Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
Clinic for Plastic, Aesthetic and Hand Surgery, Otto von Guericke University Magdeburg, 39120 Magdeburg, Germany.
Int J Mol Sci. 2020 Feb 13;21(4):1263. doi: 10.3390/ijms21041263.
Prostate cancer is one of the leading causes of cancer mortality in men worldwide. An unusual but unique environment for studying tumor cell processes is provided by microgravity, either in space or simulated by ground-based devices like a random positioning machine (RPM). In this study, prostate adenocarcinoma-derived PC-3 cells were cultivated on an RPM for time periods of 3 and 5 days. We investigated the genes associated with the cytoskeleton, focal adhesions, extracellular matrix, growth, survival, angiogenesis, and metastasis. The gene expression of signaling factors of the vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mTOR (PAM) pathways was investigated using qPCR. We performed immunofluorescence to study the cytoskeleton, histological staining to examine the morphology, and a time-resolved immunofluorometric assay to analyze the cell culture supernatants. When PC-3 cells were exposed to simulated microgravity (s-µ), some cells remained growing as adherent cells (AD), while most cells detached from the cell culture flask bottom and formed multicellular spheroids (MCS). After 3-day RPM exposure, PC-3 cells revealed significant downregulation of the , , , , and gene expression in MCS, whereas , , , , , , , , and mRNAs were not significantly changed. and were elevated in AD, and , , , , , , and mRNAs were significantly upregulated in AD and MCS after 3 days. After a 5-day culture in s-µ, the PC-3 cells showed significant downregulations of mRNA in AD and MCS, and , , and in AD and in MCS. In addition, we measured significant upregulations in , , , , , , , and mRNAs in AD and MCS, and increases in , , and in MCS as well as , , , , , and mRNAs in AD. and were not altered by s-µ. In parallel, the secretion rate of VEGFA and NGAL proteins decreased. Cytoskeletal alterations (F-actin) were visible, as well as a deposition of collagen in the MCS. In conclusion, RPM-exposure of PC-3 cells induced changes in their morphology, cytoskeleton, and extracellular matrix protein synthesis, as well as in their focal adhesion complex and growth behavior. The significant upregulation of genes belonging to the PAM pathway indicated their involvement in the cellular changes occurring in microgravity.
前列腺癌是全球男性癌症死亡的主要原因之一。微重力为研究肿瘤细胞过程提供了一个独特的环境，无论是在太空中还是通过地面设备（如随机定位机 RPM）模拟。在这项研究中，前列腺腺癌衍生的 PC-3 细胞在 RPM 上培养了 3 天和 5 天。我们研究了与细胞骨架、焦点粘连、细胞外基质、生长、存活、血管生成和转移相关的基因。使用 qPCR 研究了血管内皮生长因子 (VEGF)、丝裂原激活蛋白激酶 (MAPK) 和 PI3K/AKT/mTOR (PAM) 通路的信号因子的基因表达。我们进行免疫荧光染色以研究细胞骨架，进行组织学染色以检查形态，并进行时间分辨免疫荧光测定以分析细胞培养上清液。当 PC-3 细胞暴露于模拟微重力 (s-µ) 时，一些细胞仍然作为贴壁细胞 (AD) 生长，而大多数细胞从细胞培养瓶底部脱落并形成多细胞球体 (MCS)。经过 3 天的 RPM 暴露，PC-3 细胞在 MCS 中显著下调了 、 、 、 和 基因的表达，而 、 、 、 、 、 、 和 mRNA 没有明显变化。在 AD 中上调了 和 ，在 AD 和 MCS 中 3 天后 、 、 、 、 、 和 mRNA 显著上调。经过 5 天的 s-µ 培养，PC-3 细胞在 AD 和 MCS 中显著下调了 AD 和 MCS 中的 mRNA，以及 AD 中的 、 、 和 以及 MCS 中的 mRNA。此外，我们在 AD 和 MCS 中测量到 、 、 、 、 、 、 和 mRNA 的显著上调，以及 MCS 中 、 、 和 的上调，以及 AD 中 、 、 、 、 和 mRNA 的上调。s-µ 没有改变 和 。同时，VEGFA 和 NGAL 蛋白的分泌率下降。可见细胞骨架改变（F-肌动蛋白）和 MCS 中胶原蛋白的沉积。总之，RPM 暴露诱导了 PC-3 细胞形态、细胞骨架和细胞外基质蛋白合成以及焦点粘连复合物和生长行为的变化。PAM 通路基因的显著上调表明它们参与了微重力下发生的细胞变化。
