AP-1 transcription factor decoy reduces the TGF-beta1-induced cell growth in scleroderma fibroblasts through inhibition of cyclin E

The transforming growth factor-beta (TGF-beta) signaling pathway plays a key role in the abnormal accumulation of type I and III collagen of scleroderma. Activator protein-1 (AP-1) is a key regulatory protein in TGF-beta1-induced type I collagen synthesis. However, it is largely unknown whether AP-1 is involved in the cell proliferation of fibroblasts in scleroderma. In this study, we investigated the effects of the AP-1 oligo-deoxynucleotide (ODN) decoy on TGF-beta1-induced cell growth in scleroderma fibroblasts. To investigate the inhibition of AP-1 ODN decoy on the growth rates of scleroderma fibroblasts through the regulation of cell cycle regulatory proteins, we transfected the AP-1 ODN decoy on scleroderma fibroblasts and analyzed the cell cycle regulatory proteins by RT-PCR and Western blot analysis. We found that the growth rates of normal fibroblasts and scleroderma fibroblasts showed similar rates. It is noteworthy that the scleroderma fibroblasts grew more rapidly than normal fibroblasts in the presence of TGF-beta1. Moreover, the transfection of AP-1 decoy ODN into scleroderma fibroblasts resulted in the down-regulation of the growth rates by the down-regulation of cyclin E. These results collectively suggest that AP-1 ODN decoy can down-regulate the growth rates of scleroderma fibroblasts, thus implying that AP-1 ODN decoy is a promising therapeutic tool for overcoming scleroderma.     

Menatetrenone, a vitamin K2 analogue, inhibits hepatocellular carcinoma cell growth by suppressing cyclin D1 expression through inhibition of nuclear factor kappaB activation.

PURPOSE: Menatetrenone, a vitamin K2 analogue, plays an important role in the production of blood coagulation factors. Menatetrenone has also bee shown to have antineoplastic effects against several cancer cell lines including hepatocellular carcinoma (HCC) cells. However, the mechanisms by which vitamin K2 inhibits HCC cell growth have not bee fully clarified, and we therefore investigated the molecular basis of vitamin K2-induced growth inhibition of HCC cells. EXPERIMENTAL DESIGN: HCC cells were treated with vitamin K2 and the expression of several growth-related genes including cyclin-dependent kinase inhibitors and cyclin D1 was examined at the mRNA and protein levels. A reporter gene assay of the cyclin D1 promoter was done under vitamin K2 treatment. The regulation of nuclear factor kappaB (NF-kappaB) activation was investigated by a NF-kappaB reporter gene assay, an electrophoretic mobility shift assay, a Western blot for phosphorylated IkappaB, and an in vitro kinase assay for IkappaB kinase (IKK). We also examined the effect of vitamin K2 on the growth of HCC cells transfected with p65 or cyclin D1. RESULTS: Vitamin K2 inhibited cyclin D1 mRNA and protein expression in a dose-dependent manner in the HCC cells. Vitamin K2 also suppressed the NF-kappaB binding site-dependent cyclin D1 promoter activity and suppressed the basal, 12-O-tetradecanoylphorbol-13-acetate (TPA)-, TNF-alpha-, and interleukin (IL)-1-induced activation of NF-kappaB binding and transactivation. Concomitant with the suppression of NF-kappaB activation, vitamin K2 also inhibited the phosphorylation and degradation of IkappaBalpha and suppressed IKK kinase activity. Moreover, HCC cells overexpressing cyclin D1 and p65 became resistant to vitamin K2 treatment. CONCLUSION: Vitamin K2 inhibits the growth of HCC cells via suppression of cyclin D1 expression through the IKK/IkappaB/NF-kappaB pathway and might therefore be useful for treatment of HCC.     

Oncostatin M induces growth arrest by inhibition of Skp2, Cks1, and cyclin A expression and induced p21 expression

Oncostatin M has been characterized as a potent growth inhibitor for various tumor cells. Oncostatin M-treated glioblastoma cells cease proliferation and instigate astrocytal differentiation. The oncostatin M-induced cell cycle arrest in G(1) phase is characterized by increased level of the cyclin-dependent kinase (CDK) inhibitory proteins p21(Cip1/Waf1/Sdi1) and p27(Kip1). Induction of p21 protein corresponds to increased mRNA level, whereas p27 accumulates due to increased stability of the protein. Interestingly, stabilization of p27(Kip1) occurs even in S phase, showing that p27 stabilization is a direct consequence of oncostatin M signaling and not a result of the cell cycle arrest. Degradation of p27 in late G(1) and S phase is initiated by the ubiquitin ligase complex SCF-Skp2/Cks1. Oncostatin M inhibits expression of two components of this E3 ligase complex (Skp2 and Cks1). Although combined overexpression of Skp2 and Cks1 rescues p27 degradation in S phase, it can not override p27 accumulation in G(1) phase and cell cycle arrest by oncostatin M. In addition to increasing Cdk inhibitor level, oncostatin M also impairs cyclin A expression. Cyclin A mRNA and protein level decline shortly after oncostatin M addition. The accumulation of two CDK inhibitor proteins and the repression of cyclin A expression may explain the broad and potent antiproliferative effect of the cytokine.     

Nicotine-induced human breast cancer cell proliferation attenuated by garcinol through down-regulation of the nicotinic receptor and cyclin D3 proteins

Previous studies have demonstrated that the persistent exposure of human bronchial epithelial cells to nicotine (Nic) through nicotinic acetylcholine receptors increases cyclin D1 promoter activity and protein expression. The main purpose of this study is to elucidate the carcinogenic role of cyclin D3, which is involved in breast tumorigenesis when induced by Nic. Real-time PCR analysis revealed that cyclin D3 is highly expressed at the mRNA level in surgically dissected breast tumor tissue, compared to the surrounding normal tissue (tumor/normal fold ratio?=?17.93, n?=?74). To test whether Nic/nicotinic acetylcholine receptor (nAChR) binding could affect cyclin D3 expression in human breast cancer cells, the transformed cell line MCF-10A-Nic (DOX) was generated from normal breast epithelial cells (MCF-10A) with inducible ??9-nAChR gene expression, using the adenovirus tetracycline-regulated Tet-off system. Tet-regulated overexpression of ??9-nAChR in MCF-10A-Nic (DOX) xenografted BALB/c-nu/nu mice resulted in a significant induction of cyclin D3. In contrast, cyclin D3 expression was down-regulated in ??9-nAChR knock-down (siRNA) MDA-MB-231-xenografted tumors in NOD.CB17-PRKDC(SCID)/J(NOD-SCID) mice. Furthermore, we found that Nic-induced human breast cancer (MDA-MB-231) cell proliferation was inhibited by 1???M of garcinol (Gar), isolated from the edible fruit Garcinia indica, through down-regulation of ??9-nAChR and cyclin D3 expression. These results suggest that ??9-nAChR-mediated cyclin D3 overexpression is important for nicotine-induced transformation of normal human breast epithelial cells. The homeostatic regulation of cyclin D3 has the potential to be a molecular target for antitumor chemotherapeutic or chemopreventive purposes in clinical breast cancer patients.     

Quercetin regulates growth of Ishikawa cells through the suppression of EGF and cyclin D1

Quercetin and other polyphenols have anti-carcinogenic and anti-tumorigenic activity in various organs, however, studies of this activity are lacking in endometrial cancer. We hypothesize that quercetin has anti-proliferative activity and the mechanisms of quercetin action may be through modulation of cell cycle and cell growth regulatory genes. To test this hypothesis, we treated endometrial cancer cells (Ishikawa cell line) with quercetin, and cell proliferation, expression of growth signal genes (EGF, VEGF, and TGF-alpha), cell cycle genes (p53, p21, p73, and cyclin D1), and apoptosis-related genes (bcl-2 and bax) were analyzed. Results of these experiments demonstrate that after a 7-day exposure to 1, 10 and 100 micro M of quercetin, growth of Ishikawa cells was inhibited by 3, 51 and 87%, respectively. The gene and protein expression data suggest that quercetin treatment (100 micro M) significantly decreased EGF and cyclin D1, whereas VEGF was up-regulated in Ishiwaka cell lines. Other genes such as TGF-alpha, p53, p21, p73, bcl-2 and bax were not significantly changed with quercetin treatment in Ishiwaka cell lines. The present study suggests that quercetin can suppress proliferation of Ishikawa cells through down-regulation of EGF and cyclin D1.     

Cyclin-dependent kinase inhibition by the KLF6 tumor suppressor protein through interaction with cyclin D1

Kruppel-like factor 6 (KLF6) is a tumor suppressor gene inactivated in prostate and colon cancers, as well as in astrocytic gliomas. Here, we establish that KLF6 mediates growth inhibition through an interaction with cyclin D1, leading to reduced phosphorylation of the retinoblastoma protein (Rb) at Ser(795). Furthermore, introduction of KLF6 disrupts cyclin D1-cyclin-dependent kinase (cdk) 4 complexes and forces the redistribution of p21(Cip/Kip) onto cdk2, which promotes G(1) cell cycle arrest. Our data suggest that KLF6 converges with the Rb pathway to inhibit cyclin D1/cdk4 activity, resulting in growth suppression.     

Galectin-3 and cyclin D1 expression in non-small cell lung cancer

Introduction

Lung cancer is a major cause of mortality and morbidity worldwide. Galectin-3 is multifunctional protein, which is involved in regulation of cell growth, cell adhesion, cell proliferation, angiogenesis and apoptosis. Cyclin D1 together with other cyclin plays an important role in cell cycle control. Cyclin D1 regulates the G1-to-S phase transition. The aim of this study was the evaluation of correlations between clinicopathological findings and cyclin D1 and galectin-3 expression in non-small cell lung cancer (NSCLC). We wanted also to analyze the prognostic value of cyclin D1 and galectin-3 expression. Moreover we tried to evaluate the correlations between galectin-3 and cyclin D1 expression in tumor tissue.

Materials and methods

We used the immunochemistry method to investigate the expression of galectin-3 and cyclin D1 in the paraffin-embedded tumor tissue of 47 patients (32 men and 15 women; mean age 59.34 ± 8.90). years. We used monoclonal antibodies to cyclin D1 (NCL-L-cyclin D1-GM clone P2D11F11 NOVO CASTRA) and to galectin-3 (mouse monoclonal antibody NCL-GAL3 NOVO CASTRA).

Results

Galectin-3 expression was positive in 18 cases (38.29%) and cyclin D1 in 39 (82.97%). We showed only weak trend, that galectin-3 expression was lower in patients without lymph node involvement (p = 0.07) and cyclin D1 expression was higher in this group (p = 0.080). We didn''t reveal differences in cyclin D1 and galectin-3 expression in SCC and adenocarcinoma patients. We didn''t demonstrated also differences in galectin-3 and cyclin D1 expression depending on disease stage. Moreover we analyzed the prognostic value of cyclin D1 expression and galectin-3 in all examinated patients and separately in SCC and in adenocarcinoma and in all stages, but we didn''t find any statistical differences. We demonstrated that in galectin-3 positive tumors cyclin D1 expression was higher (96.55% vs 61.11%, Chi² Yatesa 7.53, p = 0.0061) and we revealed negative correlation between cyclin D1 and galectin-3 expression (R Spearman -0.458, p = 0.0011). In squamous cell lung cancer we didn''t observed correlations between these both examinated markers (R = -0.158, p = 0.460), and in adenocarcinoma the negative correlation was very strong (R = -0.829 p = 0.000132).

Conclusions

We didn''t reveal any important correlations between clinicopathological findings and galectin-3 and cyclin D1 expression and in non small cell lung cancer. We didn''t observed also prognostic value of cyclin D1 or galectin-3 expression. But we showed higher cyclin D1 expression in galectin-3 negative tumor tissues. We revealed also differences in correlations between galectin-3 and cyclin D1 expression in two main histopathological types of NSCLC.     

降低NF-κB的活性通过下调cyclin D1的表达抑制A549细胞增殖

Objective:To investigate the effect of decreasing activity of NF-kB on proliferation of A549 cell line and the possible molecular mechanism.Methods:The recombinant plasmid PCDNA3.1( )/IkBa expressing IkBa was constructed.The recombinant plasmid was then transfected to A549 cell.The activity of NF-kB,cell proliferation,and cyclin D1 expression were observed.Results:Our results showed that transfecting PCDNA3.1( )/IkBa inhibited activity of NF-kB in A549 cells,and decreasing activity of NF-kB inhibited proliferation of A549 cells.Decreasing activity of NF-kB was accompanied with down-regulation of cyclin D1 expression.Conclusion:Decreasing activity of NF-kB inhibited proliferation of A549 cells,and the molecular mechanism of the inhibition effect may be down-regulation of cyclin D1 expression.     

Inhibition of cyclin D1 expression by cyclin D1 shRNAs in human oral squamous cell carcinoma cells is associated with increased cisplatin chemosensitivity

Cyclin D1 is a well-known cell cycle regulator. Recently, its pro-survival function has been revealed in several tumors. Because increasing expression of cyclin D1 is a common event in oral squamous cell carcinoma (OSCC) and has been correlated with cisplatin resistance, we investigated if cyclin D1 inhibition could increase cisplatin chemosensitivity of OSCC. Five cyclin D1 shRNAs were prepared and 3 were selected for subsequent experiments. IC50 values for cisplatin were determined by an MTT assay. Cisplatin-induced apoptosis and cell cycle block were investigated. A tumor transplantation model was generated to examine the cisplatin sensitivity of Tca/cisplatin after in vivo cyclin D1 silencing. The role of nuclear factor-kappaB (NF-kappaB) and cyclin-dependent kinase 4 (CDK4) in cyclin D1-mediated cisplatin resistance was also examined. The most effective shRNA resulted in 84.51% knockdown of the cyclin D1 protein level. After the transfection with the 2 most effective shRNAs, the cisplatin IC50 decreased from 5.88 microg/ml to 1.36 microg/ml and 2.47 microg/ml, although overexpression of cyclin D1 rendered OSCC cells more resistant to cisplatin treatment (IC50 increased from 6.43 microg/ml to 12.24 microg/ml). This decreasing IC50 was correlated with the down-regulation of cisplatin-induced NF-kappaB activity in cyclin D1 knockdown cells, and was independent of CDK4 function. In vivo tumor transplantation models also confirmed a cisplatin-sensitizing effect of cyclin D1 shRNA in OSCC. A TUNEL assay validated the increase in apoptosis as induced by cisplatin in cyclin D1 knockdown cells. Cyclin D1 may be an important target for future therapy in patients with OSCC.