Combined treatment with rapamycin and bortezomib inhibited cell proliferation and enhanced cell apoptosis. (A) Proliferation of HCCLM3 cells was evaluated by using Cell Counting Kit-8 (CCK-8) at indicated time points. (B) HCCLM3 cells were treated with rapamycin (10 ng/ml), bortezomib (100 nM) or both agents. ModFit software analysis of flow cytometry histograms revealed that rapamycin treatment resulted in cell cycle arrest at G1-S phase. Bortezomib significantly increased in the percentage of cells in the G2/M phase. When the two agents were combined, no significant difference in cell cycle distribution was observed compared with bortezomib alone. (C) HCCLM3 cells were treated with rapamycin (10 ng/ml), bortezomib (100 nM) or the combination for 24 or 48 h and stained with Hoechst 33342, the apoptotic nuclear changes (arrows) were examined by fluorescence microscopy (magnification, ×400). (D, E) The quantification of apoptotic cells induced by rapamycin and bortezomib was further confirmed by flow cytometry analysis. The sub-G1 contents were designed as apoptotic cells. *
P < 0.01, versus control group; **
P < 0.001, versus control group; #
P < 0.05, versus bortezomib treatment group at 24 h; †
P < 0.01, versus bortezomib treatment group at 48 h. (F) The cells were treated with various concentrations of the drugs for 24 h, and then evaluated by using Cell Counting Kit-8. †
P < 0.01, versus bortezomib treatment group.