ZGDHu-1 induces G2/M phase arrest and apoptosis in Kasumi-1 cells

The present study examined the effects of N,N′-di-(m-methylphenyi)-3, 6-dimethyl-1, 4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1), a novel oxazine derivative, in Kasumi-1 cells. Following incubation with various concentrations of ZGDHu-1, fluorescence-activated cell sorting (FACS) was used in order to detect changes in mitochondrial membrane permeability in Kasumi-1 cells. Western blot analysis was performed in order to analyze the expression of nuclear factor-κB, inhibitor of κB and AML1/ETO. In addition FACS was used to analyze leukemia cell cycles and the expression levels of cyclin, cyclin-dependent kinases and cyclin-dependent kinase inhibitors in G2/M phase were determined using FACS and western blot analysis. The upregulation of reactive oxygen species production and mitochondrial membrane permeability was ascribed to apoptosis. The growth of Kasumi-1 cells was inhibited through the downregulation of nuclear factor-κB, degradation of AML1/ETO fusion protein and cell cycle arrest at the G2/M phase. This study documented that G2/M regulatory molecules, including cyclin B1, cell division control (cdc)2 and cdc25c were downregulated and checkpoint kinase 1 (CHK1), p53, p27, phospho-cdc25c, phospho-CHK1 and phospho-p53 were upregulated following treatment with ZGDHu-1. In the present study, pretreatment with CHIR-124, a selective CHK1 inhibitor, abrogated G2/M arrest via ZGDHu-1. These results demonstrated the anti-tumor activity of ZGDHu-1, which may therefore a potential target for further investigation and may be useful for the treatment of patients with t(8;21) acute myeloid leukemia.


Introduction
Leukemia is a type of malignant cancer of the hematopoietic stem cells and is a severe threat to human health. At present, the main therapy for leukemia includes chemotherapy, allogeneic hematopoietic stem cell transplantation, bone marrow transplantation, targeted drug therapy and immunotherapy.
Genetic abnormalities have been demonstrated to be important in leukemogenesis (1). The AML1/ETO (A/E) fusion gene is a product of the chromosome translocation t(8;21) (q22; q22), which affects the acute myeloid leukemia gene 1 (AML1) of chromosome 21 and the eight twenty one gene (ETO) of chromosome 8. The fusion gene is one of the common chromosome aberrations in acute myeloid leukemia (AML), particularly in AML with maturation (M2) (2).
Aggressive cytosine arabinoside-based chemotherapy is the standard protocol for the treatment of t(8;21) AML. However, clinical observation has demonstrated that the median survival time of patients with t(8;21) AML was >2 years, with a 5 year survival rate of <40% (2). The development of novel therapies is required in order to further improve clinical outcome and to provide therapeutic options for t(8;21) AML patients.
N, N ' -d i -(m -m e t h y l p h e n y i ) -3, 6 -d i m e t h y l -1, 4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1) is a novel oxazine derivative synthesized by Professor Wei-Xiao Hu from the College of Pharmaceutical Science, Zhejiang University of Technology (Hangzhou, China) who obtained a Patent of China (3,4). ZGDHu-1 has been demonstrated to possess anti-tumor activity (5,6) and has also been found to inhibit growth and induce apoptosis of Kasumi-1 cells (7), a cell line of the t(8;21) (q22;q22) translocation. ZGDHu-1 is also able to markedly inhibit the cell cycle at the G 2 /M phase, however, the underlying mechanisms were not discussed.
The cell cycle includes the mitotic period (M), the G 1 phase, the S phase and the G 2 phase. Its regulation predominantly depends on the regulatory network, including cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CKIs) (8,9). The checkpoint of G 2 /M is important for entrance of cells into the M phase and is also important in tumor cell resistance (10). When the cell cycle arrests at the G 2 /M phase, the expression levels of the CDK1/cyclin B1 complex are altered, leading to incomplete mitosis and ultimately mitotic catastrophe resulting in cell death (11).
Thus, in the present study, we aimed to investigate the mechanism by which ZGDHu-1 induces G2/M phase arrest and apoptosis in Kasumi-1 cells.
Cell culture and drug treatment. Kasumi-1 cells (12), derived from the peripheral blood of a 7 year old Japanese male who was diagnosed with AML-M2, were purchased from the American Type Culture Collection (Manassas, VA, USA) and were maintained in RPMI-1640 with 20% fetal bovine serum (Gibco). The genetic characteristics of this cell line include a chromosome t(8;21) (q22;q22) translocation, thus making it a good research tool for investigating this type of translocation in leukemia. The cell line was incubated in a 37˚C humidified atmosphere with 5% CO 2 . Different concentrations of ZGDHu-1 (50, 100, 200, 500 and 1,000 µg/l) and controls (negative control and DMSO as solvent control) were added to the Kasumi-1 cells.
Flow cytometric analysis. DNA Prep™ reagent system (Beckman Coulter, Indianapolis, IN, USA) was used to evaluate cell cycle alterations in Kasumi-1 cells. The cells were harvested following washing with phosphate-buffered saline (PBS; DingGuo Biotechnology Co., Ltd., Beijing, China). DNA Prep LPR (50 µl; Beckman Coulter) was added for 1 min and then 150 µl DNA Prep stain was added to the cells. Following gentle agitation, the cells were incubated for 5 min at room temperature, the results were detected using fluorescence-activated cell sorting (FACS) using a Coulter Epics XL flow cytometer (Beckman Coulter) and the proportion of cells at each stage of the cell cycle was determined.
To elucidate the underlying mechanisms of apoptosis, the expression of certain apoptosis-associated proteins were analyzed using FACS. To detect Apo 2.7, collected cells were permeabilized for 20 min at 4˚C with 100 µg/ml digitonin and then phycoerythrin-labeled Apo 2.7 mouse monoclonal (mAb) immunoglobulin G antibody (IM2088U; Beckman Coulter) was added for 15 min.
Dihydrorhodamine 123 (DHR123; Sigma-Aldrich) was used to detect the ROS levels of collected cells (13). Following being washed in PBS, 150 µl 10 µM DHR123 was added to the cells. Subsequently, the cells were incubated at 37˚C for 30 min. FACS was used to measure the changes in median fluorescence intensity (MFI).
IntraPrep permeabilization reagent (Beckman Coulter) was used to detect the expression of the following intracellular proteins: B-cell lymphoma 2 (Bcl-2), Bcl-2-associated death promoter (Bad), Bcl-2-associated X protein (Bax) and cyclin B1. A total of 50 µl fixation reagent was added to the collected cells and then incubated for 15 min at room temperature. Following being washed with PBS, 50 µl permeabilization reagent was added. After 5 min incubation, specific antibodies, including Bcl-2 (BD Biosciences, San Jose, CA, USA), Bad (Biovision, Mountain View, CA, USA), Bax (BD Biosciences) and mouse mAb cyclin B1 (1:2,000; #4135; Cell Signaling Technology, Inc., Beverly, MA, USA) were added to the cells. The cells were incubated for 15 min in the dark at room temperature and then FACS was used to determine the results.
Statistical analysis. All statistical calculations were performed using SPSS 15.0 software (SPSS, Inc., Chicago, IL, USA). Results are expressed as the mean ± standard deviation. The differences between treated and control groups were analyzed using a one-way analysis of variance or χ 2 test. P<0.05 was considered to indicate a statistically significant difference.

ZGDHu-1 induces Kasumi-1 cell apoptosis through the activation of caspase-3 and the mitochondrial apoptosis pathway.
The caspase family is important in the apoptotic signaling pathway (16). In the present study, alterations in caspase-3, cleaved caspase-3 (the active part of caspase-3) and PARP (the substrate of caspase-3) were assessed following treatment with different concentrations of ZGDHu-1 for 48 h in Kasumi-1 cells using western blotting. Caspase-3 decreased and cleaved caspase-3 markedly increased in a dose-dependent manner. The cleaved fragments of PARP (89 kDa) were also easily observed, which suggested that caspase-3 was activated following ZGDHu-1 treatment (Fig. 1A).

ZGDHu-1 induces ROS production in Kasumi-1 cells.
Overproduction of ROS may cause oxidative stress, which is a major factor leading to apoptosis. In order to investigate whether ROS accumulation had occurred, DHR123, one of the most widely used ROS probes for intracellular measurement and analysis, was used. DHR123 is not fluorescent until oxidized by ROS to the highly fluorescent product rhodamine 123, therefore, MFI was measured using flow cytometry. The MFI was increased to 26.6±1.2, 25±1.4, 24.2±1.0, 23.7±1.6 and 38.3±2.1 (P<0.05) at a concentration of 500 µg/l (Fig. 1E).
ZGDHu-1 downregulates the expression of NF-κB and upregulates the expression of IκB. Following being incubated with different concentrations of ZGDHu-1 (0, 100, 200 and 500 µg/l), the expression levels of NF-κB and IκB were detected using western blotting. The result suggested that IκB levels were induced by ZGDHu-1 to suppress the function of NF-κB and inhibit the growth of Kasumi-1 cells ( Fig. 2A).

Effect of ZGDHu-1 on the A/E fusion gene of Kasumi-1 cells at the mRNA and protein level. The A/E fusion gene is a vital
feature of Kasumi-1 cells and it also has a major role in the development of AML. Therefore, in the present study, alterations in the expression of this fusion gene following treatment with different concentrations of ZGDHu-1 were investigated. The result demonstrated that A/E fusion levels did not change at the mRNA level (data not shown), but AML and ETO genes were degraded by ZGDHU-1 (Fig. 2B).

Activation of CHK1 and p53 induces G 2 /M arrest in
Kasumi-1 cells. To further investigate the molecular mechanism for ZGDHu-1-induced G 2 /M arrest in Kasumi-1 cells, the protein levels of cyclin B1 were analyzed using FACS. Additionally, certain CDKs and CKIs were measured using western blotting. The combination of cyclin B1 and cdc2 is an important step for eukaryotic cells entering into mitosis (19). The present result implied that the protein level of cdc2 (Fig. 4A) and cyclin B1 (Fig. 4A, D and E) were markedly decreased in a concentration-and time-dependent manner, which inhibited the number of Kasumi-1 cells entering mitosis.
There are also additional CDKs and CKIs, which have important roles in G 2 /M arrest. Cdc25c is a protein phosphatase responsible for dephosphorylating and activating cdc2, while phosphorylation at Ser216 is DNA damage dependent at the G 2 /M checkpoint. Therefore, the expression of cdc25c and phospho-cdc25c at Ser216 was detected and the result revealed that the level of cdc25c was decreased in a concentration-and time-dependent manner, while phospho-cdc25c was increased in a concentration-dependent manner (Fig. 4A-C).
CHK1 and CHK2 are important in DNA damage check point control (20,21), therefore, their protein levels were evaluated in order to determine through which pathway ZGDHu-1 induced G 2 /M arrest in Kasumi-1 cells. The expression of CHK1 was significantly increased in a concentration-and time-dependent manner, while no difference was identified in the expression of CHK2 and phospho-CHK1 (Ser245), which exhibited similar results compared with CHK1 ( Fig. 4A-C).
p53, a well-known tumor suppressor, has various roles in the cellular response to damage information (22). In the present study, p53 was activated in a time-and concentration-dependent manner when treated with ZGDHu-1 (Fig. 4A-C). Phospho-p53 at Ser20 site was also upregulated via CHK1, while no difference was identified at the Ser15 site (Fig. 4B). P27, a CKI, was also upregulated (Fig. 4A).

Discussion
Our previous study revealed that ZGDHu-1 may inhibit the growth of Kasumi-1 cells in a time-and dose-dependent manner in vitro, however, the underlying mechanisms have not been discussed. In the present study, ZGDHu-1 induced apoptosis through the activation of caspase-3. Caspases are a family of cytosolic aspartate-specific cysteine proteases, which are involved in the initiation and execution of apoptosis. They are expressed as latent zymogens and are activated by an autoproteolytic mechanism or by processing by other proteases. Within the caspase family, caspase-3 is a key enzyme and it was demonstrated to be activated following treatment with ZGDHu-1 in leukemia cells (23). Overall, the present results demonstrate that ZGDHu-1 may induce Kasumi-1 cell apoptosis. Furthermore, ZGDHu-1 may arrest the cell cycle at the G 2 /M phase of leukemia cells when the concentration of ZGDHu-1 was 100 µg/l. NF-κB is a protein complex that controls the transcription of DNA, while IκB is an inhibitory factor (24); activation of the NF-κB signaling pathway is initiated by the signal-induced degradation of IκB proteins. With the degradation of IκB, the NF-κB complex is then translocated to the nucleus where it can 'turn on' the expression of specific genes that have DNA-binding sites for NF-κB. Furthermore, the present data revealed that ZGDHu-1 may upregulate the expression of IκB and downregulate the expression of NF-κB to inhibit the growth of Kasumi-1 cells. The A/E fusion gene is a vital characteristic of Kasumi-1 Figure 5. Effects of the CHK1 inhibitor on ZGDHu-1-treated Kasumi-1 cells. The CHIR-124 inhibitor reduced cell cycle arrest in ZGDHu-1-treated cells. Fluorescence-activated cell sorting was used to analyze the cell cycle of Kasumi-1 cells, which were treated with ZGDHu-1 or CHIR-124 alone or with both. ZGDHu-1, N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide; CHK, checkpoint kinase; PI, propidium iodide. cells and it also has a major role in the biology of this type of leukemia. In the present study, it was revealed that ZGDHu-1 may significantly decrease the protein level of this fusion gene, suggesting that ZGDHU-1 may effectively inhibit the development of this leukemia partly through this mechanism.
There are two major pathways able to induce apoptosis (25), which are classified as the extracellular (extrinsic inducers) or intracellular (intrinsic inducers) pathway. Mitochondrial regulation is a vital part of the intracellular pathway. The changes in mitochondrial membrane protein (Apo 2.7) and Δψm suggested that the integrity of the mitochondrial membrane was destroyed. There was also ROS accumulation in the Kasumi-1 cells. The changes in Bcl-2, Bad and Bax determined via FACS and western blotting confirmed that ZGDHu-1 induced apoptosis through the mitochondrial pathway (26).
The cell cycle is a series of events that take place in a cell leading to its division and duplication. It includes the mitotic period and interphase; interphase may be further subdivided into three phases, which include the G 1 phase, S phase and G 2 phase. In the present study, ZGDHu-1 induced G 2 /M phase arrest in Kasumi-1 cells in a concentration-dependent manner.
Numerous proteins and kinases are involved in the process of the cell cycle. The protein levels of cdc2 and cyclin B1 were markedly decreased in a concentration-dependent manner, leading to the obstruction of mitotic entry in Kasumi-1 cells. The level of cdc25c was also decreased; cdc25c is a protein phosphatase responsible for dephosphorylating and activating cdc2, while phosphorylation at Ser216 is DNA damage dependent at the G 2 /M checkpoint. The present results demonstrated that phospho-cdc25c was increased to inhibit the combination of cdc2 and cyclin B1. p53 was activated and p27, as CKIs, were upregulated to induce G 2 /M arrest.
In order to elucidate whether CHK1 is important in ZGDHu-1-induced cell cycle arrest in Kasumi-1 cells, western blotting was used to detect the protein level of CHK1 and p-CHK1, which revealed an increased concentration of this protein. In addition, a type of CHK1 inhibitor was added prior to ZGDHu-1 administration. Following being incubated with ZGDHu-1, it was revealed that the proportion of cells in the G 2 /M reduced, while the number of cells in the G 0/1 phase increased.
In conclusion, the present study demonstrated that ZGDHu-1 was able to inhibit the proliferation and induce the apoptosis of Kasumi-1 cells. Notably, this compound was able to arrest the cell cycle at the G 2 /M phase. CHK1 kinase was found to be important in these activities. The present results suggested that ZGDHu-1 may be a potential drug to treat leukemia in the future.