Hui Hui1, Xiaoxiao Zhang1, Hui Li1, Xiao Liu1, Le Shen1, Yu Zhu2, Jingyan Xu3, Qinglong Guo4, Na Lu5. 1. State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, JiangSu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, People's Republic of China. 2. Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, Jiangsu Province, People's Republic of China. 3. Department of Hematology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, 210008, People's Republic of China. xjy1967@sina.com. 4. State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, JiangSu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, People's Republic of China. anticancer_drug@163.com. 5. State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, JiangSu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, People's Republic of China. luna555@163.com.
Abstract
PURPOSE: AML1/ETO fusion gene is one of disease-causing genes of t(8;21)-positive acute myeloid leukemia (AML). Oroxylin A (OA) has showed anticancer effects on other cancer cells. Here, studies were conducted to determine the antileukemia effect of OA on t(8;21)-positive AML cells in vitro and in vivo. MATERIALS AND METHODS: The effects of OA on cell viability of t(8;21)-positive Kasumi-1 and primary AML cells were analyzed by MTT assay. Cell differentiation was examined by NBT reduction assay, flow cytometry analysis for CD11b/CD14, and Giemsa stain. Protein expressions were determined by Western blots. Immunofluorescence assay was used to verify the effect of OA on HDAC-1 expression in vivo. Immunohistochemical staining was applied to evaluate leukemic infiltration of AML-bearing NOD/SCID mice. RESULTS: OA enhanced NBT reduction activity and CD11b/CD14 expression of AML1/ETO-positive AML cells markedly. Results of Giemsa staining also demonstrated that OA could induce the morphologic changes with reduction of nuclear/cytoplasmic ratios, suggesting the cell differentiation induced by OA. Further study showed that OA decreased the expression of fusion protein AML1/ETO and down-regulated HDAC-1 protein levels in vitro and in vivo. Moreover, OA increased the expression of differentiation-related proteins C/EBPα and P21. Acetylation levels of histones were also advanced obviously after treatment of OA. In vivo study indicated that OA could prolong the survival of AML-bearing NOD/SCID mice and reduce leukocytic infiltration of the spleen. CONCLUSIONS: All these results suggested that OA might be a novel candidate agent for differentiation therapy for AML1/ETO-positive AML and the mechanism required further investigation.
PURPOSE:AML1/ETO fusion gene is one of disease-causing genes of t(8;21)-positive acute myeloid leukemia (AML). Oroxylin A (OA) has showed anticancer effects on other cancer cells. Here, studies were conducted to determine the antileukemia effect of OA on t(8;21)-positive AML cells in vitro and in vivo. MATERIALS AND METHODS: The effects of OA on cell viability of t(8;21)-positive Kasumi-1 and primary AML cells were analyzed by MTT assay. Cell differentiation was examined by NBT reduction assay, flow cytometry analysis for CD11b/CD14, and Giemsa stain. Protein expressions were determined by Western blots. Immunofluorescence assay was used to verify the effect of OA on HDAC-1 expression in vivo. Immunohistochemical staining was applied to evaluate leukemic infiltration of AML-bearing NOD/SCIDmice. RESULTS: OA enhanced NBT reduction activity and CD11b/CD14 expression of AML1/ETO-positive AML cells markedly. Results of Giemsa staining also demonstrated that OA could induce the morphologic changes with reduction of nuclear/cytoplasmic ratios, suggesting the cell differentiation induced by OA. Further study showed that OA decreased the expression of fusion protein AML1/ETO and down-regulated HDAC-1 protein levels in vitro and in vivo. Moreover, OA increased the expression of differentiation-related proteins C/EBPα and P21. Acetylation levels of histones were also advanced obviously after treatment of OA. In vivo study indicated that OA could prolong the survival of AML-bearing NOD/SCIDmice and reduce leukocytic infiltration of the spleen. CONCLUSIONS: All these results suggested that OA might be a novel candidate agent for differentiation therapy for AML1/ETO-positive AML and the mechanism required further investigation.
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