OBJECTIVETo investigate the effect of Oviductus Ranae (OR) on the expressions of CyclinD1, CDK6 and P15 in the liver of aged male rats.

METHODSEighteen male SD rats were randomly divided into 3 equal groups, namely the OR group, VE group and ageing model group. The rats received subcutaneous injection of D-galactose for 6 weeks to establish the aging models, and another 6 rats were injected daily with normal saline (NS) to serve as the normal control group. From the third week of the experiment, the rats were given oral OR or Vitamin E (VE) accordingly till the sixth week. After completion of the drug administration, all the rats were sacrificed for detecting the expressions of CyclinD1, CDK6 and P15 in the liver tissue by Western blotting.

RESULTSThe relative expression levels of CyclinD1, CDK6 and P15 in the liver of the rats in the OR group were 41.73-/+0.54, 23.29-/+0.30 and 1.49-/+0.30, respectively, significantly up-regulated as compared with those in the ageing model group (P<0.01). The expressions of the proteins were obviously down-regulated in the model group in comparison with those in the normal control group.

CONCLUSIONSOR treatment can lower the expressions of Cyclin D1 and CDK6 in the liver to enhance the liver cell proliferation in aged male rats. OR also promotes the expression of P15 through a feedback mechanism to prevent excessive proliferation of the cells. The effect of OR against ageing is mediated possibly by up-regulation of the proteins associated with the cell proliferation in the liver, a mechanism different from that of VE.

Aging ; metabolism ; Animals ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 6 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Liver ; metabolism ; Male ; Materia Medica ; pharmacology ; Rats

BACKGROUND/AIMS: Eukaryotic cell cycle is regulated by signal transduction pathways mediated by complexes of cyclin dependent kinases (CDKs) and their partner cyclins, or by interaction with CDK inhibitors. Thioacetamide (TA) is a weak hepatocarcinogen causing several types of liver damage in a dose dependent manner and ultimately producing malignant transformation. We investigated alterations of expression of cell cycle regulators in the rat liver, involved in G1 entry and progression during TA administration. METHODS: We studied expression patterns of cyclin D1, CDK4, CDK6, p21(CIP1) and p16(INK4a) during daily intraperitoneal injection of low dose TA (50 mg/kg) till 7 day. We used western blot and immunohistochemistry for detection. RESULTS: Expression of cyclin D1, CDK4, CDK6 and p21(CIP1) increased from 6 hour and peaked at 2, 3 day, then decreased next 2 days, and re-increased at 6 day. Cytoplasmo-nuclear translocation of cyclin D1 and p21(CIP1) was evident within 1 day and prominent at 2 and 7 day. Expression of p16(INK4a) increased immediately after TA treatment and remarkably increased from 3 day and progressed till 7 day, showing cytoplasmic location, suggestive of inactive form. Most of in situ immunoreactions occurred at the centrilobular hepatocytes. Concomitant nuclear translocation of p21(CIP1) and cyclin D1, different with p16(INK4a) suggests that p21(CIP1) might be a transporter for nuclear translocation rather than cell cycle inhibitor. CONCLUSIONS: Daily administration of low dose TA makes cell cycle open and G1 progress, possibly due to cyclin D1, CDK4 and CDK 6, their transporter p21(CIP1), and inactive p16(INK4a), which occur at quiescent hepatocytes, not stem cells.
Animals ; Cell Cycle Proteins/*metabolism ; Cyclin D1/metabolism ; Cyclin-Dependent Kinase 4/metabolism ; Cyclin-Dependent Kinase 6/metabolism ; Cyclin-Dependent Kinase Inhibitor p16/metabolism ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; G1 Phase ; Immunohistochemistry ; Liver/*drug effects/enzymology/metabolism ; Liver Diseases/chemically induced/metabolism/pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Thioacetamide/*toxicity

OBJECTIVETo explore the expression of CDK6 in nasopharyngeal carcinoma (NPC) and explore its clinical significance.

METHODSImmunohistochemistry was used to examine the differential expression of CDK6 protein in 101 NPC and 30 nasopharyngeal tissues, and the correlation of CDK6 expression with the clinical characteristics was analyzed in NPC cases.

RESULTSImmunohistochemistry demonstrated that CDK6 protein was a co-expressed factor in the cytoplasm and cell nuclei. CDK6 was expressed predominantly in the cytoplasm in nasopharyngeal tissues, but in NPC tissues, CDK6 was co-expressed predominantly in the cytoplasm and nuclei. Compared to the nasopharyngeal tissues, NPC tissues showed significantly up-regulated CDK6 expression (P=0.009) in positive correlation with tumor size (P=0.020) and clinical stages (P=0.0039).

CONCLUSIONSIncreased CDK6 protein expression is an unfavorable factor that promotes the development and progression of NPC.

Carcinoma ; Cell Nucleus ; Cyclin-Dependent Kinase 6 ; metabolism ; Cytoplasm ; Disease Progression ; Humans ; Immunohistochemistry ; Nasopharyngeal Neoplasms ; metabolism ; Up-Regulation

OBJECTIVETo assess the effect of small interfering RNA (siRNA)-mediated suppression of CDK6 expression on the proliferation and cell cycles of nasopharyngeal carcinoma (NPC) cells in vitro.

METHODSQRT-PCR was used to examine the differential expression of CDK6 in 30 NPC tissues and 18 normal nasopharyngeal tissues. A siRNA targeting CDK6 was transfected in NPC CNE2 cells, and MTT assay and flow cytometry were used to analyze the changes in cell proliferation and cell cycle distribution. Western blotting was used to examine the expressions of the cell cycle-related factors.

RESULTSCompared with normal nasopharyngeal tissues, NPC tissues showed an increased expression of CDK6 mRNA. Knocking down CDK6 expression obviously inhibited tumor cell growth and cell cycle transition from G1 to S phase and caused reduced expressions of CDK4, CCND1, and E2F1 and enhanced expression of the tumor suppressor p21.

CONCLUSIONNPC tissues overexpress CDK6. Knocking down CDK6 expression inhibits the growth and cell cycle transition of NPC cells in vitro by inhibiting the expressions of CDK4, CCND1, and E2F1 and upregulating tumor suppressor p21 expression.

Carcinoma ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase 6 ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Nasopharyngeal Neoplasms ; pathology ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Up-Regulation

OBJECTIVETo investigate the impact of the ischemic postconditioning on the tumor necrosis factor (TNF)-α/IL-6/signal transducers and activators of transcription (STAT)-3 signal pathway of liver regeneration.

METHODSNinety healthy clean grade male Sprague-Dawley rats weighting 230 to 280 g were selected and assigned into three groups randomly: group I subtotal hepatectomy (SH), group II ischemia reperfusion (IR), group III ischemic postconditioning (IPO). The left and middle liver was resected, and the remnant liver was treated as followed: the blood flow was not blocked in SH group, but blocked 30 minutes in IR group, then reperfused; IPO groups received three cycles of 30 s-30 s intermittent interruptions of blood flow at onset of reperfusion. At 1, 6, 12, 24, 48 h after reperfusion, the serum TNF-α, IL-6 was detected and the mRNA of cyclinD1, Cdk4, STAT-3 was assayed by real-time PCR as well as the protein expression of cyclinD1 and Cdk4 by Western blot.

RESULTSCompared with SH group, the expression of IL-6 declined at each set time point in IR group (t = 5.076 to 8.334, P = 0.000), but the content of TNF-α increased in early stage (1 to 12 h) (t = 2.972 to 7.215, P = 0.000 - 0.014). The expression of STAT-3, cyclinD1 and Cdk4 mRNA and protein of cyclinD1 and Cdk4 at 24 and 48 h after reperfusion were lower in IR group than in SH group (t = 2.857 to 6.684, P = 0.000 to 0.017). However, there was a significant decrease in TNF-α from 1 to 12 h after reperfusion (t = 2.995 to 4.112, P = 0.002 to 0.017), but a significant increase in IL-6 in IPO group than in IR group (t = 2.458 to 3.543, P = 0.005 to 0.034). The expression of STAT-3, cyclinD1, Cdk4 mRNA and protein of cyclinD1 and Cdk4 at 24 and 48 h after reperfusion were all increased in IPO group in comparison with in IR group (t = 2.383 to 6.803, P = 0.000 to 0.038).

CONCLUSIONSThe ischemic postconditioning could promote the remnant liver regeneration after subtotal hepatectomy with ischemia reperfusion injury. Its mechanism relates with the activation of the TNF-α/IL-6/STAT-3 signal pathway of and the cyclinD1-Cdk4 complex which enhances the proliferation of hepatocyte.

Animals ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Hepatectomy ; Interleukin-6 ; metabolism ; Ischemic Postconditioning ; Liver ; metabolism ; Liver Regeneration ; Male ; Rats ; Rats, Sprague-Dawley ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the expression of p16INK4a protein in breast cancer and analyze its clinical significance.

METHODSA total of 132 surgical specimens of primary breast cancer obtained between 2014 and 2015 were examined for expressions of ER, PR, CK5/6, Her-2 and p16INK4a proteins using immunohistochemistry.

RESULTSThe breast cancer samples were classified into 5 molecular subtypes, namely Luminal A (58 cases), Luminal B (32 cases), Her-2-positive (21 cases), basal-like (12 cases) and normal-like (9 cases) types. p16INK4a expression was negative in 7/132 (5.30%) cases, weakly positive in 15/132 (11.36%) cases, positive in 40/132 (30.30%) cases, and strongly positive in 70/132 (53.03%) cases. When categorizing negative and weakly positive cases into negative group and the positive and strongly positive cases into positive group, the total negative and positive expression rates of p16INK4a were 16.67% (22/132) and 83.33% (110/132) in the carcinoma tissues. Statistical analysis showed the expression intensity of p16INK4a differed significantly between the age groups (P<0.05) but was not significantly correlated with ER, PR, Her-2, molecular subtypes or metastasis of the tumors.

CONCLUSIONThe compensatory high expression of p16INK4a is the main mechanism of cell cycle deregulation in invasive breast cancer and can be an important specific molecular marker for invasive breast cancer.

Biomarkers, Tumor ; metabolism ; Breast Neoplasms ; classification ; diagnosis ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Female ; Humans ; Keratin-5 ; metabolism ; Keratin-6 ; metabolism ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism

BACKGROUND: Angiogenesis is an important process in the development of tumor. PD 0332991, a cell cycle inhibitor, can specifically inhibit CD4/6 phosphorylation and cell cycle progression. In xeongraft mice models, PD 0332991 treated mice had significantly decreased angiogenesis and vascular density compared with the control group, but the mechanism remains unknown. The purpose of this study is to investigate the role and molecular mechanism of PD 0332991 on vascular endothelial cells. METHODS: EA.hy926 cells, a kind of vascular endothelial cell, were used as the research model. The effects of PD 0332991 on the activity and proliferation of EA.hy926 cells were detected by the MTT, EdU assays. Wound-healing assays and transwell assays were used to determine the effects of PD 0332991 on the mobility of EA.hy926. The influence of PD 0332991 on cell cycle and apoptosis of endothelial cells was tested by flow cytometry, and the Western blot was applied to observe the expression of cell cycle related proteins in EA.hy926 cells treated by PD 0332991. RESULTS: PD 0332991 significantly inhibited the proliferation and mobility of EA.hy926 cells, caused cell cycle arrest and apoptosis. At the same time, PD 0332991 inhibited the expression of CDK4/6 and phosphorylation of Rb, and thus inhibited the cell cycle progression of EA.hy926 cells. CONCLUSIONS PD 0332991 can inhibit the proliferation and activity of endothelial cells and induces apoptosis.
Angiogenesis Inhibitors ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase 6 ; genetics ; metabolism ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; physiopathology ; Mice ; Piperazines ; pharmacology ; Pyridines ; pharmacology

OBJECTIVETo investigate the expression of microRNA-218 (miR-218) and its role in hepatocellular carcinoma (HCC).

METHODSForty-six pairs of fresh surgical specimens of HCC and adjacent tissues were examined for miR-218 expression using qRT-PCR. A miR-218 mimic was transfected into HepG2 cells, and the cell viability and apoptosis were analyzed by MTT assay and flow cytometry, and the potential targets of miR-218 were detected by qRT-PCR and Western blotting.

RESULTSThe expressions of miR-218 in HCC tissues were significantly down-regulated compared to those in the adjacent tissues (P<0.05). Down-regulation of miR-218 was found to correlate significantly with the tumor size (>5 cm) and an advanced TNM stage (III+IV) (P<0.05). Ectopic expression of miR-218 in HepG2 cells resulted in suppressed cell proliferation and enhanced cell apoptosis as well as the down-regulation of Bmi-1 and CDK6 mRNA and protein expressions (P<0.05).

CONCLUSIONThe low-expression of miR-218 is correlated with malignant clinicopathological characteristics of HCC, and miR-218 may inhibit cell proliferation and promote cell apoptosis by down-regulating Bmi-1 and CDK6 in HCC.

Adult ; Aged ; Apoptosis ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Proliferation ; Cyclin-Dependent Kinase 6 ; metabolism ; Female ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Polycomb Repressive Complex 1 ; metabolism

OBJECTIVETo investigate the DNA methylation status of the promoter region within the coding gene hsa-miR-124a in human gastric cancer tissue, and examine its association with the expression of Rb and CDK6 protein and clinicopathological factors.

METHODSMethylation-specific PCR (MS-PCR) was used to detect the DNA methylation status of hsa-miR-124a in gastric cancer tissues and adjacent normal tissues from 30 patients. The expression of Rb and CDK6 protein within these tissues was examined using immunohistochemistry.

RESULTSThe overall methylation rate of gastric cancer tissues was 73.3%, which was significantly higher than that in the adjacent tissues(10.0%, P<0.05). The overall positive rates of Rb and CDK6 protein in gastric cancer tissues were 86.7% and 80.0%, respectively. DNA methylation of hsa-miR-124a was positively correlated with the expression of Rb and CDK6 proteins. Significant associations were found between hypermethylation of hsa-miR-124a and tumor size, differentiation, lymphatic metastasis, and invasion depth(P<0.01).

CONCLUSIONSHypermethylation of hsa-miR-124a is present in gastric cancer, and is associated with increased expression of CDK6 and Rb protein. These may be related to the proliferation and development of malignancy in the stomach.

Adult ; Aged ; Aged, 80 and over ; Cyclin-Dependent Kinase 6 ; metabolism ; DNA Methylation ; Female ; Humans ; Male ; MicroRNAs ; genetics ; Middle Aged ; Promoter Regions, Genetic ; genetics ; Retinoblastoma Protein ; metabolism ; Stomach Neoplasms ; genetics ; metabolism

OBJECTIVETo study the methylation of O-6-methylguanine-DNA methyltransferase (MGMT) and p16 gene in the sputum cells of radon-exposed population. To provide the experimental base for finding the molecular biomarker of the high risk population of the radon-induced lung cancer.

METHODS91 radon-exposed workers were divided into 4 groups, high dosage group (> 120 WLM), middle dosage group (between 60 and 120 WLM), low dosage group (between 30 and 60 WLB) and lower dosage group (between 2 and 30 WLM) according to the accumulated exposure dosage of the radon daughters. The abnormal methylation of p16 and MGMT gene in the sputum cells of the population in the four groups was detected with the methylation specific PCR (MSP).

RESULTSThere was significantly upward trend for the p16 gene methylation rate (0.00%-20.00%), the MGMT gene methylation rate (0.00%-28.00%) and the total methylation rate (0.00%-40.00%) with the increase of the accumulated exposure dosage of the radon daughters (P < 0.01).

CONCLUSIONThe methylation of p16 and MGMT gene is related to the accumulate exposure dosage of the radon daughters.

Carcinogens, Environmental ; adverse effects ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; DNA Methylation ; Dose-Response Relationship, Radiation ; Humans ; Male ; O(6)-Methylguanine-DNA Methyltransferase ; genetics ; metabolism ; Occupational Exposure ; Radon ; adverse effects ; Radon Daughters ; adverse effects ; Sputum ; metabolism