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1.
Theranostics ; 11(20): 10047-10063, 2021.
Article in English | MEDLINE | ID: mdl-34815803

ABSTRACT

Parkin (PK) is an E3-ligase harboring tumor suppressor properties that has been associated to various cancer types including glioblastoma (GBM). However, PK is also a transcription factor (TF), the contribution of which to GBM etiology remains to be established. Methods: The impact of PK on GBM cells proliferation was analyzed by real-time impedance measurement and flow cytometry. Cyclins A and B proteins, promoter activities and mRNA levels were measured by western blot, luciferase assay and quantitative real-time PCR. Protein-protein and protein-promoter interactions were performed by co-immunoprecipitation and by ChIP approaches. The contribution of endogenous PK to tumor progression in vivo was performed by allografts of GL261 GBM cells in wild-type and PK knockout mice. Results: We show that overexpressed and endogenous PK control GBM cells proliferation by modulating the S and G2/M phases of the cell cycle via the trans-repression of cyclin A and cyclin B genes. We establish that cyclin B is regulated by both E3-ligase and TF PK functions while cyclin A is exclusively regulated by PK TF function. PK invalidation leads to enhanced tumor progression in immunocompetent mice suggesting an impact of PK-dependent tumor environment to tumor development. We show that PK is secreted by neuronal cells and recaptured by tumor cells. Recaptured PK lowered cyclins levels and decreased GBM cells proliferation. Further, PK expression is decreased in human GBM biopsies and its expression is inversely correlated to both cyclins A and B expressions. Conclusion: Our work demonstrates that PK tumor suppressor function contributes to the control of tumor by its cellular environment. It also shows a key role of PK TF function in GBM development via the control of cyclins in vitro and in vivo. It suggests that therapeutic strategies aimed at controlling PK shuttling to the nucleus may prove useful to treat GBM.


Subject(s)
Glioblastoma/therapy , Ubiquitin-Protein Ligases/therapeutic use , Animals , Brain Neoplasms/pathology , Cell Cycle , Cell Line, Tumor , Cell Proliferation/genetics , Cyclin A/drug effects , Cyclin A/metabolism , Cyclin B/drug effects , Cyclin B/metabolism , Cyclins/genetics , Female , Flow Cytometry/methods , Gene Expression/genetics , Gene Expression Regulation, Neoplastic/genetics , Genes, Tumor Suppressor/physiology , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Male , Mice , Mice, Knockout , MicroRNAs/metabolism , Models, Animal , RNA, Messenger
2.
Turk Neurosurg ; 31(1): 46-50, 2021.
Article in English | MEDLINE | ID: mdl-33216331

ABSTRACT

AIM: To investigate the relationship between paracetamol and expression levels of cyclooxygenase-2, cyclin B, cell viability and apoptosis in glioblastoma cell line. MATERIAL AND METHODS: The A172 glioblastoma cells were treated with different concentrations of paracetamol and phosphate buffer saline as a vehicle for 24, 48, and 72 hours. Cell viability was detected by MTT. Bax, procaspase 3, COX-2 and Cyclin B expressions were detected using Western blotting. RESULTS: A paracetamol treatment of 0.5 mg/mL for 24, 48, and 72 hours led to a 14%, 31%, and 37% decrease in cell viability. The expression of COX-2 and cyclin B levels decreased by 36% and 52% respectively, after treatment with 0.5 mg/mL paracetamol. Treatment with 0.5 mg/mL and 1 mg/mL paracetamol significantly induced the expression of cleaved caspase 3, procaspase 3 and Bax proteins compared to the control group (60%, 40%, 21%, %100, 18%, 17%, respectively). CONCLUSION: The results of our study showed that paracetamol has antitumoral effects on glioblastoma cells and this activity was induced by different signaling pathways.


Subject(s)
Acetaminophen/pharmacology , Glioblastoma/pathology , Signal Transduction/drug effects , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclin B/drug effects , Cyclin B/metabolism , Cyclooxygenase 2/drug effects , Cyclooxygenase 2/metabolism , Glioblastoma/metabolism , Humans
3.
Biochem Biophys Res Commun ; 510(1): 59-64, 2019 02 26.
Article in English | MEDLINE | ID: mdl-30665718

ABSTRACT

Neural stem/progenitor cells (NSPCs) express higher levels of poly(ADP-ribose) polymerase 1 (PARP1) than mouse embryonic fibroblasts (MEFs). Inhibition of PARP induces the expression of several genes in the p53 signaling pathway, including p21, which is critical for cell cycle control at the G1/S phase, triggers apoptosis, and suppresses cell cycle progression in NSPCs. However, upon the up-regulation of p21, the cell cycle does not arrest at any specific phase. In the present study, the expression of genes specific to the G1/S and G2/M phases of the cell cycle were analyzed following treatment with PJ34 (N-[6-oxo-5,6-dihydro-phenanthridin-2-yl]-N,N-dimethylacetamide), an inhibitor of PARP. PJ34 treatment dramatically down-regulated cyclin B1 expression in NSPCs, but not in MEFs, which was confirmed by a promoter assay. Down-regulation of FoxM1 and B-MYB revealed that the down-regulation of cyclin B occurs at the transcriptional level. GADD45 was also specifically up-regulated in NSPCs. Taken together, the activation of p53 by PJ34 treatment in NSPCs induced changes in the expression of genes involved in the cell cycle. Fluorescence-activated cell sorting analysis revealed that PJ34 treatment suppressed G2/M to G1 progression in NSPCs, but not in MEFs. These data indicate that PJ34 treatment inhibits cyclin expression at the mRNA level and suppresses cell cycle progression in NSPCs.


Subject(s)
Cell Cycle/drug effects , Neural Stem Cells/cytology , Phenanthrenes/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Animals , Cyclin B/drug effects , Cyclin B/genetics , Fibroblasts/drug effects , Genes, cdc/drug effects , Mice , Neural Stem Cells/drug effects , Neural Stem Cells/metabolism , RNA, Messenger/drug effects
4.
J Chemother ; 25(4): 229-38, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23816136

ABSTRACT

BACKGROUND: Few researches on increase of chemotherapy sensitivity by microRNA (miRNA) were reported. We aim to investigate exact role of miR-381 in chemotherapy sensitivity of 5-fluorouracil (5-FU) in renal cancer cells. METHODS: We investigated the cell survival, cell-cycle and apoptosis of 786-O and HK-2 cells treated with miR-381 and 5-FU. IC50 of 5-FU was calculated. To study apoptosis and G2/M arrest, we determined pHH3, mitotic index and caspase-3/7 activity. RESULTS: We showed that miR-381 combined with 5-FU inhibited proliferation and potentiated the anti-tumour efficacies of 5-FU at tolerated concentration in vitro. miR-381 combined with 5-FU led to Cdc2 activation, mitotic catastrophe, and cell apoptosis through inhibitory WEE1. WEE1 was also validated as the direct target of miR-381. IC50 of 5-FU decreased significantly in the presence of miR-381. CONCLUSION: miR-381 increases sensitivity of 786-O cells to 5-FU by inhibitory WEE1 and increase of Cdc2 activity.


Subject(s)
Carcinoma, Renal Cell/drug therapy , Cell Cycle Proteins/antagonists & inhibitors , Fluorouracil/pharmacology , Kidney Neoplasms/drug therapy , MicroRNAs/pharmacology , Molecular Targeted Therapy/methods , Nuclear Proteins/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Antineoplastic Combined Chemotherapy Protocols , Apoptosis/drug effects , CDC2 Protein Kinase , Carcinoma, Renal Cell/genetics , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cyclin B/drug effects , Cyclin-Dependent Kinases , Drug Synergism , Fluorouracil/therapeutic use , Gene Expression Regulation, Neoplastic , Humans , Kidney Neoplasms/genetics , MicroRNAs/therapeutic use , Nuclear Proteins/genetics , Protein-Tyrosine Kinases/genetics , Up-Regulation
5.
J Periodontal Res ; 48(1): 66-73, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22834967

ABSTRACT

BACKGROUND AND OBJECTIVE: Short-chain fatty acids, such as butyric acid and propionic acid, are metabolic by-products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated. MATERIAL AND METHODS: Human GFs were exposed to various concentrations of butyrate (0.5-16 mm) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide-staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2',7'-dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT-PCR using specific primers or to western blotting using specific antibodies, respectively. RESULTS: Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4-16 mm) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT-PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS. CONCLUSION: These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle-related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases.


Subject(s)
Butyrates/pharmacology , Fibroblasts/drug effects , Gingiva/drug effects , Reactive Oxygen Species/metabolism , Apoptosis/drug effects , Butyrates/toxicity , CDC2 Protein Kinase , Cell Culture Techniques , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cell Shape/drug effects , Cell Survival/drug effects , Coloring Agents , Cyclin B/drug effects , Cyclin B1/drug effects , Cyclin-Dependent Kinases , Fibroblasts/cytology , Flow Cytometry , Fluorescein-5-isothiocyanate , Fluoresceins , Fluorescent Dyes , G1 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/drug effects , Gingiva/cytology , Humans , M Phase Cell Cycle Checkpoints/drug effects , Propidium , Resting Phase, Cell Cycle/drug effects , cdc25 Phosphatases/drug effects
6.
Int Endod J ; 45(9): 848-58, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22486746

ABSTRACT

AIM: To evaluate the effect of TEGDMA on cell cycle progression as well as alterations of cell cycle-related gene and protein expression. METHODOLOGY: Human dental pulp cells were exposed to 0-5 mmol L(-1) TEGDMA for 24 h. Cytotoxicity was evaluated by 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Cell cycle progression was analysed by propidium iodide (PI) flow cytometry. Cell death pathway was surveyed by annexin V/PI dual-staining flow cytometry. The mRNA expression of cell cycle-related genes (cdc2, cyclinB1 and p21) and COX-2 was evaluated by reverse transcriptase-polymerase chain reaction, and their protein expression was evaluated by Western blotting. The production of PGE(2) and PGF(2α) in the culture medium was determined by enzyme-linked immunosorbent assay. RESULTS: Triethylene glycol dimethacrylate inhibited cellular growth and induced cell cycle deregulation in dental pulp cells. High-dose exposure provoked both necrotic and apoptotic cell death. The gene and protein expression of cdc2, cyclin B1 and cdc25C declined obviously whilst cells treated with 2.5 mmol L(-1) TEGDMA concurrent with the elevated expression of p21. The mRNA and protein expression of COX-2, along with production of PGE(2) and PGF(2α), are drastically raised by 2.5-5 mmol L(-1) TEGDMA. CONCLUSIONS: Triethylene glycol dimethacrylate induced cytotoxicity, cell cycle arrest and apoptosis in dental pulp cells, which was associated with the decline of cdc2, cyclin B1, cdc25C expression and elevation of p21 expression. Concomitantly, COX-2 expression, PGE(2) and PGF(2α) production increased. These effects may contribute to explain the pulpal damage and inflammation induced by TEGDMA after operative procedures.


Subject(s)
Cyclooxygenase 2/drug effects , Dental Materials/toxicity , Dental Pulp/drug effects , Polyethylene Glycols/toxicity , Polymethacrylic Acids/toxicity , Prostaglandins/biosynthesis , Annexin A5/pharmacology , Apoptosis/drug effects , CDC2 Protein Kinase , Cell Culture Techniques , Cell Cycle/drug effects , Cell Death/drug effects , Cell Proliferation/drug effects , Cell Shape/drug effects , Coloring Agents , Cyclin B/drug effects , Cyclin B1/drug effects , Cyclin-Dependent Kinase Inhibitor p21/drug effects , Cyclin-Dependent Kinases , Dental Pulp/cytology , Dinoprost/analysis , Dinoprostone/analysis , Enzyme Inhibitors/pharmacology , Flow Cytometry/methods , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Humans , Necrosis , Propidium , Tetrazolium Salts , Thiazoles , Time Factors , cdc25 Phosphatases/drug effects
7.
Mol Nutr Food Res ; 55(7): 1036-43, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21538854

ABSTRACT

SCOPE: We investigated the inhibition of pulmonary tumor formation through treatment with curcumin in transgenic mice. METHODS AND RESULTS: In this study, a strain of transgenic mice carrying human vascular endothelial growth factor A165 (hVEGF-A165) gene to induce pulmonary tumor was used as an in vivo cancer therapy model. We found that curcumin significantly reduced hVEGF-A165 overexpression to normal, specifically in Clara cells of the lungs of transgenic mice, and suppressed the formation of tumors. In addition, we demonstrated a relationship between curcumin treatment and the expression of VEGF, EGFR, ERK2, and Cyclin A at the transcriptional and translational levels. We also noticed a reduction of Cyclin A and Cyclin B after curcumin treatment that had an effect on the cell cycle. Curcumin-induced inhibition of Cyclin A and Cyclin B likely results in decreased progression through S and G2/M phases. These results demonstrated that the expression of proteins involved in the S to M phase transition in transgenic mice is suppressed by curcumin. CONCLUSION: A Data suggest that a blockade of the cell cycle may be a critical mechanism for the observed effects on vasculogenesis and angiogenesis following treatment with curcumin.


Subject(s)
Curcumin/pharmacology , Lung Neoplasms/drug therapy , Lung Neoplasms/physiopathology , Vascular Endothelial Growth Factor A/genetics , Animals , Blotting, Western , Cell Cycle/drug effects , Cell Cycle/physiology , Cyclin A/drug effects , Cyclin A/genetics , Cyclin B/drug effects , Cyclin B/genetics , Drug Screening Assays, Antitumor , Gene Expression Regulation, Neoplastic/drug effects , Genes, erbB-1/drug effects , Genetic Markers/drug effects , Humans , Lung Neoplasms/pathology , Mice , Mice, Transgenic , RNA, Messenger , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects
8.
Planta Med ; 77(10): 992-8, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21267808

ABSTRACT

In this study, we incubated human A549 lung cancer cells with quercetin-metabolite-enriched plasma (QMP) obtained from Mongolian gerbils 2 h after quercetin feeding (100 mg/kg body wt/week). We investigated the effects of QMP on the growth of A549 cells and the possible mechanisms for these effects. We found that QMP but not control plasma (CP) reduced the cell growth in A549 cells. QMP led to cell cycle arrest at the G (2)/M phase by downregulating the expression of cdk1 and cyclin B. QMP but not CP or quercetin itself significantly increased PPAR- γ expression (p < 0.05), which was accompanied by an increase of phosphatase and tensin homologue deleted on the chromosome ten level and a decrease of phosphorylation of Akt. Furthermore, quercetin-3-glucuronide and quercetin-3'-sulfate also significantly increased PPAR- γ expression in A549 cells. GW9662, a PPAR- γ antagonist, significantly suppressed the effects of 10 % QMP on cell proliferation and on the expression of cyclin B and cdk1. Taken together, these data suggest that the activation of PPAR- γ plays an important role, at least in part, in the antiproliferative effects of quercetin metabolites.


Subject(s)
Cell Division/drug effects , G2 Phase/drug effects , Lung Neoplasms/metabolism , PPAR gamma/metabolism , Quercetin/metabolism , Quercetin/pharmacology , Administration, Oral , Anilides/pharmacology , Animals , CDC2 Protein Kinase/drug effects , CDC2 Protein Kinase/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin B/drug effects , Cyclin B/metabolism , Gerbillinae , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Male , Oncogene Protein v-akt/drug effects , Oncogene Protein v-akt/metabolism , PPAR gamma/antagonists & inhibitors , PTEN Phosphohydrolase/drug effects , PTEN Phosphohydrolase/metabolism , Phosphorylation/drug effects , Plasma/metabolism , Quercetin/administration & dosage , Quercetin/analogs & derivatives , Up-Regulation/drug effects
9.
J Gastroenterol ; 44(10): 1055-63, 2009.
Article in English | MEDLINE | ID: mdl-19688288

ABSTRACT

BACKGROUND: In this study, we investigated the effects of 2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one (CHM-1) on cell viability, cell cycle arrest and apoptosis in CT-26 murine colorectal adenocarcinoma cells. METHODS: For determining cell viability, the MTT assay was used. CHM-1 promoted G2/M arrest by PI staining and flow cytometric analysis. Apoptotic cells were evaluated by DAPI staining. We used CDK1 kinase assay, Western blot analysis and caspase activity assays for examining the CDK1 activity and proteins correlated with apoptosis and cell cycle arrest. The in vivo anti-tumor effects of CHM-1-P were evaluated in BALB/c mice inoculated with CT-26 cells orthotopic model. RESULTS: CHM-1 induced CT-26 cell viability inhibition and morphologic changes in a dose-dependent and time-dependent manner and the approximate IC50 was 742.36 nM. CHM-1 induced significant G2/M arrest and apoptosis in CT-26 cells. CHM-1 inhibited the CDK1 activity and decreased CDK1, Cyclin A, Cyclin B protein levels. CHM-1 induced apoptosis in CT-26 cells and promoted increasing of cytosolic cytochrome c, AIF, Bax, BAD, cleavage of pro-caspase-9, and -3. The significant reduction of caspase-9 and -3 activity and increasing the viable CT-26 cells after pretreated with caspase-9 and -3 inhibitor indicated that CHM-1-induced apoptosis was mainly mediated a mitochondria-dependent pathway. CHM-1-P improved mice survival rate, and enlargement of the spleen and liver metastasis were significantly reduced in groups treated with either 10 mg/kg and 30 mg/kg of CHM-1-P and 5-FU in comparison to these of CT-26/BALB/c mice. CONCLUSIONS: Taken together, CHM-1 acted against colorectal adenocarcinoma cells in vitro via G2/M arrest and apoptosis, and CHM-1-P inhibited tumor growth in vivo.


Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , CDC2 Protein Kinase/antagonists & inhibitors , Cell Cycle/drug effects , Cell Survival/drug effects , Colorectal Neoplasms/drug therapy , Dioxoles/pharmacology , Quinolones/pharmacology , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Antineoplastic Agents/therapeutic use , Caspase 3/metabolism , Caspase 9/metabolism , Cell Division/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cyclin A/drug effects , Cyclin A/metabolism , Cyclin B/drug effects , Cyclin B/metabolism , Dioxoles/therapeutic use , Dose-Response Relationship, Drug , Flow Cytometry , G2 Phase/drug effects , Humans , Liver Neoplasms/secondary , Meiosis , Mice , Mice, Inbred BALB C , Mitochondria/drug effects , Mitochondria/metabolism , Quinolones/therapeutic use , Xenograft Model Antitumor Assays
10.
Anticancer Drugs ; 20(4): 230-7, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19288605

ABSTRACT

A number of polyamine analogs are currently used in various clinical trials as cancer treatment and it is important to investigate their effects not only on cancer cells but also on normal cells. Treatment with polyamine analogs depletes cells of polyamines and inhibits cell proliferation, but the analogs cannot take over the normal function of the natural polyamines in the cell. In this study, the normal-like breast epithelial cell line MCF-10A was treated with the polyamine analog N',N"-diethylnorspermine (DENSPM). The cells were then studied using a bromodeoxyuridine- DNA flow cytometry method as well as western blot. The ability of both normal-like and breast cancer cells to recover from DENSPM treatment was also studied. DENSPM treatment of MCF-10A cells resulted in a prolongation of the S and G2 +M phases, followed by a G1/S block. The p53/p21/RB1 pathway was involved in the G1/S block as shown by increased levels of p53 and p21 detected by western blot. Decreased levels of cyclin E1, cyclin A2, and cyclin B1 in DENSPM-treated cells can explain the prolongation of cell cycle phases that occurred before the G1/S block. We also show that MCF-10A cells rapidly recover from DENSPM-induced growth inhibition in contrast to four human breast cancer cell lines. The goal of cancer treatment is to cause minimal and reversible damage to normal cells, while cancer cells should be eliminated. Altogether, the data show that treatment with polyamine analogs spares normal cells, while negatively affecting the cancer cells.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Cell Cycle/drug effects , Spermine/analogs & derivatives , Blotting, Western , Breast Neoplasms/pathology , Bromodeoxyuridine , Cell Line , Cell Line, Tumor , Cyclin A/drug effects , Cyclin A/metabolism , Cyclin A2 , Cyclin B/drug effects , Cyclin B/metabolism , Cyclin B1 , Cyclin E/drug effects , Cyclin E/metabolism , DNA , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Flow Cytometry , Humans , Oncogene Proteins/drug effects , Oncogene Proteins/metabolism , Spermine/pharmacology
11.
Cytometry A ; 75(6): 535-46, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19235202

ABSTRACT

Using the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) for thymidine substitution instead of BrdU (5-bromo-2'-deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. We compared the effect of EdU and BrdU incorporation into SK-BR-3 and BT474 breast cancer cells and the impact on cell cycle kinetics, cell viability, and DNA damage. We found that EdU can be used not only for pulse but also for continuous cell labeling and henceforth in high resolution EdU/Hoechst quenching assays. BrdU and EdU proliferation assays based on click chemistry revealed comparable results. However, cell viability of SK-BR-3 breast cancer cells was highly affected by long term exposure to EdU. Both SK-BR-3 as well as BT474 cells show cell cycle arrests upon long term EdU treatment whereas only SK-BR-3 cells were driven into necrotic cell death by long term exposure to EdU. In contrast BT474 cells appeared essentially unharmed by EdU treatment in terms of viability. Consequently using EdU enables highly sensitive and quantitative detection of proliferating cells and facilitates even continuous cell cycle assessment. Nevertheless, potential cellular susceptibility needs to be individually evaluated.


Subject(s)
Cell Proliferation/drug effects , Deoxyuridine/analogs & derivatives , Flow Cytometry , Apoptosis , Bromodeoxyuridine/toxicity , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Line, Tumor , Cell Survival/drug effects , Cyclin B/drug effects , Cyclin B/metabolism , Cyclin B1 , DNA/metabolism , Deoxyuridine/toxicity , Female , Histones/drug effects , Histones/metabolism , Humans , Phosphorylation/drug effects , Phosphorylation/physiology
12.
Chem Biol Interact ; 179(2-3): 304-13, 2009 May 15.
Article in English | MEDLINE | ID: mdl-19159621

ABSTRACT

Arsenic compounds have been used as anti-cancer agents in traditional Chinese medicine. Ionizing radiation (IR) is one of the most effective tools in the clinical treatment of cancer. The induction of apoptotic cell death is a significant mechanism of tumor cells under the influence of radio-/chemotherapy, and resistance to these treatments has been linked to some cancer cell lines with a low propensity for apoptosis. A combination of different anti-tumoral treatment modalities is advantageous in limiting non-specific toxicity often observed by an exceedingly high dose of single regimen. The present study aimed at investigating the enhanced effects and mechanisms in cell cycle distribution and apoptosis of U937 cells, a human pre-monocytic leukemia cell line lacking functional p53 protein, after combination treatment with irradiation and As(2)O(3). Our results indicated that combined treatment led to activation of cdc-2, which is related to the expression of cyclin B. In addition, combined treatment increased apoptotic cell death in U937 cells, which is correlated with the induction of mitotic arrest, the increase in intracellular reactive oxygen species (ROS) generation, the decrease in B-cell leukemia/lymphoma 2 (Bcl-2) and B-cell leukemia/lymphoma XL (Bcl-XL) levels, the loss of mitochondria membrane potential, and the activation of caspase-3. We found that combining radiation and As(2)O(3) may be an effective strategy against p53-deficient leukemia cells.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/radiation effects , Arsenicals/pharmacology , Mitosis/drug effects , Mitosis/radiation effects , Oxides/pharmacology , Reactive Oxygen Species/metabolism , Arsenic Trioxide , Caspases/metabolism , Cell Cycle/drug effects , Cell Division/drug effects , Cell Division/radiation effects , Cyclin A/biosynthesis , Cyclin A/drug effects , Cyclin A/radiation effects , Cyclin B/biosynthesis , Cyclin B/drug effects , Cyclin B/radiation effects , Cytochromes c/drug effects , Cytochromes c/metabolism , Cytochromes c/radiation effects , Drug Screening Assays, Antitumor , G2 Phase/drug effects , G2 Phase/radiation effects , Humans , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Mitochondrial Membranes/radiation effects , Radiation, Ionizing , Tumor Cells, Cultured , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/drug effects , Tumor Suppressor Protein p53/radiation effects , U937 Cells
13.
J Nutr Biochem ; 20(8): 614-20, 2009 Aug.
Article in English | MEDLINE | ID: mdl-18835704

ABSTRACT

We recently demonstrated that treatment of three leukemic cell lines with an aqueous extract of cinnamon (CE) for 24 h produced dose-dependent arrests in the G2/M phase of the cell cycle. To accomplish the goal of understanding underlying mechanisms, we selected the cell line most responsive to the CE treatment to study the effects of the extract on signaling molecules regulating cell cycle progression. Cell cycle analyses were conducted on treated versus nontreated cells from 0-6 h. The percentages of cells in G2/M in CE-treated cells increased significantly from 11.0+/-1.0 to 23.6+/-1.4 after 6 h, while the percentage for nontreated cells remained unchanged (12.3+/-0.8). Multiparametric flow cytometric analyses were used to associate activation of p38 mitogen-activated protein kinase (MAPK) with cells arrested in G2/M, the size of these cells, and the presence or absence of cyclin B1. After 4 h, there was a 26% increase in the activated phosphorylated form of p38 MAPK in CE-treated cells compared with the nontreated control cells, with larger cells showing the greater increases. Although the proportion of CE-treated cells in G2/M was higher than controls, this population was shown to be less positive for cyclin B1 than the control G2/M population. Our results demonstrate that CE significantly modulated two signaling proteins, p38 MAPK and cyclin B, that regulate progression through G2/M. Overall, the data provide evidence that CE affects proliferation in a leukemic cell line by disrupting critical phosphorylating/dephosphorylating signaling events that propel cells through the G2/M phase.


Subject(s)
Cell Cycle/drug effects , Cinnamomum zeylanicum , Flavonoids/pharmacology , MAP Kinase Signaling System/drug effects , Phenols/pharmacology , p38 Mitogen-Activated Protein Kinases/drug effects , Cell Size/drug effects , Clone Cells , Cyclin B/drug effects , Cyclin B/genetics , Cyclin B1 , Flow Cytometry/methods , Gene Expression Regulation, Enzymologic , Humans , Jurkat Cells , Leukocyte Common Antigens/deficiency , Plant Extracts/pharmacology , Polyphenols , Tumor Cells, Cultured , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
14.
Cancer Chemother Pharmacol ; 64(3): 433-43, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19082595

ABSTRACT

PURPOSE: 17-(demethoxy), 17-allylamino geldanamycin (17-AAG) suppresses growth in some cancers by inhibiting Heat shock protein 90 (Hsp90). We examined the effects of 17-AAG-mediated Hsp90 inhibition on human hepatocellular carcinoma (HCC) growth in vitro and in vivo. METHODS: Human HCC cell lines, Hep3B and HuH7, were exposed to 17-AAG and cell viabilities and apoptosis were determined. Cell cycle profiles were analyzed and the G(2)/M cell cycle checkpoint proteins cdc2 and cyclin B1 were examined. Studies were performed to determine whether 17-AAG-mediated cdc2 decrease was due to altered gene expression, transcription, or protein degradation. The effects of 17-AAG on Hep3B and HuH7 xenograft growth in athymic nude mice were also examined. RESULTS: Hep3B and HuH7 treated with 17-AAG versus untreated controls showed decreased cell viability and increased apoptosis. Cells treated with 17-AAG also showed an increased fraction in G(2)/M phase and an associated decrease in cdc2 through protein degradation rather than through other mechanisms. Hsp90 inhibition by 17-AAG also decreased HCC xenograft growth in association with decreased cdc2 expression. CONCLUSIONS: 17-AAG-mediated inhibition of Hsp90 abrogates human HCC cell growth in vitro and in vivo through cdc2 decrease, which in turn induces G(2)/M cell cycle arrest and apoptosis. Hsp90 is a mediator of HCC growth and survival and its inhibition may serve as a potential treatment.


Subject(s)
Apoptosis/drug effects , Benzoquinones/pharmacology , Carcinoma, Hepatocellular/drug therapy , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Lactams, Macrocyclic/pharmacology , Liver Neoplasms/drug therapy , Animals , CDC2 Protein Kinase , Carcinoma, Hepatocellular/physiopathology , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Cyclin B/drug effects , Cyclin B/metabolism , Cyclin-Dependent Kinases , Female , G2 Phase/drug effects , Gene Expression Regulation/drug effects , Humans , Liver Neoplasms/physiopathology , Mice , Mice, Nude , Xenograft Model Antitumor Assays
15.
Article in English | MEDLINE | ID: mdl-19036348

ABSTRACT

We have studied in vivo, the effects of physiological androgen (11-ketotestosterone: 11-KT and testosterone: T) concentrations on the growth of cod previtellogenic oocytes and steroidogenic gene expression patterns. Immature female Atlantic cod were injected three times (days 0, 7 and 14) with 0.05, 0.5 and 5 mg/kg of 11-KT and T. The control group was injected with the carrier solvent (ethanol diluted 1:10 in sunflower oil). Quantitative histological analyses demonstrated growth and development of previtellogenic oocytes after exposure to androgens. The oocyte developmental effect of androgens was more pronounced in fish receiving 11-KT. Quantitative PCR analysis demonstrated dose- and androgen-specific modulation of mRNA expression for genes involved in steroidogenesis (StAR (steroidogenic acute regulatory) protein, P450scc (P450-mediated cholesterol side-chain cleavage), 20beta-HSD (20beta-hydroxysteroid dehydrogenase)) and cell growth control, namely--opioid growth factor receptor (OGF-R), progesterone receptor protein p23 (PR23P) and apoptosis-inducing TAF9-like domain 1 (TAF9). Messenger RNA species associated with the zona pelucida, namely--the zona pellucida protein A domain (ZPA) and egg envelope glycoprotein (EeG) were modulated based on dose and androgen type. Cyclin-B mRNA expression was not affected by androgen exposure. Interestingly, we showed recently that these transcripts were responsive to in vitro androgen exposure in previtellogenic cod ovary. In conclusion, the present study adds further information regarding the effects of androgens on the development of previtellogenic oocytes, suggesting androgen control of early oocyte growth in cod. The enhanced effects of 11-KT on oocyte growth support our hypothesis that non-aromatizable androgens play significant roles in the regulation of early previtellogenic oocyte growth and development.


Subject(s)
Cholesterol Side-Chain Cleavage Enzyme/genetics , Cortisone Reductase/genetics , Gadus morhua/genetics , Oocytes/drug effects , Phosphoproteins/genetics , Testosterone/pharmacology , Transcription, Genetic/drug effects , Androgens/chemistry , Animals , Cyclin B/drug effects , Cyclin B/genetics , Dose-Response Relationship, Drug , Egg Proteins/drug effects , Egg Proteins/genetics , Female , Gadus morhua/growth & development , Gene Expression Regulation, Developmental/drug effects , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/genetics , Oocytes/growth & development , RNA, Messenger/genetics , Receptors, Cell Surface/drug effects , Receptors, Cell Surface/genetics , Receptors, Opioid/drug effects , Receptors, Opioid/genetics , Receptors, Progesterone/drug effects , Receptors, Progesterone/genetics , Reverse Transcriptase Polymerase Chain Reaction , TATA-Binding Protein Associated Factors/drug effects , TATA-Binding Protein Associated Factors/genetics , Testosterone/analogs & derivatives , Testosterone/blood , Time Factors , Transcription, Genetic/genetics , Zona Pellucida Glycoproteins
16.
J Pharm Pharmacol ; 60(11): 1459-63, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18957166

ABSTRACT

We reported previously that oroxylin A, a natural product isolated from Scutellariae Radix, was a potent apoptosis inducer of human hepatoma HepG2 cells. In this study, cell-cycle arrest of BGC-823 human gastric carcinoma cells caused by oroxylin A has been investigated. Based on our 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometric analysis, treatment of BGC-823 cells with growth suppressive concentrations of oroxylin A caused an irreversible arrest in the G2/M phase of the cell cycle. Western blot analysis demonstrated that oroxylin A-induced cell-cycle arrest in BGC-823 cells was associated with a significant decrease in cdc2/p34, cyclin B1 and cyclin A expression. In addition, oroxylin A-treated cells decreased the expression of Cdk7, which was responsible for the low expression of M phase promoting factor (cyclin B1/Cdc2). The results suggested that oroxylin A induced G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 cells.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , CDC2 Protein Kinase/drug effects , Flavonoids/pharmacology , Gene Expression Regulation/drug effects , Blotting, Western , CDC2 Protein Kinase/metabolism , Cell Division/drug effects , Cell Line, Tumor , Cyclin A/drug effects , Cyclin A/metabolism , Cyclin B/drug effects , Cyclin B/metabolism , Cyclin B1 , Cyclin-Dependent Kinases/drug effects , Cyclin-Dependent Kinases/metabolism , Flow Cytometry , G2 Phase/drug effects , Humans , Stomach Neoplasms/drug therapy , Stomach Neoplasms/pathology , Tetrazolium Salts , Thiazoles , Cyclin-Dependent Kinase-Activating Kinase
17.
Int J Oncol ; 33(3): 613-21, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18695893

ABSTRACT

In mammalian cells, the H2AX histone is rapidly phosphorylated upon the induction of DNA double strand breaks and promotes their repair, which is required for preserving genomic integrity. Etoposide is an inhibitor of DNA topoisomerase II, which causes DNA breaks and induces H2AX phosphorylation. To elucidate whether H2AX may affect cellular sensitivity to etoposide, we studied the response to this agent in immortalized embryonic fibroblasts derived from H2AX knockout mice. Clonogenic assays in cells treated with the drug revealed a greater sensitivity of H2AX null cells compared to wild-type cells, possibly due to the persistence of a higher number of DNA breaks, as detected with the comet assay. In both cell lines, etoposide induced micronuclei formation and nuclear fragmentation; however, in H2AX deficient cells nuclear fragmentation was observed at a lower drug concentration. Flow cytometric analysis showed that etoposide induced a G2/M cell cycle arrest in both cell lines, which occurred at lower drug concentrations in H2AX deficient cells. G2/M arrest was paralleled by an accumulation of cyclin A and cyclin B1, suggesting that treated cells are not able to complete cell cycle correctly and undergo cell death. Taken together, our observations suggest that H2AX takes part to the cellular response to etoposide and confirm its role in the maintenance of genome stability.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Drug Resistance, Neoplasm/physiology , Etoposide/pharmacology , Fibroblasts/drug effects , Histones/metabolism , Animals , Apoptosis/drug effects , Blotting, Western , Cell Cycle , Comet Assay , Cyclin A/biosynthesis , Cyclin A/drug effects , Cyclin B/biosynthesis , Cyclin B/drug effects , Flow Cytometry , Histones/genetics , Mice , Mice, Knockout , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/biosynthesis , Poly(ADP-ribose) Polymerases/drug effects , Topoisomerase II Inhibitors
18.
J Neurosci Res ; 86(11): 2476-87, 2008 Aug 15.
Article in English | MEDLINE | ID: mdl-18438935

ABSTRACT

Reactivation of the cell cycle, including DNA replication, might play a major role in Alzheimer's disease. In this study, we report that the expressions of Swedish double mutation of amyloid precursor protein (Swe-APP) or of the APP intracellular domain (AICD) into nerve growth factor (NGF)-differentiated PC12 cells or rat primary cortical neurons increased mRNA and protein levels of cyclin D1 and cyclin B1. Treatment with lithium chloride (a glycogen synthase kinase-3beta inhibitor) down-regulated cyclin B1 induced by Swe-APP expression but up-regulated cyclin D1 expression induced by Swe-APP, suggesting that glycogen synthase kinase-3beta activity is involved in these expression changes of cyclins D1 and B1. Swe-APP, which is a prevailing cause of familial Alzheimer's disease, is well known to increase amyloid beta peptide production both in vitro and in vivo, but the underlying molecular means whereby it leads to the pathogenesis of AD remains unknown. The finding that cyclin D1 and B1 expressions were up-regulated by Swe-APP in in vitro cultured cells was substantiated in the brain tissues of Tg2576 mice, which harbor the Swe-APP mutation. These results suggest that some disturbances in cell cycle regulation may be involved in Swe-APP or AICD-induced neurodegeneration and that these contribute to the pathogenesis of AD.


Subject(s)
Amyloid beta-Protein Precursor/genetics , Brain/metabolism , Cyclin B/biosynthesis , Cyclin D1/biosynthesis , Neurons/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/physiopathology , Animals , Blotting, Western , Brain/physiopathology , Cell Cycle/physiology , Cyclin B/drug effects , Cyclin B1 , Cyclin D1/drug effects , Enzyme Inhibitors/pharmacology , Humans , Immunohistochemistry , Immunoprecipitation , Mice , Mice, Transgenic , Mutation , Neurons/drug effects , PC12 Cells , Rats , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation
19.
J Cell Biochem ; 104(4): 1181-91, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18247328

ABSTRACT

Lithium, a therapeutic agent for bipolar disorder, can induce G2/M arrest in various cells, but the mechanism is unclear. In this article, we demonstrated that lithium arrested hepatocellular carcinoma cell SMMC-7721 at G2/M checkpoint by inducing the phosphorylation of cdc2 (Tyr-15). This effect was p53 independent and not concerned with the inhibition of glycogen synthase kinase-3 and inositol monophosphatase, two well-documented targets of lithium. Checkpoint kinase 1 (Chk1), a critical enzyme in DNA damage-induced G2/M arrest, was at least partially responsible for the lithium action. The lithium-induced phosphorylation of cdc2 and G2/M arrest was abrogated largely by SB218078, a potent Chk1 inhibitor, as well as by Chk1 siRNA or the over-expression of kinase dead Chk1. Furthermore, lithium-induced cdc25C phosphorylation in 7721 cells and in vitro kinase assay showed that the activity of Chk1 was enhanced after lithium treatment. Interestingly, the increase of Chk1 activity by lithium may be independent of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) kinase. This is because no elevated phosphorylation on Chk1 (Ser-317 and Ser-345) was observed after lithium treatment. Moreover, caffeine, a known ATM/ATR kinase inhibitor, relieved the phosphorylation of cdc2 (Tyr-15) by hydroxyurea, but not that by lithium. Our study's results revealed the role of Chk1 in lithium-induced G2/M arrest. Given that Chk1 has been proposed to be a novel tumor suppressor, we suggest that the effect of lithium on Chk1 and cell cycle is useful in tumor prevention and therapy.


Subject(s)
Carcinoma, Hepatocellular/pathology , Cell Cycle/drug effects , Cyclin B/metabolism , Lithium/pharmacology , Protein Kinases/physiology , CDC2 Protein Kinase , Cell Division , Cell Line, Tumor , Checkpoint Kinase 1 , Cyclin B/drug effects , Cyclin-Dependent Kinases , G2 Phase , Humans , Phosphorylation
20.
Oncol Rep ; 19(1): 275-80, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18097607

ABSTRACT

Diallyl disulfide (DADS), a major organosulfur compound of garlic oil, is known to have an anticancer effect on human cancer cells. However, the exact mechanisms of this anticancer activity remain unclear. Here, we investigate the effects of DADS on cell cycle progression in human colon cancer HCT-116 cells by exploring the role played by regulatory molecules such as p53 and cyclin B1. Treatment of HCT-116 cells by DADS induced a marked growth inhibition with a slight reduction in viability and induced transient cell cycle arrest in the G2/M phase. Cyclin B1 is thought to play an important role in this process, as the DADS-induced G2/M phase arrest occurs with the increase of cyclin B1 expression. DADS also significantly induced the expression of p53, which contributes to cell cycle arrest in cancer cells, at a late time-point of 24 h. In addition, knockdown of p53 by siRNA did not affect cell cycle arrest, its reversibility, or the expression of cyclin B1 in the G2/M phase induced by DADS. Based on these results we conclude that, with the dynamic expression of cyclin B1, DADS induces reversible cell cycle arrest in the G2/M phase of HCT-116 cells through a p53-independent mechanism.


Subject(s)
Allyl Compounds/pharmacology , Antineoplastic Agents/pharmacology , Cell Division/drug effects , Colonic Neoplasms/metabolism , Disulfides/pharmacology , G2 Phase/drug effects , Tumor Suppressor Protein p53/metabolism , Blotting, Western , Cell Proliferation/drug effects , Cyclin B/drug effects , Cyclin B1 , Flow Cytometry , HCT116 Cells , Humans , RNA, Small Interfering , Transfection
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