Oridonin induces NPM mutant protein translocation and apoptosis in NPM1c+ acute myeloid leukemia cells in vitro
Abstract
Fei-fei LI2, #, Sha YI1, #, Lu WEN1, Jing HE1, Li-jing YANG1, Jie ZHAO1, Ben-ping ZHANG1, Guo-hui CUI1, *, Yan CHEN1, *
1Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; 2Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
Aim: Skewed cytoplasmic accumulation of NPM mutant protein (NPM1c+) is close related to leukemia pathogenesis. The aim of this study was to investigate whether oridonin, a diterpenoid isolated from the Chinese traditional medicine Rabdosia rubescens, was able to interfere with NPM1c+ protein trafficking and induce apoptosis in NPM1c+ acute myeloid leukemia cells in vitro.
Methods: OCI-AML3 cell line harboring a NPM1 gene mutation was examined. Cell growth was detected by MTT assay. Cell apoptosis was evaluated using flow cytometry and Hoechst 33258 staining. The expression and subcellular localization of relevant proteins were detected by Western blot and immunofluorescent staining. The mRNA expression was detected by RT-PCR.
Results: Oridonin (2–12 μmol/L) dose-dependently inhibited the viability of OCI-AML3 cells (the IC50 value was 3.27±0.23 μmol/L at 24 h). Moreover, oridonin induced OCI-AML3 cell apoptosis accompanied by activation of caspase-3 and nuclear translocation of NPM1c+ protein. Oridonin did not change the expression of Crm1 (the export receptor for nuclear export signal-containing proteins), but induced nuclear translocation of Crm1. Oridonin markedly increased the expression of nucleoporin98 (Nup98), which had an important role in Crm1-mediated nuclear protein export, and induced nuclear accumulation of Nup98. Furthermore, oridonin markedly increased the expression of p14arf and p53.
Conclusion: In NPM1c+ leukemia cells, oridonin induces NPM1c+ protein translocation into the nucleus possibly via nuclear accumulation of Crm1; the compound markedly increases p53 and p14arf expression, which may contribute to cell apoptosis.
Keywords: oridonin; acute myeloid leukemia; NPM mutation; protein trafficking; Crm1; nucleoporin98; apoptosis; p53; p14arf
The authors would like to thank Prof Brunangelo FALINI at the University of Perugia for kindly providing the NPM-mutant specific antibody. The authors would also like to thank MD MINDEN (Ontario Cancer Institute, Toronto, ON, Canada) for providing the OCI-AML3 cells. This work was supported by grants from the National Natural Science Foundation of China (No 81070429, 81372541, and 81060048).
#These two authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail yanchen981@aliyun.com (Yan CHEN); ghcui@medmail.com.cn (Guo-hui CUI)
Received 2013-12-14 Accepted 2014-03-24
Keywords:
1Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; 2Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
Aim: Skewed cytoplasmic accumulation of NPM mutant protein (NPM1c+) is close related to leukemia pathogenesis. The aim of this study was to investigate whether oridonin, a diterpenoid isolated from the Chinese traditional medicine Rabdosia rubescens, was able to interfere with NPM1c+ protein trafficking and induce apoptosis in NPM1c+ acute myeloid leukemia cells in vitro.
Methods: OCI-AML3 cell line harboring a NPM1 gene mutation was examined. Cell growth was detected by MTT assay. Cell apoptosis was evaluated using flow cytometry and Hoechst 33258 staining. The expression and subcellular localization of relevant proteins were detected by Western blot and immunofluorescent staining. The mRNA expression was detected by RT-PCR.
Results: Oridonin (2–12 μmol/L) dose-dependently inhibited the viability of OCI-AML3 cells (the IC50 value was 3.27±0.23 μmol/L at 24 h). Moreover, oridonin induced OCI-AML3 cell apoptosis accompanied by activation of caspase-3 and nuclear translocation of NPM1c+ protein. Oridonin did not change the expression of Crm1 (the export receptor for nuclear export signal-containing proteins), but induced nuclear translocation of Crm1. Oridonin markedly increased the expression of nucleoporin98 (Nup98), which had an important role in Crm1-mediated nuclear protein export, and induced nuclear accumulation of Nup98. Furthermore, oridonin markedly increased the expression of p14arf and p53.
Conclusion: In NPM1c+ leukemia cells, oridonin induces NPM1c+ protein translocation into the nucleus possibly via nuclear accumulation of Crm1; the compound markedly increases p53 and p14arf expression, which may contribute to cell apoptosis.
Keywords: oridonin; acute myeloid leukemia; NPM mutation; protein trafficking; Crm1; nucleoporin98; apoptosis; p53; p14arf
The authors would like to thank Prof Brunangelo FALINI at the University of Perugia for kindly providing the NPM-mutant specific antibody. The authors would also like to thank MD MINDEN (Ontario Cancer Institute, Toronto, ON, Canada) for providing the OCI-AML3 cells. This work was supported by grants from the National Natural Science Foundation of China (No 81070429, 81372541, and 81060048).
#These two authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail yanchen981@aliyun.com (Yan CHEN); ghcui@medmail.com.cn (Guo-hui CUI)
Received 2013-12-14 Accepted 2014-03-24