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1.
Wnt inhibitory factor 1 is epigenetically silenced in human osteosarcoma,
and targeted disruption accelerates osteosarcomagenesis in mice 总被引:1,自引:0,他引:1
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Maya Kansara Michael Tsang Laurent Kodjabachian Natalie A. Sims Melanie K. Trivett Mathias Ehrich Alexander Dobrovic John Slavin Peter F.M. Choong Paul J. Simmons Igor B. Dawid David M. Thomas 《The Journal of clinical investigation》2009,119(4):837-851
Wnt signaling increases bone mass by stimulating osteoblast lineage commitment and expansion and forms the basis for novel anabolic therapeutic strategies being developed for osteoporosis. These strategies include derepression of Wnt signaling by targeting secreted Wnt pathway antagonists, such as sclerostin. However, such therapies are associated with safety concerns regarding an increased risk of osteosarcoma, the most common primary malignancy of bone. Here, we analyzed 5 human osteosarcoma cell lines in a high-throughput screen for epigenetically silenced tumor suppressor genes and identified Wnt inhibitory factor 1 (WIF1), which encodes an endogenous secreted Wnt pathway antagonist, as a candidate tumor suppressor gene. In vitro, WIF1 suppressed β-catenin levels in human osteosarcoma cell lines, induced differentiation of human and mouse primary osteoblasts, and suppressed the growth of mouse and human osteosarcoma cell lines. Wif1 was highly expressed in the developing and mature mouse skeleton, and, although it was dispensable for normal development, targeted deletion of mouse Wif1 accelerated development of radiation-induced osteosarcomas in vivo. In primary human osteosarcomas, silencing of WIF1 by promoter hypermethylation was associated with loss of differentiation, increased β-catenin levels, and increased proliferation. These data lead us to suggest that derepression of Wnt signaling by targeting secreted Wnt antagonists in osteoblasts may increase susceptibility to osteosarcoma. 相似文献
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Seok-Jun Kim 《Journal of controlled release》2011,155(3):427-434
MicroRNAs, small non-coding RNAs, are key regulators of tumorigenesis and cancer metastasis through inhibition of gene expression. Therefore, there is increasing interest in developing anti-cancer therapies using microRNAs. In this study, we determined the therapeutic potency of microRNA-145(miR-145) against breast cancer. We found a reverse-correlation between the expression of miR-145 and its target genes, such as fascin-1, c-myc, SMAD2/3 and IGF-1R in breast cancer cell lines and breast cancer patient tissues. Transfected miR-145 mimicking double-stranded oligonucleotides was directly reduced cell proliferation and motility via interaction with 3′UTR of target gene and also indirectly regulates Wnt signaling. An inhibitor of miR-145 nullified this decreasing effect of miR-145 on cell proliferation and motility. We prepared an adenoviral constructed miR-145(Ad-miR-145) and subjected it to breast cancer cells in vitro and orthotopic breast cancer mice in vivo. Ad-miR-145 suppressed cell growth and motility in both the in vitro and in vivo systems. Furthermore, a treatment combining Ad-miR-145 with 5-FU significantly showed anti-tumor effects, compared to treating alone. In conclusion, this study demonstrated that miR-145 suppresses tumor growth by inhibition of multiple tumor survival effectors, and more we suppose that miR-145 is potentially useful in the therapy of breast cancers. 相似文献
4.
Koornneef A van Logtenstein R Timmermans E Pisas L Blits B Abad X Fortes P Petry H Konstantinova P Ritsema T 《Gene therapy》2011,18(9):929-935
RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i). U1i is a gene-silencing technique that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined effect allows usage of minimal doses, thereby avoiding toxicity while retaining high target inhibition. As a proof of concept, we investigated knockdown of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs showed additive reduction of luciferase expression up to 95% in vitro. We attained similar knockdown when RNAi and U1i constructs were hydrodynamically transfected into murine liver, demonstrating for the first time successful in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knockdown in vivo. 相似文献
5.
Kato Y Salumbides BC Wang XF Qian DZ Williams S Wei Y Sanni TB Atadja P Pili R 《Molecular cancer therapeutics》2007,6(1):70-81
Resistance to chemotherapy is a major hurdle in the treatment of malignant melanoma. Histone deacetylase (HDAC) inhibitors have been shown to have antitumor activity in different tumor types, including melanoma, and to reverse epigenetic repression of tumor suppressor genes, such as retinoic acid receptor beta (RARbeta). In this study, we tested the antitumor effect of the HDAC inhibitor LAQ824 in combination with 13-cis-retinoic acid (CRA) on two human melanoma cell lines both in vitro and in vivo. Treatment of LAQ824 showed a dose-dependent inhibitory effect on A2058 and HMV-I cell lines in a clonogenic assay. These cell lines were relatively resistance to CRA. On treatment with combination of LAQ824 and CRA, a greater inhibitory effect (up to 98%) was achieved compared with single agents. Lack of RARbeta2 gene expression was associated with histone acetylation and gene methylation at the promoter level. Treatment with LAQ824 restored retinoid sensitivity by reverting RARbeta2 epigenetic silencing. The biological effect of LAQ824 was associated with p21 induction in both cell lines but G(2) cell cycle arrest in A2058 and apoptosis in HMV-I cell line. The induction of apoptosis by LAQ824 was associated with increased reactive oxygen species and induction of SM22 gene expression in HMV-I but not in A2058 cell line. Administration of the free radical scavenger l-N-acetylcysteine blocked LAQ824 + CRA-mediated apoptosis in HMV-I cells, suggesting a primary role for reactive oxygen species generation in LAQ824 + CRA-associated lethality. Combination treatment showed 61% and 82% growth inhibition in A2058 and HMV-I tumors, respectively. Greater induction of in vivo apoptosis was observed in the HMV-I but not in the A2058 tumors treated with combination therapy compared with single agents. These results suggest that the HDAC inhibitor LAQ824 has a greater antitumor activity in combination with CRA in melanoma tumors but the degree of induced apoptosis may vary. Combination of HDAC inhibitors and retinoids represents a novel therapeutic approach for malignant melanoma that warrants clinical testing. 相似文献
6.
J. Susie Zoltewicz Amir M. Ashique Youngshik Choe Gena Lee Stacy Taylor Khanhky Phamluong Mark Solloway Andrew S. Peterson 《PLoS Clinical Trials》2009,4(7)
Precise regulation of Wnt signaling is important in many contexts, as in development of the vertebrate forebrain, where excessive or ectopic Wnt signaling leads to severe brain defects. Mutation of the widely expressed oto gene causes loss of the anterior forebrain during mouse embryogenesis. Here we report that oto is the mouse ortholog of the gpi deacylase gene pgap1, and that the endoplasmic reticulum (ER)-resident Oto protein has a novel and deacylase-independent function during Wnt maturation. Oto increases the hydrophobicities of Wnt3a and Wnt1 by promoting the addition of glycophosphatidylinositol (gpi)-like anchors to these Wnts, which results in their retention in the ER. We also report that oto-deficient embryos exhibit prematurely robust Wnt activity in the Wnt1 domain of the early neural plate. We examine the effect of low oto expression on Wnt1 in vitro by knocking down endogenous oto expression in 293 and M14 melanoma cells using shRNA. Knockdown of oto results in increased Wnt1 secretion which is correlated with greatly enhanced canonical Wnt activity. These data indicate that oto deficiency increases Wnt signaling in vivo and in vitro. Finally, we address the mechanism of Oto-mediated Wnt retention under oto-abundant conditions, by cotransfecting Wnt1 with gpi-specific phospholipase D (GPI-PLD). The presence of GPI-PLD in the secretory pathway results in increased secretion of soluble Wnt1, suggesting that the gpi-like anchor lipids on Wnt1 mediate its retention in the ER. These data now provide a mechanistic framework for understanding the forebrain defects in oto mice, and support a role for Oto-mediated Wnt regulation during early brain development. Our work highlights a critical role for ER retention in regulating Wnt signaling in the mouse embryo, and gives insight into the notoriously inefficient secretion of Wnts. 相似文献
7.
Pachynski RK Zabel BA Kohrt HE Tejeda NM Monnier J Swanson CD Holzer AK Gentles AJ Sperinde GV Edalati A Hadeiba HA Alizadeh AA Butcher EC 《The Journal of experimental medicine》2012,209(8):1427-1435
Infiltration of specialized immune cells regulates the growth and survival of neoplasia. Here, in a survey of public whole genome expression datasets we found that the gene for chemerin, a widely expressed endogenous chemoattractant protein, is down-regulated in melanoma as well as other human tumors. Moreover, high chemerin messenger RNA expression in tumors correlated with improved outcome in human melanoma. In experiments using the B16 transplantable mouse melanoma, tumor-expressed chemerin inhibited in vivo tumor growth without altering in vitro proliferation. Growth inhibition was associated with an altered profile of tumor-infiltrating cells with an increase in natural killer (NK) cells and a relative reduction in myeloid-derived suppressor cells and putative immune inhibitory plasmacytoid dendritic cells. Tumor inhibition required host expression of CMKLR1 (chemokine-like receptor 1), the chemoattractant receptor for chemerin, and was abrogated by NK cell depletion. Intratumoral injection of chemerin also inhibited tumor growth, suggesting the potential for therapeutic application. These results show that chemerin, whether expressed by tumor cells or within the tumor environment, can recruit host immune defenses that inhibit tumorigenesis and suggest that down-regulation of chemerin may be an important mechanism of tumor immune evasion. 相似文献
8.
Ai Z Fischer A Spray DC Brown AM Fishman GI 《The Journal of clinical investigation》2000,105(2):161-171
9.
Annalisa Crema Mario Ledda Daniela Fioretti Maria Grazia Lolli Massimo Sanchez Elisabetta Carico Rodolfo Marchese Monica Rinaldi Antonella Lisi 《Journal of tissue engineering and regenerative medicine》2019,13(6):1031-1043
Cell therapy represents a promising alternative strategy for end‐stage liver disease, and hepatic progenitors are the best candidates. The possibility to maximize the paracrine effects of transplanted cells represents a great potential benefit for cell therapy success. We studied how cell type and microenvironment modulate the Wnt/β‐catenin signaling in vitro and in vivo. In vitro, the onset of hepatocyte commitment was characterized by the presence of nuclear truncated β‐catenin. In vivo, we analyzed the effect of human hepatic progenitors on damage recovery and functional regeneration in a mouse model of acute liver injury, either in combination or in absence of a selected mix of hepatogenic factors. Animals injected with human hepatic progenitors and hepatogenic factors showed improved engraftment triggering the Wnt/β‐catenin signaling cascade. Human hepatic progenitors expressing the human oval cell marker OV6 displayed a consistent colocalization with β‐catenin and colocalized with Wnt1 main ligand of the canonical pathway. Wnt5a, on the contrary, was expressed in distinct liver cell populations. Epithelial mesenchymal transition‐related markers showed enhanced expression and wider distribution, and the hepato‐mesenchymal population Thy1 + CK19? was also present. Control animals injected with hepatogenic factors alone exhibited higher β‐catenin, decreased Wnt5a levels, and persistent proliferation of the hepato‐mesenchymal population. In conclusion, the combination of human hepatic progenitors with selected hepatogenic factors creates a positive synergy with local microenvironment, ameliorates cell engraftment, stimulates and accelerates regenerative process, and improves the rescue of hepatic function by modulating the Wnt/βcatenin signaling and activating hepato‐mesenchymal population. 相似文献
10.
Increasing study reports that Wnt/β-catenin signaling pathway plays an essential role in numerous cancers growth, progression and metastasis. Aberrant miR-940 expression has been studied in gastric and breast cancer. However, the molecular mechanism of miR-940 enhancing proliferation and metastatic ability in human pancreatic carcinoma is far from to know. Real-time PCR was used to quantify miR-940 expression. Luciferase reporter assays here were performed to verify the activity of Wnt/β-catenin signaling pathway and targeting gene relationships, and immunofluorescence assay was applied to observe β-catenin expressed intensity. Bioinformatics analysis together with in vivo and vitro functional analysis indicated the potential targeting genes of miR-940. Specimens from 15 pairs of patients with human pancreatic carcinoma were involoved to confirm the relationship between miR-940 expression and the GSK3β/sFRP1 through real-time PCR and western blot assays. Bioinformatics combined with cell luciferase function researches determined the possible regulation of miR-940 on the 3′-UTR of the GSK3β and sFRP1 genes, resulting in the Wnt/β-catenin signaling activation. Further, miR-940 knockdown significantly recovered GSK3β and sFRP1 expression and relieved Wnt/β-catenin-mediated cell invasion, migration, metastasis and proliferation. The ectopic up-regulation of miR-940 significantly suppressed GSK3β/sFRP1 expression and promoted pancreatic carcinoma proliferation and invasion. Our study suggested mechanistic relationship between miR-940 and Wnt/β-catenin in the development and progression of pancreatic carcinoma through regulation of GSK3β and sFRP1. 相似文献
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Interaction between CD44 and hyaluronate is directly implicated in the regulation of tumor development 总被引:28,自引:3,他引:25
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《The Journal of experimental medicine》1994,180(1):53-66
CD44 is implicated in the regulation of tumor growth and metastasis but the mechanism by which expression of different CD44 isoforms determines the rate of primary and secondary tumor growth remains unclear. In the present study we use a human melanoma transfected with wild-type and mutant forms of CD44 to determine which functional property of the CD44 molecule is critical in influencing tumor behavior. We show that expression of a wild-type CD44 isoform that binds hyaluronic acid augments the rapidity of tumor formation by melanoma cells in vivo, whereas expression of a CD44 mutant, which does not mediate cell attachment to hyaluronate, fails to do so. The importance of CD44- hyaluronate interaction in tumor development is underscored by the differential inhibitory effect of soluble wild-type and mutant CD44-Ig fusion proteins on melanoma growth in vivo. Whereas local administration of a mutant, nonhyaluronate binding, CD44-Ig fusion protein has no effect on subcutaneous melanoma growth in mice, infusion of wild-type CD44-Ig is shown to block tumor development. Taken together, these observations suggest that the tumor growth promoting property of CD44 is largely dependent on its ability to mediate cell attachment to hyaluronate. 相似文献
13.
Exploring RNA interference as a therapeutic strategy for renal disease 总被引:17,自引:0,他引:17
Takabatake Y Isaka Y Mizui M Kawachi H Shimizu F Ito T Hori M Imai E 《Gene therapy》2005,12(12):965-973
The short synthetic interfering RNA duplexes (siRNAs) can selectively suppress gene expression in somatic mammalian cells without nonselective toxic effects of double-stranded RNA (dsRNA). However, a selective in vivo delivery of siRNA transfer has not been reported in kidney. Here, we investigated whether injection of synthetic siRNAs via renal artery followed by electroporation could be effective and therapeutic in silencing specific gene in glomerulus. We investigated the effect of siRNA in rat cultured mesangial cells (MCs) and showed that siRNA sequence-specific suppression of transgene expression was over a 1000-fold more potent than that by antisense oligodeoxynucleotide (ASODN). Transfection of siRNA targeting luciferase into rat kidneys significantly inhibited expression of a cotransfected luciferase expression vector in vivo. The delivery of siRNA targeting enhanced green fluorescent protein (EGFP) in the transgenic 'green' rat reduced endogenous EGFP expression, mainly in glomerular MCs. Furthermore, RNAi targeting against TGF-beta1 significantly suppressed TGF-beta1 mRNA and protein expression, thereby ameliorated the progression of matrix expansion in experimental glomerulonephritis. In addition, vector-based RNAi also inhibited TGF-beta1 expression in vitro and in vivo. In conclusion, siRNA-directed TGF-beta1 silencing may be of therapeutic value in the prevention and treatment of fibrotic diseases. 相似文献
14.
Dihydromyricetin enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro partially via the activation of Wnt/β‐catenin signaling pathway
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Houfa Yin Erman Chen Deting Xue Qiang Zheng Xiang Gao Zhijun Pan 《Fundamental & clinical pharmacology》2016,30(6):596-606
Substantial evidence has demonstrated that the decreased osteogenic differentiation of bone mesenchymal stem cells (BMSCs) is closely related to bone metabolic diseases. Thus, it is very important to develop several potentially useful therapeutic agents to enhance BMSC osteogenesis. Flavonoids show promise in enhancing bone mass. Dihydromyricetin (DMY), a type of flavonoid, has not yet been investigated regarding its effects on BMSC osteogenesis. To investigate the effects of DMY on osteogenesis, human BMSCs were induced with or without DMY. We found that DMY (0.1–50 μm ) exhibited no cytotoxic effect on proliferation, but increased alkaline phosphatase activity, osteoblast‐specific gene expression, and mineral deposition. It also enhanced active β‐catenin expression and reduced dickkopf‐1(DKK1) and sclerostin expression. The Wnt/β‐catenin signaling pathway inhibitor (DKK1 and β‐catenin‐specific siRNA) decreased the enhanced bone mineral formation caused by DMY. Taken together, these findings reveal that DMY enhances osteogenic differentiation of human BMSCs partly through Wnt/β‐catenin in vitro. 相似文献
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Intradermal administration of DNA vaccines encoding luciferase represents a convenient method to assess gene expression in vivo. Gene silencing by intradermal gene gun administration of DNA encoding short hairpin RNA (shRNA) may represent an effective technique for the specific knockdown of gene expression in vivo. In the current study, we characterized luciferase gene expression over time in vivo by noninvasive bioluminescence imaging. Furthermore, we characterized in vivo luciferase gene silencing with DNA encoding shRNA targeting luciferase. We also characterized human papillomavirus type 16 (HPV-16) E7-specific CD8(+) T cell immune responses in mice immunized with E7 DNA and DNA encoding shRNA targeting Fas ligand (FasL), a key proapoptotic signaling protein. Our results indicated that coadministration of DNA encoding shRNA targeting luciferase significantly reduced luciferase expression in mice intradermally administered luciferase DNA. Furthermore, we observed that mice vaccinated with E7-expressing DNA coadministered with DNA encoding shRNA targeting FasL generated significantly enhanced E7-specific CD8(+) cytotoxic T cell responses as well as potent therapeutic antitumor effects against E7-expressing tumors. Thus, intradermal administration of DNA encoding shRNA represents a plausible approach to silence genes in vivo and a potentially useful tool to enhance DNA vaccine potency. 相似文献
17.
Melanoma is the most common malignant skin cancer, appears indestructible and is notoriously resistant to all current modalities of cancer treatment strategies. Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor, has shown to have radiosensitizing properties for a variety of cancers. Recently, we have shown that PTX exhibits antimetastatic and anti-angiogenic activities in B16F10 melanoma cells in vitro as well as in vivo. In the present study, we have demonstrated the anticancer and antimetastatic potential of PTX against A375 human melanoma cell line at sub-toxic doses. The results implicate that PTX at sub-toxic doses exhibited an inhibitory effect on the ability of cellular proliferation as shown by MTT and colony formation assay. It impedes migration and also induces apoptosis. A375 cells pretreated with PTX showed decrease in adhesion to both Matrigel and Collagen type IV. Further, Gelatin zymography result reveals that PTX treatment decreases the secretion of MMP2 and MMP9. Finally, PTX significantly inhibited A375 subcutaneous tumour xenograft growth without having any toxicity. Thus PTX at sub-toxic doses affected melanoma metastasis at multiple steps in vitro as well as tumour growth in vivo. These data demonstrate its antimetastatic potential and provide preclinical evidence for the development of PTX as a potential agent against metastatic melanoma. 相似文献
18.
Qiuzhen Liu Sara Tomei Maria Libera Ascierto Valeria De Giorgi Davide Bedognetti Cuilian Dai Lorenzo Uccellini Tara Spivey Zoltan Pos Jaime Thomas Jennifer Reinboth Daniela Murtas Qianbing Zhang Lotfi Chouchane Geoffrey R. Weiss Craig L. Slingluff Jr. Peter P. Lee Steven A. Rosenberg Harvey Alter Kaitai Yao Ena Wang Francesco M. Marincola 《The Journal of clinical investigation》2014,124(5):2147-2159
19.
Lopez MV Blanco P Viale DL Cafferata EG Carbone C Gould D Chernajovsky Y Podhajcer OL 《Molecular cancer therapeutics》2006,5(10):2503-2511
20.
Steg A Wang W Blanquicett C Grunda JM Eltoum IA Wang K Buchsbaum DJ Vickers SM Russo S Diasio RB Frost AR LoBuglio AF Grizzle WE Johnson MR 《The Journal of molecular diagnostics : JMD》2006,8(1):76-83
Recent studies have shown the hedgehog and Wnt families of signaling proteins to be associated with tumor initiation, growth, and survival. However, these pathways remain unexplored in ovarian endometrioid adenocarcinoma (OEA). Here, we describe a novel TaqMan low-density array to examine the expression of 26 and 20 genes in the hedgehog and Wnt pathways, respectively, in six matched snap-frozen and formalin-fixed, paraffin-embedded (FPE) OEA specimens. Expression values were normalized to uninvolved ovarian epithelium. Gene expression in matched frozen and FPE tissues demonstrated significant concordance (r = 0.92, P < 0.0001). However, comparison of amplified and unamplified RNA from frozen OEA tissues revealed an altered molecular profile in amplified RNA. Amplification of RNA from FPE tissues was not successful. The expression of Desert hedgehog (DHH), Indian hedgehog (IHH), Hedge-hog interacting protein (HHIP), Wnt10B, Wnt9B, and Wnt inhibitory factor (WIF1) were tumor-specific with no detectable expression in normal ovarian epithelium. In addition, several genes were significantly (P < 0.025) down-regulated in OEA, including cyclin E2, Porcupine, c-Myc, and Axin 2 (4.8-, 3.6-, 2.9-, and 1.9-fold, respectively). TaqMan low-density array provides an effective multivariate technique for examining gene expression in RNA isolated from either snap-frozen or archival FPE tissues and can identify tumor-specific genes, possibly leading to novel treatments. 相似文献