RESUMO
Zinc finger protein 384 (ZNF384) rearrangement defined a novel subtype of B-cell acute lymphoblastic leukemia (B-ALL). The prognostic significance of ZNF384 fusion transcript levels represented measurable residual disease remains to be explored. ZNF384 fusions were screened out in 57 adult B-ALL patients at diagnosis by real-time quantitative polymerase chain reaction and their transcript levels were serially monitored during treatment. The reduction of ZNF384 fusion transcript levels at the time of achieving complete remission had no significant impact on survival, whereas its ≥2.5-log reduction were significantly associated with higher relapse free survival (RFS) and overall survival (OS) rates after course 1 consolidation (p = 0.022 and = 0.0083) and course 2 consolidation (p = 0.0025 and = 0.0008). Compared with chemotherapy alone, allogeneic hematopoietic stem cell transplantation (allo-HSCT) significantly improved RFS and OS of patients with <2.5-log reduction after course 1 consolidation (p < 0.0001 and = 0.0002) and course 2 consolidation (p = 0.0003 and = 0.019), whereas exerted no significant effects in patients with ≥2.5-log reduction (all p > 0.05). ZNF384 fusion transcript levels after course 1 and course 2 consolidation strongly predict relapse and survival and may guide whether receiving allo-HSCT in adult B-ALL.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto , Humanos , Prognóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Fatores de Transcrição , Neoplasia Residual/diagnóstico , Recidiva , Transativadores/metabolismo , Transativadores/uso terapêuticoRESUMO
BACKGROUND: The immunomodulatory drug lenalidomide, which is now widely used for the treatment of multiple myeloma (MM), exerts pharmacological action through the ubiquitin-dependent degradation of IKZF1 and subsequent down-regulation of interferon regulatory factor 4 (IRF4), a critical factor for the survival of MM cells. IKZF1 acts principally as a tumour suppressor via transcriptional repression of oncogenes in normal lymphoid lineages. In contrast, IKZF1 activates IRF4 and other oncogenes in MM cells, suggesting the involvement of unknown co-factors in switching the IKZF1 complex from a transcriptional repressor to an activator. The transactivating components of the IKZF1 complex might promote lenalidomide resistance by residing on regulatory regions of the IRF4 gene to maintain its transcription after IKZF1 degradation. METHODS: To identify unknown components of the IKZF1 complex, we analyzed the genome-wide binding of IKZF1 in MM cells using chromatin immunoprecipitation-sequencing (ChIP-seq) and screened for the co-occupancy of IKZF1 with other DNA-binding factors on the myeloma genome using the ChIP-Atlas platform. RESULTS: We found that c-FOS, a member of the activator protein-1 (AP-1) family, is an integral component of the IKZF1 complex and is primarily responsible for the activator function of the complex in MM cells. The genome-wide screening revealed the co-occupancy of c-FOS with IKZF1 on the regulatory regions of IKZF1-target genes, including IRF4 and SLAMF7, in MM cells but not normal bone marrow progenitors, pre-B cells or mature T-lymphocytes. c-FOS and IKZF1 bound to the same consensus sequence as the IKZF1 complex through direct protein-protein interactions. The complex also includes c-JUN and IKZF3 but not IRF4. Treatment of MM cells with short-hairpin RNA against FOS or a selective AP-1 inhibitor significantly enhanced the anti-MM activity of lenalidomide in vitro and in two murine MM models. Furthermore, an AP-1 inhibitor mitigated the lenalidomide resistance of MM cells. CONCLUSIONS: C-FOS determines lenalidomide sensitivity and mediates drug resistance in MM cells as a co-factor of IKZF1 and thus, could be a novel therapeutic target for further improvement of the prognosis of MM patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Fator de Transcrição Ikaros , Lenalidomida , Mieloma Múltiplo , Proteínas Proto-Oncogênicas c-fos , Animais , Humanos , Camundongos , Medula Óssea , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Lenalidomida/farmacologia , Lenalidomida/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Transativadores/uso terapêutico , Fator de Transcrição AP-1/uso terapêutico , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
PURPOSE: The oncogenic factor ZNF217 promotes aggressive estrogen receptor (ER)+breast cancer disease suggesting that its inhibition may be useful in the clinic. Unfortunately, no direct pharmacological inhibitor is available. Dimethyl fumarate (DMF) exhibits anti-breast cancer activities, in vitro and in pre-clinical in vivo models. Its therapeutic benefits stem from covalent modification of cellular thiols such as protein cysteines, but the full profile of molecular targets mediating its anti-breast cancer effects remains to be determined. METHODS: ER+breast cancer cells were treated with DMF followed by cysteine-directed proteomics. Cells with modulated ZNF217 levels were used to probe the efficacy of DMF. RESULTS: Covalent modification of ZNF217 by DMF identified by proteomics was confirmed by using a DMF-chemical probe. Inhibition of ZNF217's transcriptional activity by DMF was evident on reported ZNF217-target genes. ZNF217 as an oncogene has been shown to enhance stem-like properties, survival, proliferation, and invasion. Consistent with ZNF217 inhibition, DMF was more effective at blocking these ZNF217-driven phenotypes in cells with elevated ZNF217 expression. Furthermore, partial knockdown of ZNF217 led to a reduction in DMF's efficacy. DMF's in vivo activity was evaluated in a xenograft model of MCF-7 HER2 cells that have elevated expression of ZNF217 and DMF treatment resulted in significant inhibition of tumor growth. CONCLUSION: These data indicate that DMF's anti-breast cancer activities in the ER+HER2+models, at least in part, are due to inhibition of ZNF217. DMF is identified as a new covalent inhibitor of ZNF217.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fumarato de Dimetilo/farmacologia , Fumarato de Dimetilo/uso terapêutico , Receptores de Estrogênio , Transativadores/genética , Transativadores/metabolismo , Transativadores/uso terapêutico , Células MCF-7RESUMO
Glioblastoma is one of the most common and invasive types of primary brain malignancies in adults, accounting for 45.5% of malignancies. Its annual prevalence is low compared to other cancers. The survival rate of this disease is about 14 months after diagnosis. Temozolomide (TMZ) is a common chemotherapy drug used to treatment of glioblastoma, but drug resistance against this drug is an important barrier to successful treatment of this cancer. Today, siRNAs play a significant role in cancer treatment. SIX4 is a transcriptional regulatory molecule that can act as a transcriptional suppressor and an activator in target genes involved in differentiation, migration, and cell survival processes. The aim of this study was to evaluate the effect of SIX4-siRNA on A-172 glioblastoma cells, its role as a tumor suppressor, and its combination with TMZ. We studied the cytotoxic effect of the SIX4-siRNA and TMZ on A-172 cells using the MTT assay investigated their effect on apoptosis and cell cycle of A-172 cells used wound healing assays to assess their effect on cell migration. Finally, we used qRT-PCR to study the mRNA expression levels of genes involved in apoptosis and migration of tumoral cells after treatments. Based on our results, silencing SIX4-siRNA expression reduced the cell viability of A-172 cells and sensitize these cells to TMZ. Furthermore, we observed an increase in apoptosis and cell cycle arrest, and a decrease in migration. Bax and caspase-9 overexpression and BCL2 and MMP9 downregulation were detected in the combination of SIX4-siRNA and TMZ. According to our results, the combination of SIX4-siRNA and TMZ can be a very useful strategy for successful glioblastoma treatment.
Assuntos
Glioblastoma , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , RNA Interferente Pequeno/genética , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Proliferação de Células , Linhagem Celular Tumoral , Apoptose , Antineoplásicos Alquilantes/farmacologia , Resistencia a Medicamentos Antineoplásicos , Transativadores/farmacologia , Transativadores/uso terapêutico , Proteínas de HomeodomínioRESUMO
PURPOSE: Hypoxia inducible factor (HIF) pathway alterations drive progression of clear cell renal cell carcinoma (ccRCC). We aim to evaluate genes within the canonical and non-canonical HIF pathways as predictors of survival in metastatic ccRCC. MATERIALS AND METHODS: Gene expression was determined from 324 archival pretreatment nephrectomy specimens from CALGB90206, a phase III trial of patients treated with interferon alpha (INF-α) vs. INF-α plus bevacizumab. TaqMan RT-qPCR was performed using RNA from tumors macrodissected based on review by genitourinary pathology. RESULTS: A total of 35 HIF-related genes were assessed by Cox regression analysis. After adjusting for sex and Memorial Sloan Kettering Cancer Center risk score (MSKCC-RS), 11 genes predicted OS: HIF2A (HR 1.059, Pâ¯=â¯0.012), EGLN3 (HR 1.089, Pâ¯=â¯0.012), VEGFC (HR 0.904, Pâ¯=â¯0.039), VEGFD (HR 1.085, Pâ¯=â¯0.016), FLT4 (HR 1.093, P = 0.038), CCND1 (HR 1.077, Pâ¯=â¯0.026), TGFA (HR 1.127, Pâ¯=â¯0.003), EGFR (HR 1.151, Pâ¯=â¯0.028), VHL (HR 0.764, Pâ¯=â¯0.002), HSP90AA1 (HR 0.845, Pâ¯=â¯0.002), and PTEN (HR 1.163, Pâ¯=â¯0.050); 7 genes predicted PFS: HIF2A (HR 1.060, Pâ¯=â¯0.011), CCND1 (HR 1.082, Pâ¯=â¯0.016), TGFA (HR 1.096, Pâ¯=â¯0.026), EP300 (HR 1.171, Pâ¯=â¯0.031), VHL (HR 0.775, Pâ¯=â¯0.007), HSP90AA1 (HR 0.871, Pâ¯=â¯0.015), and TP53 (HR 1.119, Pâ¯=â¯0.050). Most of these genes validated as significant predictors of survival in the external, TCGA dataset. In multivariate analysis of all externally validated genes, VEGFC (HR 0.906, Pâ¯=â¯0.043), TGFA (HR 1.122, Pâ¯=â¯0.003), CITED2 (HR 1.113, Pâ¯=â¯0.035) and EP300 (HR 1.136, Pâ¯=â¯0.049) predicted OS; and HIF2A (HR 1.049, Pâ¯=â¯0.036) and EP300 (HR 1.199, Pâ¯=â¯0.010) predicted PFS. EGLN3 (HR 1.156, Pâ¯=â¯0.045) and BNIP3 (HR 1.254, Pâ¯=â¯0.049) significantly interacted with treatment status and predicted PFS in patients treated with IFN-α and IFN-α+bevacizumab, respectively. CONCLUSIONS: We identified specific gene isoforms in both the canonical and non-canonical HIF pathways associated with metastatic RCC survival. EGLN3 and BNIP3 showed significant interaction with treatment arm and may be predictive of treatment response. We have identified genes for future prospective investigation as predictive biomarkers and novel drug targets.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Bevacizumab/uso terapêutico , Interferon-alfa , RNA , Hipóxia , Receptores ErbB , Proteínas Repressoras , Transativadores/uso terapêuticoRESUMO
The pathogenesis of hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) differs according to whether prior treatment with interferon (IFN) vs. direct-acting antiviral agents (DAAs) was administered. Cyclo- oxygenase-2 (COX-2), yes-associated protein 1 (YAP), and transcriptional co-activator with PDZ-binding motif (TAZ) play a crucial role in hepatocarcinogenesis. However, their roles in untreated or treated HCV-related HCC development have not been clarified. Therefore, we performed an immunohistochemical study and stained tissue from 83 HCV-related HCC cases using antibodies against COX-2, YAP, and TAZ and correlated their expression with the clinicopathological characteri stics and survival data. The cases were subdivided into 3 groups based on prior HCV treatment. In the 3 groups, COX-2 was significantly higher in HCC tissue compared with adjacent non-tumour liver tissue. However, the expression of YAP/TAZ was not significantly different between HCC and adjacent non-tumour tissue. We further grouped HCC cases into YAP+/TAZ+ and YAP-/TAZ- cases. In the YAP+/TAZ+ cases, COX-2 was significantly associated with tumour size, tumour multifocality, and late pathologic stage. No significant difference was observed in COX-2 and TAZ expression as a result of IFN or DAA treatment; however, YAP was significantly higher in IFN-treated HCC. Cyclo-oxygenase-2 overexpression may play a role in late HCC development, while YAP/TAZ could play an early role in HCC progression. Sustained expression of combined YAP/TAZ could mediate the poor prognostic role of COX-2.
Assuntos
Carcinoma Hepatocelular , Hepatite C Crônica , Hepatite C , Neoplasias Hepáticas , Humanos , Antivirais/uso terapêutico , Ciclo-Oxigenase 2/uso terapêutico , Hepacivirus/metabolismo , Hepatite C/complicações , Hepatite C/tratamento farmacológico , Hepatite C Crônica/complicações , Hepatite C Crônica/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Transativadores/metabolismo , Transativadores/uso terapêutico , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
Hepatocellular carcinoma (HCC) remains a notably global health challenge with high mortality rates and poor prognosis. The deregulation of the Hippo signalling pathway, especially the overexpression and activation of downstream effector Yes-associated protein (YAP), has been demonstrated to result in the rapid malignant evolution of HCC. In this context, multiple efforts have been dedicated to targeting YAP for HCC therapy, but effective YAP inhibitors are still lacking. In this study, through a YAP-TEAD (8×GTIIC) luciferase reporter assay, we identified fingolimod, an immunomodulatory drug approved for the treatment of multiple sclerosis, as a novel YAP inhibitor. Fingolimod suppressed the proliferation of HCC cell lines by downregulating the protein levels as well as the trans-activating function of YAP. Overall, our current study not only identifies fingolimod as a novel YAP-targeting in hibitor, but also indicates that this clinically-approved drug could be utilized as a potential and feasible therapeutic drug for HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Cloridrato de Fingolimode/farmacologia , Cloridrato de Fingolimode/uso terapêutico , Fatores de Transcrição/metabolismo , Fatores de Transcrição/uso terapêutico , Transativadores/metabolismo , Transativadores/uso terapêutico , Proteínas de Sinalização YAP , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Linhagem Celular TumoralRESUMO
Prostate cancer remains one of the most prevalent cancers in aging men. Active surveillance subpopulation of patients with prostate cancer includes men with varying cancer risk categories of precancerous disease due to prostatic intraepithelial neoplasia (PIN) heterogeneity. Identifying molecular alterations associated with PIN can provide preventable measures through finding novel pharmacologic targets for cancer interception. Targeted nutritional interception may prove to be the most appropriate chemoprevention for intermediate- and high-risk active surveillance patients. Here, we have generated two prostate-specific transgenic mouse models, one overexpressing MTA1 (R26MTA1 ) and the other overexpressing MTA1 on the background of Pten heterozygosity (R26MTA1 ; Pten+/f ), in which we examined the potential chemopreventive efficacy of dietary pterostilbene. We show that MTA1 promotes neoplastic transformation of prostate epithelial cells by activating cell proliferation and survival, leading to PIN development. Moreover, MTA1 cooperates with PTEN deficiency to accelerate PIN development by increasing cell proliferation and MTA1-associated signaling. Further, we show that mice fed with a pterostilbene-supplemented diet exhibited more favorable histopathology with decreased severity and number of PIN foci accompanied by reduced proliferation, angiogenesis, and inflammation concomitant to reduction in MTA1 and MTA1-associated CyclinD1, Notch2, and oncogenic miR-34a and miR-22 levels. PREVENTION RELEVANCE: Developing novel interceptive strategies for prostate cancer chemoprevention is a paramount goal in clinical oncology. We offer preclinical evidence for the potential of pterostilbene as a promising natural agent for MTA1-targeted interceptive strategy in future cancer prevention trials towards protecting select patients with prostate cancer under active surveillance from developing cancer.
Assuntos
Lesões Pré-Cancerosas , Neoplasia Prostática Intraepitelial , Neoplasias da Próstata , Animais , Linhagem Celular Tumoral , Dieta , Humanos , Masculino , Camundongos , Lesões Pré-Cancerosas/tratamento farmacológico , Lesões Pré-Cancerosas/prevenção & controle , Neoplasia Prostática Intraepitelial/prevenção & controle , Neoplasias da Próstata/patologia , Proteínas Repressoras , Estilbenos , Transativadores/genética , Transativadores/uso terapêuticoRESUMO
BACKGROUND: As a metabolic reprogramming feature, cancer cells derive most of their energy from aerobic glycolysis, while its regulatory mechanisms and therapeutic strategies continue to be illusive. METHODS: Integrative analysis of publically available expression profile datasets was used to identify critical transcriptional regulators and their target glycolytic enzymes. The functions and acting mechanisms of transcriptional regulators in cancer cells were investigated by using in vitro and in vivo assays. The Kaplan-Meier curve and log-rank assay were used to conduct the survival study. RESULTS: Salmonella pathogenicity island 1 (SPI1/PU.1), a haematopoietic transcription factor, was identified to facilitate glycolytic process, tumourigenesis, invasiveness, as well as metastasis of colon cancer cells, which was interplayed by tumour-associated neutrophils. Mechanistically, neutrophils delivered SPI1 mRNA via extracellular vesicles, resulting in enhanced SPI1 expression of cancer cells. Through physical interaction with SPI1-related protein (SPIB), SPI1 drove expression of glycolytic genes within cancer cells, which in turn induced polarization of neutrophils via glycolytic metabolite lactate. Depletion of neutrophils or SPIB-SPI1 interaction in cancer cells significantly inhibited glycolytic process, tumourigenesis and aggressiveness. Upregulation of SPI1 or SPIB was found to be associated with poor prognosis in patients suffering from colon cancer. CONCLUSIONS: Therapeutic targeting of SPIB/SPI1-facilitated interplay of cancerous cells and neutrophils suppresses aerobic glycolysis and progression of cancer.
Assuntos
Linhagem Celular/metabolismo , Proteínas Proto-Oncogênicas/farmacologia , Transativadores/farmacologia , Efeito Warburg em Oncologia/efeitos dos fármacos , Progressão da Doença , Humanos , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Proteínas Proto-Oncogênicas/uso terapêutico , Transativadores/uso terapêuticoRESUMO
OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by chromatin immunoprecipitation-sequencing that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2, and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. Short hairpin RNA knockdown experiments demonstrated that OCT1, OCT2, and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance, such as BCL6, are downregulated, whereas genes related to exit from the GC program, such as IRF4, are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.
Assuntos
Centro Germinativo/metabolismo , Fator 1 de Transcrição de Octâmero/fisiologia , Fator 2 de Transcrição de Octâmero/uso terapêutico , Transativadores/uso terapêutico , Transcrição Gênica/genética , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ontologia Genética , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 1 de Transcrição de Octâmero/deficiência , Fator 1 de Transcrição de Octâmero/genética , Fator 2 de Transcrição de Octâmero/deficiência , Fator 2 de Transcrição de Octâmero/genética , Proteína Proto-Oncogênica c-ets-1/análise , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Transativadores/deficiência , Transativadores/genéticaRESUMO
Chromosome translocation t(12;22)(p13;q12)/MN1-ETV6 and MN1 overexpression confer a subset of adverse prognostic AML but so far lack in-depth research. We focused on the clinical course and comprehensive genetic analysis of eight cases with t(12;22)(p13;q12) and one with t(12;17;22) (p13;q21;q13) to elucidate their molecular etiology and outcomes of allogeneic hemopoietic stem cell transplantation (allo-HSCT). The total incidence of t(12;22)(p13;q12) and related translocations was 0.32% in myeloid neoplasms. These patients were confirmed to have dismal prognosis when treated only with chemotherapy, and we firstly provided evidence that they can significantly benefit from timely allo-HSCT. Five cases were MN1-ETV6 positive, and a novel MN1-STAT3 fusion was identified in the patient with triadic translocation. Significant MN1 overexpression was observed in all three MN1-fusion-negative cases. Genetic analysis highlighted the evidence of an ectopic super-enhancer associated orchestrated mechanism of MN1 overexpression and ETV6 haploinsufficiency in t(12;22)(p13;q12) myeloid neoplasms, rather than the conventional thought of MN1-ETV6 fusion formation. We also disclosed the high concomitance of trisomy 8 and 531 Kbps focal 8q duplication in t(12;22)(p13;q12) cases. The new perspective about this entity of disease will enlighten further research to define the mechanism of tumorigenesis and discover effective treatments for MN1-driven malignancies.
Assuntos
Genômica/métodos , Síndromes Mielodisplásicas/genética , Transativadores/uso terapêutico , Fatores de Transcrição/genética , Translocação Genética/genética , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transativadores/farmacologia , Proteínas Supressoras de Tumor/farmacologia , Proteínas Supressoras de Tumor/uso terapêutico , Adulto JovemRESUMO
BACKGROUND: Stem cell therapy has revealed a promising future for treating erectile dysfunction (ED), but the fate and curative mechanism of intracavernosal transplanted stem cells are under further exploration. This study aimed to demonstrate the effects of myocardin gene modification on improving erectile function and prolonging the retention of implanted adipose-derived stem cells (ASCs) using in vivo small animal imaging. METHODS: ASCs were isolated, cultured, and identified by flow cytometry and osteogenic and adipogenic induction. The effects of gene modification on cell proliferation, apoptosis, and contraction were determined by CCK-8, EdU, flow cytometry, and collagen gel lattice contraction assays as well as confocal microscopy. A total of 20 normal and 60 diabetes mellitus ED to (DMED) Sprague-Dawley rats were recruited to the 7 day and 21 day groups. Each group contained subgroups of 10 rats each: the negative control (NC), DMED + ASCs plus Ad-Luc-Myocardin, DMED + ASCs plus Ad-Luc, and DMED + phosphate buffer solution (PBS) groups. Erectile function was evaluated with the intracavernosal pressure/mean arterial pressure (â³ICP/MAP) ratio. In vivo small animal imaging and an EdU cell tracking strategy were introduced to detect the transplanted ASCs, and IHC and WB were performed to assess smooth muscle cell protein levels. RESULTS: The ASCs expressed high CD29 and CD90 and scant CD45, while the multi-induction potential was verified by oil red O and alizarin red staining. Gene transfection of myocardin had no significant influence on ASC apoptosis but inhibited cell proliferation and promoted cell contraction. Myocardin combined with ASCs enhanced the therapeutic potential of ASCs for improving the â³ICP/MAP ratio as well as α-SMA and calponin expression. In vivo imaging confirmed that ASCs resided within the cavernous body in 21 days, while only a few red EdU dots were detected. CONCLUSIONS: Myocardin induced ASC differentiation towards smooth muscle-like cells and enhanced the therapeutic potential of ASCs for ameliorating ED in STZ-induced diabetic rats. Notably, in vivo small animal tracking was an effective strategy for monitoring the implanted stem cells, and this strategy might have advantages over traditional EdU assays.
Assuntos
Diabetes Mellitus Experimental/terapia , Disfunção Erétil/terapia , Transplante de Células-Tronco Mesenquimais , Proteínas Nucleares/genética , Transativadores/genética , Animais , Apoptose/genética , Diferenciação Celular/genética , Proliferação de Células , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Disfunção Erétil/genética , Disfunção Erétil/patologia , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Músculo Liso/metabolismo , Proteínas Nucleares/uso terapêutico , Ereção Peniana/genética , Ereção Peniana/fisiologia , Ratos , Ratos Sprague-Dawley , Transativadores/uso terapêuticoRESUMO
Hepatitis B virus (HBV) X protein (HBx) plays a key role in the initiation and progression of HBV infectioninduced hepatocellular carcinoma (HCC). Oncogenic microRNA-21 (miR-21) can be modulated by HBx protein in HCC. However, critical regulator genes in the pathway of HBx-induced miR-21 in HCC remain unclear. This study aimed to investigate the role of HBx-induced miR-21 in the apoptosis of HCC cells. In the study, interleukin-12 (IL-12) was demonstrated as a direct target of miR-21 by dualluciferase report assay, and miR-21 was highly expressed in HCC cells (HepG2 and HepG2 2.2.15) compared to L02 cells, but IL-12 was weakly expressed as detected by real-time quantitative PCR (RT-qPCR). Furthermore, miR-21 mimics, inhibitor, HBx-targeted siRNA, and the HBx overexpression vector (pHBx) were used to observe the regulatory effects of HBx-induced miR-21 via IL-12, and cell apoptosis was assessed. The results showed that overexpression of HBx resulted in the inhibition of IL-12. A high level of miR-21 resulted in a significant increase in proliferation and a decrease in IL-12 expression. Inhibition of miR-21 resulted in a significant increase in apoptosis and increased IL-12 expression. The results suggest that HCC cell apoptosis was suppressed at least partially through HBx-induced miR-21 by targeting IL-12.
Assuntos
Carcinoma Hepatocelular/genética , Interleucina-12/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , Transativadores/genética , Apoptose/genética , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/virologia , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos/uso terapêutico , Células Hep G2 , Hepatite B/complicações , Hepatite B/genética , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Humanos , Interleucina-12/biossíntese , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/virologia , MicroRNAs/biossíntese , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Transativadores/antagonistas & inibidores , Transativadores/uso terapêutico , Proteínas Virais Reguladoras e AcessóriasRESUMO
The aim of this study is to investigate the effect of hepatitis B virus X (HBx) protein on the apoptosis of placental trophoblastic cells and its potential mechanism. A pcDNA3.1 expression vector of HBx gene was built and transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines, respectively. After transfection for 48 h, RT-PCR and immunofluorescence analyses showed that HBx mRNA and protein expression was detected in JEG-3 and HTR-8 cells. Flow cytometry revealed that early apoptosis of JEG-3 and HTR-8 cells was reduced by pcDNA-HBx transfection. Immunofluorescence and Western blotting showed that PI3K and p-Akt were significantly upregulated in HTR-8 cells. HBx ectopic expression did not change the viability of JEG-3 and HTR-8 cells when the PI3K/Akt pathway was blocked by its specific inhibitor LY294002. Moreover, the pcDNA-PI3K expression vector and pcDNA-HBx were transfected individually or co-transfected into the cells. The results showed that pcDNA-PI3K/pcDNA-HBx co-transfection promoted the expression of PI3K protein compared with the pcDNA-PI3K transfection group but did not increase the expression of HBx protein compared with pcDNA-HBx transfection group. In conclusion, HBx gene can be transferred into JEG-3 and HTR-8 human placental trophoblastic cell lines and cause inhibition of cell apoptosis. Its effect of apoptosis inhibition is related to the activation of the PI3K/Akt signaling pathway.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transativadores/biossíntese , Trofoblastos/metabolismo , Apoptose/genética , Apoptose/fisiologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Feminino , Vírus da Hepatite B/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Gravidez , RNA Mensageiro/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/uso terapêutico , Transfecção , Trofoblastos/fisiologia , Proteínas Virais Reguladoras e AcessóriasRESUMO
BACKGROUND: Epstein-Barr virus (EBV) infection is a major cause of morbidity following hematopoietic stem cell transplantation. EBV-infected B cells may not respond to rituximab treatment and may lead to a life-threatening post-transplantation lymphoproliferative disorder. Adoptive cellular immunotherapy using EBV-lymphoblastoid cell lines (LCL) as stimulating antigen has proved effective in restoring specific immunity. However, EBV presents several immunodominant antigens, and developing a swift and effective clinical-grade immunotherapy relies on the definition of a Good Manufacturing Practices (GMP) universal stimulating antigen. METHODS: Peripheral blood mononuclear cells (PBMCs) from six donors with a cellular immune response against EBV were immunoselected after stimulation with a new EBV antigen associated with an EBNA3 peptide pool. RESULTS: After immunoselection, a mean of 0.53 ± 0.25 × 106 cells was recovered consisting of a mean of 24.77 ± 18.01% CD4âº-secreting interferon (IFN)-γ and 51.42 ± 26.92% CD8âº-secreting IFN-γ. The T memory stem cell sub-population was identified. EBV-specific T cells were expanded in vitro, and their ability to secrete IFN-γ and to proliferate after re-stimulation with EBV antigen was confirmed. A specific lysis was observed against autologous target cells pulsed with EBV peptide pools (57.6 ± 11.5%) and against autologous EBV-LCL (18.3 ± 7.3%). A mean decrease of 94.7 ± 3.3% in alloreactivity against third-party donor mononuclear cells with EBV-specific T cells was observed compared with PBMCs before selection. CONCLUSIONS: Our results show that a combination of peptide pools including EBNA3 is needed to generate EBV-specific T cells with good specific cytotoxicity and devoid of alloreactivity, but as yet GMP grade is not fully achieved.
Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/uso terapêutico , Imunoterapia Adotiva , Linfócitos T/metabolismo , Transativadores/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/terapia , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Herpesvirus Humano 4/imunologia , Humanos , Imunidade Celular/imunologia , Linfócitos T/virologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Transativadores/imunologiaRESUMO
PURPOSE OF REVIEW: A lack of effective treatments for advanced cancer remains a major challenge in oncology. Because cancer is a disease associated with aberrant gene expression patterns, transcription factors, which serve as the convergence points of oncogenic signaling and are functionally altered in many cancers, hold great therapeutic promise. RECENT FINDINGS: Many human cancers are dependent on the inappropriate activity of oncogenic transcription factors. By contrast, normal cells can often tolerate disruption of these proteins with little toxicity. Direct inhibition of transcription factor expression (e.g., with RNA interference or microRNAs) and DNA binding (e.g., with oligodeoxynucleotide decoys or pyrrole-imidazole polyamides) has demonstrated antitumor responses with minimal side-effects. New strategies of targeting transcription factors include disrupting critical protein-protein interactions, and restricting binding at the epigenetic level by modulating chromatin accessibility. Moreover, targeting transcription factors in tumor-associated immune cells has the potential to overcome tumor immunoresistance. SUMMARY: Transcription factors are an important target for cancer therapy, both through direct anticancer effects and immunomodulatory actions. Newly developed delivery systems that specifically target tumor cells also create opportunities for successes in targeting transcription in cancer.
Assuntos
Antineoplásicos/uso terapêutico , Marcação de Genes/métodos , Terapia Genética , Neoplasias/terapia , RNA Interferente Pequeno/uso terapêutico , Fatores de Transcrição/efeitos dos fármacos , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/imunologia , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Marcação de Genes/tendências , Terapia Genética/métodos , Humanos , Masculino , NF-kappa B/imunologia , Neoplasias/genética , Neoplasias/imunologia , RNA Interferente Pequeno/imunologia , Receptor Notch1/imunologia , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/imunologia , Transativadores/uso terapêuticoRESUMO
Pathogenic bacteria delay wound healing through several different mechanisms such as persistent production of inflammatory mediators or maintenance of necrotic neutrophils, which release cytolytic enzymes and free oxygen radicals. One of the most frequent pathogens isolated from infections in chronic wounds is Pseudomonas aeruginosa. This bacterium is extremely refractory to therapy and to host immune attack when it forms biofilms. Therefore, antibiotics and antiseptics are becoming useless in the treatment of these infections. In previous works, we demonstrated that Lactobacillus plantarum has an important antipathogenic capacity on P. aeruginosa. The aim of the present work was to elucidate the mechanism involved in the control of growth of P. aeruginosa on different surfaces by L. plantarum. For this purpose, we investigated the effects of L. plantarum supernatants on pathogenic properties of P. aeruginosa, such as adhesion, viability, virulence factors, biofilm formation, and quorum sensing signal expression. L. plantarum supernatants were able to inhibit pathogenic properties of P. aeruginosa by a quorum quenching mechanism. The antipathogenic properties mentioned above, together with the immunomodulatory, tissue repair, and angiogenesis properties in the supernatants of L. plantarum, make them an attractive option in infected chronic wound treatment.
Assuntos
Inflamação/imunologia , Lactobacillus plantarum/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/patogenicidade , Cicatrização/imunologia , Infecção dos Ferimentos/imunologia , Biofilmes/crescimento & desenvolvimento , Proteínas de Transporte/farmacologia , Proteínas de Transporte/uso terapêutico , Doença Crônica , Humanos , Inflamação/patologia , Lipoproteínas/farmacologia , Lipoproteínas/uso terapêutico , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Percepção de Quorum , Espectroscopia de Infravermelho com Transformada de Fourier , Transativadores/farmacologia , Transativadores/uso terapêuticoRESUMO
OBJECTIVE: The key pancreatic transcription factor pancreatic duodenal homeobox-1 (Pdx1), known to control development and maintenance of pancreatic beta-cells, possesses a protein transduction domain (PTD) that facilitates its entry into cells. We therefore sought to evaluate the capacity of in vivo-administered recombinant Pdx1 (rPdx1) to ameliorate hyperglycemia in mice with streptozotocin-induced diabetes. RESEARCH DESIGN AND METHODS: Cell entry and transcriptional regulatory properties of rPdx1 protein and its PTD-deletion mutant rPdx1Delta protein, as well as a PTD-green fluorescent protein, were evaluated in vitro. After intraperitoneal rPdx1 injection into mice with streptozotocin-induced diabetes, we assessed its action on blood glucose levels, insulin content, intraperitoneal glucose tolerance test (IPGTT), Pdx1 distribution, pancreatic gene expression, islet cell proliferation, and organ histology. RESULTS: Restoration of euglycemia in Pdx1-treated diabetic mice was evident by improved IPGTT and glucose-stimulated insulin release. Insulin, glucagon, and Ki67 immunostaining revealed increased islet cell number and proliferation in pancreata of rPdx1-treated mice. Real-time PCR of pancreas and liver demonstrated upregulation of INS and PDX1 genes and other genes relevant to pancreas regeneration. While the time course of beta-cell gene expression and serum/tissue insulin levels indicated that both liver- and pancreas-derived insulin contributed to restoration of normoglycemia, near-total pancreatectomy resulted in hyperglycemia, suggesting that beta-cell regeneration played the primary role in rPdx1-induced glucose homeostasis. CONCLUSIONS: rPdx1 treatment of mice with streptozotocin-induced diabetes promotes beta-cell regeneration and liver cell reprogramming, leading to restoration of normoglycemia. This novel PTD-based protein therapy offers a promising way to treat patients with diabetes while avoiding potential side effects associated with the use of viral vectors.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Proteínas de Homeodomínio/administração & dosagem , Proteínas de Homeodomínio/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Transativadores/administração & dosagem , Transativadores/uso terapêutico , Animais , Glicemia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/sangue , Camundongos , Pâncreas/metabolismo , Estrutura Terciária de Proteína , Ratos , Fatores de Tempo , Transativadores/genética , Transativadores/metabolismoRESUMO
CPPs (cell-penetrating peptides), including Tatp (transactivator of transcription peptide), have been successfully used for intracellular delivery of a wide variety of cargoes including various nanoparticulate pharmaceutical carriers such as liposomes, micelles and nanoparticles. Here, we will consider the major results obtained in this area with emphasis on Tatp-mediated delivery of liposomes and various transfection vectors. We will also address the development of 'smart' stimuli-sensitive nanocarriers, where the cell-penetrating function can only be activated when the nanocarrier is inside the biological target, thus minimizing the interaction with non-target cells.
Assuntos
Sistemas de Liberação de Medicamentos , Vetores Genéticos/administração & dosagem , Lipossomos/administração & dosagem , Nanoestruturas/administração & dosagem , Peptídeos/uso terapêutico , Transativadores/uso terapêuticoRESUMO
Circadian rhythms and the genes that make up the molecular clock have long been implicated in bipolar disorder. Genetic evidence in bipolar patients suggests that the central transcriptional activator of molecular rhythms, CLOCK, may be particularly important. However, the exact role of this gene in the development of this disorder remains unclear. Here we show that mice carrying a mutation in the Clock gene display an overall behavioral profile that is strikingly similar to human mania, including hyperactivity, decreased sleep, lowered depression-like behavior, lower anxiety, and an increase in the reward value for cocaine, sucrose, and medial forebrain bundle stimulation. Chronic administration of the mood stabilizer lithium returns many of these behavioral responses to wild-type levels. In addition, the Clock mutant mice have an increase in dopaminergic activity in the ventral tegmental area, and their behavioral abnormalities are rescued by expressing a functional CLOCK protein via viral-mediated gene transfer specifically in the ventral tegmental area. These findings establish the Clock mutant mice as a previously unrecognized model of human mania and reveal an important role for CLOCK in the dopaminergic system in regulating behavior and mood.