OBJECTIVE: To investigate the effect of CDK1 interference regulation of PLK1, Aurora B and TRF1 on the proliferation of leukemia cells. METHODS: The human myelogenous leukemia cell line HL-60 was selected as the research object, and the effect of TRF1 expression and its changes on cell proliferation and cycle was investigated by regulating intracellular CDK1 expression. The objects were divided into 5 groups, including control group, shRNA-NC group, CDK1-shRNA group, pcDNA group and pcDNA-CDK1 group. RT-PCR was used to detect the CDK1 expression of cells in each group; colony formation was used to detect the proliferation of the cells. Western blot was used to detect the expression of CDK1, PLK1, Aurora B, TRF1, and cyclin p53, p27, cyclinA. RESULTS: The phosphorylation level of PLK1, Aurora B and the expression of TRF1 in the CDK1-shRNA group were significantly down-regulated as compared with those in the control group (P<0.05). Compared with the control group, the cells in CDK1-shRNA group showed lower clone formation rate, the increasing of cycle-associated proteins p53 and p27 and the decreasing of cyclinA expression (P<0.05). It was shown that interfered CDK1 expression could inhibit the proliferation of HL-60 cells and prolong the time that they enter mitosis, thereby extending the cell cycle. Compared with the control group, the overexpressed CDK1 in the pcDNA-CDK1 group made the phosphorylation level of PLK1, Aurora B, and TRF1 expression increase significantly (P<0.05), also the colony formation rate (P<0.05). The cycle-related proteins p53 and p27 was down-regulated, while cyclinA expression was up-regulate significantly (P<0.05). The results indicted that overexpressed CDK1 could stimulate adverse reactions, thereby promoting the proliferation of HL-60 cells and shortening the cell cycle. CONCLUSION Knocking out CDK1 can inhibit the phosphorylation of PLK1 and Aurora B and negatively regulate TRF1, thereby inhibiting the proliferation of leukemia cells.
CDC2 Protein Kinase ; Cell Cycle Proteins/genetics* ; Cell Proliferation ; Humans ; Leukemia ; Mitosis ; Phosphorylation ; Proto-Oncogene Proteins/genetics*

OBJECTIVE: To investigate the clinical characteristics, diagnosis and treatment of 1 case EBV negative extranodal NK/T cell lymphoma (ENKTL) patients. METHODS: The clinical manifestations, diagnosis and treatment of one case ENKTL patients with EBV negative were analyzed retrospectively. RESULTS: A 46-year-old woman diagnosed as positive for exosanal NK/T cell lymphoma (EBER CONCLUSION EBV negative ENKTL is rare in clinic and easy to be misdiagnosed, so it should be distinguished from peripheral T cell lymphoma. This case was treated with EBV positive ENKTL regimen, with good short-term efficacy.
CDC2 Protein Kinase ; Cell Proliferation ; Female ; Humans ; Leukemia ; Lymphoma, Extranodal NK-T-Cell ; Middle Aged ; Positron Emission Tomography Computed Tomography ; Retrospective Studies

PURPOSE: Several studies have shown that the oral cavity is a secondary location for Helicobacter pylori colonization and that H. pylori is associated with the severity of periodontitis. This study investigated whether H. pylori had an effect on the periodontium. We established an invasion model of a standard strain of H. pylori in human periodontal ligament fibroblasts (hPDLFs), and evaluated the effects of H. pylori on cell proliferation and cell cycle progression. METHODS: Different concentrations of H. pylori were used to infect hPDLFs, with 6 hours of co-culture. The multiplicity of infection in the low- and high-concentration groups was 10:1 and 100:1, respectively. The Cell Counting Kit-8 method and Ki-67 immunofluorescence were used to detect cell proliferation. Flow cytometry, quantitative real-time polymerase chain reaction, and western blots were used to detect cell cycle progression. In the high-concentration group, the invasion of H. pylori was observed by transmission electron microscopy. RESULTS: It was found that H. pylori invaded the fibroblasts, with cytoplasmic localization. Analyses of cell proliferation and flow cytometry showed that H. pylori inhibited the proliferation of periodontal fibroblasts by causing G2 phase arrest. The inhibition of proliferation and G2 phase arrest were more obvious in the high-concentration group. In the low-concentration group, the G2 phase regulatory factors cyclin dependent kinase 1 (CDK1) and cell division cycle 25C (Cdc25C) were upregulated, while cyclin B1 was inhibited. However, in the high-concentration group, cyclin B1 was upregulated and CDK1 was inhibited. Furthermore, the deactivated states of tyrosine phosphorylation of CDK1 (CDK1-Y15) and serine phosphorylation of Cdc25C (Cdc25C-S216) were upregulated after H. pylori infection. CONCLUSIONS: In our model, H. pylori inhibited the proliferation of hPDLFs and exerted an invasive effect, causing G2 phase arrest via the Cdc25C/CDK1/cyclin B1 signaling cascade. Its inhibitory effect on proliferation was stronger in the high-concentration group.
Blotting, Western ; CDC2 Protein Kinase ; Cell Count ; Cell Cycle ; Cell Proliferation ; Coculture Techniques ; Colon ; Cyclin B1 ; Cytoplasm ; Fibroblasts ; Flow Cytometry ; Fluorescent Antibody Technique ; G2 Phase ; Helicobacter pylori ; Helicobacter ; Humans ; Methods ; Microscopy, Electron, Transmission ; Mouth ; Periodontal Ligament ; Periodontitis ; Periodontium ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; Serine ; Tyrosine

PURPOSE: The cyclin-dependent kinase 1 (Cdk1) and cyclin B complex performs important roles in the transition from the G2 to M phase in the cell cycle through removal of inhibitory phosphates on Cdk1, and Cdc25B, which is a dual-specific phosphatase, mediates these dephosphorylation events. However, measuring Cdc25B activity by existing methods is hampered by inadequate nonspecific substrates and the need to use a radiolabeled isotope. The present study aimed to develop an improved method with which to properly measure Cdc25B activity using a novel nonradioisotopic assay and Cdc25B overexpression cell lines. MATERIALS AND METHODS: A nonradioisotopic Cdk1 kinase assay, based on Western blotting for retinoblastoma protein and histone H1, was used to analyze Cdc25B activity. Also, stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines were constructed using the tetracycline-regulated expression system and were applied as a tool for screening for inhibitors of Cdc25B. RESULTS: The present study developed and optimized a nonradioisotopic assay method to properly measure Cdc25B activity. Furthermore, we constructed stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines for the establishment of a strong assay system with which to evaluate the specificity of Cdc25B inhibitors under conditions similar to the intracellular environment. These methods were confirmed as useful tools for measuring Cdc25B activity. CONCLUSION: The nonradioisotopic Cdk1 kinase assay and Cdc25B overexpression cell lines developed in this study can be conveniently used as tools for screening inhibitors of Cdc25B phosphatase as anticancer drugs.
Blotting, Western ; CDC2 Protein Kinase ; cdc25 Phosphatases ; Cell Cycle ; Cell Division ; Cell Line* ; Cyclin B ; HeLa Cells ; Histones ; Humans ; Mass Screening* ; Methods ; Phosphates ; Retinoblastoma Protein ; Sensitivity and Specificity

BACKGROUND: Sorbus rufopilosa, a tsema rowan, is a species of the small ornamental trees in the genus Sorbus and the family Rosaceae found in East Asia. The bioactivities of S. rufopilosa have not yet been fully determined. The objective of this study is to evaluate the antioxidant and anticancer effects of ethanol extract of S. rufopilosa (EESR) and to determine the molecular mechanism of its anticancer activity in human colon carcinoma HT29 cells. METHODS: To examine the antioxidant activity of EESR, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity assay was performed. Inhibitory effect of EESR on cancer cell growth and proliferation was determined by water-soluble tetrazolium salt assay. To investigate the mechanism of EESR-mediated cytotoxicity, HT29 cells were treated with various concentrations of EESR and the induction of cell cycle arrest and apoptosis was analyzed by flow cytometry, 4,6-diamidino-2-phenylindole staining, and Western blot analysis. RESULTS: EESR showed significant antioxidant activity and inhibitory effect on HT29 cell growth in a dose-dependent manner. EESR induced cell cycle arrest at G2/M phase in a dose-dependent manner by modulating cyclin B, cyclin-dependent kinase 1 (CDK1), and CDK inhibitor p21 expression. EESR-induced apoptosis was associated with the upregulation of p53, a death receptor Fas, and a pro-apoptotic protein Bax and the activation of caspase 3, 8, and 9, resulting in the degradation of PARP. CONCLUSIONS: EESR possessing antioxidant activity efficiently inhibits proliferation of HT29 cells by inducing both cell cycle arrest and apoptosis. EESR may be a possible candidate for the anticancer drug development.
Adenocarcinoma* ; Apoptosis* ; Blotting, Western ; Caspase 3 ; CDC2 Protein Kinase ; Cell Cycle Checkpoints* ; Cell Cycle* ; Colon* ; Cyclin B ; Ethanol ; Far East ; Flow Cytometry ; HT29 Cells* ; Humans* ; Rosacea ; Rosaceae ; Sorbus* ; Trees ; Up-Regulation

This study aimed to identify the differentially expressed genes after silencing of β-catenin in multiple myeloma transduced with β-catenin shRNA. The DNA microarray dataset GSE17385 was downloaded from Gene Expression Omnibus, including 3 samples of MM1.S (human multiple myeloma cell lines) cells transduced with control shRNA and 3 samples of MM1.S cells transduced with β-catenin shRNA. Then the differentially expressed genes (DEGs) were screened by using Limma. Their underlying functions were analyzed by employing Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. Moreover, DEGs annotation was conducted based on the databases of tumor associated genes, tumor suppressed genes and the transcriptional regulation from patterns to profiles. Furthermore, the protein-protein interaction (PPI) relationship was obtained from STRING and the protein-protein interaction network and the functional modules were visualized by Cytoscape. Then, the pathway enrichment for the DEGs in the functional module was performed. A total of 301 DEGs, including 124 up-regulated and 117 down-regulated DEGs, were screened. Functional enrichment showed that CCNB1 and CDK1 were significantly related to the function of cell proliferation. FOS and JUN were related to innate immune response-activating signal transduction. Pathway enrichment analysis indicated that CCNB1 and CDK1 were most significantly enriched in the pathway of cell cycle. Besides, FOS and JUN were significantly enriched in the Toll-like receptor signaling pathway. FOXM1 was identified as a transcription factor. Moreover, there existed interactions among CCNB1, FOXM1 and CDK1 in PPI network. The expression of FOS, JUN, CCNB1, FOXM1 and CDK1 may be affected by β-catenin in multiple myeloma.
CDC2 Protein Kinase ; Cyclin B1 ; genetics ; Cyclin-Dependent Kinases ; genetics ; Forkhead Box Protein M1 ; Forkhead Transcription Factors ; genetics ; Gene Expression Profiling ; methods ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks ; Gene Silencing ; Humans ; Multiple Myeloma ; genetics ; Oncogene Proteins v-fos ; genetics ; Protein Interaction Maps ; Proto-Oncogene Proteins c-jun ; genetics ; beta Catenin ; genetics

BACKGROUND: Osmanthus matsumuranus, a species of Oleaceae, is found in East Asia and Southeast Asia. The bioactivities of O. matsumuranus have not yet been fully understood. Here, we studied on the molecular mechanisms underlying anti-cancer effect of ethanol extract of O. matsumuranus (EEOM). METHODS: Inhibitory effect of EEOM on cell growth and proliferation was determined by WST assay in various cancer cells. To investigate the mechanisms of EEOM-mediated cytotoxicity, HepG2 cells were treated with various concentration of EEOM and analyzed the cell cycle arrest and apoptosis induction by flow cytometry, Western blot analysis, 4,6-diamidino-2-phenylindole (DAPI) staining and DNA fragmentation. RESULTS: EEOM showed the cytotoxic activities in a dose-dependent manner in various cancer cell lines but not in normal cells, and HepG2 cells were most susceptible to EEOM-induced cytotoxicity. EEOM induced G2/M arrest in HepG2 cells associated with decreased expression of cyclin-dependent kinase 1 (CDK1), cyclin A and cylcin B, and increased expression of phospho-checkpoint kinase 2, p53 and CDK inhibitor p21. Immunofluorescence staining showed that EEOM-treated HepG2 increased doublet nuclei and condensed actin, resulting in cell rounding. Furthermore, EEOM-mediated apoptosis was determined by Annexin V staining, chromatin condensation and DNA fragmentation. EEOM caused upregulation of FAS and Bax, activation of caspase-3, -8, -9, and fragmentation of poly ADP ribose polymerase. CONCLUSIONS: These results suggest that EEOM efficiently inhibits proliferation of HepG2 cells by inducing both G2/M arrest and apoptosis via intrinsic and extrinsic pathways, and EEOM may be used as a cancer chemopreventive agent in the food or nutraceutical industry.
Actins ; Annexin A5 ; Apoptosis* ; Asia, Southeastern ; Blotting, Western ; Carcinoma, Hepatocellular* ; Caspase 3 ; CDC2 Protein Kinase ; Cell Cycle Checkpoints ; Cell Line ; Chromatin ; Cyclin A ; Dietary Supplements ; DNA Fragmentation ; Ethanol ; Far East ; Flow Cytometry ; Fluorescent Antibody Technique ; Hep G2 Cells* ; Humans* ; Oleaceae ; Phosphotransferases ; Poly(ADP-ribose) Polymerases ; Up-Regulation

WEE1 is an important factor for histone transcription, chromosome condensation and regulation of cell cycle progression. WEE1 kinase can phosphorylate Cdc2 and down-regulate Cdc2 kinase activity. It can regulate G2 to M phase transition and cell mitosis. It plays a key role in chromosome condensation delay and histone synthesis, suggesting the important functions of WEE1 in the occurrence and development in cancer. At present, a multiple WEE1 inhibitors have been discovered. A great progress has been made in combination of WEE1 inhibitors with DNA damage treatment (chemotherapy or radiotherapy), which makes WEE1 an important target in cancer treatment.
CDC2 Protein Kinase ; metabolism ; Cell Cycle ; Cell Cycle Proteins ; metabolism ; DNA Damage ; Histones ; metabolism ; Humans ; Neoplasms ; metabolism ; Nuclear Proteins ; metabolism ; Phosphorylation ; Protein-Tyrosine Kinases ; metabolism

Sphingosine kinase 1 (SphK1) plays critical roles in cell biological functions. Here we investigated the effects of SphK1 inhibitor SKI II on hepatoma HepG2 cell cycle progression and invasion. Cell survival was determined by SRB assay, cell cycle progression was assayed by flow cytometry, the ability of cell invasion was measured by Matrigel-Transwell assay and protein expression was detected by Western blotting. The results showed that SKI II markedly inhibited HepG2 cell survival in a dose-dependent manner, induced G1 phase arrest in HepG2 cell and inhibited cell invasion. SKI II markedly decreased the expressions of G1-phase-related proteins CDK2, CDK4 and Cdc2 and the levels of cell invasion-associated proteins MMP2 and MMP9. The results showed that SKI II inhibited cell cycle progression and cell invasion, implying SphK1 as a potential target for hepatoma treatment.
CDC2 Protein Kinase ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinases ; metabolism ; G1 Phase ; drug effects ; Hep G2 Cells ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; antagonists & inhibitors ; Thiazoles ; pharmacology

Fucoidan is one of the main bioactive components of polysaccharides. The current study was focused on the anti-tumor effects of fucoidan on human heptoma cell line HepG2 and the possible mechanisms. Fucoidan treatment resulted in cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner detected by MTT assay, flow cytometry and fluorescent microscopy. The results of flow cytometric analysis revealed that fucoidan induced G2/M arrest in the cell cycle progression. Hoechst 33258 and Annexin V/PI staining results showed that the apoptotic cell number was increased, which was associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2 and p-Stat3. In parallel, the up-regulation of p53 and the increase in reactive oxygen species were also observed, which may play important roles in the inhibition of HepG2 growth by fucoidan. In the meantime, Cyclin B1 and CDK1 were down-regulated by fucoidan treatment. Down-regulation of p-Stat3 by fucoidan resulted in apoptosis and an increase in ROS in response to fucoidan exposure. We therefore concluded that fucoidan induces apoptosis through the down-regulation of p-Stat3. These results suggest that fucoidan may be used as a novel anti-cancer agent for hepatocarcinoma.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; CDC2 Protein Kinase ; genetics ; metabolism ; Cyclin B1 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; Flow Cytometry ; G2 Phase Cell Cycle Checkpoints ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Hep G2 Cells ; Hepatoblastoma ; genetics ; metabolism ; pathology ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Microscopy, Fluorescence ; Polysaccharides ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; STAT3 Transcription Factor ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism