Department of Clinical Biochemistry, School of Pharmacy & Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Department of Biochemistry, Arak University of Medical Sciences, Arak, Iran.
J Cell Physiol. 2019 Nov;234(11):19539-19552. doi: 10.1002/jcp.28552. Epub 2019 Apr 8.
Didscoidin domain receptor 1 (DDR1) is involved in the progression of prostate cancer metastasis through stimulation of epithelial-mesenchymal transition (EMT). So DDR1 inhibition can be a helpful target for cancer metastasis prevention. So, we studied the effects of DDR1 inhibition on EMT as well as induction of cell-cycle arrest and apoptosis in prostate cancer cell lines. DDR1 expression was evaluated using reverse-transcription polymerase chain reaction and western blot analysis. The EMT-associated protein expression was determined using the western blot analysis and immunocytochemistry following treatment with various concentrations of DDR1 inhibitor. The activation of DDR1 and also downstream-signaling molecules Pyk2 and MKK7 were determined using western blot analysis. Cell survival and proliferation after DDR1 inhibition were evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide, bromodeoxyuridine, and colony formation assays. Flow cytometry analysis was used to determine the effects of DDR1 inhibition on cell-cycle arrest and apoptosis using annexin V/propidium iodide-based flow cytometry. Results showed that the protein expression of N-cadherin and vimentin were decreased whereas protein expression of E-cadherin was increased after DDR1 inhibition. Results of our western blot analysis indicated that DDR1 inhibitor effectively downregulated P-DDR1, P-Pyk2, and P-MKK7 levels. This result also showed that DDR1 inhibition decreased cell survival and proliferation, induced G1 cell-cycle arrest, induced apoptosis by an increase in the Bax/Bcl-2 ratio and depletion of the mitochondrial membrane potential, and also by reactive oxygen species creation in prostate cancer cells. These data show that DDR1 inhibition can result in the EMT prevention via inhibition of Pyk2 and MKK7 signaling pathway and induces cell-cycle arrest and apoptosis in prostate cancer cell lines. Thus, this study identifies DDR1 as an important target for modulating EMT and induction of apoptosis in prostate cancer cells.
盘状结构域受体 1（DDR1）通过刺激上皮-间充质转化（EMT）参与前列腺癌转移的进展。因此，DDR1 抑制可能是预防癌症转移的一个有帮助的靶点。因此，我们研究了 DDR1 抑制对前列腺癌细胞系 EMT 以及细胞周期阻滞和细胞凋亡诱导的影响。使用逆转录聚合酶链反应和 Western blot 分析评估 DDR1 表达。用 Western blot 分析和免疫细胞化学法在用不同浓度的 DDR1 抑制剂处理后测定 EMT 相关蛋白表达。用 Western blot 分析测定 DDR1 和下游信号分子 Pyk2 和 MKK7 的激活。用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐、溴脱氧尿苷和集落形成测定法评估 DDR1 抑制后细胞存活和增殖。用 Annexin V/碘化丙啶基于流式细胞术测定法用流式细胞术分析评估 DDR1 抑制对细胞周期阻滞和凋亡的影响。结果表明，DDR1 抑制后 N-钙粘蛋白和波形蛋白的蛋白表达减少，而 E-钙粘蛋白的蛋白表达增加。我们的 Western blot 分析结果表明，DDR1 抑制剂可有效下调 P-DDR1、P-Pyk2 和 P-MKK7 水平。该结果还表明，DDR1 抑制降低了细胞存活和增殖，通过 G1 细胞周期阻滞诱导细胞凋亡，通过增加 Bax/Bcl-2 比值和耗竭线粒体膜电位，以及通过在前列腺癌细胞中产生活性氧来诱导细胞凋亡。这些数据表明，DDR1 抑制通过抑制 Pyk2 和 MKK7 信号通路可导致 EMT 预防，并诱导前列腺癌细胞系中的细胞周期阻滞和凋亡。因此，本研究确定 DDR1 是调节 EMT 和诱导前列腺癌细胞凋亡的重要靶点。
