D1 mRNA Levels Research Articles

Pre-Clinical Efficacy of Combined Therapy with Novel β-Catenin Antagonist BC2059 and Histone Deacetylase Inhibitor Against Cultured and Primary AML Blast Progenitor Cells

Abstract 287The canonical WNT-β-catenin pathway is essential for self-renewal, growth and survival of AML stem and early blast progenitor cells (BPCs). Deregulated WNT signaling in AML inhibits polyubiquitylation and proteasomal degradation of β-catenin by the multi-protein complex. The inactivation of the degradation complex for β-catenin results in the preservation, nuclear translocation and activity of β-catenin in AML BPCs. This is especially notable in AML BPCs expressing FLT3-ITD, where increased activity of PI3K/AKT phosphorylates and inactivates GSK3β, thereby inhibiting phospho-degradation, increasing stability and nuclear accumulation of β-catenin. Activated FLT3 kinase has also been shown to directly phosphorylate β-catenin and promote its stabilization and nuclear localization. As a co-activator, in the nucleus β-catenin interacts with the bipartite T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factor, which increases the expression of pro-growth and pro-survival genes, including cyclin D1, Myc and survivin. Aberrant expression of LEF1 in hematopoietic stem cells has been shown to induce AML. β-catenin is also required for the HOXA9 and MEIS1 or MLL-AF9-mediated transformation of hematopoietic stem/progenitor cells. Thus, reactivation of β-catenin is required for maintenance of leukemic transformation making it an attractive drug target in AML. Here, we determined the in vitro and in vivo anti-AML activity of BC2059 (β-Cat Pharmaceuticals), a potent, small molecule, anthraquinone oxime-analog, against cultured (HL-60, OCI-AML3 and MV4-11 cells) and primary AML BPCs with or without the expression of FT3-ITD. First, as compared to the normal human CD34+ cells, FLT3-ITD expressing AML BPCs showed increased levels of β-catenin, mostly in the nucleus, as determined by confocal immunofluorescence microscopy. Exposure to 100 nM of BC2059 (BC) induced β-catenin degradation through the proteasome, as well as attenuated the nuclear and cytoplasmic levels of β-catenin in the cultured and primary AML BPC. Chromatin immunoprecipitation with anti-β-catenin antibody demonstrated that treatment with BC2059 (BC) reduced the binding of β-catenin to the WNT response elements (WRE) in the promoter DNA of its target genes, including Myc, cyclin D1 and survivin. Estimation of the intracellular luciferase levels in AML cells transfected with the TOP/FLASH versus FOP/FLASH construct showed that treatment with BC2059 significantly reduced only the TOP-FLASH luciferase activity, indicating that BC inhibits the expression of genes with promoters containing WRE elements. This was associated with reduced mRNA and protein levels of cyclin D1, MYC and survivin. Treatment with BC dose-dependently induced apoptosis of cultured and primary AML BPCs (up to 70%), including apoptosis of the CD34+CD38-Lin- AML BPCs. In contrast, BC induced apoptosis in < 10% of normal CD34+ progenitor cells. We have previously reported that treatment with the histone deacetylase inhibitor panobinostat (PS) attenuates p-FLT3, p-AKT and p-GSK3β levels in AML BPCs expressing FLT3-ITD. Consistent with this, here, we determined that co-treatment with BC and PS (10 to 20 nM) synergistically induced apoptosis of cultured and primary AML BPCs. Against primary AML BPCs expressing FLT3-ITD, co-treatment with the FLT3 kinase inhibitor AC220 (100 nM) further augmented BC mediated depletion of the cytoplasmic and nuclear levels of β-catenin and significantly enhanced BC-induced apoptosis (p < 0.01). This was associated with induction of BIM and p27 with depletion of MCL-1 levels. Following tail vein infusion and establishment of AML by OCI-AML3 cells in NOD-SCID mice, treatment with BC (5 or 10 mg/kg b.i.w, IV) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). Survival was further improved upon co-treatment with BC and PS (5 mg/kg IP, MWF). BC treatment (5 or 10 mg/kg IV) also dramatically improved survival of the NOD/SCID/IL2Rγ-depleted mice with established human AML following tail-vein injection of primary AML BPCs expressing FLT3 ITD. Mice did not experience any toxicity or weight loss. These findings support a compelling rationale for further development and in vivo testing of the BC-based combination with PS and FLT3 antagonist against human AML BPCs. Disclosures:Horrigan:BetaCat Pharmaceuticals: Employment. Sharma:BetaCat Pharmaceuticals: Equity Ownership.

Canonical Wnt Signaling Pathway Plays an Essential Role in N-Methyl-N-Nitrosurea Induced Gastric Tumorigenesis of Mice

Deregulated Wnt signaling pathway is implicated in many hereditary diseases and tumorigenesis including colorectal cancer, hepatocellular carcinoma and gastric cancer. In this study, to assess the relationship between chemically induced gastric tumor and canonical Wnt signaling pathway in genetically intact mice, histopathological and quantitative mRNA analyses were performed in C57BL/6J mice given drinking water containing N-methyl- N-nitrosurea (MNU). 60.5% of gastric adenoma and 27.9% of adenocarcinoma were observed 48 weeks after first administration. Also, in immunohistochemical analysis, aberrant expressions of phospho-GSK-3β, β-catenin, cyclin D1, c-Myc, osteopontin and COX-2 were found. In double immunofluorescent-antibody stains, β-catenin accumulation was colocalized with other proteins. mRNA levels of cyclin D1, c-myc and COX-2 were relatively higher in adenocarcinoma. Altogether, canonical Wnt pathway was highly involved in MNU induced gastric neoplasia of C57BL/6J mice, and it could be a considerably suitable system for the study to examine the linkage between gastric tumorigenesis and the canonical Wnt pathway.

Role of FBXL20 in human colorectal adenocarcinoma

In the present study, in order to determine whether the FBXL20 gene plays a role in the carcinogenesis of human colorectal adenocarcinoma, the mRNA expression levels of FBXL20 were examined in 30pairs of human colorectal adenocarcinoma tissues and corresponding adjacent normal colorectal tissues by quantitative real-time PCR (qRT-PCR). The pGPU/GFP/Neo-FBXL20 siRNA expression vector was transfected into the human colon carcinoma cell lines, SW480 and SW620. The mRNA levels of β-catenin, c-Myc, cyclinD1, p53, PP2A, SET and E-cadherin were determined by qRT-PCR, whereas the protein levels of β-catenin, c-Myc, caspase-3 and SET were examined by western blot analysis. MTT assay and flow cytometry were employed to assess cell proliferation, apoptosis and the cell cycle. As shown by qRT-PCR, FBXL20 mRNA expression was upregulated in 76.7% of the tumor samples. Cell proliferation was inhibited by 33.3% in the SW480-FBXL20 cells and by 22.7% in the SW620-FBXL20 cells, compared to the corresponding control cells. Furthermore, the percentages of apoptotic cells were 21.3 and 17.1% in the SW480-FBXL20 and SW620-FBXL20 cells, whereas the percentages of G1 phase cells in the two cell lines were 90.13 and 78.13%, respectively. In addition, the mRNA expression of SET and E-cadherin was upregulated in the SW480-FBXL20 and SW620-FBXL20 cells, whereas that of β-catenin, c-Myc, cyclinD1, p53 and PP2A was downregulated. Consistently, the protein expression of β-catenin and c-Myc was downregulated, whereas that of SET and caspase-3 was upregulated. Based on these results, we can conclude that FBXL20 plays an essential role in the carcinogenesis of human colorectal adenocarcinoma. Moreover, our data suggest that FBXL20 promotes carcinogenesis through the regulation of the Wnt signaling pathway and caspase activation.

The role of CCND1 alterations during the progression of cutaneous malignant melanoma

It is well demonstrated that CCND1 amplification is a frequent event in the acral subtype of cutaneous malignant melanoma; however, its role in the other subtypes of the disease is still controversial. The objectives of this study were to evaluate genetic and expression alterations of CCND1 with a focus on primary cutaneous melanomas, to define BRAF and NRAS mutation status, and correlate the data with clinical-pathological parameters. CCND1 amplification was associated with ulceration and the localization of the metastasis. After correction for the mutation state of BRAF and NRAS genes, CCND1 amplification in samples without such mutations was associated with ulceration and sun exposure. The cyclin D1 (CCND1) mRNA level decreased in lesions with multiple metastases and was correlated with both the mRNA levels and mutation state of BRAF and NRAS genes. Primary melanomas with BRAF(V600) or NRAS(Q61 ) mutations exhibited lower CCND1 mRNA level. CCND1 protein expression was associated with Breslow thickness, metastasis formation, and shorter survival time. These observations suggest that CCND1 alterations are linked to melanoma progression and are modified by BRAF and NRAS mutations. Our data show that CCND1 amplification could have a prognostic relevance in cutaneous melanoma and highlight that altered CCND1 gene expression may influence the metastatic progression, survival, and the localization of metastases.

Puerarin suppresses proliferation of endometriotic stromal cells partly via the MAPK signaling pathway induced by 17ß-estradiol-BSA.

BackgroundPuerarin is a major isoflavonoid compound extracted from Radix puerariae. It has a weak estrogenic action by binding to estrogen receptors (ERs). In our early clinical practice to treat endometriosis, a better therapeutic effect was achieved if the formula of traditional Chinese medicine included Radix puerariae. The genomic and non-genomic effects of puerarin were studied in our Lab. This study aims to investigate the ability of puerarin to bind competitively to ERs in human endometriotic stromal cells (ESCs), determine whether and how puerarin may influence phosphorylation of the non-genomic signaling pathway induced by 17ß-estradiol conjugated to BSA (E2-BSA).MethodologyESCs were successfully established. Binding of puerarin to ERs was assessed by a radioactive competitive binding assay in ESCs. Activation of the signaling pathway was screened by human phospho-kinase array, and was further confirmed by western blot. Cell proliferation was analyzed according to the protocol of CCK-8. The mRNA and protein levels of cyclin D1, Cox-2 and Cyp19 were determined by real-time PCR and western blotting. Inhibitor of MEK1/2 or ER antagonist was used to confirm the involved signal pathway.Principal FindingsOur data demonstrated that the total binding ability of puerarin to ERs on viable cells is around 1/3 that of 17ß-estradiol (E2). E2-BSA was able to trigger a rapid, non-genomic, membrane-mediated activation of ERK1/2 in ESCs and this phenomenon was associated with an increased proliferation of ESCs. Treating ESCs with puerarin abrogated the phosphorylation of ERK and significantly decreased cell proliferation, as well as related gene expression levels enhanced by E2-BSA.Conclusions/SignificancePuerarin suppresses proliferation of ESCs induced by E2-BSA partly via impeding a rapid, non-genomic, membrane-initiated ERK pathway, and down-regulation of Cyclin D1, Cox-2 and Cyp19 are involved in the process. Our data further show that puerarin may be a new candidate to treat endometriosis.

1031P - Cyclin D1, EGFR and AKT/MTOR Signalling in Localized Laryngeal Squamous Cell Carcinoma

ABSTRACT Introduction EGFR pathway, Akt/mTOR and Cyclin D1 are activated and interact in squamous cell head and neck cancer. Patients and methods We assessed relative mRNA expression of EGFR, Akt1, 2, 3, mTOR and cyclin D1, copy number variants of EGFR and CCND1 genes and immunohistochemical (IHC) expression of EGFR, pAKT308, pAKT473, pmTOR, PTEN, p53 and cyclin D1 in paraffin-embedded tissue of localized laryngeal carcinomas. Results In 289 patients with T3-4 (77.8%), node-negative (84.1%) tumours of the larynx, high EGFR and CCND1 mRNA correlated with never or ex smoking, (p = 0.003) and (p = 0.029) respectively, while low AKT3 correlated with alcohol abuse. At a median follow-up of 74.5 months, high mTOR RNA was marginally associated with shorter DFS, HR = 1.54, p = 0.093 and high Akt3 mRNA with shorter OS, HR = 1.49, p = 0.0786 whereas high EGFR IHC expression was associated with shorter DFS HR = 1.60, p = 0.0311 and OS HR = 1.57, p = 0.056, pAKT308 with shorter OS, HR = 1.003, p = 0.0586 and pAKT473 with shorter DFS HR = 1.004, p = 0.021 and OS HR = 1.004, p = 0.033, all in univariate analysis. An interaction between mRNA levels of mTOR and cyclin D1 was found to be associated with shorter DFS, namely at high mTOR mRNA expression, one unit increase of CCND1 mRNA expression had aHR = 2.16 and this remained statistically significant in multivariate analysis (p-value for interaction = 0.0010). In multivariate analysis, node-positive status, subglottic-transglottic localization, surgery other than total laryngectomy and mTOR/cyclin D1 mRNA interaction, were independent predictors of relapse, while node-positive status, and subglottic-transglottic localization were associated with higher risk of death. Conclusions In laryngeal cancer, an interaction of high mTOR and cyclin D1 mRNA expression is associated with poor patient outcome. Disclosure All authors have declared no conflicts of interest.

Suberoylanilide hydroxamic acid (SAHA) inhibits EGF-induced cell transformation via reduction of cyclin D1 mRNA stability

Suberoylanilide hydroxamic acid (SAHA) inhibiting cancer cell growth has been associated with its downregulation of cyclin D1 protein expression at transcription level or translation level. Here, we have demonstrated that SAHA inhibited EGF-induced Cl41 cell transformation via the decrease of cyclin D1 mRNA stability and induction of G0/G1 growth arrest. We found that SAHA treatment resulted in the dramatic inhibition of EGF-induced cell transformation, cyclin D1 protein expression and induction of G0/G1 growth arrest. Further studies showed that SAHA downregulation of cyclin D1 was only observed with endogenous cyclin D1, but not with reconstitutionally expressed cyclin D1 in the same cells, excluding the possibility of SAHA regulating cyclin D1 at level of protein degradation. Moreover, SAHA inhibited EGF-induced cyclin d1 mRNA level, whereas it did not show any inhibitory effect on cyclin D1 promoter-driven luciferase reporter activity under the same experimental conditions, suggesting that SAHA may decrease cyclin D1 mRNA stability. This notion was supported by the results that treatment of cells with SAHA decreased the half-life of cyclin D1 mRNA from 6.95h to 2.57h. Consistent with downregulation of cyclin D1 mRNA stability, SAHA treatment also attenuated HuR expression, which has been well-characterized as a positive regulator of cyclin D1 mRNA stability. Thus, our study identifies a novel mechanism responsible for SAHA inhibiting cell transformation via decreasing cyclin D1 mRNA stability and induction of G0/G1 growth arrest in Cl41 cells.

4E-BP3 regulates eIF4E-mediated nuclear mRNA export and interacts with replication protein A2

In nucleus, eIF4E regulates the nucleus export of specific mRNA. In this study, altered 4E-BP3 (eIF4E-binding protein 3) expression resulted in profoundly affected cyclin D1 protein levels, partially due to changes in the cytoplasmic cyclin D1 mRNA levels in both U2OS and MCF7 cells, whereas altered 4E-BP1 expression did not affect eIF4E-mediated cyclin D1 mRNA export. 4E-BP3 also affected a subset of growth promoting mRNAs exported in an eIF4-dependent manner. Furthermore, 4E-BP3 interacted with dephosphorylated RPA2 (replication protein A2). The results indicated 4E-BP3 acts as an inhibitor of eIF4E-mediated mRNA export in the examined cells, and 4E-BP3 inhibition of eIF4E-mediated mRNA export is regulated by the phosphorylation state of RPA2. Structured summary of protein interactions4EBP3 physically interacts with RPA2 by anti bait coimmunoprecipitation (View interaction)

Cyclin D1 expression in podocytes: regulated by mitogens in collaboration with integrin–extracellular matrix interaction through extracellular signal-regulated kinase

Cyclin D1 plays significant roles in cell cycle entry and migration. We have documented that both integrin α3β1 expressions and the number of podocytes were reduced in focal segmental glomerulosclerosis. We wondered whether integrin-extracellular matrix (ECM) interaction was involved in the regulation of cyclin D1 expression, and the possible signaling pathways in mitogen-stimulating podocytes. Cultured podocytes were divided into serum (mitogens/growth factors)-starved and serum-stimulated groups. Reverse transcription polymerase chain reaction was used to detect cyclin D1 mRNA, and Western blot analysis was used to measure protein concentrations of cyclin D1 and extracellular signal-regulated kinase (ERK) activation (p-ERK/ERK). The integrin-ECM interaction was blocked by anti-β1-integrin monoclonal antibody or RGDS (Arg-Gly-Asp-Ser). The MEK inhibitor, U0126, was used to inhibit ERK activation. The results showed that there was little cyclin D1 protein in serum-starved groups, but it was abundant in serum-stimulated groups. Both cyclin D1 mRNA and protein levels were reduced in serum-stimulated podocytes after blocking integrin-ECM interaction. ERK activation in serum-stimulated podocytes was significantly decreased after blocking integrin-ECM interaction. Cyclin D1 mRNA and protein concentrations in serum-stimulated podocytes were reduced after blocking ERK activation by U0126. We demonstrate that integrin-ECM interaction collaborates with mitogens to activate ERK/mitogen-activated protein kinase pathways which are essential for cyclin D1 expression in podocytes.

Catalysis leads to posttranslational inactivation of the type 1 deiodinase and alters its conformation

Previously, it was shown that the type 1 deiodinase (D1) is subject to substrate-dependent inactivation that is blocked by pretreatment with the inhibitor of D1 catalysis, propylthiouracil (PTU). Using HepG2 cells with endogenous D1 activity, we found that while considerable D1-mediated catalysis of reverse tri-iodothyronine (rT(3)) is observed in intact cells, there was a significant loss of D1 activity in sonicates assayed from the same cells in parallel. This rT(3)-mediated loss of D1 activity occurs despite no change in D1 mRNA levels and is blocked by PTU treatment, suggesting a requirement for catalysis. Endogenous D1 activity in sonicates was inactivated in a dose-dependent manner in HepG2 cells, with a ∼50% decrease after 10 nM rT(3) treatment. Inactivation of D1 was rapid, occurring after only half an hour of rT(3) treatment. D1 expressed in HEK293 cells was inactivated by rT(3) in a similar manner. (75)Se labeling of the D1 selenoprotein indicated that after 4 h rT(3)-mediated inactivation of D1 occurs without a corresponding decrease in D1 protein levels, though rT(3) treatment causes a loss of D1 protein after 8-24 h. Bioluminescence resonance energy transfer studies indicate that rT(3) exposure increases energy transfer between the D1 homodimer subunits, and this was lost when the active site of D1 was mutated to alanine, suggesting that a post-catalytic structural change in the D1 homodimer could cause enzyme inactivation. Thus, both D1 and type 2 deiodinase are subject to catalysis-induced loss of activity although their inactivation occurs via very different mechanisms.

Abstract 1490: Impact of bone marrow microenvironment on mRNA expression of genes in the apoptotic pathway in CLL cells

Abstract Chronic lymphocytic leukemia (CLL) is thought to arise due to dysregulated apoptosis rather than uncontrolled proliferation. Recent reports revealed that CLL cells accumulate due to both an intrinsic defect in apoptosis and extrinsic signals delivered by the microenvironment. Although several entities of bone marrow stroma cell (MSC) have been unveiled, the critical components involved in CLL survival remain obscure. In the present study, we used low density microarray analysis to identify the essential apoptotic genes (n=93) that are involved in cell survival. Primary cells obtained from CLL patients (n=12) were co-cultured with MSC (Nktert, human stromal cell line that mimics bone marrow microenvironment) in a ratio of 100:1. At the end of the incubation (12hr, d1, and d3), the expression profile of 93 genes as well as the percent apoptosis were evaluated. When CLL lymphocytes were co-cultured on MSC for 6 days, the viability of CLL cells, measured by Annexin/PI staining, increased by 6-27% at d1-d6 (n=9, p&amp;lt;0.001) compared to time matched controls. Next, we investigated the effect of MSC on global transcription. There was a 1.7+0.5 (d1, n=13) and 2.5+1.0 (d3, n=7) fold increase in global RNA synthesis as measured by [3H]uridine incorporation (p&amp;lt;0.001). Examination of mRNA profile of 93 genes showed that among the BCL2 family, the mRNA levels of the pro-apoptotic members such as BCL2L13, BAK1 and BIK decreased, while mRNA levels of anti-apoptotic, BCL2, BFL-1, BCL-XL, increased at d1 (1.46+0.56, p=0.015; 1.68+0.67, p=0.005; 2.17+1.46, p=0.018; respectively). Among caspase recruitment domain targets, pro-apoptotic APAF1 mRNA level significantly decreased at d1 but NALP1 mRNA level increased significantly (1.53+0.42, p=0.001), suggesting this gene may have alternate function in apoptosis pathway. In addition, NFKB-REL complex family members demonstrated decrease with MSC; in addition, member of IKB family, inhibitor of NFKB complex, such as NFKBIA increased significantly (1.56+0.7, p=0.018) at d1. Furthermore, in tumor necrosis factor receptor super family, TNFRSF1B expression significantly decreased (d1,0.74+0.21, p=0.001; d3,0.75+0.18, p=0.005), while TNFRSF10A and TNFRSF21 mRNA expression levels significantly increased (1.85+0.72, p=0.002; 2.81+2.08, p=0.016) at d1 but returned to baseline at d3. Additionally, 4 out of 6 DED domain containing pro-apoptotic proteins, (FADD, TRADD-adaptor proteins interacting with TNFRSF proteins, DEDD, DEDD2) demonstrated increase in mRNA levels compared to control. Moreover, mRNA levels of anti-apoptotic FLIP, which inhibits Caspase8 and Caspase10, increased significantly (1.50+0.59, p=0.013) after incubation with MSC. In summary, our data shows that MSC promoted survival to CLL lymphocytes is associated with transcriptional regulation of several genes including TNFRSF and BCL2 anti-apoptotic family members. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1490. doi:1538-7445.AM2012-1490

Attenuated Wnt/β-catenin signalling mediates methotrexate chemotherapy-induced bone loss and marrow adiposity in rats

Cancer chemotherapy often causes significant bone loss, marrow adiposity and haematopoietic defects, yet the underlying mechanisms and recovery potential remain unclear. Wnt/β-catenin signalling is integral to the regulation of osteogenesis, adipogenesis and haematopoiesis; using a rat model, the current study investigated roles of this signalling pathway in changes to bone marrow stromal and haematopoietic cell differentiation after chemotherapy with methotrexate (MTX), a commonly used antimetabolite. MTX treatment in rats (5 daily administrations at 0.75mg/kg) has previously been found to decrease bone volume and increase marrow fat, which was associated with increased osteoclastogenesis in haematopoietic cells and with an osteogenesis to adipogenesis switch in bone marrow stromal cells of treated rats. In the current study, on day 6 after the first MTX dose we found that accompanying these changes as well as a suppressed haematopoietic cellularity but increased granulocyte/macrophage differentiation potential, there was an increase in mRNA expression of Wnt antagonists sFRP-1 and Dkk-1 in bone, a reduction in nuclear β-catenin protein in bone marrow stromal cells, and decreased mRNA levels of β-catenin target genes lef-1, cyclin D1 and survivin, suggesting reduced activation of Wnt/β-catenin signalling in the bone during MTX-induced damage. Concurrent administration of BIO, a GSK-3β inhibitor that stabilises β-catenin, partially abrogated the MTX-induced transient changes in osteogenic/adipogenic commitment, granulocyte/macrophage lineage differentiation and osteoclast number. These findings demonstrate a potentially important role of Wnt/β-catenin signalling in MTX chemotherapy-induced cellular changes to the bone marrow microenvironment.

T-AUCB, an improved sEH inhibitor, suppresses human glioblastoma cell growth by activating NF-κB-p65

Although sEH inhibitors are well studied in inflammatory and cardiovascular diseases, their effects on gliomas are unclear. In this study, we investigated the effects of t-AUCB, a more potent and selective sEH inhibitor, on U251 and U87 human glioblastoma cell lines and the HepG2 human hepatocellular carcinoma cell line. Our results showed that t-AUCB efficiently inhibited sEH activities in all three cell lines (the inhibition rate was more than 80% in each) and suppressed U251 and U87 cell growth in a dose-dependent manner, but exhibited no cell growth inhibition on HepG2. We detected high levels of phosphorylated NF-κB-p65 (Ser536) in t-AUCB-treated U251 and U87 cells, and then found that the NF-κB inhibitor PDTC can completely abolish t-AUCB-induced growth inhibition. This indicated that t-AUCB suppresses U251 and U87 cell growth by activating NF-κB-p65. Moreover, we found that t-AUCB induces cell-cycle G0/G1 phase arrest by regulating Cyclin D1 mRNA and protein levels and CDC2 (Thr161) phosphorylation level. We propose to further test this promising reagent for its anti-glioma activity in clinical relevant orthotopic brain glioma models.

Millimeter wave treatment promotes chondrocyte proliferation via G1/S cell cycle transition

Millimeter waves, high-frequency electromagnetic waves, can effectively alleviate the clinical symptoms in osteoarthritis patients, as a non-pharmaceutical and non-invasive physical therapy regimen. However, the molecular mechanisms of the therapeutic effects of millimeter wave treatment are not well understood. In the present study, the effect of millimeter waves on the G1/S cell cycle progression in chondrocytes and the underlying mechanism was investigated. Chondrocytes isolated from the knee of SD rats were cultured and identified using toluidine blue staining. The second generation chondrocytes were collected and stimulated with or without millimeter waves for 48 h. Chondrocyte viability was analyzed using the MTT assay. The cell cycle distribution of chondrocytes was analyzed by flow cytometry. mRNA and protein expression levels of cyclin D1, cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and p21 were detected using real-time PCR and western blotting, respectively. Millimeter wave stimulation was found to significantly enhance chondrocyte viability. Moreover, the percentage of chondrocytes in the G0/G1 phase was significantly decreased, whereas that in the S phase was significantly increased. In addition, following millimeter wave treatment, cyclin D1, CDK4 and CDK6 expression was significantly upregulated, whereas p21 expression was significantly downregulated. The results indicate that millimeter wave treatment promotes chondrocyte proliferation via cell cycle progression.

The impact of cyclin D1 mRNA isoforms, morphology and p53 in mantle cell lymphoma: p53 alterations and blastoid morphology are strong predictors of a high proliferation index

Mantle cell lymphoma is a clinically heterogeneous disease characterized by overexpression of cyclin D1 protein. Blastoid morphology, high proliferation, and secondary genetic aberrations are markers of aggressive behavior. Expression profiling of mantle cell lymphoma revealed that predominance of the 3'UTR-deficient, short cyclin D1 mRNA isoform was associated with high cyclin D1 levels, a high "proliferation signature" and poor prognosis. Sixty-two cases of mantle cell lymphoma were analyzed for cyclin D1 mRNA isoforms and total cyclin D1 levels by real-time reverse transcriptase polymerase chain reaction, and TP53 alterations were assessed by immunohistochemistry and molecular analysis. Results were correlated with proliferation index and clinical outcome. Predominance of the short cyclin D1 mRNA was found in 14 (23%) samples, including four with complete loss of the standard transcript. TP53 alterations were found in 15 (24%) cases. Predominance of 3'UTR-deficient mRNA was significantly associated with high cyclin D1 mRNA levels (P=0.009) and more commonly found in blastoid mantle cell lymphoma (5/11, P=0.060) and cases with a proliferation index of >20% (P=0.026). Both blastoid morphology (11/11, P<0.001) and TP53 alterations (15/15, P<0.001) were significantly correlated with a high proliferation index. A proliferation index of 10% was determined to be a significant threshold for survival in multivariate analysis (P=0.01). TP53 alterations are strongly associated with a high proliferation index and aggressive behavior in mantle cell lymphoma. Predominance of the 3'UTR-deficient transcript correlates with higher cyclin D1 levels and may be a secondary contributing factor to high proliferation, but failed to reach prognostic significance in this study.

The histone deacetylase inhibitor trichostatin A induces cell cycle arrest and rapid upregulation of gadd45β in LS174T human colon cancer cells

Histone deacetylase (HDAC) inhibitors are considered as promising therapeutic agents against several malignant diseases because they inhibit cancer cell proliferation. The stress sensor genes of the growth arrest and DNA damage-inducible protein (gadd45) family exhibit disordered expression in several types of malignant diseases and are thus a novel target for cancer therapy. However, there have been only few investigations of whether HDAC inhibitors affect the expression of gadd45 genes. We examined the effects of a HDAC inhibitor, trichostatin A (TSA), on the time-dependent expression of gadd45 genes in the human colon cancer cell line LS174T. Addition of TSA to LS174T cells induced inhibition of cell proliferation by arresting the cell cycle. We found that TSA treatment of LS174T cells induced rapid upregulation of gadd45β mRNA expression within 15 min, reaching a peak level at 3 h. Although the time-dependent expression pattern of gadd45β mRNA was similar to that of gadd45β mRNA, the peak level of gadd45β was lower than that of gadd45β. TSA treatment also upregulated the mRNA level of p21Waf1/Cip1, a prolif- eration inhibitor, after 3 h, but downregulated the mRNA levels of cyclin D1, a proliferation inducer, after 3 h, and of c-Myc after 1 h. TSA treatment induced a certain level of apoptosis, but the mRNA level of p53, a potent apoptosis inducer, was down-regulated after 3 h. These results suggest that the up-regulation of p21Waf1/Cip1 and apoptosis was independent of p53 and that the early upregulation of gadd45β gene, which precedes the upregulation of p21Waf1/Cip1 and the downregulation of cyclin D1, are important in TSA-treated LS174T cells.

Investigation of effects and mechanisms of total flavonoids of Astragalus and calycosin on human erythroleukemia cells.

Flavonoids are found in most parts of plants and have been shown to have multiple biological activities such as anticancer, anti-inflammation, antibacteria, antivirus, and immune-stimulation. Existing data showed that the total flavonoids of Astragalus (TFA) can provide biological system with resistance to injury and can possess antimutagenic, atherosclerotic inhibition, and other biological effects. This study investigated the effects of TFA and calycosin (a compound isolated from TFA), on apoptosis induction, and cell cycle of human erythroleukemia cell line K562 by an array of techniques, including proliferation (MTT), PI staining, Annexin V/PI double staining, and RT-PCR. The experimental data showed that TFA and calycosin could inhibit the proliferation of K562 cells. The 50% inhibiting concentrations of TFA and calycosin were 98.63 μg/mL and 130.32 μg/mL, respectively. However, TFA and calycosin could not induce apoptosis in K562 cells, but could increase the number of the cells in the G0/G1 phase. The level of cyclin D1 mRNA in K562 cells decreased after the treatment with TFA and calycosin. This study provides new insights into the functional mechanism of total flavonoids of Astragalus and calycosin on human erythroleukemia cells.

Tissue-specific expression of hormonal carcinogenesis target genes in rats treated with polycyclic aromatic hydrocarbons

We have investigated the effect of polycyclic aromatic hydrocarbons (PAHs) on estrogen- metabolizing genes CYP1A1, CYP1B1, CYP19 and ERα and cyclin D1 genes, which control of cell division in estrogen-depended tissues. Treatment of rats with benzo(a)pyren (BP) or 3-methylcholantrene (MC) significantly up-regulated CYP1A1, CYP1B1 gene expression in liver, uterus and ovary, whereas alfa-naphthoflavone (α-NF) did not have any effect. The high level of aromatase gene (CYP19) expression was detected in ovary only. Treatment of rats with BP or MC significantly down-regulated expression of this gene (15- and 5,5-fold, respectively), whereas α-NF did not have any effect. BP produced an increase in ERα and cyclin D1 gene expression in rat liver. This effect was not seen with MC and α-NF. ERα and cyclin D1 mRNA levels were unchanged in uterus of rats after PAHs treatment. On the other hand, BP treatment caused an increase of the ERα and cyclin D1 mRNA levels (3,5- and 2,5-fold, respectively) in ovary, whereas MC and α-NF did not have any effects. Thus, our results give evidence for tissue-specific effects of PAHs on expression of genes, which participate in hormonal carcinogenesis. Moreover, the fact that BP and MC treatment affects the expression of estrogen-metabolizing genes and genes, which control of cell division, supports the view that PAHs may be one of the causes of endocrine disorder and consequent hormonal carcinogenesis.

Effects and mechanisms of total flavonoids of Astragali Radix and calycosin on inhibiting human erythroleukemia cell line K562

To investigate the effects of total flavonoids of Astragali Radix (TFA) and calycosin on apoptosis induction and cell cycle in human erythroleukemia cell line K562. MTT assay was used to measure the inhibition effect on the proliferation of K562 cells cultured with TFA and calycosin. The effect of TFA and calycosin on cell cycle in K562 was detected by PI staining. The apoptosis induction effect was measured by Annexin V/PI double staining. RT-PCR was used to determine the level of Cyclin D1 mRNA in K562 cells after treated with TFA and calycosin. TFA and calycosin could inhibit the proliferation of K562 cells, the 50% inhibiting concentration of TFA and calycosin were 98.63 mg x L(-1) and 130.32 mg x L(-1) respectively. TFA and calycosin could not induce apoptosis in K562 cells, but could increase the number of cells in the G0/G1 phase. The level of Cyclin D1 mRNA in K562 cells decreased after treated with TFA and calycosin. TFA could inhibit the proliferation of K562 cells, and attribute to arrest them in the G0/G1 phase and decrease Cyclin D1 mRNA.

Peroxisome proliferator-activated receptor α (PPARα) mRNA expression in human hepatocellular carcinoma tissue and non-cancerous liver tissue

BackgroundPeroxisome proliferator-activated receptor α (PPARα) regulates lipid metabolism in the liver. It is unclear, however, how this receptor changes in liver cancer tissue. On the other hand, mouse carcinogenicity studies showed that PPARα is necessary for the development of liver cancer induced by peroxisome proliferators, and the relationship between PPARα and the development of liver cancer have been the focus of considerable attention. There have been no reports, however, demonstrating that PPARα is involved in the development of human liver cancer.MethodsThe subjects were 10 patients who underwent hepatectomy for hepatocellular carcinoma. We assessed the expression of PPARα mRNA in human hepatocellular carcinoma tissue and non-cancerous tissue, as well as the expression of target genes of PPARα, carnitine palmitoyltransferase 1A and cyclin D1 mRNAs. We also evaluated glyceraldehyde 3-phosphate dehydrogenase, a key enzyme in the glycolytic system.ResultsThe amounts of PPARα, carnitine palmitoyltransferase 1A and glyceraldehyde 3-phosphate dehydrogenase mRNA in cancerous sections were significantly increased compared to those in non-cancerous sections. The level of cyclin D1 mRNA tends to be higher in cancerous than non-cancerous sections. Although there was a significant correlation between the levels of PPARα mRNA and cyclin D1 mRNA in both sections, however the correlation was higher in cancerous sections.ConclusionThe present investigation indicated increased expression of PPARα mRNA and mRNAs for PPARα target genes in human hepatocellular carcinoma. These results might be associated with its carcinogenesis and characteristic features of energy production.