ABSTRACT
Previous studies of Jak-STAT inhibitors have shown promise in treating kidney diseases. The activation of Jak-STAT components is important in cell fate determination in many cell types, including bone marrow-derived cells, which are important contributors in renal interstitial fibrosis. In this study, we tested the effect of a new STAT3 inhibitor, BP-1-102, on monocyte-to-fibrocyte transition and the progression of renal interstitial fibrosis. We tested the effect of BP-1-102 in a mouse model of unilateral ureteral obstruction in vivo and IL-33-treated bone marrow-derived monocytes in vitro. BP-1-102 treatment alleviated renal interstitial fibrosis, reduced collagen deposition and extracellular matrix protein production, inhibited inflammatory cell infiltration, suppressed the percentage of CD45+ PDGFRß+, CD45+ CD34- Col I+ and CD45+ CD11b+ Col I+ cells within the obstructed kidney and reduced the mRNA levels of the proinflammatory and profibrotic cytokines IL-1ß, TGF-ß, TNF-α, ICAM-1, and CXCL16. In vitro, BP-1-102 inhibited the IL-33-induced phenotypic transition into fibroblast precursors in bone marrow-derived monocytes, marked by reduced CD45+ CD34- Col I+ and CD45+ CD11b+ Col I+ cell percentage. Our results indicate a potential mechanism by which the STAT3 inhibitor BP-1-102 inhibits bone marrow-derived monocyte transition into fibroblast precursors in an IL-33/STAT3-dependent manner and thereby alleviates renal interstitial fibrosis.
Subject(s)
Adaptation, Physiological/drug effects , Aminosalicylic Acids/pharmacology , Bone Marrow/metabolism , Fibroblasts/metabolism , Interleukin-33/pharmacology , Kidney/pathology , Monocytes/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , Sulfonamides/pharmacology , Ureteral Obstruction/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Fibrosis , Interleukin-33/metabolism , Male , Mice , Mice, Inbred C57BLABSTRACT
PURPOSE: Silicosis is a serious occupational disease that is characterized by pulmonary infiltrates and fibrosis and is often refractory to current treatments. New therapeutic strategies for silicosis are needed. Hepatocyte growth factor (HGF) is a latent anti-inflammatory and anti-fibrotic growth factor. METHODS: We prepared a polyethyleneimine-polyethylene glycol/pHGF/hyaluronic acid (PEG-PEI/pHGF/HA) nanomaterials loaded with plasmid DNA encoding HGF gene to increase its transfection efficiency. The characterization, including DNA entrapment efficiency, morphology, particle size, and zeta-potential of PEG-PEI/pHGF/HA was studied. And a PEG-PEI/pHGF/HA (N/P=30:1) nanoparticle with low toxicity and high transfection efficiency was used in treatment for silicosis in mice. RESULTS: The results showed that the human HGF expression in the lungs of the mice was increased, and the inflammatory cell infiltration and fibrous collagen deposition was significantly reduced. CONCLUSION: Therefore, PEG-PEI/pHGF/HA nanoparticle warrant further investigation and may be a potential therapeutic strategy for silicosis.
Subject(s)
Genetic Therapy/methods , Hepatocyte Growth Factor/administration & dosage , Nanoparticle Drug Delivery System , Silicosis/drug therapy , A549 Cells , Animals , Hepatocyte Growth Factor/genetics , Hepatocyte Growth Factor/therapeutic use , Humans , Lung/drug effects , Lung/pathology , Male , Mice , Mice, Inbred C57BL , Plasmids/genetics , Silicosis/pathology , Transfection/methodsABSTRACT
Hypoxia affects proliferation, differentiation, as well as death of cardiomyocyte, and plays an important role in the development of myocardial ischemia. However, the detailed mechanisms through which hypoxia regulates cardiomyocyte ferroptosis have not been explored. In this study, we revealed that hypoxia suppresses the proliferation, migration, and erastin-induced ferroptosis of H9c2 cells. First, we confirmed the upregulation of SENP1 in H9c2 cells cultured under hypoxic conditions. Through adenovirus-mediated SENP1 gene transfection, we demonstrated that SENP1 overexpression could enhance H9c2 cell proliferation and migration while also protecting H9c2 cells from erastin-induced ferroptosis. Furthermore, through immunoprecipitation and western blotting, we confirmed that SENP1 mediated deSUMOylation of HIF-1α and ACSL4 in H9c2 cells. In conclusion, this study describes the underlying mechanism through which hypoxia upregulates SENP1 expression, in turn protecting against ferroptosis via the regulation of HIF-1α and ACSL4 deSUMOylation. Our findings provide a theoretical foundation for the development of novel therapeutics for ischemic heart diseases.
Subject(s)
Cell Hypoxia/genetics , Cysteine Endopeptidases/metabolism , Ferroptosis/genetics , Myocytes, Cardiac/pathology , Animals , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Coenzyme A Ligases/metabolism , Cysteine Endopeptidases/genetics , Ferroptosis/drug effects , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Myocardial Ischemia/pathology , Myocytes, Cardiac/drug effects , Piperazines/pharmacology , Rats , Signal Transduction/genetics , Sumoylation/genetics , Up-RegulationABSTRACT
The mRNA precursor 3'-end modification factor NUDT21 is a major regulator of 3'UTR shortening and an important component of pre-mRNA cleavage and polyadenylation. However, its role in pathologic progress of small cell lung cancer (SCLC) remains unclear. In this study, we observed that NUDT21 expression is downregulated in SCLC tissues. Hypoxia-induced down-regulation of NUDT21 through HIF-1α. NUDT21 shRNA transduction promotes proliferation and inhibits apoptosis of A549 cells. NUDT21 inhibition also promotes tumor growth in a mouse xenograft model. Furthermore, we clarified that HIF-1α mediated NUDT21 downregulation which altered the expression patterns of two isoforms of GLS1, GAC and KGA. These results link the hypoxic tumor environments to aberrant glutamine metabolism which is important for cellular energy in SCLC cells. Therefore, NUDT21 could be considered as a potential target for the treatment of SCLC.
Subject(s)
Cleavage And Polyadenylation Specificity Factor/metabolism , Glutaminase/genetics , Lung Neoplasms/genetics , Lung Neoplasms/pathology , RNA Splicing/genetics , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/pathology , A549 Cells , Cell Proliferation/genetics , Cells, Cultured , Glutaminase/metabolism , Humans , Lung Neoplasms/metabolism , MicroRNAs/genetics , Polyadenylation , Small Cell Lung Carcinoma/metabolismABSTRACT
Chimeric antigen receptor (CAR)-modified T cell therapy evokes only modest antitumor responses in solid tumors. Meso-CAR-T cells are CAR-T cells targeted mesothelin, which are over-expressed in tumor tissues of breast cancer patients. To improve the therapeutic effects, we combined it with rAd.sT, a transforming growth factor ß signaling-targeted oncolytic adenovirus, to therapy breast cancer. In subcutaneous MDA-MB-231 xenograft of NSG mice, both rAd.sT and meso-CAR-T inhibited tumor growth, however combination therapy produced stronger inhibitory effects. Interestingly, rAd.sT reduced tumor burden at initial stage following vector treatments, while meso-CAR-T cells decreased tumor burden at a later stage. Moreover, meso-CAR-T could target tumor microenvironments, and combination therapy could enhance cytokines production, such as interleukin (IL)-6 and IL-12 in tumor microenvironment. In conclusion, combination of rAd.sT with meso-CAR-T produced much more impressive antitumor responses to breast cancer and its metastasis, which could be developed as a promising therapeutic strategy.
Subject(s)
Breast Neoplasms , Combined Modality Therapy/methods , Immunotherapy, Adoptive/methods , Oncolytic Virotherapy/methods , Adenoviridae , Animals , Antineoplastic Agents/pharmacology , Female , GPI-Linked Proteins/antagonists & inhibitors , Humans , Mesothelin , Mice , Oncolytic Viruses , Receptors, Chimeric Antigen , Transforming Growth Factor beta/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
Conditions in space, such as microgravity, may affect the hematopoietic and bone marrow-derived mesenchymal stromal cells (BM-MSCs) of astronauts. However, to date, few detailed phenotype change data about the different type of hematopoietic cells have reported. In this study, C57BL/6 mice were randomly divided into two groups: a control group (control) and a hindlimb suspension group (treated). After four weeks of hindlimb suspension, we found that this simulated microgravity (sµg) condition could increase the percentage of monocytes and macrophages and decrease the percentage of B lymphocytes and mature red cells in bone marrow. The percentage of B lymphocytes in the spleen and the red blood cell count in peripheral blood also decreased, consistent with the response of bone marrow. The cytoskeleton in the BM-MSCs was disrupted. The expression levels of hematopoietic-related genes, such as fms-like tyrosine kinase-3 ligand, granulocyte-macrophage colony stimulating factor, interleukin-3, and adipogenic differentiation associated genes, leptin and proliferator-activated receptor γ type 2, were upregulated under sµg conditions. These results indicated that simulating microgravity can affect the phenotype of certain types of hematopoietic cells and the morphology and gene expression pattern of BM-MSCs.
Subject(s)
Hematopoietic Stem Cells/cytology , Mesenchymal Stem Cells/cytology , Weightlessness/adverse effects , Adipogenesis , Animals , B-Lymphocytes , Bone Marrow , Bone Marrow Cells/cytology , Cell Differentiation , Cells, Cultured , Female , Hematopoietic Stem Cells/metabolism , Hindlimb Suspension/adverse effects , Macrophages , Male , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Monocytes , Weightlessness Simulation/methodsABSTRACT
Endothelial cell death is linked to vascular diseases such as atherosclerosis and tissue ischemia. miRNA-17-92 (miR-17-92) is a multiple functional oncogenic miRNA cluster which plays vital roles in tumor angiogenesis and tissue development. However, its role in regulation of endothelial cell ferroptosis remains unclear. In this study, we revealed that miR-17-92 protects endothelial HUVEC cells from erastin-induced ferroptosis. miR-17-92 overexpression significantly reduced erastin-induced growth inhibition and ROS generation of HUVEC cells. Furthermore, Zinc lipoprotein A20, a validated target of miR-17-92, was identified as a novel regulator of endothelial cell ferroptosis. Lentivirus mediated A20 overexpression increased ROS generation and enhanced erastin-induced ferroptosis, whereas A20 knockdown inhibited erastin-induced ferroptosis. Mechanistic studies revealed that erastin-induced ferroptosis is associated with GPX4 downregulation and ACSL4 upregulation. miR-17-92 overexpression or A20 inhibition increased the ACSL4 expression in HUVEC cells. A20 was identified to directly with and regulate ACSL4 expression by immunoprecipitation. It suggests that the A20-ACSL4 axis plays important roles in erastin-induced endothelial ferroptosis. In conclusion, this study revealed a novel mechanism through which miR-17-92 protects endothelial cells from erastin-induced ferroptosis by targeting the A20-ACSL4 axis.
Subject(s)
Coenzyme A Ligases/metabolism , Cytoprotection , Ferroptosis/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , MicroRNAs/metabolism , Piperazines/pharmacology , Signal Transduction , Tumor Necrosis Factor alpha-Induced Protein 3/metabolism , Cell Proliferation/drug effects , Cytoprotection/drug effects , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/drug effects , Humans , MicroRNAs/genetics , Signal Transduction/drug effectsABSTRACT
BACKGROUND: The gene transduction efficiency of adenovirus to hematopoietic cells, especially T lymphocytes, is needed to be improved. The purpose of this study is to improve the transduction efficiency of T lymphocytes by using fiber-modified human adenovirus 5 (HAdV-5) vectors. RESULTS: Four fiber-modified human adenovirus 5 (HAdV-5) vectors were investigated to transduce hematopoietic cells. F35-EG or F11p-EG were HAdV-35 or HAdV-11p fiber pseudotyped HAdV-5, and HR-EG or CR-EG vectors were generated by incorporating RGD motif to the HI loop or to the C-terminus of F11p-EG fiber. All vectors could transduce more than 90% of K562 or Jurkat cells at an multiplicity of infection (MOI) of 500 viral particle per cell (vp/cell). All vectors except HR-EG could transduce nearly 90% cord blood CD34+ cells or 80% primary human T cells at the MOI of 1000, and F11p-EG showed slight superiority to F35-EG and CR-EG. Adenoviral vectors transduced CD4+ T cells a little more efficiently than they did to CD8+ T cells. These vectors showed no cytotoxicity at an MOI as high as 1000 vp/cell because the infected and uninfected T cells retained the same CD4/CD8 ratio and cell growth rate. CONCLUSIONS: HAdV-11p fiber pseudotyped HAdV-5 could effectively transduce human T cells when human EF1a promoter was used to control the expression of transgene, suggesting its possible application in T cell immunocellular therapy.
Subject(s)
Adenoviruses, Human/genetics , Gene Transfer Techniques/standards , Genetic Vectors/genetics , T-Lymphocytes/metabolism , Viral Tail Proteins/genetics , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/virology , Cell Proliferation/genetics , Genetic Therapy/methods , HL-60 Cells , Humans , Jurkat Cells , K562 Cells , T-Lymphocytes/virology , Transduction, Genetic/standards , Transgenes/genetics , U937 Cells , Viral Tail Proteins/metabolismABSTRACT
Ginkgo biloba L., an ancient dioecious gymnosperm, is now cultivated worldwide for landscaping and medical purposes. A novel biflavonoid-amentoflavone 7''-O-ß-D-glucopyranoside (1)-and four known biflavonoids were isolated and identified from the male flowers of Ginkgo. The anti-proliferative activities of five biflavonoids were evaluated on different cancer lines. Bilobetin (3) and isoginkgetin (4) exhibited better anti-proliferative activities on different cancer lines. Their effects were found to be cell-specific and in a dose and time dependent manner for the most sensitive HeLa cells. The significant morphological changes validated their anticancer effects in a dose-dependent manner. They were capable of arresting the G2/M phase of the cell cycle, inducing the apoptosis of HeLa cells dose-dependently and activating the proapoptotic protein Bax and the executor caspase-3. Bilobetin (3) could also inhibit the antiapoptotic protein Bcl-2. These might be the mechanism underlying their anti-proliferation. In short, bilobetin (3) and isoginkgetin (4) might be the early lead compounds for new anticancer agents.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Biflavonoids/pharmacology , Flowers/chemistry , Ginkgo biloba/chemistry , Plant Extracts/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Biflavonoids/chemistry , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Humans , Magnetic Resonance Spectroscopy , Molecular Structure , Plant Extracts/chemistryABSTRACT
Ferroptosis is an iron- and oxidative-dependent form of regulated cell death and may play important roles in maintaining myocardium homeostasis and pathology of cardiovascular diseases. Currently, the regulatory roles of lipid signals in regulating cardiomyocytes ferroptosis has not been explored. In this study, we show that ENPP2, as a lipid kinase involved in lipid metabolism, protects against erastin-induced ferroptosis in cardiomyocytes. The classical ferroptosis inducer erastin remarkably inhibits the growth which could be rescued by the small molecule Fer-1 in H9c2 cells. Adenovirus mediated ENPP2 overexpression modestly promotes migration and proliferation and significantly inhibits erastin-induced ferroptosis of H9c2 cells. ENPP2 overexpression leads to increase the LPA level in supernatant of H9c2 cells. H9c2 cells express the LPAR1, LPAR3, LPAR4 and LPAR5 receptors. The supernatant of ENPP2 transduced cardiomyocytes could protects the cells from erastin-induced ferroptosis of H9c2 cells. Furthermore, we observed that ENPP2 overexpression regulates ferroptosis-associated gene GPX4, ACSL4 and NRF2 expression and modulates MAPK and AKT signal in H9c2 cells. Collectively, these findings demonstrated that ENPP2/LPA protects cardiomyocytes from erastin-induced ferroptosis through modulating GPX4, ACSL4 and NRF2 expression and enhancing AKT survival signal.
Subject(s)
Apoptosis/drug effects , Cytotoxins/toxicity , Iron/metabolism , Myocytes, Cardiac/drug effects , Phosphoric Diester Hydrolases/genetics , Piperazines/toxicity , Animals , Apoptosis/genetics , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Coenzyme A Ligases/genetics , Coenzyme A Ligases/metabolism , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Lentivirus/genetics , Lentivirus/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Phospholipid Hydroperoxide Glutathione Peroxidase , Phosphoric Diester Hydrolases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Rats , Receptors, Lysophosphatidic Acid/genetics , Receptors, Lysophosphatidic Acid/metabolism , Signal Transduction , Transduction, GeneticABSTRACT
Identifying targets for chimeric antigen receptor-modulated T lymphocyte (CAR-T) therapy against solid tumors is an urgent problem to solve. In this study, we showed for the first time that the receptor tyrosine kinase, AXL, is overexpressed in various tumor cell lines and patient tumor tissues including triple negative breast cancer (TNBC) cell lines and patient samples, making AXL a potent novel target for cancer therapy, specifically for TNBC treatment. We also engineered T cells with a CAR consisting of a novel single-chain variable fragment against AXL and revealed its antigen-specific cytotoxicity and ability to release cytokines in a TNBC cell line and other AXL-positive tumors in vitro. Furthermore, AXL-CAR-T cells displayed a significant anti-tumor effect and in vivo persistence in a TNBC xenograft model. Taken together, our findings indicate that AXL-CAR-T cells can represent a promising therapeutic strategy against TNBC.
Subject(s)
Immunotherapy, Adoptive/methods , Proto-Oncogene Proteins/immunology , Receptor Protein-Tyrosine Kinases/immunology , Receptors, Chimeric Antigen/immunology , Triple Negative Breast Neoplasms/therapy , Animals , Cell Line, Tumor , Cells, Cultured , Female , Humans , MCF-7 Cells , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Triple Negative Breast Neoplasms/immunology , Xenograft Model Antitumor Assays , Axl Receptor Tyrosine KinaseABSTRACT
MicroRNAs (miRNAs) regulate the hypoxia-induced erythroid differentiation of hematopoietic cells. In this study, we identified that miR-486 was a rapid response miRNA to hypoxia in erythroleukemia TF-1 cells. Hypoxia exposure increased both intracellular and miR-486 levels of TF-1 cells. Ectopic miR-486 expression enhanced the growth and erythroid differentiation of TF-1 cells, whereas miR-486 inhibition suppressed their growth and erythroid differentiation. Treatment of TF-1 and cord blood CD34+ cells with exogenous containing miR-486 resulted in an increase of intracellular miR-486 level and enhanced erythroid differentiation. Furthermore, we identified that Sirt1 is a miR-486 target gene which modulates hypoxia-induced erythroid differentiation of TF-1 cells. Thus we identified a novel miRNA regulatory network that contributes to hypoxia-induced erythroid differentiation of hematopoietic cells.
Subject(s)
Erythroid Precursor Cells/cytology , Erythropoiesis , Leukemia, Erythroblastic, Acute/metabolism , MicroRNAs/genetics , Sirtuin 1/genetics , Cell Hypoxia , Cell Line, Tumor , Cells, Cultured , Erythroid Precursor Cells/metabolism , Humans , Oxygen/metabolism , Sirtuin 1/metabolismABSTRACT
miR-17-92 cluster are overexpressed in hematological malignancies including chronic myeloid leukemia (CML). However, their roles and mechanisms that regulate BCR-ABL induced leukemogenesis remain unclear. In this study, we demonstrated that genomic depletion of miR-17-92 inhibited the BCR-ABL induced leukemogenesis by using a mouse model of transplantation of BCR-ABL transduced hematopoietic stem cells. Furthermore, we identified that miR-19b targeted A20 (TNFAIP3). A20 overexpression results in inactivation of NF-κB activity including decrease of phosphorylation of P65 and IκBα, leads to induce apoptosis and inhibit proliferation and cycle in CML CD34 + cells. Thus we proved that miR-17-92 is a critical contributor to CML leukemogenesis via targeting A20 and activation of NF-κB signaling. These findings indicate that miR-17-92 will be important resources for developing novel treatment strategies of CML and better understanding long-term disease control.
Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , MicroRNAs/metabolism , NF-kappa B/metabolism , Signal Transduction , Tumor Necrosis Factor alpha-Induced Protein 3/metabolism , Animals , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , RNA, Long NoncodingABSTRACT
MicroRNAs (miRNAs) are key regulators of hematopoietic cell differentiation and may contribute to altered growth of leukemic stem cells. Using microarray-based miRNA profiling, we found that miRNA 486 (miR-486) is significantly upregulated in chronic myeloid leukemia (CML) compared with normal CD34(+) cells, particularly in the megakaryocyte-erythroid progenitor population. miR-486-5p expression increased during erythroid differentiation of both CML and normal CD34(+) cells. Ectopic miR-486-5p expression enhanced in vitro erythroid differentiation of normal CD34(+) cells, whereas miR-486-5p inhibition suppressed normal CD34(+) cell growth in vitro and in vivo and inhibited erythroid differentiation and erythroid cell survival. The effects of miR-486-5p on hematopoietic cell growth and survival are mediated at least in part via regulation of AKT signaling and FOXO1 expression. Using gene expression and bioinformatics analysis, together with functional screening, we identified several novel miR-486-5p target genes that may modulate erythroid differentiation. We further show that increased miR-486-5p expression in CML progenitors is related to both kinase-dependent and kinase-independent mechanisms. Inhibition of miR-486-5p reduced CML progenitor growth and enhanced apoptosis following imatinib treatment. In conclusion, our studies reveal a novel role for miR-486-5p in regulating normal hematopoiesis and of BCR-ABL-induced miR-486-5p overexpression in modulating CML progenitor growth, survival, and drug sensitivity.
Subject(s)
Cell Proliferation/genetics , Drug Resistance, Neoplasm/genetics , Erythropoiesis/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Megakaryocyte-Erythroid Progenitor Cells/physiology , MicroRNAs/physiology , Animals , Cell Differentiation/genetics , Cell Proliferation/drug effects , Cells, Cultured , HEK293 Cells , Hep G2 Cells , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Mice, TransgenicABSTRACT
In advanced prostate cancer, small ubiquitin-like modifier (SUMO)-specific cysteine protease 1 (SENP1) is up-regulated. However, the role of SENP1 in regulating deSUMOylation of TGF-ß/SMADs signaling is unknown. In this study, we developed a lentiviral vector, PLKO.1-shSENP1, to silence SENP1 in prostate cancer cells with high metastatic characteristics (PC3M). Likewise, we also created an adenovirus vector, Ad5/F11p-SENP1 to over-express SENP1 in prostate cancer cells with low metastatic potential (LNCaP). We showed that silencing of SENP1 promoted cellular apoptosis, and inhibited proliferation and migration of PC3M cells. Moreover, SENP1 silencing increased the SMAD4 expression at protein level, up-regulated E-cadherin and down-regulated Vimentin expression, indicating the inhibition of epithelial mesenchymal transition (EMT). Furthermore, SMAD4 interference abolished SENP1-mediated up-regulation of E-cadherin, suggesting that SENP1 regulated E-cadherin expression via SMAD4. SENP1 over-expression in LNCaP cells reduced SMAD4 protein, and promoted EMT via decreasing E-cadherin and increasing Vimentin. Moreover, down-regulation of SMAD4 and E-cadherin were blocked, after transfection with two SUMOylation sites mutated SMAD4, suggesting that SENP1 might reduce SMAD4 levels to regulate E-cadherin expression via deSUMOylation of SMAD4. In conclusion, SENP1 deSUMOylated SMAD4 to promote EMT via up-regulating E-cadherin in prostate cancer cells. Therefore, SENP1 is a potential target for treatment of advanced prostate cancer.
Subject(s)
Endopeptidases/metabolism , Epithelial-Mesenchymal Transition , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Smad4 Protein/metabolism , Androgens , Apoptosis/genetics , Cell Line, Tumor , Cell Movement/genetics , Cysteine Endopeptidases , Epithelial-Mesenchymal Transition/genetics , Gene Expression , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Male , Prostatic Neoplasms/genetics , RNA, Small Interfering/genetics , Signal Transduction , Sumoylation , Transforming Growth Factor beta/metabolismABSTRACT
Mesenchymal stem cells (MSCs) possess an immunoregulatory capacity and are a therapeutic target for many inflammation-related diseases. However, the detailed mechanisms of MSC-mediated immunosuppression remain unclear. In this study, we provide new information to partly explain the molecular mechanisms of immunoregulation by MSCs. Specifically, we found that A20 expression was induced in MSCs by inflammatory cytokines. Knockdown of A20 in MSCs resulted in increased proliferation and reduced adipogenesis, and partly reversed the suppressive effect of MSCs on T cell proliferation in vitro and inhibited tumour growth in vivo. Mechanistic studies indicated that knockdown of A20 in MSCs inhibited activation of the p38 mitogen-activated protein kinase (MAPK) pathway, which potently promoted the production of tumour necrosis factor (TNF)-α and inhibited the production of interleukin (IL)-10. Collectively, these data reveal a crucial role of A20 in regulating the immunomodulatory activities of MSCs by controlling the expression of TNF-α and IL-10 in an inflammatory environment. These findings provide novel insights into the pathogenesis of various inflammatory-associated diseases, and are a new reference for the future development of treatments for such afflictions.
Subject(s)
Mesenchymal Stem Cells/immunology , Tumor Necrosis Factor alpha-Induced Protein 3/metabolism , Adipogenesis , Animals , Carcinogenesis/metabolism , Carcinogenesis/pathology , Cell Differentiation , Cell Proliferation , Cell Shape , Cytokines/metabolism , Dinoprostone/metabolism , Gene Knockdown Techniques , Immunosuppression Therapy , Inflammation Mediators/metabolism , Intercellular Adhesion Molecule-1/metabolism , MAP Kinase Signaling System , Male , Mesenchymal Stem Cells/cytology , Mice, Inbred C57BL , Nitric Oxide/metabolism , Phenotype , Programmed Cell Death 1 Receptor/metabolism , RNA, Small Interfering/metabolism , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
MicroRNA-486 (miR-486) was first identified from human fetal liver cDNA library and validated as a regulator of hematopoiesis. Its roles in regulating the biological function of bone marrow-derived mesnechymal stem cells (BM-MSCs) under hypoxia have not been explored yet. In this study, we demonstrated that exposure to hypoxia upregulates miR-486 expression in BM-MSCs. Lentivirus-mediated overexpression of miR-486 resulted in increase of hepatocyte growth factor (HGF) and vascular endothelial growth factor(VEGF) in both mRNA and protein levels. MiR-486 expression also promotes proliferation and reduces apoptosis of BM-MSCs. Whereas MiR-486 knockdown downregulated the secretion of HGF and VEGF and induced apoptosis of BM-MSCs. Furthermore, PTEN-PI3K/AKT signaling was validated to be involved in changes of BM-MSC biological functions regulated by miR-486. These results suggested that MiR-486 mediated the hypoxia-induced angiogenic activity and promoted the proliferation and survival of BM-MSCs through regulating PTEN-PI3K/AKT signaling. These findings might provide a novel understanding of effective therapeutic strategy for hypoxic-ischemic diseases.
Subject(s)
Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/physiology , MicroRNAs/metabolism , Neovascularization, Physiologic/physiology , Oncogene Protein v-akt/metabolism , Oxygen/metabolism , Angiogenic Proteins/metabolism , Cell Differentiation/physiology , Cell Hypoxia/physiology , Cell Movement/physiology , Cell Survival/physiology , Cells, Cultured , Gene Expression Regulation/physiology , Humans , Signal Transduction/physiologyABSTRACT
Hypoxia provokes metabolism misbalance, mitochondrial dysfunction and oxidative stress in both human and animal cells. However, the mechanisms which hypoxia causes mitochondrial dysfunction and energy metabolism misbalance still remain unclear. In this study, we presented evidence that mitochondrial phosphatase Ptpmt1 is a hypoxia response molecule that regulates cell proliferation, survival and glucose metabolism in human erythroleukemia TF-1 cells. Exposure to hypoxia or DFO treatment results in upregulation of HIF1-α, HIF-2α and Ptpmt1. Only inhibition of HIF-2α by shRNA transduction reduces Ptpmt1 expression in TF-1 cells under hypoxia. Ptpmt1 inhibitor suppresses the growth and induces apoptosis of TF-1 cells. Furthermore, we demonstrated that Ptpmt1 inhibition reduces the Glut1 and Glut3 expression and decreases the glucose consumption in TF-1 cells. In additional, Ptpmt1 knockdown also results in the mitochondrial dysfunction determined by JC1 staining. These results delineate a key role for HIF-2α-induced Ptpmt1 upregulation in proliferation, survival and glucose metabolism of erythroleukemia cells. It is indicated that Ptpmt1 plays important roles in hypoxia-induced cell metabolism and mitochondrial dysfunction.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Glucose/metabolism , Leukemia, Erythroblastic, Acute/metabolism , PTEN Phosphohydrolase/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Hypoxia , Cell Line, Tumor , Cell Proliferation , Down-Regulation/genetics , Glucose Transporter Type 1/genetics , Glucose Transporter Type 1/metabolism , Glucose Transporter Type 3/genetics , Glucose Transporter Type 3/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Leukemia, Erythroblastic, Acute/pathology , PTEN Phosphohydrolase/geneticsABSTRACT
The deregulation of HGF/c-Met signaling is implicated in epithelial-mesenchymal transition (EMT) and progress of hepatocellular carcinoma (HCC). However, the epigenetic mechanisms that HGF/c-Met regulates EMT and metastasis of HCC cells are less explored. In this study, we demonstrated that HCC cells express a high level of SUMO/sentrin-specific protease 1 (Senp1) which is induced by HGF/c-Met signals. Lentivirus-mediated small hairpin RNA (shRNA) transduction results in Senp1 silence in HCC cells. Senp1 silence reduces the HGF-induced proliferation and migration of HCC cells. Senp1 inhibition also induces HCC cell apoptosis and growth arrest. Furthermore, Senp1 knockdown inhibits epithelial-to-mesenchymal transition, with increase of E-cadherin and ZO-1 expression, decrease of fibronectin and N-cadherin expression. The EMT-related transcription factor Zeb1 was SUMO-modified and decreased in Senp1-silenced HCC cells. These results delineate that senp1 might play an important role in the regulation of HGF-induced invasion and migration of HCC cells.
Subject(s)
Carcinoma, Hepatocellular/pathology , Endopeptidases/physiology , Epithelial-Mesenchymal Transition/genetics , Liver Neoplasms/pathology , Neoplasm Proteins/physiology , Apoptosis , Carcinoma, Hepatocellular/genetics , Cell Adhesion Molecules/biosynthesis , Cell Adhesion Molecules/genetics , Cell Cycle Checkpoints , Cell Division , Cell Line, Tumor , Cell Movement , Cysteine Endopeptidases , Endopeptidases/genetics , Hepatocyte Growth Factor/physiology , Humans , Liver Neoplasms/genetics , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-met/physiology , RNA Interference , RNA, Small Interfering/genetics , Signal Transduction/genetics , Sumoylation , Zinc Finger E-box-Binding Homeobox 1/metabolismABSTRACT
Liposarcoma(LPS) is the most common type of soft tissue sarcoma accounting for 20 % of all adult sarcomas. However, the molecular pathogenesis of this malignancy is still poorly understood. Here, we showed that GPS2 expression was downregulated in LPS and correlated with the prognosis of this disease. In vitro study showed that knockdown of GPS2 resulted in enhanced proliferation and migration of LPS cell line SW872, without significant influence of cell death. Conclusively, our results suggest that GPS2 may act as a tumor suppressor in LPS and serve as a potential prognosis marker for this disease.