Luteolin induces myelodysplastic syndrome-derived cell apoptosis via the p53-dependent mitochondrial signaling pathway mediated by reactive oxygen species

摘要

Luteolin, a common dietary flavonoid, induces the apoptosis of cells in several types of cancer. However, its role in myelodysplastic syndrome (MDS) and the potential underlying mechanisms remain to be elucidated. To evaluate the potential benefit and underlying mechanisms of luteolin in MDS cells, the viability of SKM-1 cells and primary bone marrow (PBM) mononuclear cells from patients with intermediate- or high-risk MDS were assessed using a Cell Counting Kit-8 assay. The apoptotic features of cell morphology were assessed using Wright-Giemsa staining, DNA fragmentation was analyzed by agarose gel electrophoresis, and the extent of apoptosis was quantified by flow cytometry (FCM). Reactive oxygen species (ROS) were measured by FCM with 2,7-dichlorodihydrofluorescein diacetate staining and mitochondrial membrane potential (Delta Psi m) was determined using 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocy-anine iodide staining. Caspase activity was detected using a fluorometric protease assay. Furthermore, the effects of luteolin on the expression of apoptosis-related proteins were analyzed using western blot analysis. The resulting data revealed that luteolin significantly inhibited the proliferation of SKM-1 cells in vitro, and its half maximal inhibitory concentration was 139.41 mu M at 24 h and 23.95 mu M at 72 h. Luteolin also markedly inhibited the proliferation of mononuclear cells from patients with intermediate- or high-risk MDS. Luteolin suppressed cell proliferation, mainly as a result of the induction of apoptosis, as demonstrated by typical apoptotic morphological features, the ladder pattern of genomic DNA fragmentation, and the results of FCM using Annexin V-FITC/PI double staining. It was also found that short-term exposure of SKM-1 cells to luteolin led to a marked increase in the accumulation of ROS. The increased intracellular level of ROS appeared to induce the activation of p53 and elevate the B-cell lymphoma 2 (Bcl-2)-associated X protein/Bcl-2 ratio, which modulates Delta Psi m and triggers the release of cytochrome c, and may increase the activities of apoptotic protease activating factor 1, caspase-3, -8 and -9 to further trigger the destruction of structural and specific proteins and thereby cell apoptosis. Notably, the inhibition of ROS generation by the antioxidant N-acetyl-L-cysteine significantly attenuated the luteolin-induced loss of Delta Psi m and activities of caspase-3, -8 and -9. These data suggested that luteolin exerts its pro-apoptotic action partly through the p53-dependent mitochondrial signaling pathway mediated by intracellular ROS, which provides a promising therapeutic candidate for patients with MDS.