The pro-apoptotic effect of rapamycin plus bortezomib is dependent on p53 activity. (A, B) The mRNA expression levels of p53, p27, p21 and Bcl-2 family in HCCLM3 cells were evaluated by RT-PCR. Bcl-2 mRNA was significantly suppressed by the combined treatment of rapamycin and bortezomib. The expression of p53, p27 p21and Bax mRNA was significantly up-regulated by monotherapy or the combination therapy. (C) HCCLM3 cells were treated with rapamycin (10 ng/ml), bortezomib (100 nM) or the combination with or without PFT-α for 48 h and stained with Hoechst 33342, the apoptotic nuclear changes were examined by fluorescence microscopy (magnification, ×400). (D, E) The quantification of apoptotic cells was further confirmed by flow cytometry analysis. The sub-G1 contents were designed as apoptotic cells. (F) The rapamycin plus bortezomib mediated up-regulation of p53 protein could be significantly suppressed by p53 inhibitor PFT-α. And the level of p21 protein was also down-regulated during this process. *
P < 0.05, versus bortezomib group without PFT-α treatment; **
P < 0.01, versus combined treatment group without PFT-α.