Inhibitory effect of combination of rapamycin and 5-Aza-dC on Nalm-6 cells and the underlying mechanisms

Abstract

Objective To investigate the effects and underlying mechanisms of combination of mTOR inhibitor rapamycin(RAPA)and DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine(5-Aza-dC)on the cell proliferation and apoptosis of human acute leukemia cell lines Nalm-6 for providing a new promising targeted therapeutic strategy for acute leukemia. Methods The recovered Nalm-6 cells were cultured to exponential phase, and divided into groups in which RAPA and 5-Aza-dC with different concentrations or their combinations. The effects on cell proliferation were analyzed by CCK-8 assay. Apoptosis was detected by flow cytometry with Annexin V-PE/7-AAD staining. Western blot analysis was performed to examine the expression of TSC2 protein and phosphorylation of mTOR. Results RAPA and 5-Aza-dC could inhibit the proliferative activity of Nalm-6 cells in a dose dependent manner(P < 0.05). Combination of 5-Aza-dC with RAPA exerted a synergistic anti-poliferative effect on Nalm-6 cells(CI < 1). apoptosis was induced by RAPA and 5-Aza-dC in Nalm-6 cells(P < 0.05). Compared with RAPA alone groups, combined groups could observably increase the apoptosis rate of Nalm-6 cells(P < 0.05). In Nalm-6 cells, 5-Aza-dC could increase the expression of TSC2 protein. Though RAPA and 5-Aza-dC individually inhibited the phosphorylation of mTOR, combination of them were more significantly effective in inhibiting mTOR phosphorylation. Conclusions Combination of RAPA and 5-Aza-dC exerted a synergistic effect on inhibition of cell proliferation and induction of cell apoptosis in Nalm-6 cells. The mechanisms may be associated with the regulation of the proteins expression of mTOR pathway. Key words: Leukemia; Rapamycin; 5-aza-2'-deoxycytidine; Mammalian target of rapamycin