Notch1 mRNA Research Articles

Hepatic Notch2 deficiency leads to bile duct agenesis perinatally and secondary bile duct formation after weaning

Notch signaling plays an acknowledged role in bile-duct development, but its involvement in cholangiocyte-fate determination remains incompletely understood. We investigated the effects of early Notch2 deletion in Notch2fl/fl/Alfp-Cretg/− (“Notch2-cKO”) and Notch2fl/fl/Alfp-Cre−/− (“control”) mice. Fetal and neonatal Notch2-cKO livers were devoid of cytokeratin19 (CK19)-, Dolichos-biflorus agglutinin (DBA)-, and SOX9-positive ductal structures, demonstrating absence of prenatal cholangiocyte differentiation. Despite extensive cholestatic hepatocyte necrosis and growth retardation, mortality was only ~15%. Unexpectedly, a slow process of secondary cholangiocyte differentiation and bile-duct formation was initiated around weaning that histologically resembled the ductular reaction. Newly formed ducts varied from rare and non-connected, to multiple, disorganized tubular structures that connected to the extrahepatic bile ducts. Jaundice had disappeared in ~30% of Notch2-cKO mice by 6 months. The absence of NOTCH2 protein in postnatally differentiating cholangiocyte nuclei of Notch2-cKO mice showed that these cells had not originated from non-recombined precursor cells. Notch2 and Hnf6 mRNA levels were permanently decreased in Notch2-cKO livers. Perinatally, Foxa1, Foxa2, Hhex, Hnf1β, Cebpα and Sox9 mRNA levels were all significantly lower in Notch2-cKO than control mice, but all except Foxa2 returned to normal or increased levels after weaning, coincident with the observed secondary bile-duct formation. Interestingly, Hhex and Sox9 mRNA levels remained elevated in icteric 6 months old Notch2-cKOs, but decreased to control levels in non-icteric Notch2-cKOs, implying a key role in secondary bile-duct formation. ConclusionCholangiocyte differentiation becomes progressively less dependent on NOTCH2 signaling with age, suggesting that ductal-plate formation is dependent on NOTCH2, but subsequent cholangiocyte differentiation is not.

Short hairpin RNA targeting Notch2 inhibits U87 human glioma cell proliferation by inducing cell cycle arrest and apoptosis in vitro and in vivo.

Notch signaling has been reported to be oncogenic or tumor suppressive, depending on the tissue context. To investigate the effects of Notch2 knockdown on U87 human glioma cell proliferation in vitro and in vivo, and the associated mechanisms, U87 cells were stably transfected with p green fluorescent protein (GFP)-V-RS Notch2 short hairpin (sh) RNA plasmid and pGFP-V-RS scramble-shRNA plasmid. The former was referred to as the Notch2-shRNA group and the latter as the negative-shRNA group. mRNA and protein expression, cell proliferation, cell cycle and apoptosis were measured by reverse transcription-polymerase chain reaction, western blot analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and flow cytometry using propidium iodide, respectively. Tumor volume, tumor weight and cumulative survival rate were determined in a nude mouse xenograft tumor model. Notch2 mRNA and protein expression in the Notch2-shRNA group were reduced by 87.6 and 94.5% compared with the negative-shRNA group (P<0.001). Notch2 knockdown significantly inhibited U87 cell proliferation after three days of culture (P<0.05). Notch2 silencing induced cell cycle arrest at G0/G1 phase by upregulation of p21 protein expression and downregulation of mini chromosome maintenance complex 2 and cyclin-D1 protein expression. Furthermore, knockdown of Notch2 also induced U87 cell apoptosis. On day 50 after inoculation, tumor weight in the Notch2-shRNA group was significantly lower than that in the negative-shRNA group (0.55±0.10 vs. 1.23±0.52 g; P<0.01). The cumulative survival rate was significantly longer in the Notch2-shRNA group compared with the negative-shRNA group (log rank test P=0.01). In conclusion, Notch2 silencing inhibited U87 glioma cell proliferation by inducing cell cycle arrest and apoptosis in vitro and in vivo. Thus, Notch2 may be a key therapeutic target for the treatment of glioma.

Molecular mechanism of Doxorubicin resistance in multiple myeloma cell line

This study was aimed to investigate the molecular mechanism of doxorubicin resistance in multiple myeloma cell line and certify the effect of Notch signal over-expression on drug resistance of myeloma cells. The doxorubicin RPMI 8226 cell line (RPMI8226/DOX) was established by culturing 8226 cells with continuous low concentration and intermittent gradually-increasing-concentration of doxorubicin in vitro, the mRNA expression of Notch2,Jagged1, Jagged2, HES1 were measured by RT-PCR and the P-170 protein expression was detected by Western blot in RPMI 8226 cell line; the changes of IL-6 and VEGF were tested by ELISA. The results showed that the Notch mRNA expression (Notch2, Jagged1, Jagged2 increased gradually along with the increase of chemotherapeutic drug resistance, but the expression of HESI mRNA gradually decreased along with the increase of drug resistance. The expression level of P-170 protein was upregulated gradually along with the increase of drug resistance. The level of VEGF and IL-6 in culture supernatants of RPMI8226/DOX was higher than that in RPMI 8226. It is concluded that the establishment of RPMI 8226/DOX cell line is a useful model to analyze the mechanism of chemotherapeutic drug resistance in multiple myeloma, Notch activation is closely correlated with the drug resistance of multiple myeloma and Notch signaling may to be used as a new target for multiple myeloma treatment.

Abstract 910: NOTCH3 expression is predictive of efficacy in pancreas tumor models treated with OMP-59R5, a monoclonal antibody targeting the NOTCH2 and NOTCH3 receptors

Abstract The Notch signaling pathway regulates key functions during embryonic development, stem cell maintenance and differentiation in adult tissues, and is implicated in many human cancers. OMP-59R5 is a ligand-blocking antibody targeting both the NOTCH2 and NOTCH3 receptors. We have developed a series of primary human xenograft models from patients with pancreatic cancer and used these models to examine efficacy response to OMP-59R5. We found that anti-NOTCH2/3, either as a single agent or in combination with chemotherapeutic agents, was efficacious in pancreatic tumor models. Expression of NOTCH3 mRNA by next-generation sequencing in ten baseline pancreatic tumors correlates with response to OMP-59R5, where growth of tumors with moderate to high expression of NOTCH3 was significantly reduced compared to tumors with low expression. We developed a Research-Use-Only (RUO) qPCR assay for measuring NOTCH3 mRNA expression using Formalin-Fixed, Paraffin-Embedded (FFPE) samples. This assay shows consistent NOTCH3 expression data with the next-generation sequencing data in the ten pancreatic xenograft tumors. Expression levels of NOTCH3 were also examined in ∼120 human metastatic pancreatic specimens to determine the reportable range of the assay and to identify association with clinical factors. This analysis showed that NOTCH3 gene expression maintained the same distribution across different specimen types, such as biopsy, surgical biopsy and surgical resection, etc. Samples with clinically relevant sites of recurrence also showed a similar range in NOTCH3 gene expression. Moreover, we developed an immunohistochemistry (IHC) assay for NOTCH3 protein expression. The correlation between the IHC assay and the qPCR assay was examined in both the metastatic pancreatic human specimens and the primary human pancreatic xenograft models. A significant correlation was found between the gene and protein levels, suggesting that both NOTCH3 gene expression and protein expression may predict the response to OMP-59R5 in pancreatic cancer. We are evaluating NOTCH3 levels and patient response in ALPINE, a Ph1b/2 Anti-NOTCH2/3 trial in first-line advanced pancreatic cancer patients. Citation Format: Belinda Cancilla, Wan-Ching Yen, Chun Zhang, Marcus M. Fischer, May Ji, Tracy Tang, Yu-Wang Liu, Raymond S. Tam, Min Wang, Austin Gurney, Timothy Hoey, John Lewicki, Ann M. Kapoun. NOTCH3 expression is predictive of efficacy in pancreas tumor models treated with OMP-59R5, a monoclonal antibody targeting the NOTCH2 and NOTCH3 receptors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 910. doi:10.1158/1538-7445.AM2014-910

Abstract 1945: TORC inhibitors increase the cancer stem cell (CSC) population and Notch signaling in triple negative breast cancer

Abstract Tumor-initiating cells (TICs) or cancer stem cells (CSCs) are resistant to chemotherapy and have been associated with metastatic recurrences and poor patient outcome particularly among patients with triple negative breast cancer (TNBC). Genomic and proteomic data have indicated more than 30% of TNBC patients have PI3K/mTOR pathway lesions making this pathway a promising therapeutic target. Recent publications have demonstrated mechanisms of resistance (JAK2/STAT5 and MYC amplification) to PI3K pathway inhibitors. We hypothesized that resistance to TORC inhibition is due to the survival of a CSC population and that targeting pathways that sustain these cells can provide a significant therapeutic benefit. Treatment of TNBC cell lines with the PI3K/mTOR inhibitor NVP-BEZ235 or the TORC1/2 inhibitor MLN128 resulted in a significant reduction of proliferation in vitro. However, we observed that both BEZ235 and MLN128 enriched for a CSC population as assessed by FACS analysis of cancer stem-like markers and mammosphere formation. This observation was specific to TNBC cell lines since BEZ235 and MLN128 significantly abrogated the CSC population in ER+ (MCF7) and HER2+ (HCC1954) breast cancer cell lines. To determine the mechanisms involved in this CSC enrichment we used a Stem Cell specific PCR Array. We observed an increase in Notch1, FGF1 and ABCG2 mRNA levels in TNBC cells treated with BEZ235 and MLN128. Treatment with these inhibitors also increased the expression of the active Notch intracellular domain, the Notch ligand Jagged1, and the Notch1 target genes Hes1 and Hey1 by qRT-PCR and transcriptional reporter activity. In addition to Hes1 and Hey1, c-myc, another Notch target gene, expression was augmented in 2 of the 3 TNBC cell lines tested. Treament with the γ-secretase inhibitor, DAPT and transfection with Notch1 siRNA oligonucleotides abrogated BEZ235 and INK128-mediated enrichment of CSC populations as measured by FACS analysis and mammosphere formation assays. To determine whether inhibition of either TORC1 or TORC2 enriched for the CSC population, we used RNAi against Rictor (TORC2), Raptor (TORC2) or both. We observed that only the combined knockdown of Rictor and raptor increased the CSC population in TNBC cell lines. These observations suggest that treatment of TNBC harboring PI3K pathway aberrations with TORC1/2 inhibitors results in an initial reduction of tumor burden but do not eradicate the drug-resistant, slow cycling CSC population driven by Notch signaling. Thus, combination of a Notch inhibitor with TORC1/2 inhibitors and chemotherapy may be an effective therapeutic strategy to decrease primary tumor growth and prevent recurrences in patients with TNBC. Citation Format: Neil E. Bhola, Valerie Jansen, Carlos Arteaga. TORC inhibitors increase the cancer stem cell (CSC) population and Notch signaling in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1945. doi:10.1158/1538-7445.AM2014-1945

MiR-429 represses cell proliferation and induces apoptosis in HBV-related HCC

MicroRNAs are a class of endogenous non-coding RNAs that regulate gene expression at post-transcriptional level, thus participating in diverse biological pathways. Increasing miRNAs are found to dysregulate hepatocellular carcinoma (HCC) and are involved in liver tumorigenesis. In this study, miR-429 was found to obviously downregulate much more in hepatitis B virus (HBV)-related hepatocellular carcinoma. To evaluate the effects of miR-429, miR-429 was over-expressed in HepG2.2.15 cells. The results have proved that overexpression of miR-429 decreased cell proliferation and induced cell apoptosis. Further, overexpression of miR-429 can suppress the secretion of HBsAg and HBeAg. In concordance to this, the level of NOTCH1 expression was high in human HBV-related HCC tissues and HepG2.2.15 cells. MiR-429 directly targeted NOTCH1 and reduced both mRNA and protein levels of NOTCH1 which stimulated proliferation and suppressed apoptosis in HCC cells. Our results provide new insight into the function of miR-429 in HBV-related HCC. It is beneficial to insight into the mechanism of HBV infection and pathophysiology of HBV-related HCC.

Notch-1 regulates proliferation and differentiation of human bladder cancer cell lines by inhibiting expression of Krüppel-like factor 4.

Inhibition of Notch signaling pathways, consisting of 4 highly conserved receptors (Notch 1-4), induces expression of Krüppel-like transcription factors (KLFs) linked to bladder cancer tumorigenesis and metastasis. Effects of Notch-1 knockdown on cell proliferation, differentiation and KLF4 levels in bladder cancer cell lines were investigated. PsiRNA1‑mediated Notch-1 and KLF4 knockdown models and control model without the psiRNA1 vector were constructed using bladder cancer cell lines T24 and BIU87. Cell proliferation, apoptosis and expression of Notch-1 and KLF4 were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry assay with AnnexinV-FITC/PI staining, and reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis, respectively. Proliferation was assessed in Notch-1 and/or KLF4 knockdown. The results showed that Notch-1 mRNA and protein expression levels were significantly lower following psiRNA1 vector transfection in both cell lines (P<0.05). Growth and proliferation of both cell lines were significantly inhibited by Notch-1 knockdown (P<0.05), and more G0/G1 arrest and apoptosis were observed compared to those in the control groups (P<0.05). The effects were time-dependent, peaking between 24-48h and declining by 72h. KLF4 expression was significantly higher in the Notch-1 knockdown group than in control cells (P<0.05). Notch-1 knockdown cell proliferation was significantly lower than that of Notch-1 and KLF4 knockdown (P<0.05). In conclusion, Notch-1 may act as an oncogene, regulating the proliferation and differentiation of bladder cancer cells by inhibiting KLF4. Pending further exploration of pathway variations and crosstalk, these pathways may be useful targets for bladder cancer therapy.

Abstract 50: Therapeutic Silencing of miRNA-652 Restores Cardiac Function and Attenuates Pathological Remodeling in a Mouse Model of Pressure Overload

Introduction: Targeting microRNAs differentially regulated in settings of stress and protection could represent a new approach for the treatment of heart failure. miR-652 expression increased in hearts of a cardiac stress mouse model and was downregulated in a model of cardiac protection. Aim: To assess the therapeutic potential of silencing miR-652 in a mouse model with established pathological hypertrophy and cardiac dysfunction due to pressure overload. Methods: Mice were subjected to a sham operation (n=10) or transverse aortic constriction (TAC, n=14) for 4 weeks to induce hypertrophy and cardiac dysfunction. Mice were subcutaneously administered a locked nucleic acid (LNA)-antimiR-652 or LNA-control. Cardiac function was assessed by echocardiography before and 8 weeks post treatment, followed by molecular and histological analyses. Results: Expression of miR-652 increased in hearts subjected to pressure overload compared to sham operated mice (2.9 fold, n=3-5, P&lt;0.05), but was silenced in hearts of mice administered LNA-antimiR-652 (95% decrease, n=3-7, P&lt;0.05). In mice subjected to pressure overload, inhibition of miR-652 improved cardiac function (29±1% at 4 weeks post TAC compared to 35±1% post treatment, n=7, P&lt;0.001) and attenuated cardiac hypertrophy. Functional and morphologic improvements in hearts of treated mice were associated with reduced cardiac fibrosis, apoptosis, cardiomyocyte size; decreased B-type natriuretic peptide gene expression; and preserved angiogenesis (all P&lt;0.05, n=4-7/group). Mechanistically, we identified Jagged1, a Notch1 ligand, as a direct target of miR-652 by luciferase assay. Jagged1 and Notch1 mRNA were upregulated in hearts of TAC treated mice (1.2-1.7 fold, n=7, P&lt;0.05). Importantly, chronic knockdown of miR-652 was not associated with any notable toxicity in other tissues. Conclusion: Therapeutic silencing of miR-652 protects the heart against pathological cardiac remodeling and improves heart function via mechanisms that are associated with preserved angiogenesis, decreased fibrosis and upregulation of a miR-652 target, Jagged1. These studies provide the first evidence that targeted inhibition of miR-652 could represent an attractive approach for the treatment of heart failure.

Increased leukemia-associated gene expression in benzene-exposed workers

Long-term exposure to benzene causes several adverse health effects, including an increased risk of acute myeloid leukemia. This study was to identify genetic alternations involved in pathogenesis of leukemia in benzene-exposed workers without clinical symptoms of leukemia. This study included 33 shoe-factory workers exposed to benzene at levels from 1 ppm to 10 ppm. These workers were divided into 3 groups based on the benzene exposure time, 1- < 7, 7- < 12, and 12- < 24 years. 17 individuals without benzene exposure history were recruited as controls. Cytogenetic analysis using Affymetrix Cytogenetics Array found copy-number variations (CNVs) in several chromosomes of benzene-exposed workers. Expression of targeted genes in these altered chromosomes, NOTCH1 and BSG, which play roles in leukemia pathogenesis, was further examined using real-time PCR. The NOTCH1 mRNA level was significantly increased in all 3 groups of workers, and the NOTCH1 mRNA level in the 12- < 24 years group was significantly higher than that in 1- < 7 and 7- < 12 years groups. Compared to the controls, the BSG mRNA level was significantly increased in 7- < 12 and 12- < 24 years groups, but not in the 1- < 7 years group. These results suggest that CNVs and leukemia-related gene expression might play roles in leukemia development in benzene-exposed workers.

Effect of spinal cord extracts after spinal cord injury on proliferation of rat embryonic neural stem cells and Notch signal pathway in vitro

ObjectiveTo investigate the effect of the spinal cord extracts (SCE) after spinal cord injuries (SCIs) on the proliferation of rat embryonic neural stem cells (NSCs) and the expressions of mRNA of Notch1 as well as of Hes1 in this process in vitro. MethodsThe experiment was conducted in 4 different mediums: NSCs+PBS (Group A-blank control group), NSCs+SCE with healthy SD rats (Group B-normal control group), NSCs+SCE with SD rats receiving sham-operation treatment (Group C-sham-operation group) and NSCs+ SCE with SCIs rats (Group D-paraplegic group). Proliferative abilities of 4 different groups were analyzed by MTT chromatometry after co-culture for 1, 2, 3, 4 and 5 d, respectively. The expressions of Notch1 and Hes1 mRNA were also detected with RT-PCR after co-culture for 24 and 48 h, respectively. ResultsAfter co-culture for 1, 2, 3, 4 and 5 d respectively, the MTT values of group D were significantly higher than those of group A, group B and group C (P<0.05). However, there were no significantly differences regarding MTT values between group A, group B and group C after co-culture for 1, 2, 3, 4 and 5 d, respectively (P>0.05). Both the expressions of Notch1 and Hes1 mRNA of group D were significantly higher than those of other 3 groups after co-culture for 24 h and 48 h as well (P<0.05). But there was no difference oin expressions of Notch1 and Hes1 mRNA among group A, group B and group C after co-culture for 24 h and 48 h (P>0.05). There was no difference in expressions of Notch1 and Hes1 mRNA between 24 h and 48 h treatment in group D. ConclusionsSCE could promote the proliferation of NSCs. It is demonstrated that the microenvironment of SCI may promote the proliferation of NSCs. Besides, SCE could increase the expression of Notch1 and Hes1 mRNA of NSC. It can be concluded that the Notch signaling pathway activation is one of the mechanisms that locally injured microenvironment contributes to the proliferation of ENSC after SCIs. This process may be performed by up-regulating the expressions of Notch1 and Hes1 gene.

DAPT suppresses the proliferation of human glioma cell line SHG-44

ObjectiveTo explore the suppressing effect of γ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism. MethodsThe SHG-44 cell was treated by DAPT with different concentration. The proliferation of cells was detected by MTT assay; cell cycle and TSC of CD133+ were determined by flow cytometry analysis technique; the key factor in Notch signaling pathway (Notch-1, Delta-1, Hes-1) was measured by reverse transcrip tase-polymerase chain reaction and western blotting. ResultsDAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05). And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner. DAPT increased the rate of cells in G0/G1 phase of SHG-44 cells, while it decreased the rate of cells in S phase. TSC of CD133+ was significantly reduced after DAPT treated SHG-44 cells. The expression of protein and mRNA of Notch-1, Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses. ConclusionsDAPT can downregulate these key factor in Notch signaling pathway, reduce the TSC of CD133+ and inhibit the proliferation of SHG-44 cells.

The role of RhoC in epithelial-to-mesenchymal transition of ovarian carcinoma cells

BackgroundRhoC is a small G protein/GTPase and involved in tumor mobility, invasion and metastasis. Previously, up-regulated RhoC expression is found to play an important role in ovarian carcinogenesis and subsequent progression by modulating proliferation, apoptosis, migration and invasion.MethodsWe transfected RhoC-expressing plasmid and RhoC siRNA into CAOV3 and OVCAR3 cells respectively. These cells and transfectants were exposed to vascular epithelial growth factor (VEGF), transforming growth factor (TGF)-β1 or their receptor inhibitors with the phenotypes and their related-molecules examined.ResultsTGF-β1R or VEGFR inhibitor suppressed the proliferation, migration, invasion and lamellipodia formation, the expression of N-cadherin, α-SMA, snail and Notch1 mRNA or protein, and enhanced E-cadherin mRNA and protein expression in CAOV3 and its RhoC-overexpressing transfectants, whereas both growth factors had the opposite effects in OVCAR3 cells and their RhoC-hypoexpressing transfectants. Ectopic RhoC expression enhanced migration, invasion, lamellipodia formation and the alteration in epithelial to mesenchymal transition (EMT) markers of CAOV3 cells regardless of the treatment of VEGFR or TGF-β1R inhibitor, whereas RhoC knockdown resulted in the converse in OVCAR3 cells even with the exposure to VEGF or TGF-β1.ConclusionRhoC expression might be involved in EMT of ovarian epithelial carcinoma cells, stimulated by TGF-β1 and VEGF.

CD24 regulates stemness and the epithelial to mesenchymal transition through modulation of Notch1 mRNA stability by p38MAPK

We report here that CD24 knockdown resulted in decreased expression of Notch1 in MCF-7 cells. CD24-downstream p38MAPK was shown to regulate Notch1 at the level of mRNA stability. We also found that CD24-mediated cell migration, invasion, mammosphere formation, and drug resistance was regulated by its downstream target Notch1. Together, our results indicate that CD24 may regulate the epithelial to mesenchymal transition and stemness through Notch1 signaling in breast cancer cells.

Visfatin promotes cell and tumor growth by upregulating Notch1 in breast cancer.

Overexpression of Notch1 has been associated with breast cancer. We recently showed that visfatin stimulates breast cancer cell proliferation and invasion. The present study was undertaken to determine whether Notch1 signaling is affected by visfatin and to characterize the functional role of the visfatin-Notch1 axis in breast cancer. Visfatin and Notch1 were expressed at higher levels in breast tumors than in matched control tissues. Visfatin induced Notch1 expression in MDA-MB-231 breast cancer cell line and in nontransformed MCF10A mammary epithelial cells, whereas visfatin depletion reduced Notch1 mRNA and protein levels. Depletion of Notch1 in MDA-MB-231 cells attenuated cell growth in vitro and in vivo; visfatin depletion produced similar effects, but was less potent. Additionally, Notch1 depletion inhibited cell proliferation induced by visfatin. Analysis of the signaling pathways underlying visfatin-mediated Notch1 upregulation revealed that visfatin activated NF-κB p65. Blockade of NF-κB signaling suppressed the effects of visfatin on Notch1 upregulation and breast cancer cell proliferation. Breast tumors expressing high levels of NF-κB p65 exhibited increased expression of Notch1. Our results demonstrate that the visfatin-Notch1 axis contributes to breast tumor growth through the activation of the NF-κB pathway. Study of the visfatin-Notch1 axis may offer new therapeutic directions for breast cancer.

AB0051 Toll-Like Receptor 2 Activation Induces Pro-Inflammatory, Inflammasome and Notch Signalling Pathways in Rheumatoid Arthritis

BackgroundRheumatoid arthritis (RA) is characterised by inflammation and proliferation of synovial tissue, leading to progressive degradation of articular cartilage and bone.ObjectivesThis study examines the effect of Toll-like Receptor (TLR) 2...

Drosophila p53 controls Notch expression and balances apoptosis and proliferation.

A balance between cell proliferation and apoptosis is important for normal development and tissue homeostasis. Under stress conditions, the conserved tumor suppressor and transcription factor Dp53 induces apoptosis to contribute to the maintenance of homeostasis. However, in some cases Dp53-induced apoptosis results in the proliferation of surrounding non-apoptotic cells. To gain insight into the Dp53 function in the control of apoptosis and proliferation, we studied the interaction between the Drosophila Dp53 and Notch genes. We present evidence that simultaneous reduction of Dp53 and Notch function synergistically increases the wing phenotype of Notch heterozygous mutant flies. Further, we found that a Notch cis-regulatory element is responsive to loss and gain of Dp53 function and that over-expression of Dp53 up-regulates Notch mRNA and protein expression. These findings suggest not only that Dp53 and Notch act together to control wing development but also indicate that Dp53 transcriptionally regulates Notch expression. Moreover, using Notch gain and loss of function mutations we examined the relevance of Dp53 and Notch interactions in the process of Dp53-apoptosis induced proliferation. Results show that proliferation induced by Dp53 over-expression is dependent on Notch, thus identifying Notch as a new player in Dp53-induced proliferation. Interestingly, we found that Dp53-induced Notch activation and proliferation occurs even under conditions where apoptosis was inhibited. Our findings highlight the conservation between flies and vertebrates of the Dp53 and Notch cross-talk and suggest that Dp53 has a dual role regulating cell death and proliferation gene networks to control the homeostatic balance between apoptosis and proliferation.

Down-regulation of Notch1 by small interfering RNA enhances chemosensitivity to gemcitabine in pancreatic cancer cells through activating apoptosis activity

To investigate the effect of down-regulation of Notch1 by Notch1 small interfering RNA (siRNA) on chemosensitivity to gemcitabine in pancreatic cancer cells and its mechanism. Notch1 siRNA was transfected to pancreatic cancer cell lines AsPC-1, BxPC-3, MIAPaCa-2 and Panc-1. The transfected pancreatic cancer cells were treated with 10 μmol/L gemcitabine in vitro. The relative quantity of Notch1 mRNA of pancreatic cancer cells was detected by real-time PCR. The inhibition rates of gemcitabine-treated cells were evaluated by CCK-8 method. The expression of Bax protein was examined by Western blot, and the caspase 3 activity was detected by CaspACETM assay system kit. The relative quantity of Notch1 mRNA was the highest in BxPC-3 cell line and the lowest in Panc-1 cells. The inhibition rates of gemcitabine treated-cells were significantly higher in Notch1 siRNA transfection groups than in corresponding siRNA control groups (AsPC-1: 67.5±6.7 vs 47.5±6.8; BxPC-3: 90.5±4.4 vs 70.2±4.2; MIAPaCa-2: 80.9±5.7 vs 58.1±6.0; Ps<0.05), with the overexpression of protein Bax. The activity of caspase 3 was also significantly increased in Notch1 siRNA transfection groups compared with corresponding siRNA control groups (AsPC-1: 28.90±2.70 vs 12.82±3.44; BxPC-3: 59.87±6.77 vs 27.27±11.88; MIAPaCa-2: 29.34±4.06 vs 14.59±4.25; P<0.05). Inhibition of Notch signaling pathway by Notch1 siRNA can enhance chemosensitivity to gemcitabine in pancreatic cancer cells through activating apoptosis activity．

Crosstalk between miR-34a and Notch Signaling Promotes Differentiation in Apical Papilla Stem Cells (SCAPs)

Stem cells from the apical papilla (SCAPs) are important for the formation and regeneration of root dentin. Here, we examined the expression of Notch signaling components in SCAPs and investigated crosstalk between microRNA miR-34aand Notch signaling during cell differentiation. We found that human SCAPs express NOTCH2, NOTCH3, JAG2, DLL3, and HES1, and we tested the relationship between Notch signaling and both cell differentiation and miR-34a expression. NOTCH activation in SCAPs inhibited cell differentiation and up-regulated the expression of miR-34a, whereas miR-34a inhibited Notch signaling in SCAPs by directly targeting the 3′UTR of NOTCH2 and HES1 mRNA and suppressing the expression of NOTCH2, N2ICD, and HES1. DSPP, RUNX2, OSX, and OCN expression was consequently up-regulated. Thus, Notch signaling in human SCAPs plays a vital role in maintenance of these cells. miR-34a interacts with Notch signaling and promotes both odontogenic and osteogenic differentiation of SCAPs.

Protective Effect of Curcumin on Acute Airway Inflammation of Allergic Asthma in Mice Through Notch1–GATA3 Signaling Pathway

Curcumin, a natural product derived from the plant Curcuma longa, has been found to have anti-inflammatory, antineoplastic and antifibrosis effects. It has been reported that curcumin attenuates allergic airway inflammation in mice through inhibiting NF-κB and its downstream transcription factor GATA3. It also has been proved the antineoplastic effect of curcumin through down-regulating Notch1 receptor and its downstream nuclear transcription factor NF-κB levels. In this study, we aimed to investigate the anti-inflammatory effect of curcumin on acute allergic asthma and its underlying mechanisms. 36 male BALB/c mice were randomly divided into four groups (normal, asthma, asthma+budesonide and asthma+curcumin groups). BALF (bronchoalveolar lavage fluid) and lung tissues were analyzed for airway inflammation and the expression of Notch1, Notch2, Notch3, Notch4 and the downstream transcription factor GATA3. Our findings showed that the levels of Notch1 and Notch2 receptors were up-regulated in asthma group, accompanied by the increased expression of GATA3. But the expression of Notch2 receptor was lower than Notch1 receptor. Curcumin pretreatment improved the airway inflammatory cells infiltration and reversed the increasing levels of Notch1/2 receptors and GATA3. Notch3 receptor was not expressed in all of the four groups. Notch4 receptor protein and mRNA expression level in the four groups had no significant differences. The results of the present study suggested that Notch1 and Notch2 receptor, major Notch1 receptor, played an important role in the development of allergic airway inflammation and the inhibition of Notch1–GATA3 signaling pathway by curcumin can prevent the development and deterioration of the allergic airway inflammation. This may be a possible therapeutic option of allergic asthma.

Resveratrol Inhibits Phenotypic Switching of Neointimal Vascular Smooth Muscle Cells After Balloon Injury Through Blockade of Notch Pathway

Phenotypic switching of vascular smooth muscle cells (VSMCs) plays an initial role in neointimal hyperplasia, the main cause of many occlusive vascular diseases. The aim of this study was to measure the effects of resveratrol (RSV) on the phenotypic transformation of VSMCs and to investigate its mechanism of action. Cultured VSMCs isolated from rat thoracic aorta were prepared with serum starvation for 72 hours followed by RSV treatment (50-200 μmol/L) and 10% serum stimulation. Male Sprague-Dawley rats, subjected to carotid arteries injury from a balloon catheter, were exposed to intraperitoneal injection of RSV (1 mg/kg) or saline and were killed after 7 or 28 days. Compared with cells in the serum-induced group, VSMCs in the RSV or N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment group exhibited significant decreases of proliferation and migration. The total and cytoplasmic Notch-1 levels were declined by RSV, accompanied by a significant increase in smooth muscle α-actin and smooth muscle myosin heavy chain protein. The expression of Notch-1, Jagged-1, Hey-1, and Hey-2 mRNA in balloon-injured arteries at 7 days was decreased by RSV treatment. Arteries from RSV-treated rats showed less neointimal hyperplasia, lower collagen content, and a lower rate of cells positive for proliferating cell nuclear antigen 28 days after injury, compared with saline controls. The results indicate that RSV can attenuate phenotypic switching of VSMCs after arterial injury through inhibition of the Notch pathway.