This study aimed to observe the effect of terpinen-4-ol(T4O) on the proliferation of vascular smooth muscle cells(VSMCs) exposed to high glucose(HG) and reveal the mechanism via the Krüppel-like factor 4(KLF4)/nuclear factor kappaB(NF-κB) signaling pathway. The VSMCs were first incubated with T4O for 2 h and then cultured with HG for 48 h to establish the model of inflammatory injury. The proliferation, cell cycle, and migration rate of VSMCs were examined by MTT method, flow cytometry, and wound healing assay, respectively. The content of inflammatory cytokines including interleukin(IL)-6 and tumor necrosis factor-alpha(TNF-α) in the supernatant of VSMCs was measured by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to determine the protein levels of proliferating cell nuclear antigen(PCNA), Cyclin D1, KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. The KLF4 expression in VSMCs was silenced by the siRNA technology, and then the effects of T4O on the cell cycle and protein expression of the HG-induced VSMCs were observed. The results showed that different doses of T4O inhibited the HG-induced proliferation and migration of VSMCs, increased the percentage of cells in G_1 phase, and decreased the percentage of cells in S phase, and down-regulated the protein levels of PCNA and Cyclin D1. In addition, T4O reduced the HG-induced secretion and release of the inflammatory cytokines IL-6 and TNF-α and down-regulated the expression of KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. Compared with si-NC+HG, siKLF4+HG increased the percentage of cells in G_1 phase, decreased the percentage of cells in S phase, down-regulated the expression of PCNA, Cyclin D1, and KLF4, and inhibited the activation of NF-κB signaling pathway. Notably, the combination of silencing KLF4 with T4O treatment further promoted the changes in the above indicators. The results indicate that T4O may inhibit the HG-induced proliferation and migration of VSMCs by down-regulating the level of KLF4 and inhibiting the activation of NF-κB signaling pathway.
NF-kappa B/metabolism* ; Interleukin-18/metabolism* ; Proliferating Cell Nuclear Antigen/genetics* ; Cyclin D1/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Muscle, Smooth, Vascular ; Cell Proliferation ; Signal Transduction ; Cytokines/metabolism* ; Glucose/metabolism*

<b>OBJECTIVEb>To study expression of CD68, cyclin D1 protein and rearrangement of bcl-6 gene impact on the prognosis of diffuse large B-cell lymphoma (DLBCL).

<b>METHODSb>Gets paraffin samples of the 105 cases DLBCL with the detailed follow-up information, and were studied by using immunohistochemical EnVision method for CD3, CD10, CD20, CD68, cyclin D1, bcl-6, MUM 1, SOX-11 immunolabeling. The DLBCL were classified into germinal center B cell-like (GCB) subtypes and non-germinal center B cell-like (non-GCB) subtypes according to Hans'algorithm. Application of fluorescence in situ hybridization (FISH) technique to detect the bcl-6 gene rearrangement. The relationship between CD68, cyclin D1 protein, the bcl-6 gene and the curative effect of chemotherapy and survival was analyzed using statistical software. Respectively by GCB type, non-GCB type immune phenotype and CHOP, R-CHOP chemotherapy group, compare the curative effects.

<b>RESULTSb>105 patients had GCB 19 cases (18.1%), non-GCB 86 cases (81.9%), CD68 expression was 18 cases (17.1%), cyclin D1 high expression 36 cases (34.3%), bcl-6 gene rearrangement in 21 cases (21.9%), there is no correlation among the three (P > 0.05). One-way analysis of variance showed that age ≤ 60 years, clinical stage I-II, IPI score 0 to 2 points, LDH (U/L) < 245 IU/L,GCB subtypes, R-CHOP therapy, the prognosis of patients with better (P < 0.05), But gender, primary site no correlation with prognosis (P > 0.05). CD68, cyclin D1 high expression, bcl-6 rearrangement had poor prognosis (P < 0.05). Stratification analysis results show GCB-type or non-GCB type with high expression of CD68 contrast alloimmune phenotype groups had a poor prognosis, non-GCB type with high expression of cyclin D1 and rearrangement of bcl-6 gene had a poor prognosis (P < 0.001, P = 0.02). Treatment scheme of layered display, the CHOP treatment, significantly correlated with overall survival with high expression of CD68, cyclin D1 (P < 0.05), the R-CHOP treatment, there was no statistically significant difference between CD68, cyclin D1 high expression and overall survival (P = 0.428 and 0.168). Multivariate COX model analysis showed that high expression of CD68 (P = 0.026), high expression of cyclin D1 (P = 0.003) and high levels of LDH (P = 0.005) were adverse prognostic factors independent.

<b>CONCLUSIONSb>high expression of CD68, cyclin D1 and rearrangement of bcl-6 gene suggests poor prognosis, CD68, cyclin D1 protein and bcl-6 gene can be used as a prognostic indicator in patients with DLBCL.

Antibodies, Monoclonal, Murine-Derived ; Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Antineoplastic Combined Chemotherapy Protocols ; B-Lymphocytes ; classification ; Cyclin D1 ; metabolism ; Cyclophosphamide ; DNA-Binding Proteins ; genetics ; Doxorubicin ; Gene Rearrangement ; Germinal Center ; cytology ; Humans ; In Situ Hybridization, Fluorescence ; Lymphoma, Large B-Cell, Diffuse ; diagnosis ; Prednisone ; Prognosis ; Proto-Oncogene Proteins c-bcl-6 ; Vincristine

BACKGROUND/AIMS: 5'-Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a cellular energy sensor that monitors intracellular AMP/adenosine triphosphate (ATP) ratios and is a key regulator of the proliferation and survival of diverse malignant cell types. In the present study, we investigated the effect of activating AMPK by 5-aminoimidazole-4-carboxamide-ribonucleotide (AICAR) in thyroid cancer cells. METHODS: We used FRO thyroid cancer cells harboring the BRAF(V600E) mutation to examine the effect of AICAR on cell proliferation and cell survival. We also evaluated the involvement of mitogen-activated protein kinase (MAPK) pathways in this effect. RESULTS: We found that AICAR treatment promoted AMPK activation and suppressed cell proliferation and survival by inducing p21 accumulation and activating caspase-3. AICAR significantly induced activation of p38 MAPK, and pretreatment with SB203580, a specific inhibitor of the p38 MAPK pathway, partially but significantly rescued cell survival. Furthermore, small interfering RNA targeting AMPK-alpha1 abolished AICAR-induced activation of p38 MAPK, p21 accumulation, and activation of caspase-3. CONCLUSIONS: Our findings demonstrate that AMPK activation using AICAR inhibited cell proliferation and survival by activating p38 MAPK and proapoptotic molecules in FRO thyroid cancer cells. These results suggest that the AMPK and p38 MAPK signaling pathways may be useful therapeutic targets to treat thyroid cancer.
AMP-Activated Protein Kinases/genetics/metabolism ; Aminoimidazole Carboxamide/*analogs & derivatives/pharmacology ; Antineoplastic Agents/*pharmacology ; Caspase 3/metabolism ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Dose-Response Relationship, Drug ; Enzyme Activation ; Enzyme Activators/pharmacology ; Humans ; Mutation ; Protein Kinase Inhibitors/pharmacology ; Proto-Oncogene Proteins B-raf/genetics ; RNA Interference ; Ribonucleotides/*pharmacology ; Signal Transduction/*drug effects ; Thyroid Neoplasms/*enzymology/genetics/pathology ; Time Factors ; Transfection ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism

<b>OBJECTIVEb>To investigate the effects of E2F-1 gene silencing on multidrug resistance of human gastric cancer SGC7901/DDP cells and its possible mechanisms.

<b>METHODSb>Gastric cancer SGC7901/DDP cells were seeded in 6 well plates and divided into three groups: the experimental group, blank control and the negative control groups. For the experimental group, the SGC7901/DDP cells were transfected with recombinant lentivirus vector (Lv-shRNA-E2F-1), while the negative control with an control lentiviral vector (Lv-shRNA-NC) and the blank control with no treatment. The E2F-1 protein level was analyzed by Western blot. MTT assay was used to detect the half maximal inhibitory concentration (IC50) of three chemotherapy drugs including adriamycin, 5-fluorouracil (5-Fu) and cisplatine (DDP) of the three cell groups. Flow cytometry (FCM) was used to detect the pump-out rate of adriamycin and apoptosis rate of the three cell groups. Semi-quantitative RT-PCR and Western blot were also used to detect the protein and mRNA levels of multidrug resistance-associated genes (MDR1, MRP) and apoptosis-related genes (c-Myc, Skp2, cyclinD1).

<b>RESULTSb>The expression of E2F-1 protein in the experimental group was significantly lower than that in the negative control and blank control groups (0.794 ± 0.033 vs. 1.487 ± 0.082 vs. 1.511 ± 0.084, P < 0.01). The IC50 of the three chemotherapy drugs (adriamycin, 5-Fu and cisplatine) in the experimental group was significantly lower than that of the negative control and blank control groups, respectively (P < 0.01). Compared with the negative control and blank control groups, the pump-out rate of adriamycin of the experimental group was significantly declined [(0.16 ± 0.01)% vs. (0.37 ± 0.01)% vs. (0.35 ± 0.02)%, P < 0.01]. However, the apoptosis rate of the experimental group was significantly higher than that of the negative control and blank control groups [(33.82 ± 1.26)% vs. (17.34 ± 0.81)% vs. (13.16 ± 1.06)%, P < 0.01]. The results of RT-PCR and Western blot assays showed that mRNA and protein expressions of five genes (MDR1, MRP, CyclinD1, c-Myc, Skp2) in the experimental group were significantly lower than that in the negative control and blank control groups, respectively (P < 0.01).

<b>CONCLUSIONSb>E2F-1 gene silencing enhances the chemosensitivity of gastric cancer SGC7901/DDP cells to the chemotherapeutic drugs, directly or indirectly downregulated the expression of MDR1 and MRP, and finally reverses the multidrug resistance of the gastric cancer cells. The mechanism may be associated with the suppression of cyclinD1, c-Myc and Skp2.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Antibiotics, Antineoplastic ; pharmacology ; Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Cyclin D1 ; genetics ; metabolism ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; E2F1 Transcription Factor ; genetics ; metabolism ; Fluorouracil ; pharmacology ; Gene Silencing ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; S-Phase Kinase-Associated Proteins ; genetics ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; Transfection

Ataxia telangiectasia mutated (ATM) kinase plays an essential role in the maintenance of genomic stability. ATM-deficient (ATM(-/-)) mice exhibit hematopoietic stem cell (HSC) dysfunction and a high incidence of lymphoma. Gadd45a controls cell cycle arrest, apoptosis and DNA repair, and is involved in the ATM-p53 mediated DNA damage response. However, the role of Gadd45a in regulating the functionality of ATM(-/-) HSCs is unknown. Here we report that Gadd45a deletion did not rescue the defects of T-cells and B-cells development in ATM(-/-) mice. Instead, ATM and Gadd45a double knockout (ATM(-/-) Gadd45a(-/-)) HSCs exhibited an aggravated defect in long-term self-renewal capacity compared to ATM(-/-) HSCs in HSC transplantation experiments. Further experiments revealed that the aggravated defect of ATM(-/-) Gadd45a(-/-) HSCs was due to a reduction of cell proliferation, associated with an accumulation of DNA damage and subsequent activation of DNA damage response including an up-regulation of p53-p21 signaling pathway. Additionally, ATM(-/-) Gadd45a(-/-) mice showed an increased incidence of hematopoietic malignancies, as well as an increased rate of metastasis than ATM(-/-) mice. In conclusion, Gadd45a deletion aggravated the DNA damage accumulation, which subsequently resulted in a further impaired self-renewal capacity and an increased malignant transformation in ATM(-/-) HSCs.
Animals ; Ataxia Telangiectasia Mutated Proteins ; genetics ; B-Lymphocytes ; pathology ; Cell Cycle Proteins ; genetics ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; DNA Damage ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; metabolism ; pathology ; Leukemia ; genetics ; pathology ; Lymphoma ; genetics ; pathology ; Mice, Knockout ; Neoplasm Metastasis ; Nuclear Proteins ; genetics ; T-Lymphocytes ; pathology ; Tumor Suppressor Protein p53 ; metabolism

<b>OBJECTIVEb>To investigate the effects and related mechanisms of hepatitis B virus X (HBx) protein on cell cycle and growth in hepatocellular carcinoma.

<b>METHODSb>A human hepatocyte HepG2 cell line stably expressing a green fluorescent protein (GFP)-tagged HBx (HepG2/GFP-HBx cells) was used for the experiment, and HepG2 parental and HepG2/GFP cells was used as the controls. Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of cells with or without treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 5 pmol/L). Effect of HBx expression on promoter methylation status of the p16INK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting.

<b>RESULTSb>The HepG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs of culture (3.225+/-0.038 A490) than either control (HepG2: 2.012+/-0.022 A490, t = -46.86, P less than 0.001; HepG2/GFP: 2.038+/-0.029 A490, t = 42.51, P less than 0.001). The HepG2/GFP-HBx cells also showed significantly lower proportion of cells in the G0/G1 phase (16.45%+/-0.45%) than either control (HepG2: 44.81%+/-1.36%, t = -34.202, P less than 0.001; HepG2/GFP: 42.76%+/-1.58%, t = -28.88, P less than 0.001). However, 5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/G1 phase (33.25%+/-0.79%, t = 31.85, P less than 0.001). The p16INK4A promoter was methylated in the HepG2/GFP-HBx cells, and became demethylation after treatment with 5-Aza-CdR. However, no methylation of p16INK4A promoter was observed in both HepG2 and HepG2/GFP cells. The p16 protein level was significantly lower in the HepG2/GFP-HBx (vs. HepG2 and HepG2/GFP cells) and this level increased after treatment with 5-Aza-CdR.

<b>CONCLUSIONb>HBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase, and the underlying mechanism may involve inducing p16INK4A promoter methylation and downregulating p16 protein expression.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Genes, p16 ; Hep G2 Cells ; Hepatitis B virus ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Promoter Regions, Genetic ; Trans-Activators ; pharmacology

Squamous cell carcinoma(SCC) is a significant cause of cancer morbidity and mortality worldwide, with an incidence of up to 166 cases per 100 000 population. It arises in the skin, upper aerodigestive tract, lung, and cervix and affects more than 200 000 Americans each year. We report here that a microarray experiment comparing 41 SCC and 13 normal tissue specimens showed that Id2, a gene that controls the cell cycle, was significantly up-regulated in SCC. Enforced expression of Id2 in vitro stimulated the proliferation of SCC cells and up-regulated the transcription of nuclear factor kappa B (NF-κB) and cyclin D1. Enhancement of the NF-κB activity with p65 significantly increased the cell proliferation and the transcription of cyclin D1, whereas inhibition of the NF-κB activity with I kappa B alpha mutant (IκBαM) and pyrroline dithiocarbamate (PDTC) abrogated cell proliferation and transcription of cyclin D1. Furthermore, a mutated NF-κB binding site in the cyclin D1 promoter fully abrogated the Id2-induced transcription of cyclin D1. Taken together, these data indicate that Id2 induces SCC tumor growth and proliferation through the NF-κB/cyclin D1 pathway.
Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Head and Neck Neoplasms ; metabolism ; pathology ; Humans ; I-kappa B Proteins ; metabolism ; Inhibitor of Differentiation Protein 2 ; genetics ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; Transcription Factor RelA ; metabolism ; Transcription, Genetic ; Up-Regulation

<b>OBJECTIVEb>To investigate the essential role and mechanism of TRPC6 gene in the development of gastric cancer.

<b>METHODSb>The expression of TRPC6 protein was assessed in gastric cancer tissues and normal tissues adjacent to the cancer from 30 patients with gastric cancer. The inhibiting effect of TRPC6 activity on cell growth, cell cycle of a human gastric cancer cell line AGS cells, tumor progression and development of xenografted human gastric cancer in a mouse model was tested using dominant-negative mutant TRPC6 (DNC6). The survival of mice bearing xenografted tumors in the GFP and DNC6 was compared using Kaplan-Meier analysis. All statistical tests were two-sided.

<b>RESULTSb>The TRPC6 protein in the tumor tissues and para-tumor tissues was (21.60 ± 8.32)% versus (7.14 ± 2.24)%. After transfection of DNC6 virus for 24 hours, 48 hours, 72 hours and 96 hours, the growth inhibition rates of gastric cancer cells were (36.90 ± 1.13)%, (44.06 ± 2.17)%, (52.12 ± 2.76)% and (50.89 ± 1.97)%, respectively. The clone formation rates of control group and DNC6 group were (14.70 ± 3.00)% versus (43.80 ± 7.00)%. After transfection with DNC6 virus for 0, 24, 36 and 48 hours, the G(2)/M phase arrest was (20.34 ± 1.98)%, (24.31 ± 2.37)%, (27.70 ± 2.36)%, (35.10 ± 3.0)% in the DNC6 group and (18.40 ± 2.01)%, (18.0% ± 1.72)%, (17.50 ± 1.74)%, (16.80 ± 1.71)% in the control group, respectively. Inhibition of TRPC6 activity also reduced the subcutaneous tumor volume in the mouse models with xenografted human tumors (P < 0.05).

<b>CONCLUSIONb>In the preclinical models tested, TRPC6 channels are essential for gastric cancer development via regulation of G(2)/M phase transition.

Adenoviridae ; genetics ; Animals ; CDC2 Protein Kinase ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin B ; metabolism ; Cyclin-Dependent Kinases ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Recombinant Proteins ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; TRPC Cation Channels ; metabolism ; TRPC6 Cation Channel ; Transfection ; Tumor Burden

To identify the integration sites in the host genome for the hepatitis B virus (HBV)-encoded X protein (HBx) in hepatocellular carcinoma (HCC) biopsies that are positive for hepatitis B surface antigen (HBsAg). HCC biopsies were obtained from six patients that were HBV carriers, as demonstrated by the presence of HBsAg in their serum and sero-negativity for antibody to HBsAg. DNA was extracted from the tissue, fractionated, and circularized. Primers were designed according to the HBx sequence and used to amplify the circularized DNA templates by inverse polymerase chain reaction (IPCR). The amplified DNA fragments were checked by electrophoresis, cloned into the PMD18-T expression vector, and sequenced. Sequence alignment was performed by the Blast algorithms. Seven electrophoresis bands yielded 22 sequencing results, which represented a total of three HBx integration sites in the host genome: 19q12, 2q32.2, 22q12. The 19q12 integration site encompasses the CCNE1 gene, which encodes a G1/S-specific cyclin-E1. HBx-related integration sites exist in HBsAg-positive HCC biopsies. The CCNE1 gene may play a role in the development of HBx-related HCC.
Carcinoma, Hepatocellular ; blood ; genetics ; Cyclin E ; genetics ; DNA Primers ; DNA, Viral ; genetics ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; blood ; genetics ; Oncogene Proteins ; genetics ; Trans-Activators ; genetics ; Virus Integration

PURPOSE: The molecular mechanisms that are responsible for the initiation and progression of breast cancers are largely unknown. This study was to analyze the cyclin B1, cdc2, p53 and p16 tumor suppressor genes in human breast cancer. MATERIALS AND METHODS: To investigate the role of cyclin B1, cdc2, p53 and p16 in the pathogenesis and progression of breast carcinomas, 98 cases of breast cancers were examined by immunohistochemical method. The correlations of cyclin B1, cdc2, p53 and p16 expression with various clinico-pathologic findings were analysed. RESULTS: In the normal breast tissues, cyclin B1, cdc2 and p16 were weakly expressed, while p53 was not expressed. On the other hand, cyclin B1, cdc2, p53 and p16 were overexpressed in breast cancer, showing correlation between the expression of cyclin B1 and cdc2 and breast cancers (p=0.00). The overexpressions of cdc2 and p16 were correlated with an infiltrative tumor border pattern and this was statistically significant (p<0.05). In addition, the overexpression of cdc2 was correlated with histologic high grade carcinomas (p=0.00). CONCLUSION: Cyclin B1 and cdc2 appeared to be involved in the genesis or progression of breast cancers. In addition, the overexpressions of p16 and p53 may play important roles in more aggressive tumor and the overexpression of cdc2 is associated with progression of tumor to a higher grade of breast carcinomas. The deranged overexpressions of cyclin B1, cdc2, p16 and p53 may play an important role in human breast carcinogenesis.
Adult ; Aged ; Breast Neoplasms/*genetics/metabolism/pathology ; Cyclin B/*genetics/metabolism ; Cyclin B1/*genetics/metabolism ; Cyclin-Dependent Kinase Inhibitor p16/*genetics/metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Middle Aged ; Tumor Suppressor Protein p53/*genetics/metabolism