. V-ATPase levels vary in time for both control and inhibited cells, and inhibition reduces cellular ATP.
CONCLUSIONS: Proton pumps promote F-actin polymerization and decrease heterochromatin, facilitating invasion. These pumps also upregulate both mTORC1 and mTORC2, thus highlighting the relevance of vacuolar proton pumps as metastatic cancer targets.