ABSTRACT
Aim To investigate the molecular mechanisms of the dual inhibitor of mammalian rapamycin target protein (mTOR) AZD8055 in migration and EMT process inhibition of the human cholangiocarcinoma cell line HuCCTl. Methods The viability of HuCCTl cells treated with different concentrations of AZD8055 was measured by MTT assay, and the colony formation ability of HuCCTl was detected by colony formation assay. The effect of AZD8055 on the motility of HuCCTl cells was examined by wound healing assay and Tran-swell assay. The expression levels of the protein associated with Akt/mTOR pathway, epithelial-mesenchymal transition (EMT) process and DEK were detected by Western blot. The interaction relationship between AZD8055, DEK and Akt signaling pathway was analyzed by STITCH and GeneMania databases. Cholangiocarcinoma cells'proliferation, migration capacities and Akt/mTOR signaling pathway-related protein expression levels were detected after DEK gene silencing. Results Compared with control group, AZD8055 inhibited the proliferation and migration capacities of cholangiocarcinoma cells, and suppressed the expression levels of Akt/mTOR signaling pathway-related markers, down-regulated DEK expression and inhibited EMT process. DEK silence significantly inhibited cell proliferation, migration and significantly decreased the phosphorylation levels of Akt, S6, and 4EBP1. Conclusions AZD8055 treatment inhibits the migration and EMT progression of HuCCTl cells, and its mechanism is associated with DEK down-regulation and inhibition of Akt/mTOR signaling pathway.
ABSTRACT
AIM:To explore the effects of mammalian target of rapamycin (mTOR) double inhibitor AZD8055 on autophagy and apoptosis of human cholangiocarcinoma cell line HuCCT1. METHODS:The effect of AZD8055 on the viability of HuCCT1 cells was detected by MTT assay. Autophagosome was detected by acridine orange (AO) staining. Af-ter treated with AZD8055, the expression levels of apoptosis-related proteins Bcl-2, Bax and cleaved caspase-3 and auto-phagy marker proteins beclin 1, LC3 and p62 were determined by Western blot. Apoptotic rate was analyzed by flow cyto-metry with Annexin V-FITC/PI double staining. RESULTS:AZD8055 significantly inhibited the viability of HuCCT1 cells (P<0.05). AO staining showed that AZD8055 significantly increased orange granules in the cytoplasm. After treated with AZD8055, compared with the control group, the protein level of beclin 1 and the ratio of LC3-Ⅱ/LC3-Ⅰ were enhanced, while p62 was attenuated (P<0.05). The protein expression level of pro-apoptotic regulator Bax was down-regulated and anti-apoptotic regulator Bcl-2 was increased. The protein level of cleaved caspase-3 was reduced (P<0.05). The results of flow cytometry showed that AZD8055 inhibited cell apoptosis. CONCLUSION:AZD8055 inhibits the viability of cholangiocarcinoma cells, and the mechanism is closely related with autophagy induced by AZD8055.