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1.
Background
Chemoresistance is the principal reason for poor survival and disease recurrence in osteosarcoma patients. Inosine 5′-monophosphate dehydrogenase type II (IMPDH2) encodes the rate-limiting enzyme in the de novo guanine nucleotide biosynthesis and has been linked to cell growth, differentiation, and malignant transformation. In a previous study we identified IMPDH2 as an independent prognostic factor and observed frequent IMPDH2 overexpression in osteosarcoma patients with poor response to chemotherapy. The aim of this study was to provide evidence for direct involvement of IMPDH2 in the development of chemoresistance.Methodology/Principal Findings
Stable cell lines overexpressing IMPDH2 and IMPDH2 knock-down cells were generated using the osteosarcoma cell line Saos-2 as parental cell line. Chemosensitivity, proliferation, and the expression of apoptosis-related proteins were analyzed by flow cytometry, WST-1-assay, and western blot analysis. Overexpression of IMPDH2 in Saos-2 cells induced strong chemoresistance against cisplatin and methotrexate. The observed chemoresistance was mediated at least in part by increased expression of the anti-apoptotic proteins Bcl-2, Mcl-1, and XIAP, reduced activation of caspase-9, and, consequently, reduced cleavage of the caspase substrate PARP. Pharmacological inhibition of IMPDH induced a moderate reduction of cell viability and a strong decrease of cell proliferation, but no increase in chemosensitivity. However, chemoresistant IMPDH2-overexpressing cells could be resensitized by RNA interference-mediated downregulation of IMPDH2.Conclusions
IMPDH2 is directly involved in the development of chemoresistance in osteosarcoma cells, suggesting that targeting of IMPDH2 by RNAi or more effective pharmacological inhibitors in combination with chemotherapy might be a promising means of overcoming chemoresistance in osteosarcomas with high IMPDH2 expression. 相似文献2.
3.
Background
The signaling pathways that may modulate the pathogenesis of diseases induced by expanded polyglutamine proteins are not well understood.Methodologies/Principal Findings
Herein we demonstrate that expanded polyglutamine protein cytotoxicity is mediated primarily through activation of p38MAPK and that the atypical PKC iota (PKCι) enzyme antagonizes polyglutamine-induced cell death through induction of the ERK signaling pathway. We show that pharmacological blockade of p38MAPK rescues cells from polyglutamine-induced cell death whereas inhibition of ERK recapitulates the sensitivity observed in cells depleted of PKCι by RNA interference. We provide evidence that two unrelated proteins with expanded polyglutamine repeats induce p38MAPK in cultured cells, and demonstrate induction of p38MAPK in an in vivo model of neurodegeneration (spinocerebellar ataxia 1, or SCA-1).Conclusions/Significance
Taken together, our data implicate activated p38MAPK in disease progression and suggest that its inhibition may represent a rational strategy for therapeutic intervention in the polyglutamine disorders. 相似文献4.
5.
Farshid Dayyani Nila U. Parikh Andreas S. Varkaris Jian H. Song Shhyam Moorthy Tanushree Chatterji Sankar N. Maity Adam R. Wolfe Joan M. Carboni Marco M. Gottardis Christopher J. Logothetis Gary E. Gallick 《PloS one》2012,7(12)
Background
Treatment of metastatic prostate cancer (PCa) with single agents has shown only modest efficacy. We hypothesized dual inhibition of different pathways in PCa results in improved tumor inhibition. The Src family kinases (SFK) and insulin-like growth factor-1 (IGF-1) signaling axes are aberrantly activated in both primary PCa and bone metastases and regulate distinct and overlapping functions in PCa progression. We examined the antitumor effects of combined inhibition of these pathways.Materials and Methods
Src andIGF-1 receptor (IGF-1R) inhibition was achieved in vitro by short hairpin (sh)RNA and in vitro and in vivo by small molecule inhibitors (dasatinib and BMS-754807, against SFK and IGF-1R/Insulin Receptor(IR), respectively).Results
In vitro, inhibition of IGF-1 signaling affected cell survival and proliferation. SFK blockade alone had modest effects on proliferation, but significantly enhanced the IGF-1R blockade. These findings correlated with a robust inhibition of IGF-1-induced Akt1 phophorylation by dasatinib, whereas Akt2 phosphorylation was SFK independent and only inhibited by BMS-754807. Thus, complete inhibition of both Akt genes, not seen by either drug alone, is likely a major mechanism for the decreased survival of PCa cells. Furthermore, dasatinib and BMS-754807 inhibited in vivo growth of the primary human xenograft MDA PCa 133, with corresponding inhibition of Akt in tumors. Also, both orthotopic and intratibial tumor growth of PC-3 cells were more potently inhibited by dual SFK and IGF-1R/IR blockade compared to either pathway alone, with a corresponding decrease in bone turnover markers.Conclusions
Dual IGF-1R/IR and SFK inhibition may be a rational therapeutic approach in PCa by blocking both independent and complementary processes critical to tumor growth. 相似文献6.
7.
Rastegar F Gao JL Shenaq D Luo Q Shi Q Kim SH Jiang W Wagner ER Huang E Gao Y Shen J Yang K He BC Chen L Zuo GW Luo J Luo X Bi Y Liu X Li M Hu N Wang L Luther G Luu HH Haydon RC He TC 《PloS one》2010,5(12):e14182
Background
Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated.Methodology/Principal Findings
Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma.Conclusions/Significance
Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective pharmacological inhibitors. 相似文献8.
Background
Most mitochondrial mRNAs in Trypanosoma brucei require RNA editing for maturation and translation. The edited RNAs primarily encode proteins of the oxidative phosphorylation system. These parasites undergo extensive changes in energy metabolism between the insect and bloodstream stages which are mirrored by alterations in RNA editing. Two U-specific exonucleases, KREX1 and KREX2, are both present in protein complexes (editosomes) that catalyze RNA editing but the relative roles of each protein are not known.Methodology/Principal Findings
The requirement for KREX2 for RNA editing in vivo was assessed in both procyclic (insect) and bloodstream form parasites by methods that use homologous recombination for gene elimination. These studies resulted in null mutant cells in which both alleles were eliminated. The viability of these cells demonstrates that KREX2 is not essential in either life cycle stage, despite certain defects in RNA editing in vivo. Furthermore, editosomes isolated from KREX2 null cells require KREX1 for in vitro U-specific exonuclease activity.Conclusions
KREX2 is a U-specific exonuclease that is dispensable for RNA editing in vivo in T. brucei BFs and PFs. This result suggests that the U deletion activity, which is required for RNA editing, is primarily mediated in vivo by KREX1 which is normally found associated with only one type of editosome. The retention of the KREX2 gene implies a non-essential role or a role that is essential in other life cycle stages or conditions. 相似文献9.
Jingyue Liu Mingxiang Zhang Chao Niu Zhengxiu Luo Jihong Dai Lijia Wang Enmei Liu Zhou Fu 《PloS one》2013,8(4)
Background
Glucocorticoids (GCs) are a first-line treatment for asthma for their anti-inflammatory effects, but they also hinder the repair of airway epithelial injury. The anti-inflammatory protein GC-induced leucine zipper (GILZ) is reported to inhibit the activation of the mitogen-activated protein kinase (MAPK)-extracellular-signal-regulated kinase (ERK) signaling pathway, which promotes the repair of airway epithelial cells around the damaged areas. We investigated whether the inhibition of airway epithelial repair imposed by the GC dexamethasone (DEX) is mediated by GILZ.Methods
We tested the effect of DEX on the expressions of GILZ mRNA and GILZ protein and the MAPK-ERK signaling pathway in human airway epithelial cells, via RT-PCR and Western blot. We further evaluated the role of GILZ in mediating the effect of DEX on the MAPK-ERK signaling pathway and in airway epithelium repair by utilizing small-interfering RNAs, MTT, CFSE labeling, wound-healing and cell migration assays.Results
DEX increased GILZ mRNA and GILZ protein levels in a human airway epithelial cell line. Furthermore, DEX inhibited the phosphorylation of Raf-1, Mek1/2, Erk1/2 (components of the MAPK-ERK signaling pathway), proliferation and migration. However, the inhibitory effect of DEX was mitigated in cells when the GILZ gene was silenced.Conclusions
The inhibition of epithelial injury repair by DEX is mediated in part by activation of GILZ, which suppressed activation of the MAPK-ERK signaling pathway, proliferation and migration. Our study implicates the involvement of DEX in this process, and furthers our understanding of the dual role of GCs. 相似文献10.
Chun-Hua Dai Jian Li Ping Chen He-Guo Jiang Ming Wu Yong-Chang Chen 《Journal of biomedical science》2015,22(1)
Background
Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance.In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin.Result
Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage.Conclusion
Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway. 相似文献11.
Background
Osteosarcoma is characterized by a high malignant and metastatic potential. CCL5 (previously called RANTES) was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. However, the effect of CCL5 on migration activity and integrin expression in human osteosarcoma cells is mostly unknown.Methodology/Principal Findings
Here we found that CCL5 increased the migration and expression of αvβ3 integrin in human osteosarcoma cells. Stimulation of cells with CCL5 increased CCR5 but not CCR1 and CCR3 expression. CCR5 mAb, inhibitor, and siRNA reduced the CCL5-enhanced the migration and integrin up-regulation of osteosarcoma cells. Activations of MEK, ERK, and NF-κB pathways after CCL5 treatment were demonstrated, and CCL5-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK, and NF-κB cascades. In addition, over-expression of CCL5 shRNA inhibited the migratory ability and integrin expression in osteosarcoma cells.Conclusions/Significance
CCL5 and CCR5 interaction acts through MEK, ERK, which in turn activates NF-κB, resulting in the activations of αvβ3 integrin and contributing the migration of human osteosarcoma cells. 相似文献12.
13.
Background
Generating autologous pluripotent stem cells for therapeutic applications will require the development of efficient DNA-free reprogramming techniques. Transfecting cells with in vitro-transcribed, protein-encoding RNA is a straightforward method of directly expressing high levels of reprogramming proteins without genetic modification. However, long-RNA transfection triggers a potent innate immune response characterized by growth inhibition and the production of inflammatory cytokines. As a result, repeated transfection with protein-encoding RNA causes cell death.Methodology/Principal Findings
RNA viruses have evolved methods of disrupting innate immune signaling by destroying or inhibiting specific proteins to enable persistent infection. Starting from a list of known viral targets, we performed a combinatorial screen to identify siRNA cocktails that could desensitize cells to exogenous RNA. We show that combined knockdown of interferon-β (Ifnb1), Eif2ak2, and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. Using this technique, we were able to transfect primary human fibroblasts every 24 hours with RNA encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utf1. We provide evidence that the encoded protein is active, and we show that expression can be maintained for many days, through multiple rounds of cell division.Conclusions/Significance
Our results demonstrate that suppressing innate immunity enables frequent transfection with protein-encoding RNA. This technique represents a versatile tool for investigating expression dynamics and protein interactions by enabling precise control over levels and timing of protein expression. Our finding also opens the door for the development of reprogramming and directed-differentiation methods based on long-RNA transfection. 相似文献14.
Bas Brinkhof Helena TA van Tol Marian JA Groot Koerkamp Frank M Riemers Sascha G IJzer Kaveh Mashayekhi Henk P Haagsman Bernard AJ Roelen 《BMC genomics》2015,16(1)
Background
Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed.Results
Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated.Conclusion
The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1448-x) contains supplementary material, which is available to authorized users. 相似文献15.
16.
Background and Aims
The enterocytes have the potential to absorb noxious substances, such as microbial products, from the gut lumen. How the enterocytes process the substances to harmless materials is not fully understood. This study aims to elucidate the role of ubiquitin E3 ligase TNFAIP3 (TNFAIP3) in facilitating the degradation of endocytic microbial products in enterocytes.Methods
Human intestinal epithelial cell line, HT-29 cells, was cultured to monolayers using as an in vitro model to observe the endocytosis and degradation of microbial products, Staphylococcal enterotoxin B (SEB) in epithelial cells. The RNA interference was employed to knock down the TNFAIP3 gene in HT-29 cells to observe the role of TNFAIP3 in the degradation of endocytic SEB. The role of TNFAIP3 in facilitating the endosome/lysosome fusion was observed by immunocytochemistry.Results
Upon the absorption of SEB, the expression of TNFAIP3 was increased in HT-29 cells. Silencing the TNFAIP3 gene in HT-29 cells resulted in a large quantity of SEB to be transported across the HT-29 monolayers to the transwell basal chambers; the transportation was via the intracellular pathway. TNFAIP3 was required in the fusion of SEB-carrying endosomes and lysosomes.Conclusions
TNFAIP3 plays a critical role in the degradation of endocytic SEB in enterocytes. 相似文献17.
Pierre Perrot Julie Rousseau Anne-Laure Bouffaut Fran?oise Rédini Elisabeth Cassagnau Frédéric Deschaseaux Marie-Fran?oise Heymann Dominique Heymann Franck Duteille Valérie Trichet Fran?ois Gouin 《PloS one》2010,5(6)
Background
Osteosarcoma is the most common malignant primary bone tumour in young adult treated by neo adjuvant chemotherapy, surgical tumor removal and adjuvant multidrug chemotherapy. For correction of soft tissue defect consecutive to surgery and/or tumor treatment, autologous fat graft has been proposed in plastic and reconstructive surgery.Principal Findings
We report here a case of a late local recurrence of osteosarcoma which occurred 13 years after the initial pathology and 18 months after a lipofilling procedure. Because such recurrence was highly unexpected, we investigated the possible relationship of tumor growth with fat injections and with mesenchymal stem/stromal cell like cells which are largely found in fatty tissue. Results obtained in osteosarcoma pre-clinical models show that fat grafts or progenitor cells promoted tumor growth.Significance
These observations and results raise the question of whether autologous fat grafting is a safe reconstructive procedure in a known post neoplasic context. 相似文献18.
Yunling Wang Geneviève Lacroix Jeffery Haines Evgueni Doukhanine Guillermina Almazan Stéphane Richard 《PloS one》2010,5(9)
Background
The quaking viable (qkv) mouse has several developmental defects that result in rapid tremors in the hind limbs. The qkI gene expresses three major alternatively spliced mRNAs (5, 6 and 7 kb) that encode the QKI-5, QKI-6 and QKI-7 RNA binding proteins that differ in their C-terminal 30 amino acids. The QKI isoforms are known to regulate RNA metabolism within oligodendrocytes, however, little is known about their roles during cellular stress.Methodology/Principal Findings
In this study, we report an interaction between the QKI-6 isoform and a component of the RNA induced silencing complex (RISC), argonaute 2 (Ago2). We show in glial cells that QKI-6 co-localizes with Ago2 and the myelin basic protein mRNA in cytoplasmic stress granules.Conclusions
Our findings define the QKI isoforms as Ago2-interacting proteins. We also identify the QKI-6 isoform as a new component of stress granules in glial cells. 相似文献19.
Background
The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD). Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters.Methodology/Principal Findings
We developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of ∼2∶1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR).Conclusions/Significance
Our technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR. 相似文献20.
Yan Liu Fei Zhang Xiao-fang Zhang Li-sha Qi Lei Yang Hua Guo Ning Zhang 《Journal of biomedical science》2012,19(1):53