Cyclin D1 mRNA Expression Research Articles

Oxytocin effects on the inhibition of the NF-κB/miR195 pathway in mice breast cancer

Oxytocin (OT) has the suppressive effects on breast tumor formation and development. We hypothesized that OT through the NF-κB inhibition can induce the miR-195 up-regulation which it can promote the cell apoptosis and inhibit the cell proliferation. Thirty-two BALB/c female mice were equally divided into four groups to study the effects of OT and atosiban (ATO) (an oxytocin receptor antagonist) on the mammary tumor growth. The animal weight, OT plasma concentration, and the tumor weight and volume were measured. Moreover, the tumor-related signaling pathways including NF-κB, miR-195, and Cyclin D1 were evaluated by qPCR assays, and Akt and ERK proteins were assessed by western blot at the end of the study. The volume and weight of tumors were significantly decreased after OT administration. The phosphorylated Akt and ERK expressions were significantly decreased in the OT group compared to the tumor group. In contrast, the dephosphorylated Akt and ERK expressions were significantly increased in the OT group in comparison with the tumor group. The mRNA expressions of miR-195, OTR, and Bax genes were significantly increased, and the mRNA expression of ERα, PI3K, NF-κB, cyclin D1 and Bcl-2 genes were decreased in the OT group in comparison with the tumor group. Interestingly, ATO administration reversed these effects. These results can exhibit a new therapeutic potential for OT on the down-regulation of the NF-κB and up-regulation of miR-195 and consequently, decrease of the tumor volume and weight in a mouse model of breast cancer.

10-gingerol inhibits proliferation of hepatocellular carcinoma HepG2 cells via Src/STAT3 signaling pathway

To study the inhibitory effect of 10-gingerol on the proliferation of hepatocellular carcinoma HepG2 cells and the role of Src/STAT3 signaling pathway in mediating the effect. SYBYL-X2.1 software was used to simulate the interaction between 10-gingerol and Src. HepG2 cells treated with 10-gingerol at 1, 3, 10 or μol/L for 24 h were assessed for cell viability using MTT assay, and EdU staining was used to detect the cell proliferation and calculate the number of positive cells. The expressions of p-Src and p-STAT3 were detected using Western blotting, and the mRNA expressions of the target genes of STAT3 (cyclin D1 and CMCC) were detected using qPCR. 10-gingerol was capable of forming hydrogen bond with such Src residues as TRY-340, MET-341, MET-314, ASP-404, and ILE-336. MTT assay showed that 10-gingerol at 3 and 10 μmol/L significantly lowered the viability of HepG2 cells (P < 0.001). Treatment with 1, 3, and 10 μmol/L 10-gingerol significantly reduces the number of EdU-positive HepG 2 cells (P < 0.001). Western blotting showed that 10-gingerol at 3 and 10 μmol/L significantly decreased the phosphorylation levels of Src and STAT3 in HepG2 cells (P < 0.01). 10-gingerol at 1, 3, and 10 μmol/L significantly decreased the mRNA expressions of cyclin D1 and CMCC as shown by qPCR (P < 0.01). 10-gingerol can dose-dependently inhibit the proliferation of HepG2 cells and suppress the activation of Src and STAT3.

Abstract 3452: TAK228, a kinase inhibitor of mTORC1 and mTORC2, is highly effective in ER+ breast cancer model

Abstract Background: mTOR is an atypical Ser/Thr kinase involved in regulating major cellular functions, such as nutrients sensing, growth, proliferation and angiogenesis. Current FDA approved clinical mTOR inhibitors only inhibit mTORC1 and have several limitations. First, inhibiting mTORC1 releases the negative feedback of PI3K-AKT signaling and therefore activates upstream AKT signaling. Secondly, allosteric inhibitors of rapalogs block the phosphorylation of some but not all mTORC1 downstream substrates. In particular phosphorylation of 4EBP1 is largely insensitive to rapalogs. In order to address these issues, “second generation” ATP competitive catalytic inhibitors including TAK228 against mTORC1/C2 have also been developed and are now in active in various stages of clinical trials. Purpose: Resistance of breast cancers (BC) to targeted hormone receptor inhibitors often occurs through deregulation of the PI3K-AKT-mTOR pathway. Mutations or loss of function of upstream regulators such as TSC1/2, LKB1, or components of the PI3K pathway such as PIK3CA, AKT, or PTEN have been reported in most types of human tumors including BC. Here, we investigated the effects of TAK228 alone or in conjunction with letrozole in ER+ BC cell lines. Methodology: Anti-proliferative, apoptotic, cell cycle, and intracellular signaling effects of TAK228 alone and in combination with letrozole were evaluated in ER +/PIK3CA mutated and ER+/PTEN mutated BC cell lines. Results: 1) TAK228 caused a strong differential growth inhibition in ER+/PIK3CA or ER+/PTEN mutated or ER+ (WT of PI3K pathway molecules) BC cell lines by 3D-ON-TOP clonogenic assay and real-time monitoring in an IncuCyte Zoom. Inhibition is greater when TAK228 was combined with letrozole. 2) Administration of TAK228 induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner. 3) qRT-PCR data showed that pro-apoptotic transcript, PUMA and cell survival transcripts SURVIVIN &amp; STATHMIN mRNA expressions were significantly increased and decreased respectively following the treatment of TAK228. 4) CYCLIN D1 mRNA expression was significantly decreased in both 24 &amp; 48 hrs following the treatment of TAK228. 5) TAK228 is mechanistically distinct from everolimus by a more potent and comprehensive inhibition of the PI3K-AKT-mTOR signaling network: a) avoided S6K inhibition-mediated feedback activation of PI3K-AKT and b) abrogated 4EBP1 phosphorylation. Conclusion: Data showed that TAK228 effectively inhibited proliferation and enhanced apoptosis via inhibition of the PI3K-AKT-mTORC1/C2 signaling pathway and dual TORC1/2 inhibition also mitigated the feedback activation of AKT caused by selective inhibition of mTORC1, and because of this, TAK228 may yield greater therapeutic benefit in BC patients. Citation Format: Pradip De, Jennifer H. Carlson, Tyler Jepperson, Casey Williams, Nandini Dey, Brian Leyland Jones. TAK228, a kinase inhibitor of mTORC1 and mTORC2, is highly effective in ER+ breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3452.

Hyperphosphorylation of protein Tau in hippocampus may cause cognitive dysfunction of propofol-anesthetized rats.

We aimed to explore the effect of hyperphosphorylation of Tau on cognitive function of propofol-anesthetized rats. Thirty 2-month-old male Wistar rats weighing 180-220 g were randomly divided into 3 groups (n=10): group of treating with saline (C group), group of treating with propofol for 1 hour (P1) and 24 h (P24 group). The cognitive function of rats was tested by Morris water maze before and 1 h or 24 h after drug administration. The rats were then sacrificed. The protein and mRNA expression levels of GSK-3β, total and phosphorylated Tau, cyclin D1, p27kip1 and c-caspase 3 in hippocampus were determined by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Compared with group C, the incubation period of P1 group and P24 group was prolonged, and the target quadrant retention time was shortened (p<0.05). There was no statistical difference between P1 and P24 group (p>0.05). Immunohistochemistry showed that compared with group C, p-Tau in hippocampus of P1 group and P24 group was highly expressed, with statistical difference (p<0.05). Western blot and RT-PCR showed that protein and mRNA expressions of GSK-3β, phosphorylated Tau, cyclin D1 and c-caspase 3 in hippocampus of P1 and P24 groups were up-regulated (p<0.05). Propofol-induced cognitive dysfunction in rats may be related to the hyperphosphorylation of Tau that causes neuronal cells to re-enter the cell cycle, thus leading to apoptosis.

Bioinformatic identification of key genes and analysis of prognostic values in clear cell renal cell carcinoma.

The present study aimed to identify new key genes as potential biomarkers for the diagnosis, prognosis or targeted therapy of clear cell renal cell carcinoma (ccRCC). Three expression profiles (GSE36895, GSE46699 and GSE71963) were collected from Gene Expression Omnibus. GEO2R was used to identify differentially expressed genes (DEGs) in ccRCC tissues and normal samples. The Database for Annotation, Visualization and Integrated Discovery was utilized for functional and pathway enrichment analysis. STRING v10.5 and Molecular Complex Detection were used for protein-protein interaction (PPI) network construction and module analysis, respectively. Regulation network analyses were performed with the WebGestal tool. UALCAN web-portal was used for expression validation and survival analysis of hub genes in ccRCC patients from The Cancer Genome Atlas (TCGA). A total of 65 up- and 164 downregulated genes were identified as DEGs. DEGs were enriched with functional terms and pathways compactly related to ccRCC pathogenesis. Seventeen hub genes and one significant module were filtered out and selected from the PPI network. The differential expression of hub genes was verified in TCGA patients. Kaplan-Meier plot showed that high mRNA expression of enolase 2 (ENO2) was associated with short overall survival in ccRCC patients (P=0.023). High mRNA expression of cyclin D1 (CCND1) (P<0.001), fms related tyrosine kinase 1 (FLT1) (P=0.004), plasminogen (PLG) (P<0.001) and von Willebrand factor (VWF) (P=0.008) appeared to serve as favorable factors in survival. These findings indicate that the DEGs may be key genes in ccRCC pathogenesis and five genes, including ENO2, CCND1, PLT1, PLG and VWF, may serve as potential prognostic biomarkers in ccRCC.

Inhibitory effects of XAV939 on the proliferation of small-cell lung cancer H446 cells and Wnt/β-catenin signaling pathway in vitro.

Lung cancer, including small-cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), are the most common tumor types, which represent 13% of newly diagnosed cancer cases worldwide. SCLC represents 15% of all lung cancer cases. Although an increasing number of novel targeted drugs are employed for the treatment of NSCLC, including Iressa, Tarceva and Conmana, there have been almost no major breakthroughs in SCLC over the last 30 years. Therefore, new drug targets are required to treat or prevent SCLC. Aberrant Wnt signaling is associated with numerous types of tumors, and it plays a key role in cell proliferation and survival. Recent preclinical studies suggested that XAV939 is a small-molecule inhibitor of the Wnt signaling pathway. In the present study, whether XAV939 is able to inhibit the proliferation of SCLC cells and the underlying mechanism were investigated. The inhibition of cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. The mRNA expression of β-catenin and cyclin D1 were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the protein expression of β-catenin and cyclin D1 was determined by western blotting. The results from the CCK-8 cell viability assay confirmed that XAV939 is able to inhibit the proliferation of SCLC cells in a dose-dependent manner. However, the effects of XAV939 were not time-dependent. By contrast, the effect of DDP treatment was time- and dose-dependent. Furthermore, the effect of combination treatment with XAV939 and DDP was antagonistic at low doses and synergistic at high doses. It was also observed that the mRNA and protein expression of β-catenin and cyclin D1 was significantly in SCLC cells following XAV939 treatment compared with the control group. These findings suggested that XAV939 is able to inhibit the proliferation of H446 cells, at least partially, through downregulating the Wnt/β-catenin signaling pathway. All of these results may provide potential therapeutic approaches for the treatment of SCLC.

The role of sex-determining region Y-box 6 in melanogenesis in alpaca melanocytes

BackgroundSex-determining region Y-box (SOX) proteins function as transcriptional regulators. The derivation of melanocytes from nerve crest cells has been reported to depend on SOX proteins, including SOX10 and SOX5. Whether SOX6 is expressed and has a functional role in melanocytes is unknown. ObjectiveWe aimed to study the effect of transcription factor SOX6 on melanogenesis in alpaca melanocytes. MethodsWe verified the role of SOX6 in melanogenesis by overexpressing and inhibiting SOX6 in melanocytes. Co-immunoprecipitation (co-IP) experiments were performed to further explore the function of SOX6 in melanogenesis and its mechanism of melanin production. We found that SOX6 interacted with cyclin-dependent kinase （CDK5）, β-catenin, and Cyclin D1. ResultsBioinformatics analysis suggested that SOX6 has a phosphorylation site for CDK5, which regulates melanogenesis, suggesting that SOX6 might play a role in melanogenesis. Co-IP experiments indicated that SOX6 interacted with CDK5, β-catenin, and Cyclin D1. Quantitative real-time polymerase chain reaction and western blot analyses of SOX6-overexpressing melanocytes revealed increased mRNA and protein expression of Cyclin D1, CDK5, microphthalmia transcription factor (MITF), tyrosinase (TYR), tyrosine related protein-1 (TYRP1), and dopachrome-tautomerase (DCT), whereas β-catenin levels decreased in SOX6-overexpressing melanocytes. The opposite results were observed upon SOX6 knockdown. The melanin content was significantly increased or decreased, respectively, by SOX6 overexpression or knockdown. ConclusionOur results suggest that SOX6 might enhance melanogenesis by binding with β-catenin to increase Cyclin D1 and MITF expression.

Molecular mechanism and cytotoxicity of allicin and all-trans retinoic acid against CD44+ versus CD117+ melanoma cells

BackgroundAll-trans retinoic acid (ATRA) is a differentiating agent that inhibits cancer cell growth during the cell cycle. However, despite its potent antitumor properties, some melanoma cells are resistant to ATRA therapy. PurposeHere, we hypothesized that allicin can sensitize malignant melanoma cells to ATRA treatment. To clarify this mechanism, we determined the sensitivity to ATRA, allicin and allicin/ATRA in CD44+ and CD117+ melanoma cell subpopulations. MethodsThe CD44+and CD117+cells were sorted from A375 melanoma cell line using the magnetic-activated cell sorting (MACS). The potential anticancer effects of ATRA, allicin and allicin/ATRA were examined using cell proliferation MTT assay. In addition, flow cytometry was used to detect cell cycle arrest. The efficacy of the treatments in controlling cancer cell proliferation was assessed by quantitative realtime polymerase chain reaction (RT-PCR). ResultsHere, we demonstrated that CD44+ melanoma cells were more resistant to allicin and ATRA than CD117+ cells. Importantly, we observed that allicin sensitized melanoma cell to ATRA-induced cell death. The combination treatment with allicin and ATRA significantly reduced the IC50 value obtained for ATRA alone in CD44+ melanoma cells. In CD44+ cells, the IC50 value of ATRA was 37.43 ± 0.54, while the IC50 value of allicin/ATRA treatment was 17.53 ± 0.2 µM. Allicin treatment resulted in significant increases in the percentage of cells at the G2/M and G0/G1 phases in the CD44+ and CD117+ cells, respectively. The combination treatment caused the inhibition of CD44+ and CD117+ melanoma cells at the S phases compared to ATRA alone. Allicin, ATRA, and allicin/ATRA increased the expression of cyclin D1 mRNA in both CD44+ and CD117+ cells. Allicin combination with ATRA increased the mRNA level of RARβ in CD117+ cells. Furthermore, allicin alone caused a remarkable reduction of MMP-9 mRNA expression in both CD44+ and CD117+ cells. In contrast, ATRA and the combination treatment significantly increased MMP-9 gene expression in CD44+ cells. ConclusionOverall, our results indicate that allicin reinforces the ATRA-mediated inhibitory effects on CD44+ and CD117+ melanoma cells and may provide a new approach for the treatment of malignant melanoma.

Metronomic vinorelbine is directly active on Non Small Cell Lung Cancer cells and sensitizes the EGFRL858R/T790M cells to reversible EGFR tyrosine kinase inhibitors

Metronomic vinorelbine (mVNR) has been described primarily as an antiangiogenic therapy, and no direct effects of mVNR on Non Small Cell Lung Cancer (NSCLC) cells has yet been demonstrated. The aims of this study were i) to establish the direct activity of mVNR on NSCLC cells either EGFR wt or EGFRL858R/T790M, and ii) to quantify the synergism of the combination with reversible EGFR tyrosine kinase inhibitors (TKIs), investigating the underlying mechanism of action. Proliferation assays were performed on A-549 (wt EGFRhigh), H-292 (EGFR-wt), H-358 (EGFR-wt), H-1975 (EGFRL858R/T790M) NSCLC cell lines exposed to mVNR, its active metabolite deacetyl-VNR (D-VNR), gefitinib and erlotinib for 144 h treatments. The synergism between mVNR and EGFR TKIs was determined by the combination index (CI) in EGFR-wt and H-1975 NSCLC cells. Cyclin-D1 and ABCG2 genes expression and protein levels were measured by RT-PCR and ELISA assays, as well as the phosphorylation of ERK1/2 and Akt. Intracellular concentrations of EGFR TKIs and VNR were investigated with a mass spectrometry system. mVNR, and its active metabolite D-VNR, were extremely active on NSCLC cells, in particular on H-1975 (IC50 = 13.56 ± 2.77 pM), resistant to TKIs. mVNR inhibited the phosphorylation of ERK1/2 and Akt and significantly decreased the expression of both cyclin-D1 and ABCG2 m-RNA and protein. The simultaneous combination of VNR and reversible EGFR TKIs showed a strong synergism on EGFR-wt NSCLC cells and on H-1975 cells (e.g. CI = 0.501 for 50% of affected cells), increasing the intracellular concentrations of EGFR TKIs (e.g. +50.5% vs. gefitinib alone). In conclusions, mVNR has direct effects on NSCLC cells and sensitizes resistant cells to EGFR TKIs, increasing their intracellular concentrations.

Abstract P1-13-08: Extended adjuvant neratinib/fulvestrant blocks ER/HER2 crosstalk and maintains complete responses of ER+/HER2+ tumors following treatment with chemotherapy and anti-HER2 therapy

Abstract Background: Neratinib is a potent, irreversible pan-HER tyrosine kinase inhibitor. The phase III trial ExteNET showed improved disease-free survival in patients (pts) with HER2+ breast cancer treated with neratinib vs placebo after trastuzumab-based adjuvant therapy. The benefit from neratinib appeared to be greater in pts with ER+ tumors. Thus, we sought to elucidate mechanisms that may explain the benefit from extended adjuvant therapy with neratinib in pts with ER+/HER2+ breast cancer using a human-in-mouse model that simulates the clinical outcomes seen in ExteNET. Results: Mice with established ER+/HER2 amplified MDA-361 tumors were treated with trastuzumab (tz) + paclitaxel (pac) for 4 weeks, and then randomized to fulvestrant (fulv) ± neratinib for 4 weeks. All MDA-361 tumors exhibited a prompt and marked reduction in volume after tz/pac treatment; 10 mice achieved a complete response (CR) before receiving 'extended adjuvant' therapy with fulv (n=5) or neratinib/fulv (n=5). A CR was maintained with neratinib/fulv following tz/pac. However, mice treated with fulv alone, relapsed rapidly (p&amp;lt;0.05 at week 8) despite of a complete downregulation of tumor ER levels. In a second experiment, nude mice with established MDA-361 xenografts were treated with pertuzumab/tz/pac for 4 weeks. Following a CR, mice were randomized to neratinib/fulv vs. fulv. Again, mice treated with neratinib/fulv maintained a CR, while mice in the fulv alone arm exhibited tumor progressions within a week. In three ER+/HER2+ cell lines (MDA-361, BT474 and UACC893) but not in ER+/HER– MCF7 cells, treatment with neratinib induced ER reporter transcriptional activity whereas treatment with fulv resulted in an increase in HER2 phosphorylation, suggesting compensatory crosstalk between the ER and HER2 pathways. To further understand the molecular basis of this crosstalk, MDA-361 tumor-bearing mice were treated with either fulv, neratinib or the combination for 7 days, after which tumors were harvested and analyzed using a Nanostring breast cancer ER panel consisting of 196 ER-regulated genes. Compared to vehicle or fulv-treated tumors, tumors treated with neratinib alone and neratinib/fulv showed marked downregulation of cyclin D1 mRNA expression. Similarly, in MDA-361, BT474 and UACC893 cells but not in MCF7 cells, only neratinib/fulv downregulated cyclin D1, P-AKT and P-ERK. Finally, treatment with neratinib/fulv but not fulv alone reduced cyclin D1 transcriptional reporter activity and cyclin D1 protein levels, and induced cell cycle arrest, suggesting double blockade is required to overcome compensatory crosstalk between ER and amplified HER2. Conclusions: Neratinib/fulv but not fulv alone maintained complete responses of ER+/HER+ tumors following treatment with tz/pac or pertuzumab/tz/pac, reminiscent of the results in ExteNET. Neratinib treatment promoted ER transcriptional activity whereas ER downregulation with fulv was associated with increased HER2 signaling. In ER+/HER2+ breast cancer cells and tumors, neratinib/fulv synergistically inhibited growth, cyclin D1 expression, and AKT and MAPK activation, thus providing a plausible mechanism to explain the results in the ExteNET trial. Citation Format: Sudhan DR, Schwarz LJ, Guerrero-Zotano AL, Nixon M, Formisano L, Croessmann S, Gonzalez Ericsson PI, Sanders ME, Balko JM, Avogadri-Connors F, Cutler RE, Lalani AS, Bryce R, Auerbach A, Arteaga CL. Extended adjuvant neratinib/fulvestrant blocks ER/HER2 crosstalk and maintains complete responses of ER+/HER2+ tumors following treatment with chemotherapy and anti-HER2 therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-13-08.

Effects of different starch source of starter on small intestinal growth and endogenous GLP-2 secretion in preweaned lambs.

The objective of this study was to investigate the effects of different sources of starch in starter feed on small intestinal growth and endogenous glucagon-like peptide 2 (GLP-2) secretion in preweaned lambs. Twenty-four 10-d-old lambs were divided into three groups that were treated with different iso-starch diets containing purified cassava starch (CS, n = 8), maize starch (MS, n = 8), and pea starch (PS, n = 8). At 56 d old, there was no significant difference in final body weight (BW) of lambs among the three groups. However, different starch source in starter significantly affected the average daily feed intake (ADFI) and average daily gain (ADG) of lambs among three groups. Compared with the CS and MS diets, the PS diet significantly increased the GLP-2 concentration in blood plasma (P < 0.001), the crypt depth of the jejunum (P = 0.006), and the villus height of the ileum (P = 0.039). Meanwhile, PS diet significantly increased the mRNA expression of proglucagon and the glucagon-like peptide 2 receptor (GLP-2R) in the jejunum and ileum (P < 0.001). Furthermore, the PS diet significantly upregulated the mRNA expression of cyclin D1 (P < 0.001), cyclin E (P = 0.006), and cyclin-dependent kinases 6 (CDK6) (P = 0.048) in the jejunum and cyclin A (P < 0.001), cyclin D1 (P < 0.001), and CDK6 (P = 0.002) in the ileum. Correlation analysis showed that endogenous GLP-2 secretion was positively related to the mRNA levels of cell cycle proteins in small intestinal mucosa. In summary, all results showed that PS in starter feed promoted small intestinal growth that may, in part, be related to cell cycle acceleration and endogenous GLP-2 secretion in preweaned lambs. These findings provide new insights into nutritional interventions that promote the development of small intestines in young ruminants.

Research of the inhibitory effect and the mechanisms of plumbagin on large cell lung cancer NL9980 cell line

Objective To investigate the effects of plumbagin on human NL9980 large-cell lung cancer cells and the role of interleukin-6/signal transducers and activators of transcription 3 (IL-6/STAT3) signaling pathway in the anticancer procedure of plumbagin. Methods Different concentrations of plumbagin were administrated in NL9980 cell line. Inhibitory effects of plumbagin on NL9980 cells were analyzed using methyl thiazol tetrazolium (MTT) assay. Groups of different drug doses (5.0, 7.5, 10.0 μmol/L) and blank control (without administration of plumbagin) were set with simple random sampling method. Cells were collected after 24 h of administration. Expression of IL-6 and STAT3 and the downstream genes B-cell lymphoma/leukemia-2 (bcl-2), vascular endothelial growth factor (VEGF), and Cyclin D1 were assessed by real-time quantitative polymerase chain reaction (Real-time PCR). Enzyme linked immunosorbent assay (ELISA) was used to measure secreted IL-6 levels and western blotting to measure intracellular phospho-STAT3 levels. Results Plumbagin significantly inhibited NL9980 cells via suppressing IL-6/STAT3 signaling. Expression of all genes was inhibited by plumbagin in a dose-dependent manner (F=32.170, P=0.000). As the concentration of plumbagin increased, the expression of IL-6 mRNA decreased significantly (F=173.067, P=0.000) to 0.50±0.02, 0.44±0.1, 0.34±0.04 respectively and IL-6 protein decreased to 4.71±0.02, 4.08±0.14, 2.01±0.07 significantlly (F=1 805.177, P=0.000). Besides, the expression of STAT3 mRNA (0.86±0.08, 0.72±0.03, 0.52±0.06, F=230.918, P=0.000) and protein (0.65±0.19, 0.45±0.22, 0.31±0.11, F=17.332, P=0.000) were reduced with the increase of concentration of plumbagin. Compared with the control group, expression of downstream genes bcl-2, VEGF and Cyclin D1 mRNA were decreased significantly after administration of plumbagin (F=33.698, P=0.000; F=337.743, P=0.000; F=180.136, P=0.000), and declined gradually as drug concentration increased. Moreover, bcl-2, VEGF, and Cyclin D1 mRNA levels positively correlated with levels of IL-6 and STAT3 gene expressions. Conclusion IL-6-mediated STAT3 suppression decreased bcl-2, VEGF, and Cyclin D1 expression. Thus, plumbagin may be an effective therapy for large-cell lung cancer that acts by targeting the IL-6/STAT3 signaling pathway. Key words: Plumbagin; Interleukin-6; Signal transducers and activators of transcription 3; Large cell lung cancer

Total flavonoids from Ampelopsis megalophylla suppress proliferation of vascular smooth muscle cells in vivo and in vitro

ABSTRACT Various benefits of flavonoids for ameliorating cardiovascular diseases have been demonstrated. However, the lowering effects on blood pressure caused by antiproliferative potentials of flavonoids in vascular smooth muscle cells are rare. In this study, the antihypertensive effects of total flavonoids from Ampelopsis megalophylla were investigated. The dynamic pressure values and the rate of media thickness versus lumen diameter were measured by the tail-cuff system and H&E staining in vivo, respectively. The mRNA expressions of ACE, Ang II, eNOS, c-Myc, cyclin D1 and p27Kip1 in thoracic aorta or A7r5 cells were measured by qPCR, respectively. The protein expressions of c-Myc, Cyclin D1, p27Kip1 and β-catenin in tissues or A7r5 cells were measured by Western blot assay. Total flavonoids of A. megalophylla (TFAM) reduced the expressions of ACE and Ang II, and elevated the content of eNOS in thoracic aorta cells of SHRs. Furthermore, TFAM decreased the mRNA and protein expressions of c-Myc and cyclin D1 by repressing the Wnt/β-catenin-mediated TCF/LEF transcriptional activation both in vivo and in vitro, which is synergetic with the up-regulation of p27Kip1 expression. Our study provided evidence for developing flavonoids from A. megalophylla as herbal supplements to prevent against cardiovascular diseases by suppressing vascular remodeling.

KSHV vPK inhibits Wnt signaling via preventing interactions between β-catenin and TCF4

Viral factors interact with host cellular proteins, leading to dysregulation of signaling pathways. The Wnt pathway is known to participate in embryonic development and oncogenesis under dysregulation conditions. A downstream factor of the Wnt signaling pathway, β-catenin, activates T-cell factor (TCF)-dependent transcription, which contributes to cell proliferation and tumorigenesis. In this study, we demonstrated that viral protein kinase (vPK) encoded by Kaposi's sarcoma-associated herpesvirus inhibits the Wnt signaling pathway without affecting nuclear localization and expression of β-catenin. Coimmunoprecipitation and chromatin immunoprecipitation assays revealed that vPK interacts with β-catenin, reducing the binding affinity on TCF binding regions as well as interactions of β-catenin with TCF4. Overexpression of vPK led to reduced mRNA expression of cyclin D1, a well-known transcriptional product of Wnt signaling, suggesting that vPK effectively regulates the host signaling pathway through direct interactions with cellular proteins.

Stimulatory effect of icariin on the proliferation of neural stem cells from rat hippocampus

BackgroundIcariin (ICA), a major ingredient of Epimediumbrevicornum, has various pharmacological activities including central nervous system protective functions such as the improvement of learning and memory function in mice models of Alzheimer’s disease. It has been reported that ICA can promote regeneration of peripheral nerve and functional recovery. The purpose of this study was to investigate the potentiating effect of ICA on the proliferation of rat hippocampal neural stem cells, and explore the possible mechanism involved.MethodsPrimary neural stem cells were prepared from the hippocampus of newly born SD rats, and cells were cultured in special stem cell culture medium. Neural stem cells were confirmed by immunofluorescence detection of nestin, NSE and GFAP expression. The effect of ICA on the growth and proliferation of the neural stem cells was evaluated by 5-ethynyl-2-deoxyuridine (EdU) labeling of proliferating cells, and photomicrographic images of the cultured neural stem cells. Further, the mechanism of ICA-induced cell proliferation of neural stem cells was investigated by analyzing the gene and protein expression of cell cycle related genes cyclin D1 and p21.ResultsThe present study showed that icariin promotes the growth and proliferation of neural stem cells from rat hippocampus in a dose-dependent manner. Incubation of cells with icariin resulted in significant increase in the number of stem cell spheres as well as the increased incorporation of EdU when compared with cells exposed to control vehicle. In addition, it was found that icariin-induced effect on neural stem cells is associated with increased mRNA and protein expression of cell cycle genes cyclin D1 and p21.ConclusionsThis study evidently demonstrates the potentiating effect of ICA on neural stem cell growth and proliferation, which might be mediated through regulation of cell cycle gene and protein expression promoting cell cycle progression.

Protective effect of epigenetic silencing of CyclinD1 against spinal cord injury using bone marrow-derived mesenchymal stem cells in rats.

This study focuses on the protective effect of epigenetic silencing of CyclinD1 against spinal cord injury (SCI) using bone marrow-derived mesenchymal stem cells (BMSCs) in rats. Eighty-eight adult female Wistar rats were randomly assigned into the sham group, the control group, the si-CyclinD1 + BMSCs group and the BMSCs group. CyclinD1 protein and mRNA expressions after siRNA transfection were detected by Western blotting and qRT-PCR. The siRNA-CyclinD1 BMSCs were transplanted into rats in the si-CyclinD1 + BMSCs group using stereotaxic method 6 hr after SCI. Hindlimb locomotor performance was determined using inclined plane test and Basso-Beattie-Bresnahan (BBB) locomotor rating scale. Expressions of glial fibrillary acidic protein (GFAP) and nerve growth factor (NGF) were detected by immunohistochemistry. Inclined plane and BBB scores in the control, si-CyclinD1 + BMSCs, and BMSCs groups were significantly lower than the sham group, but these scores were evidently decreased in the control group and increased in the si-CyclinD1 + BMSCs group compared with the BMSCs group. The repair degree of spinal cord tissues of rats in the si-CyclinD1 + BMSCs group was obvious than the BMSCs group. GFAP and NGF protein expressions were markedly decreased in the control, si-CyclinD1 + BMSCs and BMSCs groups when compared with the sham group. GFAP- and NGF-positive cells were significantly increased in the si-CyclinD1 + BMSCs group while decreased in the control group. Our study provides evidence that epigenetic silencing of CyclinD1 using BMSCs might accelerate the repair of SCI in rats.

Experimental study of miRNA200a regulating Wnt/β-catenin signaling pathway in silica-induced mouse lung epithelial cells

Objective: To observe the effect of overexpression of miRNA200a (miR-200a) recombinant lentivirus on the expression of Wnt/β-catenin signaling pathway in mouse lung epithelial cell line MLE-12 induced by silica (SiO(2)) . Methods: The mice were divided into SiO(2) control group (SiO(2)) , virus control group (SiO(2)+Lv-NC) group and overexpressing miR-200a virus group (SiO(2)+Lv-miR-200a). The expression of β-catenin, MMP2, MMP9, TCF-4 and Cyclin D1 mRNA and protein were detected by realtime-PCR and western blot after incubating cells for 18 h stimulating at the final concentration of 200 μg/ml of SiO(2). Results: The expression of miR-200a in MLE-12 cells of SiO(2)+Lv-miR-200 a group was significantly higher than that in SiO(2) group and SiO(2)+Lv-NC group. The mRNA and protein expression of β-catenin, MMP2, MMP9, TCF-4 and Cyclin D1 in MLE-12 cells of SiO(2)+Lv-miR-200a group were significantly lower than those in SiO(2) group and SiO(2)+Lv-NC group (P<0.05) . Conclusion: Overexpression of miR-200a can inhibit the expression of related genes of Wnt/β-catenin signaling pathway in silica-induced mouse lung epithelial cells.

DKK3 Overexpression Increases the Malignant Properties of Head and Neck Squamous Cell Carcinoma Cells.

DKK3, a member of the dickkopf Wnt signaling pathway inhibitor family, is believed to be a tumor suppressor because of its reduced expression in cancer cells. However, our previous studies have revealed that DKK3 expression is predominantly observed in head and neck/oral squamous cell carcinoma (HNSCC/OSCC). Interestingly, HNSCC/OSCC patients with DKK3 expression showed a high rate of metastasis and poorer survival, and siRNA-mediated knockdown of DKK3 in HNSCC-derived cancer cell lines resulted in reduced cellular migration and invasion. From these data, it was hypothesized that DKK3 might exert an oncogenic function specific to HNSCC. In the present research, the DKK3 overexpression model was established, and its influences were investigated, together with molecular mechanism studies. The DKK3 expression profile in cancer cell lines was investigated, including HNSCC/OSCC, esophageal, gastric, colorectal, pancreatic, prostatic, and lung cancers. DKK3 overexpression was performed in HNSCC-derived cells by transfection of expression plasmid. The effects of DKK3 overexpression were assessed on cellular proliferation, migration, invasion, and in vivo tumor growth. The molecular mechanism of DKK3 overexpression was investigated by Western blotting and microarray analysis. DKK3 overexpression significantly elevated cellular proliferation, migration, and invasion, as well as increased mRNA expression of cyclin D1 and c-myc. However, reporter assays did not show TCF/LEF activation, suggesting that the increased malignant property of cancer cells was not driven by the Wnt/β-catenin pathway. For the investigation of the pathways/molecules in DKK3-mediated signals, the Western blot analyses revealed that phosphorylation of Akt (S473) and c-Jun (Ser63) was elevated. The application of a PI3K kinase inhibitor, LY294002, on HSC-3 DKK3 cells significantly decreased tumor cell proliferation, migration, and invasion. From these results, we demonstrated that DKK3 might contribute to cellular proliferation, invasion, migration, and tumor cell survival in HNSCC cells through a mechanism other than the canonical Wnt signaling pathway, which might be attributed to PI3K–Akt signaling.

Effect of miR-103b on the growth of renal cell carcinoma by activating the expression of P21 protein

Objective To investigate the effect of miR-103b on the expression of P21 protein in renal cell carcinoma cell line 769-P and ACHN cells, and its effect on the growth of renal cell carcinoma. Methods Renal cancer cells were divided into two groups according to the transfected RNA, miR-103b (experimental group) and dsControl (control group), respectively. Real-time PCR and Western blotting were used to detect the expression of P21, cell cycle-dependent kinase 6, Cyclin D1 mRNA and protein expression. Flow cytometry was used to detect the cell cycle distribution. MTT assay was used to detect cell viability and colony formation assay was used to detect cell proliferation. Measurement data were represented as ±s. Comparison between groups was analyed using t test. Results Real-time PCR results showed that the relative expression levels of P21, cell cycle-dependent kinase 6 and Cyclin D1 mRNA in 769-P and ACHN which belong to control group cells were 1.00±0.10 and 1.02±0.27, 1.00±0.08 and 1.01±0.17, 1.01±0.19 and 1.00±0.02. The experimental group was 2.36±0.51 and 2.03±0.49, 0.33±0.20 and 0.58±0.22, 0.48±0.11 and 0.60±0.23, respectively, and the difference was statistically significant (P<0.05). Western blotting results were consistent with Real-time PCR results. Flow cytometry results showed that compared with the control group, the proportion of cells located in G0/G1 phase in the experimental group increased (P<0.05), suggesting that the cells were arrested in G0/G1 phase. MTT assay showed that the viability of 769-P and ACHN cells in the experimental group was significantly lower than that in the control group. Colony formation experiments showed that the number of colony formation in the experimental group was significantly less, suggesting that the cell proliferation capacity decreased. Conclusion miR-103b can inhibit the growth of renal cell carcinoma cells by activating the expression of P21 protein and blocking the progression of the renal cell cycle, which provides a theoretical basis for the molecular targeted therapy of renal cell carcinoma. Key words: MicroRNA; Renal tumor; Cell proliferation; RNA activation

Calcium propionate supplementation improves development of rumen epithelium in calves via stimulating G protein-coupled receptors

In the present study, calcium propionate (CaP) was used as feed additive in the diet of calves to investigate their effects on rumen fermentation and the development of rumen epithelium in calves. To elucidate the mechanism in which CaP improves development of calf rumen epithelium via stimulating the messenger RNA (mRNA) expression of G protein-coupled receptors, a total of 54 male Jersey calves (age=7±1 days, BW=23.1±1.2 kg) were randomly divided into three treatment groups: control without CaP supplementation (Con), 5% CaP supplementation (5% CaP) and 10% CaP supplementation (10% CaP). The experiment lasted 160 days and was divided into three feeding stages: Stage 1 (days 0 to 30), Stage 2 (days 31 to 90) and Stage 3 (days 91 to 160). Calcium propionate supplementation percentages were calculated on a dry matter basis. In total, six calves from each group were randomly selected and slaughtered on days 30, 90 and 160 at the conclusion of each experimental feeding stage. Rumen fermentation was improved with increasing concentration of CaP supplementation in calves through the first 30 days (Stage 1). No effects of CaP supplementation were observed on rumen fermentation in calves during Stage 2 (days 31 to 90). Supplementation with 5% CaP increased propionate concentration, but not acetate and butyrate in calves during Stage 3 (days 91 to 160). The rumen papillae length of calves in the 5% CaP supplementation group was greater than that of Con groups in calves after 160 days feeding. The mRNA expression of G protein-coupled receptor 41 (GPR41) and GPR43 supplemented with 5% CaP were greater than the control group and 10% CaP group in feeding 160 days calves. 5% CaP supplementation increased the mRNA expression of cyclin D1, whereas did not increase the mRNA expression of cyclin-dependent kinase 4 compared with the control group in feeding 160-day calves. These results indicate that propionate may act as a signaling molecule to improve rumen epithelium development through stimulating mRNA expression of GPR41 and GPR43.