Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, United States of America.
Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.
PLoS One. 2020 Aug 7;15(8):e0237222. doi: 10.1371/journal.pone.0237222. eCollection 2020.
Epithelial-mesenchymal transition (EMT) is a critical early step in cancer metastasis and a complex process that involves multiple factors. In this study, we used proteomics approaches to investigate the secreted proteins (secretome) of paired human androgen-repressed prostate cancer (ARCaP) cell lines, representing the epithelial (ARCaP-E) and mesenchymal (ARCaP-M) phenotypes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses showed high levels of proteins involved in bone remodeling and extracellular matrix degradation in the ARCaP-M cells, consistent with the bone metastasis phenotype. Furthermore, LC-MS/MS showed a significantly higher level of the serine protease granzyme B (GZMB) in ARCaP-M conditioned media (CM) compared to that of ARCaP-E. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to detect mRNA and Western blot to detect protein expression, we further demonstrated that the GZMB gene was expressed by ARCaP-M and the protein was secreted extracellularly. ARCaP-M cells with GZMB gene knockdown using small interfering RNA (siRNA) have markedly reduced invasiveness as demonstrated by the Matrigel invasion assay in comparison with the scrambled siRNA negative control. This study reports that GZMB secretion by mesenchymal-like androgen-repressed human prostate cancer cells promotes invasion, suggesting a possible extracellular role for GZMB in addition to its classic role in immune cell-mediated cytotoxicity.
上皮-间充质转化（EMT）是癌症转移的关键早期步骤，是一个涉及多种因素的复杂过程。在这项研究中，我们使用蛋白质组学方法研究了配对的人雄激素抑制前列腺癌细胞系（ARCaP）的分泌蛋白（分泌组），代表上皮（ARCaP-E）和间充质（ARCaP-M）表型。液相色谱-串联质谱（LC-MS/MS）分析显示，ARCaP-M 细胞中涉及骨重塑和细胞外基质降解的蛋白质水平较高，与骨转移表型一致。此外，LC-MS/MS 显示 ARCaP-M 条件培养基（CM）中丝氨酸蛋白酶 granzyme B（GZMB）的水平明显高于 ARCaP-E。通过定量逆转录聚合酶链反应（qRT-PCR）检测 mRNA 和 Western blot 检测蛋白表达，我们进一步证明 GZMB 基因在 ARCaP-M 中表达，并且蛋白被分泌到细胞外。使用小干扰 RNA（siRNA）对 ARCaP-M 中的 GZMB 基因进行敲低后，通过 Matrigel 侵袭实验，与 scramble siRNA 阴性对照相比，细胞侵袭性显著降低。这项研究报告称，间充质样雄激素抑制的人前列腺癌细胞中 GZMB 的分泌促进了侵袭，这表明 GZMB 除了在免疫细胞介导的细胞毒性中的经典作用外，还可能具有细胞外作用。
