Proliferation Inhibition and Its Mechanism of Total Coumarins on Leukemia Cells
CHEN Zhi1, WANG Bo2, QIU Mei3,LIN Sheng-yun2*,XIONG Hao1
1. Department of Hematology, Wuhan Children��s Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430016, China; 2. Department of Hematology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, China; 3. Shanghan Teaching and Research Section, Clinical College of Chinese Medicine, Hubei University of Traditional Chinese Medicine, Wuhan 430061, China
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.
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CHEN Zhi, WANG Bo, QIU Mei,LIN Sheng-yun,XIONG Hao. Proliferation Inhibition and Its Mechanism of Total Coumarins on Leukemia Cells. Chinese Pharmaceutical Journal, 2017, 52(17): 1503-1509.
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2017, Vol. 52 
