Objectives: This study aimed to study the effect of the protein kinase D (PKD) inhibitor CRT0066101 on the cell migration of salivary adenoid cystic carcinoma (SACC) cells in vitro and explore its related mechanisms to provide new strategies into the clinical treatment of SACC cells.
Methods: SACC-LM cells were treated with different concentrations of CRT0066101, and the effect of active phospho-PKD was detected through Western blot and cell immunofluorescence staining. Transwell assay was performed to test cell migration. The effect of CRT0066101 on the protein expression related to the epithelial mesenchymal transition (EMT) was detected through Western blot, cell immunofluorescence staining, and quantitative real-time polymerase chain reaction (qRT-PCR). The cells were treated with the proteasome inhibitor after CRT0066101 administration, and the expression of Snail protein was detected by Western blot.
Results: CRT0066101 inhibited PKD activity and reduced the number of invaded cells in SACC-LM cells. CRT0066101 decreased the expression of N-cadherin and Snail and increased the expression of E-cadherin in SACC-LM cells. The regulation of snail protein degradation by CRT0066101 was dependent on the proteasome pathway.
Conclusions: CRT0066101 can inhibit the migration of SACC-LM cells in SACC and regulate the expression of proteins and genes related to EMT. The mechanism may be associated with the proteasome-dependent degradation of Snail.
目的: 观察蛋白激酶D（PKD）的抑制剂CRT0066101对唾液腺腺样囊性癌（SACC）细胞迁移能力的影响，并探讨其相关机制，为SACC治疗提供新策略。方法: 将不同浓度的CRT0066101作用于SACC-LM细胞，通过蛋白质印迹（Western blot）和细胞免疫荧光染色检测CRT0066101对细胞PKD磷酸化活化的作用；Transwell实验检测CRT0066101对细胞迁移能力的影响；利用Western blot、细胞免疫荧光染色和实时定量聚合酶链反应（qRT-PCR）检测药物对上皮间充质转化（EMT）相关指标蛋白的影响；用蛋白酶体抑制剂处理CRT0066101作用后的细胞和对照细胞，Western blot检测锌指转录因子（Snail）蛋白的表达。结果: CRT0066101能抑制PMA诱导的SACC-LM细胞PKD磷酸化；降低细胞迁移数。CRT0066101能降低N-钙黏着蛋白和Snail蛋白表达量，增加E-钙黏着蛋白和CDH1基因的表达。CRT0066101能调控蛋白酶体介导的Snail蛋白降解。结论: CRT0066101抑制SACC-LM细胞迁移能力，调节EMT相关蛋白和基因表达，机制可能与蛋白酶体介导的Snail蛋白降解有关。.
Keywords: CRT0066101; cell migration; epithelial mesenchymal transition; salivary adenoid cystic carcinoma.