Silencing RICTOR gene enhances sensitivity of esophageal squamous cell carcinoma to everolimus / 肿瘤

ABSTRACT

Objective: To investigate the effect of silencing rapamycin insensitive companion of mammalian target of rapamycin (RICTOR) gene expression on the sensitivity of esophageal squamous cell carcinoma cells to everolimus, and to explore its possible molecular mechanism. Methods: The expression level of RICTOR protein in esophageal squamous cell carcinoma TE1, ECa109, EC9706, KYSE450 and KYSE790 cells were detected by Western blotting. RICTOR-shRNA or the Control-shRNA was transfected into ECa109 cells by LipofectAMINE, and the ECa109 cells stably expressing RICTOR-shRNA or the Control-shRNA were screened and named as ECa 109-RICTOR-shRNA or ECa109-control-shRNA cells. The effect of everolimus on the proliferation of ECa109-RICTOR-shRNA cells was detected by CCK-8 assay. The expression levels of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, Akt)/mammalian target of rapamycin (mTOR) signal pathwayrelated RICTOR, Akt, phospho-Akt (p-Akt) (Ser473), ribosome protein subunit 6 kinase of 70 kDa (p70S6K), phospho-p70S6K (p-p70S6K), proline-rich Akt substrate of 40 kDa (PRAS40) and phospho-PRAS40 (p-PRAS40) (Thr246) proteins in everolimus-treated ECa109-control-shRNA and ECa109-RICTOR-shRNA cells were detected by Western blotting. The nude mouse xenograft tumor models of ECa1 09-RICTOR-shRNA and ECa109-control-shRNA cells were established and treated with everolimus, then the effect of everolimus on tumor growth in nude mice was evaluated. Results: RICTOR protein was expressed in five esophageal squamous cell carcinoma cell lines, especially in ECa1 09 cells. Compared with the Control-shRNA, RICTOR-shRNA inhibited the proliferation of ECa1 09 cells. Everolimus inhibited the proliferation of ECa1 09-RICTOR-shRNA and ECa109-control-shRNA cells, especially to the former; the values of half maximal inhibitory concentration (IC50) were (17.68± 1.25) μmol/L and (36.84±1.57) μmol/L, respectively. The RICTOR-shRNA decreased the expression levels of p-Akt (Ser473) and p-PRAS40 (Thr246) (both P 0.05), which indicated that RICTOR-shRNA inhibited the phosphorylated activation of Akt and PRAS40 induced by everolimus. Both RICTOR-shRNA and everolimus inhibited the growth of ECa109 cell xenografts in nude mice (all P < 0.05), while the inhibitory effect was strongest in RICTOR-shRNA+everolimus group (P < 0.001). Conclusion: Silencing RICTOR gene can improve the sensitivity of esophageal squamous cell carcinoma cells to everolimus, and the molecular mechanism may be associated with the down-regulation of RICTOR expression to inhibit the phosphorylated activation of Akt and PRAS40 induced by everolimus.