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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2019, Vol. 13 Issue (3) : 388-397    https://doi.org/10.1007/s11684-018-0624-1
RESEARCH ARTICLE
Homoharringtonine synergy with oridonin in treatment of t(8; 21) acute myeloid leukemia
Weina Zhang, Ying Lu, Tao Zhen, Xinjie Chen, Ming Zhang, Ping Liu, Xiangqin Weng, Bing Chen, Yueying Wang()
State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
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Abstract

Collaboration of c-KIT mutations with AML1–ETO (AE) has been demonstrated to induce t(8; 21) acute myeloid leukemia (AML). Targeted therapies designed to eliminate AE and c-KIT oncoproteins may facilitate effective treatment of t(8; 21) AML. Homoharringtonine (HHT) features activity against tumor cells harboring c-KIT mutations, whereas oridonin can induce t(8; 21) AML cell apoptosis and AE cleavage. Therefore, studies should explore the efficacy of combination therapy with oridonin and HHT in t(8; 21) AML. In this study, we investigated the synergistic effects and mechanism of oridonin combined with HHT in t(8; 21) AML cell line and mouse model. The two drugs synergistically inhibited cell viability and induced significant mitochondrial membrane potential loss and apoptosis. Oridonin and HHT induced significant downregulation of c-KIT and its downstream signaling pathways and promoted AE cleavage. HHT increased intracellular oridonin concentration by modulating the expressions of MRP1 and MDR1, thus enhancing the effects of oridonin. The combination of oridonin and HHT prolonged t(8; 21) leukemia mouse survival. In conclusion, oridonin and HHT exert synergistic effects against t(8; 21) leukemia in vivo and in vitro, thereby indicating that their combination may be an effective therapy for t(8; 21) leukemia.

Keywords AML1–ETO      c-KIT      homoharringtonine      oridonin      t(8      21) AML      synergistic effect     
Corresponding Authors: Yueying Wang   
Just Accepted Date: 25 July 2018   Online First Date: 13 September 2018    Issue Date: 05 June 2019
 Cite this article:   
Weina Zhang,Ying Lu,Tao Zhen, et al. Homoharringtonine synergy with oridonin in treatment of t(8; 21) acute myeloid leukemia[J]. Front. Med., 2019, 13(3): 388-397.
 URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-018-0624-1
http://academic.hep.com.cn/fmd/EN/Y2019/V13/I3/388
Fig.1  HHT-induced reduction in CD117 expression and apoptosis in Kasumi-1 cells. (A) Cell viability in Kasumi-1 cells treated with different concentrations of HHT (0.005, 0.01, 0.02, and 0.05 mmol/L) for 48 h. (B) FACS analysis of CD117, CD34, and CD11b surface expression. After HHT treatment (0.01, 0.02, and 0.05 mmol/L) for 48 h, the cells were stained with monoclonal antibodies against CD117 (APC-conjugated), CD34 (PE-Cy7), and CD11b (PE) and then analyzed by flow cytometry. (C) Apoptotic effects of HHT in Kasumi-1 cells. After incubating the cells with 0.01, 0.02, and 0.05 mmol/L HHT, apoptotic cell percentages were assayed by flow cytometry after Annexin V/7-AAD double-staining.
Fig.2  Oridonin synergized with HHT to inhibit Kasumi-1 cell viability. (A) The combined cytotoxic effects of oridonin and HHT were determined by CCK-8 assay. Kasumi-1 cells were treated with different concentrations of oridonin and HHT at a fixed ratio of 150:1 for 48 h. (B) The combined cytotoxic effects of 3.0 mmol/L oridonin and 0.02 mmol/L HHT on Kasumi-1 cells at 24, 48, and 72 h. (C) Synergistic curve of HHT and oridonin. (D) CI values of oridonin and HHT after 48 h incubation at ED75, ED90, and ED95. CI values were calculated using CompuSyn software and expressed as mean±SD from four separate experiments.
Fig.3  Oridonin and HHT induced MMP loss and apoptosis in Kasumi-1 cells. (A) Kasumi-1 cells treated with different concentrations of oridonin and HHT were labeled with DiOC6 and then assayed by flow cytometry. Mean fluorescence of DiOC6, as shown in the histograms, represents MMP. (B) FACS analysis of apoptotic effects of oridonin and HHT. After treatment for 24 h, the cells were subjected to Annexin V/7-AAD double-staining and flow cytometry analysis. (C) Effects of oridonin and HHT on caspase-3, PARP, and Mcl-1 expressions. After treating the cells with the indicated concentrations of oridonin and HHT for 24 h, caspase-3 activation, PARP cleavage, and Mcl-1 downregulations were detected by immunoblotting. The concentration unit “mmol/L” was used for oridonin and HHT treatment (e.g., ori3.0 represents 3.0 mmol/L oridonin).
Fig.4  Expressions of c-KIT oncoprotein and its downstream signaling pathway were downregulated in oridonin+ HHT-treated Kasumi-1 cells. (A) c-KIT expression on cell surface. Mean fluorescence of c-KIT in Kasumi-1 cells exposed to 4.5 mmol/L oridonin, 0.03 mmol/L HHT, or both was analyzed by FACS and was compared with that of untreated control cells. **, P<0.01; ***, P<0.001. (B) Significant c-KIT pathway inhibition was induced by the combination of oridonin and HHT. Phosphorylated and total c-KIT, STAT3, STAT5, ERK, and AKT protein expression levels were evaluated by Western blot analysis using specific antibodies. Equal loading was confirmed with b-actin. These blots are representative of multiple independent experiments. (C) Changes in c-KIT expression after treatment with oridonin and HHT. The chart shows changes in c-KIT mRNA levels, which were normalized to GAPDH levels. Data are mean and SD from three independent experiments performed in triplicate. The concentration unit “mmol/L” was used for oridonin and HHT treatment (e.g., ori3.0 represents 3.0 mmol/L oridonin).
Fig.5  By downregulating MDR1 and MRP1 expressions, HHT increased intracellular oridonin concentrations. (A) Immunoblot analysis of AE status. Changes in AE and ?AE expression in Kasumi-1 cells treated with increasing doses of oridonin, HHT, or both are presented. b-actin was used as internal control. (B) Histograms of FACS analysis of bio-ori. Kasumi-1 cells treated with 50 µmol/L bio-ori with or without 30 nmol/L HHT were fixed, permeabilized, and stained with Sav-APC-Cy7. Fluorescence intensity of Sav was indicative of the bio-ori amount detected by flow cytometry. These results are representative of three independent experiments. (C) MRP1 and MDR1 protein expression levels decreased with increasing dose of HHT. Rat monoclonal antibody to MRP1 and polyclonal MDR1 antibody were used, and b-actin was used as loading control. (D) Cytotoxic effects of oridonin in the presence of specific inhibitors. Oridonin was added to Kasumi-1 cells treated with 50 mmol/L MRP1 inhibitor MK571 or 5 mmol/L MDR1 inhibitor PSC833. After 24 h exposure, the cytotoxic effects of oridonin were measured by CCK-8 assay. FACS analysis of the increases in bio-ori levels (E) and apoptosis (F) facilitated by MK571, PSC833, and HHT. *, P<0.05; **, P<0.01; ***, P<0.001, compared with bio-ori alone. The concentration unit “mmol/L” was used for oridonin and HHT treatment (e.g., ori3.0 represents 3.0 mmol/L oridonin).
Fig.6  Oridonin in combination with HHT exerted distinct antileukemia efficacy in treatment of t(8; 21) AML mouse model. A total of 1 × 105 leukemic cells harboring AE and c-KIT N822K were injected into 8-week-old sublethally irradiated female BALB/c mice. On day 5, the tumor-bearing mice were randomly assigned to groups receiving saline solution (vehicle, n = 7), HHT (0.5 mg/kg per day, n = 7), oridonin (40 mg/kg per day, n = 7), or HHT and oridonin (n = 7). (A) Percentages of GFP-positive cells in the peripheral blood at day 19 were analyzed by FACS. (B) Kaplan–Meier survival curves of leukemic mice treated with vehicle, oridonin, and/or HHT.
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