Effects of itraconazole plus doxorubicin on proliferation and apoptosis in acute myeloid leukemia cells

Abstract

To explore the effects of itraconazole (ITC) plus adriamycin (ADM) on proliferation and apoptosis of acute myeloid leukemia cells in vitro. The growth inhibition effects of different concentrations of ITC, ADM or ITC (2, 6, 15 µmol/L) plus ADM (0.30, 0.75 µmol/L) were detected by CCK8 assay in KG1α and primary adult acute leukemia cells. Different concentrations of ITC for 7, 14 and 21 days were applied for observing the effect on colony formation. After 48 h treatments with 6 µmol/L ITC, 0.75 µmol/L ADM or ITC (6 µmol/L) plus ADM (0.75 µmol/L), the morphological changes of cells were observed by Wright staining. Flow cytometry was used to detect cell apoptotic rate, mitochondrial membrane potential and cell cycle arrest. And the expression levels of Sonic Hedgehog (Shh) signal pathway-related proteins Shh and glima-associated oncogene homdog1 (Gli1) were determined by Western blot. ITC and ADM inhibited the proliferation of KG1α and primary adult acute leukemia cells and ITC reduced the colony-formation ability of KG1α cells both in dose-dependent manners. Compared with control or single drug group, the changes of cell morphology were more apparent in combined group. Weeb coefficient test revealed a synergistic effect (D ≤ 70%C) of 0.75 µmol/L ADM plus 6 µmol/L ITC. When KG1α cells were treated with 6 µmol/L ITC plus 0.75 µmol/L ADM, the apoptotic rate was 31.72% ± 1.58%. And it was significantly higher than that in control, ITC and ADM groups (4.17% ± 0.74%, 4.33% ± 1.12%, 9.53% ± 1.15%, P < 0.01). Detection of mitochondrial membrane potential showed that low red-fluorescent cells (P3) of combined group were obviously higher than that in control, ADM and ITC single drug groups (18.80% ± 0.96% vs 5.00% ± 0.38%, 9.70% ± 0.43%, 7.10% ± 0.77%, P < 0.01) . KG1α cells were obviously arrested in G2 phase in combined group and it showed no statistical significance compared with control or single drug group. Western blot showed that the expression levels of Shh and Gli1 decreased with rising concentration of ITC. ITC plus ADM can obviously inhibit cell proliferation and increase KG1α cell apoptotic rate, mitochondrial damage and G2 phase retardation. And ITC inhibits the Shh signal pathways.