Long-term spaceflight affects numerous organ systems in the body, including metabolic dysfunction. Recently, ample evidence has demonstrated that the liver is a vulnerable organ during spaceflight. However, the changes in hepatocyte proliferation and cell cycle control under microgravity remain largely unexplored. In the present study, we first confirmed that the serum levels of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, biochemical markers of liver function, were altered in rats under tail suspension (TS) conditions to simulate microgravity, as shown in previous reports. Next, we demonstrated that the cell proliferation activity, determined by Ki67, PCNA and PH3, was significantly decreased at the different TS time points (TS for 14, 28 and 42 days) compared with that in the control group. Consistently, the positive cell cycle regulators Ccna2, Ccnd1, Cdk1, Cdk2 and cyclin D3 were also significantly decreased in the TS groups as shown by quantitative real-time PCR and western blotting analysis. Subsequent analysis revealed that the aberrant hepatocyte proliferation inhibition under simulated microgravity was associated with the upregulation of miR-223 in the liver. We further found that miR-223 inhibited the proliferation of Hepa1-6 cells and identified CDK2 and CUL1 as its direct targets. In addition, the decreased expression of CDK2 and CUL1 was negatively correlated with the level of p27 in vitro and in vivo, which may have been responsible for retarding hepatocyte proliferation. Collectively, these data indicate that upregulation of miR-223 was associated with the inhibition of liver cell growth and reveal the role of miR-223 in rat hepatocyte proliferation disorders and the pathophysiological process under simulated microgravity.
Alanine Transaminase ; Alkaline Phosphatase ; Animals ; Aspartate Aminotransferases ; Biomarkers ; Blotting, Western ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Proliferation ; Cyclin D3 ; Hepatocytes* ; Hindlimb Suspension ; In Vitro Techniques ; Liver* ; Proliferating Cell Nuclear Antigen ; Rats* ; Real-Time Polymerase Chain Reaction ; Space Flight ; Up-Regulation* ; Weightlessness*

OBJECTIVETo investigate the effects of eukaryotic translation initiation factor 5A2 (EIF5A2) down-regulation by small interfering RNA (siRNA) on aggressiveness of human gastric cancer cell and its potential mechanisms.

METHODSThe expressions of EIF5A2 in human gastric cancer cell lines (MKN28 and HGC27) and immortalized gastric mucosal epithelial cells (GES-1) were measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting. EIF5A2 gene in MKN28 cells was silenced by RNA interference and the inhibitory effect was evaluated by both qRT-PCR and Western blotting. Cell proliferation was assessed by CCK-8 assay. Cell migration and invasion were assessed by Transwell assay. The possible downstream targets of EIF5A2, such as CyclinD1, CyclinD3, matrix metallopeptidase-9 (MMP-9), E-cadherin, vimintin, C-myc, and metastasis-associated protein 1 (MTA1) expression levels, were examined by Western blotting.

RESULTSHigh expressions of EIF5A2 were found in MKN28 cells and human gastric adenocarcinoma tissues. Both EIF5A2 mRNA and protein expression in MKN28 cells were significantly down-regulated by siRNA#1 and siRNA#2, especially siRNA#1. Knockdown of EIF5A2 caused an apparent suppression of MKN28 cell proliferation (all P<0.01), migration (P<0.001), and invasion (P<0.001). After the knockdown of EIF5A2 in MKN28 cells, E-cadherin levels were upregulated, whereas vimentin, Cyclin D1, Cyclin D3, C-myc and MTA1 levels were downregulated.

CONCLUSIONKnockdown of EIF5A2 may inhibit MKN28 cell proliferation by downregulating the CyclinD1 and CyclinD3 and suppressing the cell migration and invasion by inhibiting MTA1, C-myc and epithelial-mesenchymal transition.

Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin D3 ; metabolism ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Genes, myc ; Histone Deacetylases ; metabolism ; Humans ; Peptide Initiation Factors ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; RNA-Binding Proteins ; genetics ; Repressor Proteins ; metabolism ; Stomach Neoplasms ; pathology

BACKGROUNDClinical outcome in patients with primary central nervous lymphoma (PCNSL) is variable and poorly predictable. This study investigated the association of clinical features and immune markers with prognosis of patients with PCNSL.

METHODSOne hundred and fifteen newly diagnosed PCNSL patients at the study institution were considered eligible for this study. Clinical characteristics and biochemical assay data were collected. Immunohistochemical staining of Cyclin D3, Cyclin E, Foxp1, and LMO2 were performed. All cases were followed-up regularly.

RESULTSThe common sites of involvement were frontal lobe (54.8%) and thalamus (16.5%). Diffuse large B-cell lymphoma composed of 96.5% of the cases. The median overall survival was 22 (4 - 41) months, and the 5-year survival rate was 22.8%. Age > 65 years, serum globulin > 40 g/L, large size of tumor, lymphocyte count ≥ 1 × 10(9)/L, and expression of Cyclin D3 and Cyclin E were associated with poor prognosis of PCNSL. Expressions of Foxp1, LMO2, and CD44 were not related to the survival. Expression of Cyclin E, large tumor size, and high serum globulin were independent prognostic factors for PCNSL.

CONCLUSIONSPCNSL prognosis is relatively poor. Age, high tumor burden, higher lymphocyte count, expression of Cyclin D3, and Cyclin E are inferior prognostic factors for PCNSL.

Adaptor Proteins, Signal Transducing ; metabolism ; Adult ; Aged ; Central Nervous System Neoplasms ; metabolism ; pathology ; Cyclin D3 ; metabolism ; Cyclin E ; metabolism ; Female ; Forkhead Transcription Factors ; metabolism ; Humans ; Immunohistochemistry ; LIM Domain Proteins ; metabolism ; Lymphoma ; metabolism ; pathology ; Male ; Middle Aged ; Prognosis ; Proto-Oncogene Proteins ; metabolism ; Repressor Proteins ; metabolism ; Retrospective Studies ; Young Adult

PURPOSE: Cyclins, and their associated cyclin dependent kinases, regulate progression of the cell cycle through the G1 phase and into the S-phase during the DNA replication process. Cyclin E regulation is an important event in cell proliferation. Despite its importance, abnormalities of these genes and their protein products have yet to be found in lits asoociation with lung cancer. MATERIALS AND METHODS: The relationships between expression of cyclin A, cyclin B1, cyclin D1, cyclin D3, and cyclin E and clinicopathologic factors were investigated in 103 cases with non-small cell carcinomas, using immunohistochemical analysis. RESULTS: The positive immunoreactivity was observed in 51 cases (50%) for cyclin A, 33 cases (32%) for cyclin B1, 83 cases (81%) for cyclin D1, 19 cases (18%) for cyclin D3, and 11 cases (11%) for cyclin E. Expression of cyclin E was significant for lymph node metastasis (p=0.004, Chi-square test). There was no relationship between cyclin A, B1, D1, and E and histological typing, tumor size, lymph node metastasis, or pathological tumor, node and metastasis staging. CONCLUSION: These findings suggest that the expression of cyclin E played a role, to some degree, in the lymph node metastasis.
Adenocarcinoma ; Carcinoma, Squamous Cell ; Cell Cycle ; Cell Proliferation ; Cyclin A ; Cyclin B1 ; Cyclin D1 ; Cyclin D3 ; Cyclin E ; Cyclin-Dependent Kinases ; Cyclins ; DNA Replication ; G1 Phase ; Lung ; Lung Neoplasms ; Lymph Nodes ; Neoplasm Metastasis

Eugenol is an essential oil found in cloves and cinnamon that is used widely in perfumes. However, the significant anesthetic and sedative effects of this compound have led to its use also in dental procedures. Recently, it was reported that eugenol induces apoptosis in several cancer cell types but the mechanism underlying this effect has remained unknown. In our current study, we examined whether the cytotoxic effects of eugenol upon human melanoma G361 cells are associated with cell cycle arrest and apoptosis using a range of methods including an XTT assay, Hoechst staining, immunocytochemistry, western blotting and flow cytometry. Eugenol treatment was found to decrease the viability of the G361 cells in both a time- and dose-dependent manner. The induction of apoptosis in eugenol-treated G361 cells was confirmed by the appearance of nuclear condensation, the release of both cytochrome c and AIF into the cytosol, the cleavage of PARP and DFF45, and the downregulation of procaspase-3 and -9. With regard to cell cycle arrest, a time-dependent decrease in cyclin A, cyclin D3, cyclin E, cdk2, cdk4, and cdc2 expression was observed in the cells after eugenol treatment. Flow cytometry using a FACScan further demonstrated that eugenol induces a cell cycle arrest at S phase. Our results thus suggest that the inhibition of G361 cell proliferation by eugenol is the result of an apoptotic response and an S phase arrest that is linked to the decreased expression of key cell cycle-related molecules.
Apoptosis ; Blotting, Western ; Caspase 3 ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Proliferation ; Cinnamomum zeylanicum ; Cyclin A ; Cyclin D3 ; Cyclin E ; Cyclins ; Cytochromes c ; Cytosol ; Down-Regulation ; Eugenol ; Flow Cytometry ; Humans ; Hypnotics and Sedatives ; Immunohistochemistry ; Melanoma ; S Phase ; Syzygium

BACKGROUND AND OBJECTIVEPrecursor T lymphoblastic lymphoma (T-LBL) is a highly aggressive lymphoma. Myeloid antigen expression was found in some of the patients, and its clinical significance is worth studying. This study was to compare the clinical features, short-term efficacy and survival of T-LBL patients with or without myeloid antigen expression so as to evaluate its prognostic significance.

METHODSForty-five T-LBL patients, with a median age of 14 years, were treated at Sun Yet-sen University Cancer Center between January 2000 and July 2008. These patients were divided into myeloid antigen-positive group (My(+) group) and myeloid antigen-negative group (My(-) group) based on the flow cytometric (FCM) analysis in bone marrow or pleural fluid. Myeloid antigen expression and its correlation with the short-term efficacy and overall survival were assessed in the two groups.

RESULTSThere were 18 patients (40.0%) in the My(+) group and 27 (60.0%) in the My(-) group. The myeloid antigen expression was negatively correlated with the initial level of lactate dehydrogenase (LDH), but not with other clinical features. The remission rate was lower in the My(+) group than in the My(-) group (38.8% vs. 70.3%, P = 0.028). The 2-year overall survival rate was lower in the My(+) group than in the My(-) group (51.9% vs. 78.7%, P = 0.036). By age subgroup analysis, there were no differences in response and survival rate among children and adolescents with or without myeloid antigen expression. But the remission rate and the 2-year overall survival rate were significantly lower in adult patients with myeloid antigen expression than in patients without it. Univariate and multivariate analysis demonstrated that age and myeloid antigen expression were adverse prognostic factors.

CONCLUSIONMyeloid antigen expression is a predictor of a poor response to chemotherapy, and adverse prognostic factor in adult T-LBL, but not in children with T-LBL.

Adolescent ; Adult ; Age Factors ; Aged ; Antigens, CD7 ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Asparaginase ; therapeutic use ; Child ; Cyclin D3 ; metabolism ; Cyclophosphamide ; therapeutic use ; Cytarabine ; therapeutic use ; Daunorubicin ; therapeutic use ; Doxorubicin ; therapeutic use ; Etoposide ; therapeutic use ; Female ; Follow-Up Studies ; Humans ; Male ; Mercaptopurine ; therapeutic use ; Methotrexate ; therapeutic use ; Middle Aged ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; immunology ; Prednisone ; therapeutic use ; Proportional Hazards Models ; Remission Induction ; Survival Rate ; Transcription Factors ; metabolism ; Vincristine ; therapeutic use ; Young Adult

Chios gum mastic (CGM) is obtained from the stem and leaves of Pistacia lentiscus trees and has been extensively used for centuries in Mediterranean and Middle Eastern countries, both as a dietary supplement and herbal remedy. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying modulation of cell cycle and induction of apoptosis in YD9 human oral squamous carcinoma cell line treated with CGM. The viability of YD9 cells and human normal keratinocyes (HaCaT cells), and the growth inhibition of YD9 cells were assessed by the MTT assay and clonogenic assay respectively. The hoechst staining and DNA electrophoresis were conducted to observe the YD9 cells undergoing apoptosis. YD9 cells were treated with CGM, and Western blotting, immunocytochemistry, confocal microscopy and FACScan flow cytometry were conducted. Mitochondrial membrane potential change and proteasome activity were measured. CGM treatment on YD9 cells resulted in a does-dependent inhibition of cell growth and induced apoptotic cell death. And tested YD9 cells showed several lines of apoptotic manifestation. Flow cytometric analysis revealed that CGM resulted in G1 arrest in cell cycle progression which was associated with decrease in the protein expression of cyclin D1, cyclin D3, Cdk2 and Cdk4, and increase in the protein expression of p21(WAF1/CIP1) and p53. These results demonstrate that CGM induces G1 the cell cycle arrest via the modulation of cell cycle-related proteins, and apoptosis via mitochondria and caspase pathway in YD9 cells, suggesting that CGM can be considered as a novel therapeutic strategy for human oral squamous cell carcinoma from its strong cell cycle arrest and apoptosis-inducing activity.
Apoptosis ; Blotting, Western ; Carcinoma, Squamous Cell ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Death ; Cell Line ; Cyclin D1 ; Cyclin D3 ; Dietary Supplements ; DNA ; Electrophoresis ; Flow Cytometry ; Gingiva ; Humans ; Immunohistochemistry ; Membrane Potential, Mitochondrial ; Microscopy, Confocal ; Mitochondria ; Pistacia ; Proteasome Endopeptidase Complex ; Proteins ; Resins, Plant ; Trees

OBJECTIVETo investigate the anticancer effects of betulinic acid (BA) on Jurkat cells in vitro and its molecular mechanism.

METHODSThe effects of betulinic acid on the growth of Jurkat cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was assessed by Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effect of betulinic acid on the cell cycle of Jurkat cells was studied by propidium iodide staining. RT-PCR and Western blot were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid.

RESULTSThe proliferation of Jurkat cells was decreased in betulinic acid-treated group at a 24 h IC50 value of 70.0 micromol/L. The effect of betulinic acid to induce apoptosis in Jurkat cells was in a time- and dose-dependent manner. Jurkat cells treated with betulinic acid showed an increase of G0/G1 phase and decrease of S phase. The Jurkat cells treated with 0, 20, 60, 100 micromol/L betulinic acid for 24 h, showed an increased G0/G1 phase from 31.0% to 58.8%, whereas decreased S phase from 61.5% to 35.8%, respectively. PBMC was less sensitive to the cytotoxic effect of betulinic acid than Jurkat cells. The expression of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid.

CONCLUSIONBetulinic acid can inhibit the proliferation of Jurkat cells by regulating the cell cycle that arrests cells at G0/G1 phase and induces apoptosis in Jurkat cells. The antitumor effects of betulinic acid may be related to down-regulation of the expression of cyclin D3 and bcl-xl.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Betula ; chemistry ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin D3 ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Jurkat Cells ; Plant Bark ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; metabolism ; Triterpenes ; pharmacology ; bcl-X Protein ; genetics ; metabolism

OBJECTIVETo investigate the role of bcl-6 gene rearrangement and bcl-6 expression in three molecular subgroups of diffuse large B-cell lymphoma (DLBCL) and its clinicopathological significance.

METHODSTissue microarray including 163 newly diagnosed DLBCL was constructed. Fluorescence in situ hybridization (FISH) was performed to detect the bcl-6 gene rearrangement and immunohistochemistry (EnVision method) was used to evaluate the expression of bcl-6, Ki-67, cyclin D3, Geminin and P27(Kip1) proteins in DLBCL. The association with clinicopathological features was analyzed.

RESULTSOne hundred and forty nine of 163 cases were further classified into three molecular subgroups: 40 cases of germinal center B-cell-like (GCB) type, 75 cases of activated non-germinal center B-cell-like (ABC) type, 34 cases of Type 3. Of these 149 cases, FISH for bcl-6 gene rearrangement was successful in 118 cases. bcl-6 gene rearrangement was observed in 33 of 118 (28.0%) cases. The bcl-6 gene rearrangement was more frequently seen in the ABC subgroup (22/62, 35.5%) than in GCB (6/31, 19.4%) and Type 3 subgroups (5/25, 20.0%, P=0.16). The correlation of bcl-6 gene rearrangement and expression of its encoded protein was further analyzed. Most of DLBCL (26/33, 78.8%) with bcl-6 gene rearrangement presented with overexpression of its encoded protein, which was higher than those without bcl-6 gene rearrangement (53/84, 62.4%, P=0.088). DLBCL with bcl-6 gene rearrangement (24/33, 72.7%) more frequently expressed cyclin D3, and had a higher proliferative activity than those without bcl-6 gene rearrangement (37/81, 45.7% , P=0.009). Twenty-nine of 33 (87.9%) cases of DLBCL with bcl-6 gene rearrangement presented with advanced stage (Ann Arbor stage III/IV), which was higher than those without bcl-6 gene rearrangement (65/85, 76.5% , P=0.167). Univariate Cox proportional hazards regression analysis showed that bcl-6 gene rearrangement was associated with an increased relative risk (at 1.842) of death in DLBCL cases compared with those without bcl-6 gene rearrangement.

CONCLUSIONOverexpression of bcl-6 protein caused by bcl-6 gene rearrangement may play some important roles in the development and/or progression of a subset of DLBCL.

B-Lymphocytes ; pathology ; Chromosomes, Human, Pair 14 ; Cyclin D3 ; genetics ; Gene Rearrangement ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lymphoma, B-Cell ; diagnosis ; genetics ; Lymphoma, Large B-Cell, Diffuse ; diagnosis ; genetics ; metabolism ; pathology ; Neoplasm Staging ; Prognosis ; Proto-Oncogene Proteins c-bcl-6 ; genetics ; Translocation, Genetic

The anti-cancer effects of betulinic acid (BA) on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 mumol/L. Betulinic acid induced apoptosis of Jurkat cells in a time- and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G(0)/G(1) phase and decrease in S phase. After treatment with 0, 20, 60, 100 mumol/L betulinic acid for 24 h, the number of Jurkat cells was increased from (31.00+/-1.25)% to (58.84+/-0.32)% in G(0)/G(1) phase, whereas it was decreased from (61.45+/-1.04)% to (35.82+/-1.95)% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is concluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle, arrest cells at G(0)/G(1) phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl.
Antineoplastic Agents, Phytogenic/*pharmacology ; Apoptosis/*drug effects ; Cell Proliferation/*drug effects ; Cyclin D3/metabolism ; Down-Regulation/drug effects ; Jurkat Cells ; Triterpenes/*pharmacology ; bcl-X Protein/metabolism