Department of Life Science and Biochemical Engineering, Sun Moon University, Asan 31460, Korea.
Department of Pharmaceutical Engineering and Biotechnology, Sun Moon University, Asan 31460, Korea.
Int J Mol Sci. 2021 Mar 1;22(5):2473. doi: 10.3390/ijms22052473.
We recently discovered a novel nargenicin A1 analog, 23-demethyl 8,13-deoxynargenicin (compound 9), with potential anti-cancer and anti-angiogenic activities against human gastric adenocarcinoma (AGS) cells. To identify the key molecular targets of compound 9, that are responsible for its biological activities, the changes in proteome expression in AGS cells following compound 9 treatment were analyzed using two-dimensional gel electrophoresis (2-DE), followed by MALDI/TOF/MS. Analyses using chemical proteomics and western blotting revealed that compound 9 treatment significantly suppressed the expression of cyclophilin A (CypA), a member of the immunophilin family. Furthermore, compound 9 downregulated CD147-mediated mitogen-activated protein kinase (MAPK) signaling pathway, including c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) by inhibiting the expression of CD147, the cellular receptor of CypA. Notably, the responses of AGS cells to CypA knockdown were significantly correlated with the anticancer and antiangiogenic effects of compound 9. CypA siRNAs reduced the expression of CD147 and phosphorylation of JNK and ERK1/2. In addition, the suppressive effects of CypA siRNAs on proliferation, migration, invasion, and angiogenesis induction of AGS cells were associated with G2/M cell cycle arrest, caspase-mediated apoptosis, inhibition of MMP-9 and MMP-2 expression, inactivation of PI3K/AKT/mTOR pathway, and inhibition of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression. The specific interaction between compound 9 and CypA was also confirmed using the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA) approaches. Moreover, in silico docking analysis revealed that the structure of compound 9 was a good fit for the cyclosporin A binding cavity of CypA. Collectively, these findings provide a novel molecular basis for compound 9-mediated suppression of gastric cancer progression through the targeting of CypA.
我们最近发现了一种新型的那格列汀 A1 类似物，23-去甲基 8,13-去氧那格列汀（化合物 9），它对人胃腺癌（AGS）细胞具有潜在的抗癌和抗血管生成活性。为了确定化合物 9 负责其生物学活性的关键分子靶标，我们使用二维凝胶电泳（2-DE）分析了 AGS 细胞在化合物 9 处理后的蛋白质组表达变化，然后使用 MALDI/TOF/MS 进行分析。化学蛋白质组学和 Western blot 分析表明，化合物 9 处理显著抑制了亲环素 A（CypA）的表达，CypA 是免疫亲和素家族的一员。此外，化合物 9 通过抑制 CypA 的细胞受体 CD147 的表达，下调了 CD147 介导的丝裂原活化蛋白激酶（MAPK）信号通路，包括 c-Jun N-末端激酶（JNK）和细胞外信号调节蛋白激酶 1/2（ERK1/2）。值得注意的是，AGS 细胞对 CypA 敲低的反应与化合物 9 的抗癌和抗血管生成作用显著相关。CypA siRNA 降低了 CD147 的表达和 JNK 和 ERK1/2 的磷酸化。此外，CypA siRNA 对 AGS 细胞增殖、迁移、侵袭和血管生成诱导的抑制作用与 G2/M 细胞周期阻滞、半胱天冬酶介导的细胞凋亡、MMP-9 和 MMP-2 表达抑制、PI3K/AKT/mTOR 通路失活以及缺氧诱导因子-1α（HIF-1α）和血管内皮生长因子（VEGF）表达抑制有关。还使用药物亲和反应靶标稳定性（DARTS）和细胞热转移分析（CETSA）方法证实了化合物 9 与 CypA 之间的特异性相互作用。此外，计算机对接分析表明，化合物 9 的结构非常适合 CypA 的环孢菌素 A 结合腔。总之，这些发现为化合物 9 通过靶向 CypA 抑制胃癌进展提供了新的分子基础。
