通过改变 V-ATPase 对 F-肌动蛋白、异染色质、ETV7 和 mTORC2 信号的作用来抑制乳腺癌。
Curbing Breast Cancer by Altering V-ATPase Action on F-Actin, Heterochromatin, ETV7 and mTORC2 Signaling.
机构信息
Department of Mechanical Engineering, Texas Tech University, 2703 7th Street, Lubbock, 79409, Texas, USA.
Department of Mechanical Engineering, Texas Tech University, 2703 7th Street, Lubbock, 79409, Texas, USA,
出版信息
Cell Physiol Biochem. 2024 Jun 12;58(3):250-272. doi: 10.33594/000000706.
BACKGROUND/AIMS: 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.
CONCLUSION
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.
背景/目的：受液泡质子泵在癌症中的重要性的启发，我们研究了质子泵抑制对乳腺癌细胞迁移和增殖、F-肌动蛋白聚合、核纤层 A/C、异染色质、ETV7 表达、核大小和形状以及 AKT/mTOR 信号的影响。
方法
用 120 nM 的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。用划痕实验研究细胞迁移，用化学发光法测定 ATP 水平；用细胞面积扩张测定法定量细胞增殖。用 DAPI 核染色和荧光显微镜测定核大小和形状。用免疫细胞化学和 Western blot 定量 F-肌动蛋白、核纤层 A/C、异染色质和 ETV7 的水平；用 Western blot 测定 p-mTORC1、p-mTORC2、mTOR、p-AKT 和 AKT。
结果
我们发现质子泵抑制减少了低转移性和高转移性细胞中的 F-肌动蛋白聚合、细胞迁移和增殖，并增加了异染色质。令人惊讶的是，巴弗洛霉素在两种细胞系中都降低了核纤层 A/C。抑制对低转移性和高转移性细胞中的 ETV7 表达以及核面积、周长和圆形度有不同的影响。巴弗洛霉素还显著降低了两种细胞系中 p-mTORC1、p-MTORC2 和 MTOR 的表达，而在低转移性细胞中显著降低了 p-AKT 的表达，在高转移性细胞中则显著增加了 p-AKT 的表达。我们的质子泵抑制方案在三小时内将 V-ATPase 水平降低了约 25%。对照和抑制细胞的 V-ATPase 水平随时间而变化，抑制降低了细胞内的 ATP。
结论
质子泵促进 F-肌动蛋白聚合并减少异染色质，从而促进侵袭。这些泵还上调了 mTORC1 和 mTORC2，从而突出了液泡质子泵作为转移性癌症靶点的相关性。
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