Nimbolide-Induced Apoptosis in Acute Promyelocytic Leukemia Cells and Its Underlying Mechanisms
TAN Jing, YANG Mengnan, LI Qing, DAI Kesheng*, YAN Rong*
This study aims to investigate the effects of nimbolide on acute promyelocytic leukemia cells and to elucidate the underlying mechanisms. Nimbolide-associated targets were identified through the CTD, TargetNet, SwissTarget-Predict, and PharmMapper databases, and acute promyelocytic leukemia-related targets were obtained from the GeneCards, MalaCards, and Open Targets databases. The overlapping targets of the two sets were identified and subjected to KEGG pathway enrichment analysis. A protein-protein interaction network was then constructed based on the overlapping targets, followed by topological analysis to identify core targets. A drug-disease-target-pathway regulatory network was constructed. NB4 cells were treated with different concentrations of nimbolide. Cell viability was determined using the CCK-8 assay. Flow cytometry was used to analyze cell cycle distribution, apoptosis rate, and mitochondrial membrane potential. The expression levels of apoptosis-related proteins and PI3K-Akt signaling pathway-associated proteins were detected by Western blot. In addition, apoptosis of peripheral blood mononuclear cells from healthy donors was assessed following nimbolide treatment. In rescue experiments, NB4 cells were pretreated with the Akt activator SC79 and then co-incubated with nimbolide, followed by analysis of apoptosis rate, mitochondrial membrane potential, and apoptosis-related protein expression. A total of 225 overlapping targets between acute promyelocytic leukemia and nimbolide were identified. KEGG enrichment analysis showed that the overlapping targets were significantly enriched in apoptosis and the PI3K-Akt signaling pathway. Protein-protein interaction network analysis identified 32 core targets, including Caspase-3, Bcl-2, and PARP1. Nimbolide inhibited the proliferation of NB4 cells and induced cell cycle arrest at the G2/M phase. With increasing concentrations of nimbolide, apoptosis rate in NB4 cells increased in a dose-dependent manner, accompanied by a decrease in mitochondrial membrane potential. Nimbolide treatment increased the expression levels of Cleaved Caspase-3 and Cleaved PARP1, along with decreasing the expression levels of Caspase-3, PARP1 and Bcl-2 in NB4 cells, whereas Bax showed no significant change. However, under the same concentration, nimbolide treatment did not increase the apoptosis rate of peripheral blood mononuclear cells from healthy donors. Nimbolide treatment decreased the phosphorylation level of Akt (Ser473) in NB4 cells. The Akt activator SC79 significantly attenuated nimbolide-induced increase in apoptosis rate, decrease in mitochondrial membrane potential, and changes in apoptosis-related protein expression in NB4 cells. In conclusion, nimbolide induces apoptosis in acute promyelocytic leukemia cell line NB4 via the mitochondrial pathway, and this effect is associated with inhibition of the PI3K-Akts ignaling pathway.
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