RESUMO
PURPOSE: Glioblastoma (GBM) patients currently face poor survival outcomes with an average survival period of <15 months, while only 3-5% of patients survive longer than 36 months. Although the mechanisms of tumorigenesis are still being elucidated, miRNAs are promising candidates to explore as novel and prognostic biomarkers in GBM. In this study, we identified the association between miR-575 expression and overall survival (OS) of primary GBM patients and undertook functional studies to discern the contribution of miR-575 to GBM tumorigenesis. METHODS: Total RNAs were isolated from 254 FFPE GBM tumor samples and miR expression was assayed (simultaneously) using NanoString Technologies. To determine the association between miR-575 and patients' prognosis, Kaplan-Meier, univariable and multivariable Cox regression analyses were performed. Cell proliferation, colony formation, migration assays were conducted to investigate the function of miR-575 in vitro and in vivo. In silico target gene network analysis was performed to identify the putative targets of miR-575 in GBM, which were further verified by luciferase reporter assay, as well as qPCR and immunoblotting. RESULTS: Our clinical data (n = 254) show that miR-575 is associated with worse GBM OS by univariable analysis (UVA, HR = 1.27, p-value<0.001) and multivariable (MVA, HR = 1.23, p = 0.007) analysis incorporating critical clinical variables. Functional studies indicated that overexpression of miR-575 significantly increased cell proliferation and migration of GBM cells in vitro, as well as tumor growth in vivo. Subsequent in silico target gene network and mechanistic studies identified CDKN1B/p27 and PTEN, as potential targets of miR-575 in GBM. MicroRNA-575 can also regulate the activity of AKT and ERK pathways in GBM. CONCLUSION: miR-575 has prognostic value in GBM, with higher expression associating with worse OS of patients, and contributes to GBM tumorigenesis by regulating multiple signaling pathways in GBM.
Assuntos
Neoplasias Encefálicas , Glioblastoma , MicroRNAs , Humanos , Glioblastoma/patologia , Neoplasias Encefálicas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Movimento Celular/genética , Linhagem Celular Tumoral , MicroRNAs/genética , MicroRNAs/metabolismo , Oncogenes , Proliferação de Células/genética , Transdução de Sinais/genética , Carcinogênese/genética , Luciferases/genética , Luciferases/metabolismo , Biomarcadores , Regulação Neoplásica da Expressão Gênica/genéticaRESUMO
Piperine (PPR) is the representative alkaloid component of the piper species (family: Piperaceae). Our rapid screening study found PPR caused time-dependent inhibition of cytochrome P450s (CYP) 3A and 2D6, and CYP3A was inactivated the most. Further study demonstrated that PPR is a time-, concentration-, and NADPH-dependent inhibitor of CYP3A, and significant loss (49.5% ± 3.9%) of CYP3A activity was observed after 20minute incubations with 80 µM PPR at 37°C. The values of K I and k inact were 30.7 µM and 0.041 minutes-1, respectively. CYP3A competitive inhibitor ketoconazole showed protective effect against the enzyme inactivation. Superoxide dismutase/catalase and GSH displayed minor protection against the PPR-caused enzyme inactivation. Ferricyanide partially reduced the enzyme inhibition by PPR. Additionally, NADPH-dependent formation of reactive metabolites from PPR were found in human liver microsomal incubation mixtures. An ortho-quinone intermediate was trapped by NAC in microsomal incubations with PPR. DM-PPR, demethylene metabolite of PPR, showed weak enzyme inactivation relative to that caused by PPR. It appears that both carbene and ortho-quinone intermediates were involved in the inactivation of CYP3A caused by PPR. SIGNIFICANCE STATEMENT: CYP3A subfamily members (mainly CYP3A4 and CYP3A5) play a critical role in drug metabolism. Piperine (PPR), a methylenedioxyphenyl derivative combined with an unsaturated ketone, is the major active ingredient of pepper. PPR revealed time-, concentration-, and NADPH-dependent inhibitory effect on CYP3A. Carbene and quinone metabolites were both involved in the observed CYP3A inactivation by PPR. Apparently, the unsaturated ketone moiety did not participate in the enzyme inactivation. The present study sounds an alert of potential risk for food-drug interactions.
Assuntos
Alcaloides/metabolismo , Benzodioxóis/metabolismo , Inibidores do Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP3A/metabolismo , Piperidinas/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Catalase/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , Cetoconazol/metabolismo , Cinética , Taxa de Depuração Metabólica/fisiologia , Microssomos Hepáticos , NADP/metabolismo , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: Plakophilin 1 (PKP1) is an important plaque component of desmosomes, major intercellular adhesive junctions that act as anchorage points for intermediate filaments. Abnormal expression of PKP1 was observed in various types of cancer, however so far its function in lung cancer has not yet been elucidated. METHODS: The expression of PKP1 was analyzed by RT-PCR and western blotting in lung cancer cell lines. The protein expression of PKP1 was evaluated by immunohistochemistry in tissue microarray. The epigenetic mechanism of PKP1 was explored by demethylation test, bisulfite sequencing and Methylation-Specific-PCR. The function of PKP1 was investigated by stable transfection with an expression vector. RESULTS: We found that PKP1 was downregulated in 6 out of 8 lung cancer cell lines, and downregulation of PKP1 was associated with DNA hypermethylation. In advanced primary lung tumor samples, higher expression of PKP1 was significantly associated with favorable clinical outcome (pâ¯=â¯.003). Ectopic expression of PKP1 inhibited cell proliferation, colony formation, migration/invasion and enhanced apoptosis. These phenomena are accompanied by increased caspase 3/7 activities and cleaved PARP-1 as well as decreased extracellular signal-regulated kinase (ERK) activity. CONCLUSION: Taken together, our data suggest that PKP1 is a novel tumor suppressor and its protein expression might be a potential prognostic marker for patients with advanced lung cancer.
Assuntos
Metilação de DNA , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Placofilinas/genética , Idoso , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Placofilinas/metabolismoRESUMO
Chelidonine (CHE) is a major bioactive constituent of greater celandine, a plant used in traditional herbal medicines. CHE has widely been used as an analgesic in clinical settings. We evaluated the inhibitory effects of CHE on human cytochrome P450 enzymes. CHE produced time-, concentration-, and NADPH-dependent inhibition of CYP2D6, with K I and k inact values of 20.49 µM and 11.05 min -1 , respectively. Approximately 76% of CYP2D6 activity was suppressed after 9 minute incubation with CHE (50 µM). The loss of enzyme activity was not restored following dialysis. The estimated partition ratio of the inactivation was about 156. Quinidine, a competitive inhibitor of CYP2D6, attenuated the CHE-mediated enzyme inactivation, while glutathione and catalase/superoxide dismutase did not markedly ameliorate the inhibitory effect. Upon oxidation using potassium ferricyanide, the 15.1% activity of CYP2D6 was restored. These findings indicate that CHE acted as a mechanism-based inactivator of CYP2D6 and the observed effects may induce potential drug-drug interactions.
Assuntos
Benzofenantridinas/química , Inibidores do Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/química , HumanosRESUMO
Yellow vein clearing disease (YVCD) causes significant economic losses in lemon and other species of citrus. Usually, citrus yellow vein clearing virus (CYVCV) is considered to be the causal agent of YVCD. However, mixed infection of CYVCV and Indian citrus ringspot virus (ICRSV) or other pathogens is often detected in citrus plants with YVCD. In this study, we re-examined the causal agent of YVCD to fulfill Koch's postulates. First, the full-length genome of CYVCV isolate AY (CYVCV-AY) was amplified by long-distance RT-PCR from a Eureka lemon (Citrus limon) tree with typical YVCD symptoms. The genomic cDNAs were then cloned into a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector, pCY, using transformation-associated recombination (TAR) strategy, and 15 full-length cDNA clones of CYVCV-AY were obtained. Subsequently, four of these clones were selected randomly and inoculated on Jincheng (C. sinensis) seedlings through Agrobacterium-mediated vacuum-infiltration, and it was found that 80 to 100% of inoculated plants were infected with CYVCV by RT-PCR at 20 to 40 days postinoculation (dpi) and by direct tissue blot immunoassay at 60 dpi. The progeny of CYVCV-AY from cDNA clones caused typical symptoms of YVCD such as yellow vein clearing, leaf distortion, and chlorosis, which were the same as that elicited by wild-type virus. Finally, the regeneration of CYVCV-AY genome was confirmed by long-distance RT-PCR in lemon trees inoculated with the infectious cDNA clone. These results proved that CYVCV was the primary causal agent of YVCD. This is the first report on the development of infectious cDNA clones of CYVCV, which lays the foundation for further studies on viral gene functions and virus-host interactions.
Assuntos
Citrus/virologia , DNA Complementar/genética , Flexiviridae/genética , Clonagem Molecular , DNA Viral , Doenças das Plantas/virologiaRESUMO
Cancer stem cells (CSCs) with enhanced tumorigenicity and chemoresistance are believed to be responsible for treatment failure and tumor relapse in ovarian cancer patients. However, it is still unclear how CSCs survive DNA-damaging agent treatment. Here, we report an elevated expression of DNA polymerase η (Pol η) in ovarian CSCs isolated from both ovarian cancer cell lines and primary tumors, indicating that CSCs may have intrinsically enhanced translesion DNA synthesis (TLS). Down-regulation of Pol η blocked cisplatin-induced CSC enrichment both in vitro and in vivo through the enhancement of cisplatin-induced apoptosis in CSCs, indicating that Pol η-mediated TLS contributes to the survival of CSCs upon cisplatin treatment. Furthermore, our data demonstrated a depletion of miR-93 in ovarian CSCs. Enforced expression of miR-93 in ovarian CSCs reduced Pol η expression and increased their sensitivity to cisplatin. Taken together, our data suggest that ovarian CSCs have intrinsically enhanced Pol η-mediated TLS, allowing CSCs to survive cisplatin treatment, leading to tumor relapse. Targeting Pol η, probably through enhancement of miR-93 expression, might be exploited as a strategy to increase the efficacy of cisplatin treatment.
Assuntos
Cisplatino/química , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias Ovarianas/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , DNA/química , Dano ao DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Regulação para Baixo , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , Metástase Neoplásica , Recidiva Local de Neoplasia , Transplante de Neoplasias , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/metabolismo , RecidivaRESUMO
The expression of DNA damage-binding protein 2 (DDB2) has been linked to the prognosis of ovarian cancer and its underlying transcription regulatory function was proposed to contribute to the favorable treatment outcome. By applying gene microarray analysis, we discovered neural precursor cell expressed, developmentally downregulated 4-Like (NEDD4L) as a previously unidentified downstream gene regulated by DDB2. Mechanistic investigation demonstrated that DDB2 can bind to the promoter region of NEDD4L and recruit enhancer of zeste homolog 2 histone methyltransferase to repress NEDD4L transcription by enhancing histone H3 lysine 27 trimethylation (H3K27me3) at the NEDD4L promoter. Given that NEDD4L plays an important role in constraining transforming growth factor ß signaling by targeting activated Smad2/Smad3 for degradation, we investigated the role of DDB2 in the regulation of TGF-ß signaling in ovarian cancer cells. Our data indicate that DDB2 enhances TGF-ß signal transduction and increases the responsiveness of ovarian cancer cells to TGF-ß-induced growth inhibition. The study has uncovered an unappreciated regulatory mode that hinges on the interaction between DDB2 and NEDD4L in human ovarian cancer cells. The novel mechanism proposes the DDB2-mediated fine-tuning of TGF-ß signaling and its downstream effects that impinge upon tumor growth in ovarian cancers.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Fator de Crescimento Transformador beta/farmacologia , Ubiquitina-Proteína Ligases/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Histonas/metabolismo , Humanos , Ubiquitina-Proteína Ligases Nedd4 , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
Discretely orchestrated chromatin condensation is important for chromosome protection from DNA damage. However, it is still unclear how different chromatin states affect the formation and repair of nucleotide excision repair (NER) substrates, e.g. ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPD) and the pyrimidine (6-4) pyrimidone photoproducts (6-4PP), as well as cisplatin-induced intrastrand crosslinks (Pt-GG). Here, by using immunofluorescence and chromatin immunoprecipitation assays, we have demonstrated that CPD, which cause minor distortion of DNA double helix, can be detected in both euchromatic and heterochromatic regions, while 6-4PP and Pt-GG, which cause major distortion of DNA helix, can exclusively be detected in euchromatin, indicating that the condensed chromatin environment specifically interferes with the formation of these DNA lesions. Mechanistic investigation revealed that the class III histone deacetylase SIRT1 is responsible for restricting the formation of 6-4PP and Pt-GG in cells, probably by facilitating the maintenance of highly condensed heterochromatin. In addition, we also showed that the repair of CPD in heterochromatin is slower than that in euchromatin, and DNA damage binding protein 2 (DDB2) can promote the removal of CPD from heterochromatic region. In summary, our data provide evidence for differential formation and repair of DNA lesions that are substrates of NER. Both the sensitivity of DNA to damage and the kinetics of repair can be affected by the underlying level of chromatin compaction.
Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Eucromatina/química , Heterocromatina/química , Células Cultivadas , Imunoprecipitação da Cromatina , Imunofluorescência , Humanos , Immunoblotting , Dímeros de Pirimidina/metabolismo , Interferência de RNARESUMO
The level of microRNA-93 (miR-93) in tumors has been recently reported to be negatively correlated with survival of lung cancer patients. Considering that the most devastating aspect of lung cancer is metastasis, which can be promoted by transforming growth factor-ß (TGF-ß)-induced epithelial-to-mesenchymal transition (EMT), we sought to determine whether miR-93 is involved in this process. Here, we report that a previously unidentified target of miR-93, neural precursor cell expressed developmentally downregulated gene 4-like (NEDD4L), is able to mediate TGF-ß-mediated EMT in lung cancer cells. miR-93 binds directly to the 3'-UTR of the NEDD4L messenger RNA (mRNA), leading to a downregulation of NEDD4L expression at the protein level. We next demonstrated that the downregulation of NEDD4L enhanced, while overexpression of NEDD4L reduced TGF-ß signaling, reflected by increased phosphorylation of SMAD2 in the lung cancer cell line after TGF-ß treatment. Furthermore, overexpression of miR-93 in lung cancer cells promoted TGF-ß-induced EMT through downregulation of NEDD4L. The analysis of publicly available gene expression array datasets indicates that low NEDD4L expression correlates with poor outcomes among patients with lung cancer, further supporting the oncogenic role of miR-93 in lung tumorigenesis and metastasis.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Fator de Crescimento Transformador beta/genética , Ubiquitina-Proteína Ligases/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Complexos Endossomais de Distribuição Requeridos para Transporte/biossíntese , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Masculino , MicroRNAs/biossíntese , Ubiquitina-Proteína Ligases Nedd4 , Metástase Neoplásica , Estadiamento de Neoplasias , Proteína Smad2/biossíntese , Ubiquitina-Proteína Ligases/biossínteseRESUMO
Radiotherapy resistance is one of the major factors limiting the efficacy of radiotherapy in lung cancer patients. The extensive investigations indicate the diversity in the mechanisms underlying radioresistance. Here, we revealed that DNA damage binding protein 2 (DDB2) is a potential regulator in the radiosensitivity of non-small cell lung cancer (NSCLC) cells. DDB2, originally identified as a DNA damage recognition factor in the nucleotide excision repair, promotes the survival and inhibits the apoptosis of NSCLC cell lines upon ionizing radiation (IR). Mechanistic investigations demonstrated that DDB2 is able to facilitate IR-induced phosphorylation of Chk1, which plays a critical role in the cell cycle arrest and DNA repair in response to IR-induced DNA double-strand breaks (DSBs). Indeed, knockdown of DDB2 compromised the G2 arrest in the p53-proficient A549 cell line and reduced the efficiency of homologous recombination (HR) repair. Taken together, our data indicate that the expression of DDB2 in NSCLC could be used as a biomarker to predict radiosensitivity of the patients. Targeting Chk1 can be used to increase the efficacy of radiotherapy in patients of NSCLC possessing high levels of DDB2.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Neoplasias Pulmonares/radioterapia , Tolerância a Radiação/genética , Reparo de DNA por Recombinação/genética , Apoptose/efeitos da radiação , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Proteínas de Ligação a DNA/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fosforilação , Radiação Ionizante , Reparo de DNA por Recombinação/efeitos da radiação , Células Tumorais CultivadasRESUMO
HOPX acts as a tumour suppressor in various cancers. However, the regulation of HOPX in human lung cancer as well as the mechanism underlying its tumour-suppressive function has not yet been well elucidated. Here we investigated the epigenetic regulation and molecular mechanism by which HOPX exerts growth inhibitory effects. We found that HOPX was down-regulated in 12 out of 13 lung cancer cell lines and in 69 out of 120 primary lung tumours at mRNA and protein levels. Patients with lung adenocarcinoma (ADC) exhibited significantly more positive staining of HOPX protein compared with lung squamous cell carcinoma (SCC) (p =0.036). Again in ADC, patients with higher HOPX expression had a significantly longer disease-free survival (p =0.001). Methylation analysis showed that down-regulation of HOPX was associated with DNA methylation (p =0.011). To analyse the function of HOPX in lung cancer cells, stable transfection with an expression vector of HOPX was performed. It turned out that HOPX inhibited tumour cell proliferation rate, migration, and invasion, and, more interestingly, forced expression of HOPX enhanced cellular senescence via activation of oncogenic Ras and the downstream MAPK pathway, which in turn led to decreased MDM2 and increased p21. On the contrary, knockdown of HOPX by siRNA resulted in reduced Ras activity, inactivation of the MAPK pathway, and decreased p21 levels, accompanied by reduced cellular senescence. Additionally, the HOPX-induced senescence pathway was also active in human bronchial epithelial cells. Taken together, our data suggest that down-regulation of HOPX was related to DNA methylation and that HOPX exerts tumour-suppressive activity by oncogenic Ras-induced cellular senescence in lung cancer cells.
Assuntos
Senescência Celular/fisiologia , Metilação de DNA/fisiologia , Proteínas de Homeodomínio/fisiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas ras/fisiologia , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/fisiopatologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Transdução de Sinais/fisiologia , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/fisiopatologia , Proteínas Supressoras de Tumor/genéticaRESUMO
Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious diseases affecting the swine industry worldwide; however, there is no efficient control strategies against PRRSV at present. Therefore, development of new antiviral treatment strategies is urgently needed. As reported, germacrone can efficiently impair influenza virus replication. In this study, we exploited whether germacrone has the potential to inhibit PRRSV infection. Our results showed that the germacrone significantly inhibited replication of PRRSV in vitro and repressed the synthesis of viral RNA and protein. However, it did not block PRRSV binding and entry. Further studies confirmed that germacrone impaired PRRSV replication at an early stage, and inhibited infection of both classic and highly pathogenic type II PRRSV strains. Collectively, our findings imply that the germacrone has the potential to be used as an anti-PRRSV drug.
Assuntos
Antivirais/farmacologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Sesquiterpenos de Germacrano/farmacologia , Animais , Síndrome Respiratória e Reprodutiva Suína/tratamento farmacológico , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Suínos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
In 2006, a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV) emerged in China and caused lasting damage to the swine industry. To analyze the genetic variation of PRRSV in Southern China, 126 tissue samples were collected; 41 ORF5 and partial Nsp2 genes were sequenced and analyzed. The results showed that the PRRSV positive rate was 32.54% over the last four years, that there are two main subgenotypes in Southern China, and that the dominant strain is HP-PRRSV. An amino acid analysis of Nsp2 showed that 40 strains contained a 30-amino acid deletion in the hypervariable region. However, the 13YJ6-8 mutant exhibited a unique amino acid deletion at positions 508-514 of Nsp2. A phylogenetic analysis of ORF5 revealed that this mutant and five other strains, belong to an intermediate subgenotype (inter-subgenotype), which is characterized by extensive mutations, especially in the signal peptide and N-glycosylation sites. The results of this study demonstrate the genetic diversity of PRRSV in Southern China and provide basic knowledge of the PRRSV epidemic in this region.
Assuntos
Evolução Molecular , Variação Genética , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Sequência de Aminoácidos , Animais , China , Análise por Conglomerados , Genótipo , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , RNA Viral/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Deleção de Sequência , Homologia de Sequência , Suínos , Proteínas Virais/genéticaRESUMO
Human serum albumin (HSA) improves the pharmacokinetic profile of drugs attached to it, making it an attractive carrier with proven clinical success. In our previous studies, we have shown that Caveolin-1 (Cav-1) and caveolae-mediated endocytosis play important roles in the uptake of HSA and albumin-bound drugs. Doxorubicin is an FDA-approved chemotherapeutic agent that is effective against multiple cancers, but its clinical applicability has been hampered by its high toxicity levels. In this study, a doxorubicin-prodrug was developed that could independently and avidly bind HSA in circulation, called IPBA-Dox. We first developed and characterized IPBA-Dox and confirmed that it can bind albumin in vitro while retaining a potent cytotoxic effect. We then verified that it efficiently binds to HSA in circulation, leading to an improvement in the pharmacokinetic profile of the drug. In addition, we tested our prodrug for Cav-1 selectivity and found that it preferentially affects cells that express relatively higher levels of Cav-1 in vitro and in vivo. Moreover, we found that our compound was well tolerated in vivo at concentrations at which doxorubicin was lethal. Altogether, we have developed a doxorubicin-prodrug that can successfully bind HSA, retaining a strong cytotoxic effect that preferentially targets Cav-1 positive cells while improving the general tolerability of the drug.
RESUMO
Ex vivo red blood cell (RBC) production generates unsatisfactory erythroid cells. A deep exploration into terminally differentiated cells is required to understand the impairments for RBC generation and the underlying mechanisms. Here, we mapped an atlas of terminally differentiated cells from umbilical cord blood mononuclear cells (UCBMN) and pluripotent stem cells (PSC) and observed their dynamic regulation of erythropoiesis at single-cell resolution. Interestingly, we detected a few progenitor cells and non-erythroid cells from both origins. In PSC-derived erythropoiesis (PSCE), the expression of haemoglobin switch regulators (BCL11A and ZBTB7A) were significantly absent, which could be the restraint for its adult globin expression. We also found that PSCE were less active in stress erythropoiesis than in UCBMN-derived erythropoiesis (UCBE), and explored an agonist of stress erythropoiesis gene, TRIB3, could enhance the expression of adult globin in PSCE. Compared with UCBE, there was a lower expression of epigenetic-related proteins (e.g., CASPASE 3 and UBE2O) and transcription factors (e.g., FOXO3 and TAL1) in PSCE, which might restrict PSCE's enucleation. Moreover, we characterized a subpopulation with high proliferation capacity marked by CD99high in colony-forming unit-erythroid cells. Inhibition of CD99 reduced the proliferation of PSC-derived cells and facilitated erythroid maturation. Furthermore, CD99-CD99 mediated the interaction between macrophages and erythroid cells, illustrating a mechanism by which macrophages participate in erythropoiesis. This study provided a reference for improving ex vivo RBC generation.
Assuntos
Diferenciação Celular , Eritropoese , Sangue Fetal , Leucócitos Mononucleares , Células-Tronco Pluripotentes , Humanos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/citologia , Células Cultivadas , Proliferação de CélulasRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is characterized by its nutrient-scavenging ability, crucial for tumor progression. Here, we investigated the roles of caveolae-mediated endocytosis (CME) in PDAC progression. Analysis of patient data across diverse datasets revealed a strong association of high caveolin-1 (Cav-1) expression with higher histologic grade, the most aggressive PDAC molecular subtypes, and worse clinical outcomes. Cav-1 loss markedly promoted longer overall and tumor-free survival in a genetically engineered mouse model. Cav-1-deficient tumor cell lines exhibited significantly reduced proliferation, particularly under low nutrient conditions. Supplementing cells with albumin rescued the growth of Cav-1-proficient PDAC cells, but not in Cav-1-deficient PDAC cells under low glutamine conditions. In addition, Cav-1 depletion led to significant metabolic defects, including decreased glycolytic and mitochondrial metabolism, and downstream protein translation signaling pathways. These findings highlight the crucial role of Cav-1 and CME in fueling pancreatic tumorigenesis, sustaining tumor growth, and promoting survival through nutrient scavenging.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Camundongos , Animais , Humanos , Cavéolas/metabolismo , Cavéolas/patologia , Neoplasias Pancreáticas/patologia , Endocitose , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Transdução de Sinais , Linhagem Celular TumoralRESUMO
Aromatic azo dyes possess inherent resistance and are known to be carcinogenic, posing a significant threat to human and ecosystems. Enhancing the biodegradation of azo dyes usually requires the presence of co-metabolic substrates to optimize the process. In addressing the issue of excessive waste activated sludge (WAS) generation, this study explored the potential of utilizing alkaline-thermal hydrolysate of WAS as a co-metabolic substrate to boost the degradation of reactive black 5 (RB5) dyes. The acclimated microbial consortium, when supplemented with the WAS hydrolysate obtained at a hydrolysis temperature of 30 °C, achieved an impressive RB5 decolorization efficiency of 90.3% (pH = 7, 35 °C) with a corresponding COD removal efficiency of 45.0%. The addition of WAS hydrolysate as a co-substrate conferred the consortium with a remarkable tolerance to high dye concentration (1500 mg/L RB5) and salinity levels (4-5%), surpassing the performance of conventional co-metabolic sugars in RB5 degradation. 3D-EEM analysis revealed that protein-like substances rich in tyrosine and tryptophan, present in the WAS hydrolysate, played a crucial role in promoting RB5 biodegradation. Furthermore, the microbial consortium community exhibited an enrichment of dye-degrading species, including Acidovorax, Bordetella, Kerstersia, and Brevundimonas, which dominated the community. Notably, functional genes associated with dye degradation and intermediates were also enriched during the RB5 decolorization and biodegradation process. These findings present a practical strategy for the simultaneous treatment of dye-containing wastewater and recycling of WAS.
RESUMO
Recent studies have documented a rich phenomenology in twisted bilayer graphene (TBG), which is significantly relevant to interlayer electronic coupling, in particular to the cases under an applied electric field. While polarizability measures the response of electrons against applied fields, this work adopts a unique strategy of decomposing global polarizability into distributional contributions to access the interlayer polarization in TBG, as a function of varying twisting angles (θ). Through the construction of a model of twisted graphene quantum dots, we assess distributional polarizability at the first-principles level. Our findings demonstrate that the polarizability perpendicular to the graphene plates can be decomposed into intralayer dipoles and interlayer charge-transfer (CT) components, the latter of which provides an explicit measurement of the interlayer coupling strength and charge transfer potential. Our analysis further reveals that interlayer polarizability dominates the polarizability variation during twisting. Intriguingly, the largest interlayer polarizability and CT driven by an external field occur in the misaligned structures with a size-dependent small angle corresponding to the first appearance of AB stacking, rather than the well-recognized Bernal structures. A derived equation is then employed to address the size dependence on the angle corresponding to the largest values in interlayer polarizability and CT. Our investigation not only characterizes the CT features in the interlayer polarizability of TBG quantum dots, but also sheds light on the existence of the strongest interlayer coupling and charge transfer at small twist angles in the presence of an external electric field, thereby providing a comprehensive understanding of the novel properties of graphene-based nanomaterials.
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Albumin is an attractive candidate carrier for the development of novel therapeutic drugs. Gemcitabine has been FDA approved for the treatment of solid tumors; however, new drugs that optimize gemcitabine delivery are not available for clinical use. The aim of this study was to test the efficacy of a novel albumin-encapsulated gemcitabine prodrug, JNTX-101, and investigate whether Cav-1 expression predicts the therapeutic efficacy of JNTX-101. We first determined the treatment efficacy of JNTX-101 in a panel of pancreatic/lung cancer cell lines and found that increases in Cav-1 expression resulted in higher uptake of albumin, while Cav-1 depletion attenuated the sensitivity of cells to JNTX-101. In addition, decreased Cav-1 expression markedly reduced JNTX-101-induced apoptotic cell death in a panel of cells, particularly in low-serum conditions. Furthermore, we tested the therapeutic efficacy of JNTX-101 in xenograft models and the role of Cav-1 in JNTX-101 sensitivity using a Tet-on-inducible tumor model in vivo. Our data suggest that JNTX-101 effectively inhibits cell viability and tumor growth, and that Cav-1 expression dictates optimal sensitivity to JNTX-101. These data indicate that Cav-1 correlates with JNTX-101 sensitivity, especially under nutrient-deprived conditions, and supports a role for Cav-1 as a predictive biomarker for albumin-encapsulated therapeutics such as JNTX-101.
RESUMO
Glioblastoma (GBM) is an aggressive malignant primary brain tumor. Radioresistance largely contributes to poor clinical outcomes in GBM patients. We targeted ribonucleotide reductase subunit 2 (RRM2) with triapine to radiosensitize GBM. We found RRM2 is associated with increasing tumor grade, is overexpressed in GBM over lower grade gliomas and normal tissue, and is associated with worse survival. We found silencing or inhibition of RRM2 by siRNA or triapine sensitized GBM cells to ionizing radiation (IR) and delayed resolution of IR-induced γ-H2AX nuclear foci. In vivo, triapine and IR reduced tumor growth and increased mouse survival. Intriguingly, triapine led to RRM2 upregulation and CHK1 activation, suggesting a CHK1-dependent RRM2 upregulation following RRM2 inhibition. Consistently, silencing or inhibition of CHK1 with rabusertib abolished the triapine-induced RRM2 upregulation. Accordingly, combining rabusertib and triapine resulted in synthetic lethality in GBM cells. Collectively, our results suggest RRM2 is a promising therapeutic target for GBM, and targeting RRM2 with triapine sensitizes GBM cells to radiation and independently induces synthetic lethality of GBM cells with CHK1 inhibition. Our findings suggest combining triapine with radiation or rabusertib may improve therapeutic outcomes in GBM.