The Deubiquitinating Enzyme USP14 Regulates Leukemic Chemotherapy Drugs-Induced Cell Apoptosis by Suppressing Ubiquitination of Aurora Kinase B

Background/Aims: Aurora kinase B is a mitotic checkpoint kinase that plays a pivotal role in mitosis by ensuring correct chromosome segregation and normal progression through mitosis. Aurora B has been found to be amplified and overexpressed in several types of leukemia. The deubiquitinating enzyme USP14 is one of three proteasome-associated deubiquitinating enzymes and plays critical roles in diverse biological processes including cancer. However, whether USP14 has a role in leukemia cells remains elusive. Methods: Leukemic U937, NB4 and Jurkat cells were treated with diverse apoptosis-inducing drugs. The interaction between USP14 and Aurora B were determined by Western blot. The effect of USP14 in the regulation of Aurora B was detected by cycloheximide (CHX) and deubiquitination assays. FACS assay was used to determine the apoptosis ratio of cells after treatments. Results: We found that Aurora B was ubiquitinated and degraded during leukemic chemotherapy drugs-induced cell apoptosis. FBXW7 mediated Aurora B ubiquitination and degradation during chemotherapeutic drugs-induced apoptosis. USP14 associated with Aurora B and prevented Aurora B degradation. Functionally, overexpression of USP14 inhibits chemotherapeutic drugs-induced apoptosis in leukemia cells. On the contrary, administration of b-AP15, a specific inhibitor of USP14, significantly increased leukemia cells apoptosis in a dose-dependent manner. Conclusion: Thus, our data suggest that USP14 plays a novel critical role of in leukemia cells apoptosis through Aurora B stabilization and USP14 could be a potential therapeutic target for leukemia.

Download Full-text

Development as Anti-Leukemic Agents, a Novel Synthesis of Phospha Sugar Derivatives in Therapy for Leukemias, and Analysis of Their Characterizations.

Blood ◽

10.1182/blood.v110.11.4174.4174 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4174-4174

Author(s):

Satoki Nakamura ◽

Keiji Okinaka ◽

Isao Hirano ◽

Takaaki Ono ◽

Kazuyuki Shigeno ◽

...

Keyword(s):

Cell Proliferation ◽

Oxygen Atom ◽

Cyclin D1 ◽

Leukemia Cell ◽

Aurora Kinase ◽

Leukemia Cells ◽

Aurora Kinase B ◽

Leukemia Cell Lines ◽

Induced Apoptosis ◽

Sugar Derivatives

Abstract [Background] Sugar derivatives whose oxygen atom in the hemiacetal ring is replaced by a carbon, nitrogen, sulfur atom, and etc., are celled as pseudo sugars. The synthesis and bioassay of these pseudo sugars are well investigated and are known as potential bioactive materials. Phospha sugar, which is one of pseudo sugars, having a phosphorus atom instead of the oxygen atom in the hemiacetal ring, is little known. The synthesis and bioassay of phospha sugars are not well studied in detail. We synthesized the 2, 3, 4-tribromo-3-methyl-1-phenyl phospholane 1-oxide (TMPP) and 2, 3-dibromo-3-methyl-1-phenyl phospholane 1-oxide (DMPP) by the nucleophilic substitution reactions, and found their anti-leukemic activities. [Purpose] The aims of the present study were to evaluate the inhibition of proliferation and induction of apoptosis in leukemia cell treated with TMPP or DMPP, and define the target molecules for TMPP in leukemia cells. [Methods] The cells used in this study were human leukemia cell lines, K562, U937, and YRK2 cells. For proliferation analysis, MTT assays were performed in leukemia cells treated with TMPP. For cell cycle analysis, flow cytometory analysis was performed in leukemia cells treated with TMPP or DMPP by PI staining. For analysis of mitotic regulatory proteins (p27, p21, Skp2, Cdc25B, Cyclin D1, Survivin, and Aurora kinase B), Western blotting was performed in leukemia cells treated with TMPP. [Results] In leukemia cell lines, DMPP and TMPP significantly inhibited the cell proliferation, and TMPP more strongly inhibited the cell proliferation than DMPP. 10 μM TMPP significantly induced G2/M phase arrest, and 25 μM TMPP induced apoptosis in leukemia cells. In leukemia cells, 10 μM TMPP reduced protein of Aurora kinase B, Survivin, Cyclin D1, Skp2 KIS, and FoxM1, while increased protein expression of p21and p27 by western blot analysis. Moreover, 25 μM TMPP activated caspase-3 and caspase-9, cleaved PARP, and reduced Bcl-2. [Conclusions] In this study, we report in the first time the possibility of phospha sugar derivatives as anti-leukemic agents in therapy for leukemias, and analysis of their characterizations. DMPP significantly inhibited the proliferation, and induced apoptosis of leukemia cells. These results demonstrated that the FoxM1, which is an essential transcription factor for cell growth and regulates expression of the mitotic regulators, is one of the targets for DMPP. Therefore, DMPP or TMPP has possibility as new agents for FoxM1 in leukemia therapy.

Download Full-text

Development and Characterization of Phospha Sugar Derivatives, DMPP and TMPP, with Anti-Leukemic Effects

Blood ◽

10.1182/blood.v112.11.5057.5057 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5057-5057

Author(s):

Satoki Nakamura ◽

Mitsuji Yamashita ◽

Daisuke Yokota ◽

Isao Hirano ◽

Takaaki Ono ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Leukemia Cell ◽

Aurora Kinase ◽

Leukemia Cells ◽

Aurora Kinase B ◽

Leukemia Cell Lines ◽

Foxm1 Expression ◽

Induced Apoptosis ◽

Sugar Derivatives

Abstract [Background] Sugar derivatives, whose oxygen atom in the hemiacetal ring is replaced by a carbon, nitrogen, sulfur atom, and etc., are celled as pseudo sugars. The synthesis and bioassay of these pseudo sugars are well investigated and are known as potential bioactive materials. Phospha sugar, which is one of pseudo sugars, having a phosphorus atom instead of the oxygen atom in the hemiacetal ring, is little known. The synthesis and bioassay of phospha sugars are not well studied in detail. We synthesized the 2,3,4-tribromo-3-methyl-1-phenyl phospholane 1-oxide (TMPP) and 2,3-dibromo-3-methyl-1-phenyl phospholane 1-oxide (DMPP) by the nucleophilic substitution reactions, and found their anti-leukemic activities. [Purpose] The aims of the present study were to evaluate the inhibition of proliferation and induction of apoptosis in leukemia cell treated with TMPP or DMPP, and defines the target molecules for TMPP in leukemia cells. [Methods] The cells used in this study were human leukemia cell lines, K562, U937, and YRK2 cells. For proliferation analysis, MTT assays were performed in leukemia cells treated with TMPP. For cell cycle analysis, flow cytometory analysis was performed in leukemia cells treated with TMPP or DMPP by PI staining. For analysis of mitotic regulatory proteins (p27, p21, Skp2, Cdc25B, Cyclin D1, Survivin, and Aurora kinase B), Western blotting was performed in leukemia cells treated with TMPP. For colony analysis, the colonies of CFU-GEMM, CFU-GM, and BFU-E were counted in AML stem/progenitor cells treated with TMPP. [Results] In leukemia cell lines, DMPP and TMPP significantly inhibited the cell proliferation, and TMPP more strongly inhibited the cell proliferation than DMPP. 10 μM TMPP significantly induced G2/M phase arrest, and 25 μM TMPP induced apoptosis in leukemia cells. In leukemia cells, 10μM TMPP reduced protein of Aurora kinase B, Survivin, Cyclin D1, Skp2 KIS, and FoxM1, while increased protein expression of p21and p27 by western blot analysis. Moreover, 25μM TMPP activated caspase-3 and caspase-9, cleaved PARP, and reduced Bcl-2. Moreover, the treatment with TMPP decreased the counts of CFU-GEMM, CFU-GM and BFU-E by depletion of FoxM1 expression. [Conclusions] In this study, we report in the first time the possibility of phospha sugar derivatives as anti-leukemic agents in therapy for leukemias, and analysis of their characterizations. DMPP and TMPP significantly inhibited the proliferation, and induced apoptosis of leukemia cells. DMPP and TMPP induced cell cycle arrest by suppression of FoxM1 expression and apoptosis by Bcl-2 down-regulation in leukemia cells. Moreover, TMPP inhibited colony formation of leukemia progenitors. Therefore, TMPP has new agents with anti-leukemic effects by regulation of cell cycle and apoptosis in leukemia therapy.

Download Full-text

Identification of Target Molecules and Anti-Leukemic Effects of Novel Phospha Sugar Derivative, TMPP.

Blood ◽

10.1182/blood.v114.22.4814.4814 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4814-4814

Author(s):

Satoki Nakamura ◽

Mitsuji Yamashita ◽

Tomonari Takemura ◽

Daisuke Yokota ◽

Isao Hirano ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Leukemia Cell ◽

Docking Simulation ◽

Aurora Kinase ◽

Leukemia Cells ◽

Aurora B ◽

Aurora Kinase B ◽

Leukemia Cell Lines ◽

Target Molecules

Abstract Abstract 4814 Background and Aims We synthesized two phospha sugar derivatives, 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP) by reacting 3-methyl-1-phenyl-2-phospholene 1-oxide with bromine, and we has reported their potential as an antileukemic agent in cell lines. In this study, we have investigated that TMPP have antileukemic effects as shown in various leukemia cell lines as well as primary AML patient specimens. Moreover, we have identified the target molecules of TMPP by using docking simulation. Methods In human leukemia cell lines (HL60, NB4, U937, NOMO-1, CEM, MOLT4, SUP-B15, and YRK2 cells), the anti-leukemic effects were elevated by cell proliferation assay, Cell cycle analysis, Western blotting. In AML cells derived from AML patients and normal hematopoietic progenitor cells derived from healthy volunteers, the changes of viability by treatment with TMPP were evaluated. By using docking simulation, the target molecules were identified in leukemia cells treated with TMPP. Results TMPP showed inhibitory effects on leukemia cell proliferation, with mean IC50 values of 10.2 nmol/L for TMPP, indicating that inhibition appeared to be dependent on the number of bromine atoms in the structure. Further, TMPP at 8.4 nmol/L induced G2/M cell cycle block in leukemia cells, and TMPP at 17.3 nmol/L induced apoptosis in these cells. TMPP treatment reduced cell cycle progression signals (FoxM1, KIS, Cdc25B, Cyclin D1, Cyclin A, and Aurora-B) and tumor cell survival (Bcl-2, p27Kip1 and p21Cip1). Further, treatment with TMPP significantly reduced the viability of AML specimens derived from AML patients, but only slightly reduced the viability of normal ALDHhi progenitor cells. We also found that TMPP showed the strong activity against Aurora kinase B and Bcl-2 by using docking simulation. Conclusion We demonstrate that TMPP inhibits Aurora kinase B and Bcl-2 and shows a dominant Aurora B kinase inhibition-related cell cycle arrest and the induction of mitochondrial potential catastrophe-related apoptosis in acute leukemia cells. Moreover, we identified Aurora kinase B and Bcl-2 as the target molecules of TMPP. We conclude that TMPP has great therapeutic potential in anticancer therapy in a wide range of cancers. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Elevated FoxM1 Expression Promotes Cell Cycle Progression by Induction of KIS Expression and Contributes to the Development of Leukemia Cells.

Blood ◽

10.1182/blood.v110.11.888.888 ◽

2007 ◽

Vol 110 (11) ◽

pp. 888-888 ◽

Cited By ~ 1

Author(s):

Okinaka Keiji ◽

Satoki Nakamura ◽

Isao Hirano ◽

Takaaki Ono ◽

Shinya Fujisawa ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Leukemia Cell ◽

Aurora Kinase ◽

Leukemia Cells ◽

Clinical Samples ◽

Acute Leukemias ◽

Aurora Kinase B ◽

Leukemia Cell Lines ◽

Foxm1 Expression

Abstract [Background] FoxM1, a member of the Fox transcription factor family, plays an important cell cycle regulator of both the transition from G1 to S phase and progression to mitosis. FoxM1 expression was also found to be up-regulated in some solid tumors (basal cell carcinomas, hepatocellular carcinoma, and primary breast cancer). These results suggested that FoxM1 plays a role in the oncogenesis of malignancies. However, it is unknown whether FoxM1 expression contributes to the development or progression of leukemia cells. Therefore, we investigated how FoxM1 regulated the cell cycle of leukemia cells and the expression analysis of the FoxM1 gene in patients with acute leukemias. [Methods] The cells used in this study were human acute leukemia cell lines, U937 and YRK2 cells. Primary acute myeloblastic (25 AML (4 M1, 11 M2, 6 M4, 4 M5)) cells were obtained from the peripheral blood. Human normal mononuclear cells (MNCs) were isolated from peripheral blood (PB) of healthy volunteers after obtaining informed consents. For analysis of proliferation and mitotic regulatory proteins (p27, p21, Skp2, Cdc25B, Cyclin D1, Survivin, Aurora kinase B, and KIS) in leukemia cells, MTT assays and western blot were performed in all cell lines, which untransfected or transfected with siRNA FoxM1, respectively. For cell cycle analysis, flow cytometory analysis was performed in leukemia cells untransfected or transfected with siRNAFoxM1 by PI staining. For analysis of FoxM1 mRNA, quantitative RT-PCR was performed in all cell lines and clinical samples. [Results] In all leukemia cell lines, the expression of FoxM1B mRNA were significantly higher than normal MNCs. When transfected with the siRNA FoxM1 in leukemia cells, suppression of FoxM1 caused a mean 71% (range 62 to 80%) reduction in S phase cells and a mean 4.4-fold (range 3.2 to 5.6-fold) increase in G2/M phase cells compared to controls. MTT assay demonstrated that the proliferation of the siRNA FoxM1 transfected cells was inhibited compared to the untransfected cells. Moreover, FoxM1 knockdown by siRNA in leukemia cells reduced protein and mRNA expression of Aurora kinase B, Survivin, Cyclin D1, Skp2 and Cdc25B, while increased protein expression of p21and p27. In the clinical samples obtained from patients with acute leukemias, the FoxM1B gene was overexpressed in 22/25 (88%). The relative folds of FoxM1B gene expression were for AML: 2.83 compared to normal MNCs. [Conclusions] In this study, we report in the first time that FoxM1 is overexpressed in myeloid leukemia cells. These results demonstrated that expression of FoxM1 is an essential transcription factor for growth of leukemia cells, and regulate expression of the mitotic regulators. Moreover, we showed that FoxM1 induced the expression of KIS protein. Therefore, FoxM1 might be one of moleculer targets of therapy for acute leukemias.

Download Full-text

Aurora kinase B (AURKB; aurora-B)

Science-Business eXchange ◽

10.1038/scibx.2012.1202 ◽

2012 ◽

Vol 5 (46) ◽

pp. 1202-1202

Keyword(s):

Aurora Kinase ◽

Aurora B ◽

Aurora Kinase B

Download Full-text

Analysis of FoxM1 Expression and Regulation of Cell Cycle, and Anti-Leukemic Effectss by FoxM1 Knockdown on Leukemia Cells Transfected with siRNA FoxM1.

Blood ◽

10.1182/blood.v108.11.4320.4320 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4320-4320

Author(s):

Satoki Nakamura ◽

Takaaki Ono ◽

Yuya Sugimoto ◽

Miki Kobayashi ◽

Naohi Sahara ◽

...

Keyword(s):

Cell Cycle ◽

Cyclin D1 ◽

Cell Lines ◽

Leukemia Cell ◽

Aurora Kinase ◽

Leukemia Cells ◽

Rt Pcr ◽

Aurora Kinase B ◽

Leukemia Cell Lines ◽

Foxm1 Expression

Abstract [Background] FoxM1, a member of the Fox transcription factor family, plays an important cell cycle regulator of both the transition from G1 to S phase and progression to mitosis. FoxM1 expression was also found to be up-regulated in some solid tumors (basal cell carcinomas, hepatocellular carcinoma, and primary breast cancer). These results suggested that FoxM1 plays a role in the oncogenesis of malignancies. However, it is unknown whether FoxM1 expression contributes to the development or progression of leukemia cells. Therefore, we investigated whether and how FoxM1 regulated the cell cycle of leukemia cells. [Methods] The cells used in this study were human leukemia cell lines, K562, HL60, U937 cells. For analysis of FoxM1 mRNA, RT-PCR was performed in all cell lines. For analysis of proliferation and mitotic regulatory proteins (p27, p21, Skp2, Cdc25B, Cyclin D1, Survivin, and Aurora kinase B) in leukemia cells, MTT assays and western blot were performed in all cell lines untransfected or transfected with siRNA FoxM1, respectively. For cell cycle analysis, flow cytometory analysis was performed in leukemia cells untransfected or transfected with siRNAFoxM1 by PI staining. [Results] In all leukemia cell lines, the expression of FoxM1B mRNA were significantly higher than normal MNCs. In K562, HL60, and U937 cells transfected with the siRNA FoxM1, suppression of FoxM1 caused a mean 71% (range 62 to 80%) reduction in S phase cells and a mean 4.4-fold (range 3.2 to 5.6-fold) increase in G2/M phase cells compared to untransfected cells. MTT assay demonstrated that the proliferation of the siRNA FoxM1 transfected cells was inhibited compared to the untransfected cells at 2, 3, 4, or 5 days after siRNA FoxM1 transfection. FoxM1 has been reported to regulate transcription of essential mitotic regulatory genes. We showed that FoxM1 knockdown by siRNA in leukemia cells reduced protein and mRNA expression of Aurora kinase B, Survivin, Cyclin D1, Skp2 and Cdc25B, while increased protein expression of p21and p27 in RT-PCR and western blot analysis. [Conclusions] In this study, we report in the first time that FoxM1 is overexpressed in myeloid leukemia cells. These results demonstrated that expression of FoxM1 is an essential transcription factor for growth of leukemia cells, and regulate expression of the mitotic regulators, Cdc25B, Cyclin D1, Survivin, Aurora kinase B, and p21. Moreover, we showed that FoxM1 regulated the expression of Skp2 protein, which is known to promote degradation of the cell cycle regulator p27. Our study found that inhibition of FoxM1 expression in leukemia cells suppressed their growth in vitro. Therefore, FoxM1 might be a new potential target of therapy for leukemias. We will have further study whether the level of FoxM1 expression in leukemia cells is correlated with patient survival or sensitivity for chemotherapy.

Download Full-text

Aurora kinase B (AURKB; aurora-B); c-Myc (MYC)

Science-Business eXchange ◽

10.1038/scibx.2012.1137 ◽

2012 ◽

Vol 5 (43) ◽

pp. 1137-1137

Keyword(s):

Aurora Kinase ◽

Aurora B ◽

Aurora Kinase B

Download Full-text

A Vector-Based Short Hairpin RNA Targeting Aurora B Suppresses Human Prostatic Carcinoma Growth

Technology in Cancer Research & Treatment ◽

10.1177/1533034616673534 ◽

2016 ◽

Vol 16 (1) ◽

pp. 112-119 ◽

Cited By ~ 2

Author(s):

Mei Cao ◽

Panpan Qi ◽

Chong Chen ◽

Liju Song ◽

Xuege Wang ◽

...

Keyword(s):

Tumor Growth ◽

Prostate Carcinoma ◽

Cell Apoptosis ◽

Prostatic Carcinoma ◽

Aurora Kinase ◽

Carcinoma Cells ◽

Control Groups ◽

Hairpin Rna ◽

Aurora Kinase B ◽

Short Hairpin

Aurora kinase B, playing a vital, important role in mitosis, is frequently detected to be overexpressed in many cancer cell lines and various tumor tissues, including prostatic carcinoma. Given the essential function of Aurora kinase B in mitosis and its association with tumorigenesis, it might be a drug target for prostatic carcinoma treatment. In our study, short hairpin RNA targeting Aurora kinase B was cloned into a pGPU6 plasmid vector and then transfected into human prostatic carcinoma cells. The expression level of Aurora kinase B was verified by reverse transcription-polymerase chain reaction and Western blot. At the same time, cell apoptosis was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, fluorescent staining, and flow cytometric analysis. Furthermore, prostate carcinoma cells were injected into mice to establish a tumor xenograft model. Previous studies have shown the effect of pGPU6-shAURKB plasmid on tumor growth in a prostate carcinoma xenogenic implantation model. From the study, we knew that the Aurora kinase B was significantly downregulated in prostate carcinoma cells, and cell apoptosis was also detected higher in treated groups than that in control groups. Moreover, in the prostate carcinoma xenogenic implantation model, compared with the control groups, the tumor growth was inhibited about 78.7% in the pGPU6-shAURKB plasmid–treated group, and cell apoptosis in the experimental group was notably higher than that in control groups. The average duration of tumor-bearing mice was prolonged to about 35 days. The results of experiment indicated that specific knockdown of Aurora kinase B led to prostate carcinoma cells apoptosis and inhibited tumor growth. Our data clearly confirmed that specific knockdown of Aurora kinase B expression by vector-based short hairpin RNA/liposome may be a potential new approach to treat human prostatic carcinoma.

Download Full-text

Molecular Mechanism Analysis the Apoptosis of Leukemia Cells Induced By CRM1 Selective Inhibitor KPT-330

Blood ◽

10.1182/blood.v128.22.5901.5901 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5901-5901 ◽

Cited By ~ 1

Author(s):

Jun Lu ◽

Shaoyan Hu ◽

Weiwei DU ◽

Jian Pan ◽

Junli Ren ◽

...

Keyword(s):

Cell Proliferation ◽

Signaling Pathway ◽

Molecular Mechanism ◽

Differentially Expressed Genes ◽

Cell Apoptosis ◽

Leukemia Cells ◽

Glutathione Metabolism ◽

Toll Like Receptor ◽

Receptor Signaling Pathway ◽

Induced Apoptosis

Abstract Objective: To investigate the effect and mechanisms of KPT-330 on cell apoptosis of human leukemia cells. Methods: CCK-8 assay was used to quantify the growth inhibition of cells after exposure to KPT-330 for 24 hours. Fluorescence microscope was used to observe the morphological change of leukemia cell when treated with KPT330 for 24 hours. Annexin V/propidium iodide staining followed by flow cytometric analysis was used to detect apoptosis of leukemia cells. Then Western blot was used to detect the expression level of CRM1 and the activation of apoptosis related protein Caspase-9、Caspase-3 and PARP level. LncRNA microarray was used to analyze the differentially expressed genes from the cluster analyses. Results: The inhibition of cell proliferation of leukemia cells was in a dose-dependent manner and KPT-330 treatment induced expression of CRM1 down-regulated .Abnormal cells were observed under fluorescence microscopy. Cell apoptosis, cell cycle and activation of apoptosis markers demonstrates that KPT-330 induced apoptosis in leukemia cells. LncRNA microarray analysis showed differently expressed mRNAs and lncRNAs in KPT-330 treated HL-60 cells compared with control group. Molecular function analysis showed that KPT-330 induced apoptosis in leukemia cells partially related with Glutathione metabolism and Toll-like receptor signaling pathway. Conclusion: In this study, KPT-330 treatment resulted in inhibition of cell proliferation and induction of apoptosis in leukemia cells. LncRNA microarray analysis showed differentially expressed genes and lncRNAs in KPT-330 treated HL-60 cells and we demonstrated that KPT-330 induced apoptosis in leukemia cells partially related with Glutathione metabolism and Toll-like receptor signaling pathway. These results may provide new clue about molecular mechanism of KPT-330 induced apoptosis; however, underlying details will be required to determine further. Taken together, our findings suggest that for the first time that KPT-330 may act as new candidate's drug for leukemia. Disclosures No relevant conflicts of interest to declare.

Download Full-text