RESUMO
BACKGROUND: Glioblastoma (GBM) is a therapeutic challenge, associated with high mortality. More effective GBM therapeutic options are urgently needed. Hence, we screened a large multi-class drug panel comprising the NIH clinical collection (NCC) that includes 446 FDA-approved drugs, with the goal of identifying new GBM therapeutics for rapid entry into clinical trials for GBM. METHODS: Screens using human GBM cell lines revealed 22 drugs with potent anti-GBM activity, including serotonergic blockers, cholesterol-lowering agents (statins), antineoplastics, anti-infective, anti-inflammatories, and hormonal modulators. We tested the 8 most potent drugs using patient-derived GBM cancer stem cell-like lines. Notably, the statins were active in vitro; they inhibited GBM cell proliferation and induced cellular autophagy. Moreover, the statins enhanced, by 40-70 fold, the pro-apoptotic activity of irinotecan, a topoisomerase 1 inhibitor currently used to treat a variety of cancers including GBM. Our data suggest that the mechanism of action of statins was prevention of multi-drug resistance protein MDR-1 glycosylation. This drug combination was synergistic in inhibiting tumor growth in vivo. Compared to animals treated with high dose irinotecan, the drug combination showed significantly less toxicity. RESULTS: Our data identifies a novel combination from among FDA-approved drugs. In addition, this combination is safer and well tolerated compared to single agent irinotecan. CONCLUSIONS: Our study newly identifies several FDA-approved compounds that may potentially be useful in GBM treatment. Our findings provide the basis for the rational combination of statins and topoisomerase inhibitors in GBM.
Assuntos
Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Aprovação de Drogas , Glioblastoma/tratamento farmacológico , United States Food and Drug Administration , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Autofagia/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Camptotecina/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Intervalo Livre de Doença , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Irinotecano , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Quinolinas/administração & dosagem , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Esferoides Celulares/patologia , Estados Unidos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mutations of RUNX1 are detected in patients with myelodysplastic syndrome (MDS). In particular, C-terminal truncation mutations lack a transcription regulatory domain and have increased DNA binding through the runt homology domain. The expression of the runt homology domain, RUNX1(41-214), in mouse hematopoietic cells induced progression to MDS and acute myeloid leukemia. Analysis of premyelodysplastic animals found expansion of c-Kit(+)Sca-1(+)Lin(-) cells and skewed differentiation to myeloid at the expense of the lymphoid lineage. These abnormalities correlate with the phenotype of Runx1-deficient animals, as expected given the reported dominant-negative role of C-terminal mutations over the full-length RUNX1. However, MDS is not observed in Runx1-deficient animals. Gene expression profiling found that RUNX1(41-214) c-Kit(+)Sca-1(+)Lin(-) cells have an overlapping yet distinct gene expression profile from Runx1-deficient animals. Moreover, an unexpected parallel was observed between the hematopoietic phenotype of RUNX1(41-214) and aged animals. Genes deregulated in RUNX1(41-214), but not in Runx1-deficient animals, were inversely correlated with the aging gene signature of HSCs, suggesting that disruption of the expression of genes related to normal aging by RUNX1 mutations contributes to development of MDS. The data presented here provide insights into the mechanisms of development of MDS in HSCs by C-terminal mutations of RUNX1.
Assuntos
Transformação Celular Neoplásica/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Animais , Apoptose/genética , Medula Óssea/patologia , Ciclo Celular/genética , Linhagem Celular , Análise por Conglomerados , Perfilação da Expressão Gênica , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas , Homeostase/genética , Humanos , Leucemia Experimental , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Transdução GenéticaRESUMO
Chromosome translocation 8q22;21q22 [t(8;21)] is commonly associated with acute myeloid leukemia (AML), and the resulting AML1-ETO fusion proteins are involved in the pathogenesis of AML. To identify novel molecular and therapeutic targets, we performed combined gene expression microarray and promoter occupancy (ChIP-chip) profiling using Lin(-)/Sca1(-)/cKit(+) cells, the major leukemia cell population, from an AML mouse model induced by AML1-ETO9a (AE9a). Approximately 30% of the identified common targets of microarray and ChIP-chip assays overlap with the human t(8;21)-gene expression molecular signature. CD45, a protein tyrosine phosphatase and a negative regulator of cytokine/growth factor receptor and JAK/STAT signaling, is among those targets. Its expression is substantially down-regulated in leukemia cells. Consequently, JAK/STAT signaling is enhanced. Re-expression of CD45 suppresses JAK/STAT activation, delays leukemia development, and promotes apoptosis of t(8;21)-positive cells. This study demonstrates the benefit of combining gene expression and promoter occupancy profiling assays to identify molecular and potential therapeutic targets in human cancers and describes a previously unappreciated signaling pathway involving t(8;21) fusion proteins, CD45, and JAK/STAT, which could be a potential novel target for treating t(8;21) AML.
Assuntos
Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , DNA de Neoplasias/metabolismo , Perfilação da Expressão Gênica , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Translocação Genética , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Imunoprecipitação da Cromatina , Ativação Enzimática , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes/genética , Genes Neoplásicos/genética , Humanos , Janus Quinases/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/genéticaRESUMO
BACKGROUND AIMS: The protocols for differentiation of hepatocyte-like cells (HLCs) from mesenchymal stromal cells (MSCs) have been well established. Previous data have shown that MSCs and their derived HLCs were able to engraft injured liver and alleviate injuries induced by carbon tetrachloride. The goal of the current study was to determine the differences of MSCs and their derived HLCs in terms of therapeutic functions in liver diseases. METHODS: After hepatic differentiation of umbilical cord-derived MSCs in vitro, we detected both MSC and HLC expressions of adhesion molecules and chemokine receptor CXCR4 by flow cytometry; immunosuppressive potential and hepatocyte growth factor expression were determined by means of enzyme-linked immunosorbent assay. We compared the therapeutic effect for fulminant hepatic failure in a mouse model. RESULTS: MSC-derived-HLCs expressed lower levels of hepatocyte growth factor, accompanied by impaired immunosuppression in comparison with MSCs. Furthermore, undifferentiated MSCs showed rescuing potentials superior to those in HLCs for the treatment of fulminant hepatic failure. CONCLUSIONS: After differentiation, HLCs lost several major properties in comparison with undifferentiated MSCs, which are beneficial for their application in liver diseases. Undifferentiated MSCs may be more appropriate than are HLCs for the treatment of liver diseases.
Assuntos
Hepatócitos/metabolismo , Falência Hepática Aguda/terapia , Fígado/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Células Cultivadas , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/citologia , Humanos , Fígado/patologia , Fígado/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptores CXCR4/metabolismo , Cordão Umbilical/citologiaRESUMO
Dendritic cells (DCs) represent the key cells linking innate and adaptive immune responses. It is critical to understand the molecular factors regulating DC differentiation. Usp18 is an IFN-inducible member of the ubiquitin-specific protease family, which deconjugates ubiquitin-like modifier ISG15 from target proteins and competitively inhibits IFN-α/ß-induced JAK/STAT activation. This study demonstrates that the frequency of conventional CD11b(+) DCs in the spleen of Usp18(-/-) mice was significantly reduced, whereas the frequencies of conventional CD8(+) DCs and plasmacytoid DCs remained normal. In addition, Usp18(-/-) bone marrow (BM) cells generate DCs less efficiently in GM-CSF-supplemented culture, demonstrating a fundamental defect throughout the DC differentiation pathway. Usp18(-/-) BM cells were rescued by exogenous expression of either wild-type or deconjugation-inactive Usp18, and superimposition of an IFN-α/ß receptor knockout returned in vivo DC populations to normal, clearly showing that the defect seen is due solely to Usp18's effect on IFN signaling. Finally, Usp18(-/-) BM-derived DCs expressed high levels of SOCS1/SOCS3, known inhibitors of GM-CSF signaling, providing a mechanistic explanation for the phenotype. In conclusion, we have identified a novel role of Usp18 in modulating conventional CD11b(+) DC development via its inhibitory effect on type I IFN signaling.
Assuntos
Antígeno CD11b/biossíntese , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Endopeptidases/fisiologia , Animais , Antígenos CD8/biossíntese , Contagem de Células , Diferenciação Celular/genética , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/enzimologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Endopeptidases/deficiência , Endopeptidases/genética , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Substâncias de Crescimento/deficiência , Substâncias de Crescimento/genética , Substâncias de Crescimento/fisiologia , Masculino , Camundongos , Camundongos Knockout , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Ubiquitina TiolesteraseRESUMO
Human mesenchymal stem cells (hMSCs) are one of the most promising candidates for cell-based therapeutic products. Nonetheless, their biomechanical phenotype afterin vitroexpansion is still unsatisfactory, for example, restricting the efficiency of microcirculation of delivered hMSCs for further cell therapies. Here, we propose a scheme using maleimide-dextran hydrogel with locally varied stiffness in microscale to modify the biomechanical properties of hMSCs in three-dimensional (3D) niches. We show that spatial micro-variation of stiffness can be controllably generated in the hydrogel with heterogeneously cross-linking via atomic force microscopy measurements. The result of 3D cell culture experiment demonstrates the hydrogels trigger the formation of multicellular spheroids, and the derived hMSCs could be rationally softened via adjustment of the stiffness variation (SV) degree. Importantly,in vitro, the hMSCs modified with the higher SV degree can pass easier through capillary-shaped micro-channels. Further, we discuss the underlying mechanics of the increased cellular elasticity by focusing on the effect of rearranged actin networks, via the proposed microscopic model of biomechanically modified cells. Overall, this work highlights the effectiveness of SV-hydrogels in reprogramming and manufacturing hMSCs with designed biomechanical properties for improved therapeutic potential.
Assuntos
Hidrogéis , Células-Tronco Mesenquimais , Técnicas de Cultura de Células , Diferenciação Celular , Humanos , Esferoides CelularesRESUMO
Following bone marrow transplantation, donor stem cells are recruited from their quiescent status to promote the rapid reconstitution in recipients. This dynamic process is tightly regulated by a complex of internal and external signals. Protein modification by the ubiquitin like modifier ISG15 (ISGylation) is strongly induced by type I interferons (IFNs). There are higher levels of type I IFNs and protein ISGylation in the bone marrow of recipients shortly after transplantation. In order to clarify the physiological function of protein ISGylation, we generated a mouse model that lacks protein ISGylation due to deficiency of ISG15 conjugating enzyme Ube1L (Ube1L(-/-)). In this report, we focused on the analysis of the hematopoietic system in Ube1L(-)(/)(-) mice in steady-state hematopoiesis and its potential protective role during bone marrow reconstitution. Here we demonstrated that In Ube1L(-/-) mice, steady-state hematopoiesis was unperturbed. However, transplantation experiment revealed a 50% reduction in repopulation potential of Ube1L-deficient cells at 3weeks posttransplantation, but no differences at 6 and 12weeks. A competitive transplantation experiment magnified and extended this phenotype. Cell cycle analysis revealed that under the condition with high levels of IFNs and protein ISGylation, the Ube1L deficiency can cause G2/M phase block of cell cycle in hematopoietic multipotential progenitors. These observations indicate that although protein ISGylation is dispensable for steady-state hematopoiesis, it plays a significant role during interferon related stress response, such as bone marrow transplantation.
Assuntos
Transplante de Medula Óssea , Citocinas/metabolismo , Hematopoese/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Animais , Ciclo Celular , Proliferação de Células , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Enzimas Ativadoras de Ubiquitina/deficiência , Enzimas Ativadoras de Ubiquitina/genética , Ubiquitinas/metabolismoRESUMO
Genetic lesions in chromosomal region 3p21.3 marks one of the earliest events in human lung cancer development. It is hypothesized that one or more tumor suppressor genes reside in this region. Identification and characterization of these genes are important for the understanding of lung cancer initiation. UBE1L (UBA7) is a long-suspected 3p21.3 residing tumor suppressor gene. It encodes the key enzyme that activates ISGylation, a novel, ubiquitination-like, post-translational protein modification system that is inducible by interferon. It has been implicated that ISGylation plays a variety of biological roles ranging from viral defense to tumor surveillance. Here we tested the possible function of ISGylation during lung cancer development by using the Ube1l-deficient mice and the K-ras(LA2) lung cancer mice. Protein ISGylation levels were largely unchanged during lung cancer progression. Ube1l deficiency neither altered the lung cancer progression nor affected the overall survival of K-ras(LA2) lung cancer mice. Our study suggests that Ube1l is not a tumor suppressor gene in K-ras(LA2) lung cancer mouse model. However, as described in the discussion, additional studies with other lung cancer mouse models will be necessary to elucidate the potential tumor suppressor function of UBE1L in K-RAS mutation independent human lung cancers.
Assuntos
Genes Supressores de Tumor , Genes ras , Neoplasias Pulmonares/genética , Enzimas Ativadoras de Ubiquitina/genética , Animais , Citocinas/análise , Neoplasias Pulmonares/patologia , Linfoma/metabolismo , Camundongos , Timo/metabolismo , Enzimas Ativadoras de Ubiquitina/fisiologia , Ubiquitinas/análiseRESUMO
Thymic microenvironments are essential for the maturation of thymocytes, which can be anatomically compartmentalized into cortical and medullar regions. The absence of the gene encoding the transcription factor forkhead box n1 (Foxn1) causes epithelial differentiation to stall in the precursor stage, resulting in the formation of an abnormal thymus. In this study, we used human umbilical cord-derived mesenchymal stem cells (UC-MSCs) to treat Foxn1(-/-) mice, and then analyzed the maturation and distribution of thymic epithelial cells in the Foxn1(-/-) thymic rudiment and the thymopoiesis of this newly developed rudiment. Our data showed a well-organized cortex-medulla architecture and an obvious improvement in the maturation of thymic epithelial cells along with the appearance of UEA-1(+)MHCII(hi) thymic epithelial cells in the rudiment. We further demonstrated improved thymopoiesis and the enhanced export of mature T cells with increased numbers of regulatory T cells into the peripheral blood. Furthermore, we observed that MSCs can engraft into thymic tissue and express many cytokines or proteins, particularly keratinocyte growth factor (KGF) and CD248, which are essential for thymic development. Collectively, our data identified a new mechanism for MSCs, which may provide a proper microenvironment for the reconstitution and functional maturation of the thymus in Foxn1(-/-) mice. Additionally, we elicited additional insights into the therapeutic efficacy of MSCs in several autoimmune diseases.
Assuntos
Células Epiteliais/citologia , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Timo/citologia , Cordão Umbilical/citologia , Animais , Contagem de Células , Diferenciação Celular , Separação Celular , Citocinas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Linfócitos T Reguladores/citologiaRESUMO
The theory of cancer immunoediting refers to mechanisms by which the immune system can suppress or promote tumour progression. A major challenge for the development of novel cancer immunotherapies is to find ways to exploit the immune system's antitumour activity while concomitantly reducing its protumour activity. Using the PyVmT model of mammary tumourigenesis, we show that lack of the Usp18 gene significantly inhibits tumour growth by creating a tumour-suppressive microenvironment. Generation of this antitumour environment is driven by elevated secretion of the potent T-cell chemoattractant Cxcl10 by Usp18 deficient mammary epithelial cells (MECs), which leads to recruitment of Th1 subtype CD4(+) T cells. Furthermore, we show that Cxcl10 upregulation in MECs is promoted by interferon-λ and that Usp18 is a novel inhibitor of interferon-λ signalling. Knockdown of the interferon-λ specific receptor subunit IL-28R1 in Usp18 deficient MECs dramatically enhances tumour growth. Taken together, our data suggest that targeting Usp18 may be a viable approach to boost antitumour immunity while suppressing the protumour activity of the immune system.
Assuntos
Neoplasias da Mama/imunologia , Mama/patologia , Quimiocina CXCL10/imunologia , Endopeptidases/genética , Células Epiteliais/imunologia , Interferon gama/imunologia , Microambiente Tumoral , Animais , Mama/irrigação sanguínea , Mama/imunologia , Mama/metabolismo , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linfócitos T CD4-Positivos/imunologia , Quimiocina CXCL10/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Camundongos , Camundongos Knockout , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Células Tumorais Cultivadas , Ubiquitina Tiolesterase , Regulação para CimaRESUMO
This study was designed to assess the safety and efficacy of human umbilical cord mesenchymal stem cells (UC-MSCs) in the treatment of rheumatoid arthritis (RA). In this ongoing cohort, 172 patients with active RA who had inadequate responses to traditional medication were enrolled. Patients were divided into two groups for different treatment: disease-modifying anti-rheumatic drugs (DMARDs) plus medium without UC-MSCs, or DMARDs plus UC-MSCs group (4×10(7) cells per time) via intravenous injection. Adverse events and the clinical information were recorded. Tests for serological markers to assess safety and disease activity were conducted. Serum levels of inflammatory chemokines/cytokines were measured, and lymphocyte subsets in peripheral blood were analyzed. No serious adverse effects were observed during or after infusion. The serum levels of tumor necrosis factor-alpha and interleukin-6 decreased after the first UC-MSCs treatment (P<0.05). The percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells of peripheral blood was increased (P<0.05). The treatment induced a significant remission of disease according to the American College of Rheumatology improvement criteria, the 28-joint disease activity score, and the Health Assessment Questionnaire. The therapeutic effects maintained for 3-6 months without continuous administration, correlating with the increased percentage of regulatory T cells of peripheral blood. Repeated infusion after this period can enhance the therapeutic efficacy. In comparison, there were no such benefits observed in control group of DMARDS plus medium without UC-MSCs. Thus, our data indicate that treatment with DMARDs plus UC-MSCs may provide safe, significant, and persistent clinical benefits for patients with active RA.
Assuntos
Artrite Reumatoide/terapia , Terapia Baseada em Transplante de Células e Tecidos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Adulto , Artrite Reumatoide/sangue , Artrite Reumatoide/patologia , Feminino , Humanos , Injeções Intravenosas , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Segurança , Inquéritos e Questionários , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/sangueRESUMO
Activation of erythropoietin receptor allows erythroblasts to generate erythrocytes. In a search for genes that are up-regulated during this differentiation process, we have identified ISG15 as being induced during late erythroid differentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins by the enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we show that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6 are induced during erythroid differentiation. Loss of ISG15 in mice results in decreased number of BFU-E/CFU-E in bone marrow, concomitant with an increased number of these cells in the spleen of these animals. ISG15(-/-) bone marrow and spleen-derived erythroblasts show a less differentiated phenotype both in vivo and in vitro, and over-expression of ISG15 in erythroblasts is found to facilitate erythroid differentiation. Furthermore, we have shown that important players of erythroid development, such as STAT5, Globin, PLC γ and ERK2 are ISGylated in erythroid cells. This establishes a new role for ISG15, besides its well-characterized anti-viral functions, during erythroid differentiation.