Proliferation Inhibition and Its Mechanism of Total Coumarins on Leukemia Cells / 中国药学杂志

ABSTRACT

OBJECTIVE: To explore the effect of total coumarins isolated from Hedyotis diffusa (total coumarins from Hedyotis diffusa, TCHD) on proliferation inhibition of leukemia cells, and to explore its related mechanism. METHODS: The purity of TCHD prepared by ethanol reflux extraction was tested by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system. The cells (KG-1 Kasumi-1, THP 1 cells, U937 cells and K562 cells) were treated with TCHD(0.02, 0.04, 0.06, 0.08, 0.10 mg·mL-1) for 24 or 48 h, the inhibitive effect of TCHD on cells growth were determined by MTT method. After Kasumi-1 cells were incubated with TCHD for 24 h, the apoptosis of cells were analyzed by flow cytometry stained with Annexin V/PI. The expression levels of caspase-3, caspase-8, caspase-9, PARP and Bcl-2 family protein were assayed by Western blot. RESULTS: TCHD in certain concentration range could markedly inhibit the proliferation of AML cells, their IC50 on Kasumi-1, THP-1 KG-1, U937 and K562 cells were 0.077, 0.083, 0.096, 0.087, 0.096 mg·mL-1 for 24 h, and 0.059, 0.067, 0.072, 0.064, 0.068 mg·mL-1 for 48 h. TCHD has significant inhibitory effect on Kasumi-1, which was stronger than those on other cell lines, and showed a dose- and time-dependent manner(r=0.357, P<0.05). The apoptotic proportion of Kasumi-1cells in 0, 0.02, 0.04, 0.06, 0.08, 0.10 mg·mL-1 TCHD treatment groups for 24 h were (5.33±0.41)%, (7.99±0.45)%, (10.22±0.32)%, (20.10±1.99)%, (28.66±0.67)% and (33.24±2.12)%, respectively. After treated with TCHD(0.02-0.06 mg·mL-1) for 24 h, G0/G1 phase ratio of Kasumi-1 detected by flow cytometry were (51.43±3.21)%, (62.91±2.35)% and (76.42±4.14)%, respectively, which were significantly higher than that of the control group (35.8±5.25)% (P<0.05).Western blot results showed that different concentrations of TCHD could activate caspase-8, caspase-9, caspase-3 and PARP, promote the expression of cyto-C, down-regulate the cyclin E and CDK6, CDK2, p-CDK2 and cyclin D1 protein, and up-regulate the expression of p21 proteinin concentration- dependent manner(P<0.01). CONCLUSION: TCHD can obviously inhibit the proliferation of Kasumi-1 in a dose- and time-dependent manner, which may relate to the apoptosis of Kasumi 1 induced by activating caspase-3, 9, PARP protein through the mitochondrial pathways and Kasumi-1 cell block in G0/G1 phase through the influence of CDK2, p-CDK2, CDK4/6, cyclin E, cyclin D1 and p21.