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

To investigate the effect of RAD18-siRNA on cell proliferation and chemotherapy sensitivity of esophageal squamous cell carcinoma (ESCC) ECA-109 cells.RAD18-siRNA was transfected into human ECA-109 cells by Lipofectamine 3000. Quantitative PCR and Western blot were performed to detect RAD18 and CyclinD1 expression; CCK-8 assay was used to determine cell proliferation and chemotherapy drug sensitivity; flow cytometry was used to determine cell cycle. Correlation between RAD18 and CyclinD1 mRNA expression was analyzed by Pearson's correlation.Compared with non-transfected cells, the expression of RAD18 in RAD18-siRNA group was significantly decreased (<0.05). The cell proliferation was inhibited (<0.05) and the cell number of G1 phase was increased, G2/M phase cells decreased (<0.05) in RAD18-siRNA group. After treatment with different concentrations of cisplatin or 5-FU, the survival rate of the two cell groups was reduced (all<0.05), and the IC50 of RAD18-siRNA group was significantly lower than that of non-transfected group (<0.05). The mRNA expression of RAD18 was positively correlated with CyclinD1 expression in ESCC tissues(=0.478,<0.01).Down-regulated expression of RAD18 can decrease the cell proliferation and increase chemo-sensitivity of ESCC cells, and CyclinD1 may participate in the process.
Adjuvants, Pharmaceutic ; pharmacology ; Carcinoma, Squamous Cell ; drug therapy ; physiopathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Cyclin D1 ; drug effects ; genetics ; DNA-Binding Proteins ; administration & dosage ; pharmacology ; Down-Regulation ; drug effects ; genetics ; Drug Resistance, Neoplasm ; drug effects ; Drug Screening Assays, Antitumor ; methods ; Drug Synergism ; Esophageal Neoplasms ; drug therapy ; physiopathology ; Fluorouracil ; pharmacology ; G1 Phase ; drug effects ; G2 Phase ; drug effects ; Humans ; Metaphase ; drug effects ; RNA, Small Interfering ; administration & dosage ; pharmacology ; Transfection ; Ubiquitin-Protein Ligases ; administration & dosage ; pharmacology

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

Ac-Phe-Lys-PABC-DOX (PDOX) is a smart doxorubicin (DOX) prodrug designed to decrease toxicities while maintaining the potent anticancer effects of DOX. This study was aimed at elucidating the effectiveness and toxicities of DOX and PDOX in patient-derived MCF-7 breast cancer cells in vitro. The MCF-7 cells were exposed to both PDOX and DOX, and cytotoxicities, cell cycle and P53/P21 signaling alterations were studied. Abundant cathepsin B was found in the MCF-7 cells, and treatment with PDOX and DOX triggered dose- and time-dependent cytotoxicity and resulted in a significant reduction in cell viability. The IC50 of PDOX and DOX was 3.91 and 0.94 μmol/L, respectively. Both PDOX and DOX caused an up-regulation of the P53/P21-related signal pathway, and PDOX significantly increased expression of P53 and caspase 3, and arrested the cell cycle at the G1/G2 phase. As compared with DOX, PDOX reduced toxicities, and it may have different action mechanisms on breast cancer cells.
Antibiotics, Antineoplastic ; pharmacology ; Breast Neoplasms ; drug therapy ; metabolism ; pathology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Doxorubicin ; analogs & derivatives ; pharmacology ; Drug Screening Assays, Antitumor ; methods ; Female ; G1 Phase ; drug effects ; G2 Phase ; drug effects ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Oligopeptides ; pharmacology ; Signal Transduction ; drug effects ; Tumor Suppressor Protein p53 ; biosynthesis

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

Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27Kip1 protein level specifically increased after KIF14 knockdown. The increase in p27Kip1 was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27Kip1 accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27Kip1 for degradation by the 26S proteasome, leading to accumulation of p27Kip1. The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.
Carcinoma, Hepatocellular/*metabolism ; Cyclin-Dependent Kinase Inhibitor p27/genetics/*metabolism ; Cyclins/genetics/metabolism ; *Cytokinesis ; Gene Silencing ; Hep G2 Cells ; Humans ; Kinesin/genetics/*metabolism ; Liver Neoplasms/*metabolism ; Oncogene Proteins/genetics/*metabolism ; Proteasome Endopeptidase Complex/metabolism ; RNA, Messenger/genetics/metabolism ; S-Phase Kinase-Associated Proteins/genetics/metabolism ; *Ubiquitination

OBJECTIVETo investigate the inhibitory effect of trichostatin A (TSA) on the proliferation of HepG2 cells and explore the underlying mechanism.

METHODSHepG2 cells exposed to different concentrations of TSA for 24, 48, or 72 h were examined for cell growth inhibition using a cell counting kit, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under inverted microscope. The expressions of beta-catenin, HDAC1, HDAC3, H3K9, cyclinD1 and Bax proteins in the exposed cells were detected by Western blotting, and the expressions of HDAC1 and HDAC3 mRNAs by quantitative fluorescent PCR.

RESULTSExposure to TSA caused significant dose- and time-dependent inhibition of HepG2 cell proliferation (P<0.05) and resulted in increased cell percentage in G0/G1 and G2/M phases and decreased cell percentage in S phase. The apoptotic index in the control group was (6.22 ± 0.25)%, which increased to (7.17 ± 0.20)% and (18.14 ± 0.42)% after exposure to 250 and 500 nmol/L TSA, respectively. Exposure to 250 and 500 nmol/L TSA also caused cell morphology changes with numerous floating cells. The expressions of beta-catenin, H3K9 and Bax proteins were significantly increased and CyclinD1, HDAC1, and HDAC3 protein expressions decreased in TSA-treated cells, but the expressions of HDAC1 and HDAC3 mRNAs showed no significant changes.

CONCLUSIONSTSA can inhibit the proliferation of HepG2 cells and induce cell cycle arrest and apoptosis by inhibiting HDAC activity, promoting histone acetylation, and activating Wnt/beta-catenin signaling pathway.

Acetylation ; Apoptosis ; Cell Cycle Checkpoints ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Hep G2 Cells ; drug effects ; Histone Deacetylase 1 ; metabolism ; Histone Deacetylases ; metabolism ; Histones ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; Wnt Signaling Pathway ; bcl-2-Associated X Protein ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo observe the effect of bear bile powder (BBP) on the STAT3 pathway and its downstream target genes of nude mice hepatocellular carcinoma (HCC) xenograft, and to explore its mechanism for treating HCC.

METHODSThe subcutaneous xenograft model was established using HepG2 cells. When the subcutaneous transplanted tumor was formed, naked mice were randomly divided into two groups, the BBP group and the control group. Mice in the BBP group were administered with BBP by gastrogavage, once daily for 3 consecutive weeks, while mice in the control group were administered with normal saline by gastrogavage, once daily for 3 consecutive weeks. The body weight and the tumor volume were measured once per week. By the end of medication, the tumor weight was weighed and the tumor inhibition ratio calculated. The apoptosis of the tumor tissue was detected by TdT-mediated dUTP nick end labeling (TUNEL). The expression of Bcl2-associated X protein (Bax), B cell lymphoma/eukemina-2 (Bcl-2), cyclin-dependent protein kinase (CDK4), cyclinD1 were detected by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression levels of signal transducers and transcription activators 3 (p-STAT3), proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, CDK4, and cyclinD1 were determined by immunohistochemistry.

RESULTSBBP could inhibit the tumor volume and tumor weight, showing statistical difference when compared with the control group (P < 0.01). Results of TUNEL showed that BBP could significantly induce the apoptosis of hepatoma carcinoma cells. Results of RT-PCR showed that BBP could up-regulate the expression of Bax and down-regulate mRNA expression of Bcl-2, CDK4, and cyclinD1. Immunohistochemical results showed that BBP could up-regulate the expression of Bax and inhibit the protein expression of p-STAT3, PCNA, Bcl-2, CDK4, and cyclinD1.

CONCLUSIONBBP could induce the apoptosis of hepatoma carcinoma cells and inhibit their proliferation by regulating STAT3 pathway.

Animals ; Bile ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Ursidae ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo study the effect of Biejiajian Pills on Wnt signal pathway and the mechanisms underlying its action to suppress the invasiveness of hepatocellular carcinoma.

METHODSHepG2 cells cultured in the serum of rats fed with Biejiajian Pills for 48 h were examined for β-catenin expression using immunofluorescence, β-catenin/TCF4 complex activity with luciferase, and expressions of the downstream proteins cyclin D1 and MMP-2 using qRT-PCR.

RESULTSBiejiajian Pills-treated sera significantly reduced the expressions of cytoplasmic and nuclear β-catenin protein, cyclin D1 and MMP-2 proteins and lowered the activities of β-catenin/TCF4 complex.

CONCLUSIONBiejiajian Pills may serve as a potential anti-tumor agent, whose effect might be mediated by inhibiting the Wnt/β-catenin pathway.

Animals ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; Cyclin D1 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Rats ; Transcription Factor 4 ; Transcription Factors ; metabolism ; Wnt Proteins ; Wnt Signaling Pathway ; beta Catenin ; 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