RESUMO
Glioblastoma (GBM) is an aggressive brain cancer with a poor prognosis. While surgical resection is the primary treatment, adjuvant temozolomide (TMZ) chemotherapy and radiotherapy only provide slight improvement in disease course and outcome. Unfortunately, most treated patients experience recurrence of highly aggressive, therapy-resistant tumours and eventually succumb to the disease. To increase chemosensitivity and overcome therapy resistance, we have modified the chemical structure of the PFI-3 bromodomain inhibitor of the BRG1 and BRM catalytic subunits of the SWI/SNF chromatin remodelling complex. Our modifications resulted in compounds that sensitized GBM to the DNA alkylating agent TMZ and the radiomimetic bleomycin. We screened these chemical analogues using a cell death ELISA with GBM cell lines and a cellular thermal shift assay using epitope tagged BRG1 or BRM bromodomains expressed in GBM cells. An active analogue, IV-129, was then identified and further modified, resulting in new generation of bromodomain inhibitors with distinct properties. IV-255 and IV-275 had higher bioactivity than IV-129, with IV-255 selectively binding to the bromodomain of BRG1 and not BRM, while IV-275 bound well to both BRG1 and BRM bromodomains. In contrast, IV-191 did not bind to either bromodomain or alter GBM chemosensitivity. Importantly, both IV-255 and IV-275 markedly increased the extent of DNA damage induced by TMZ and bleomycin as determined by nuclear γH2AX staining. Our results demonstrate that these next-generation inhibitors selectively bind to the bromodomains of catalytic subunits of the SWI/SNF complex and sensitize GBM to the anticancer effects of TMZ and bleomycin. This approach holds promise for improving the treatment of GBM.
Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Domínios Proteicos , Temozolomida/farmacologia , Morte Celular , Bleomicina/farmacologia , Dano ao DNARESUMO
Despite advances in molecular characterization, glioblastoma (GBM) remains the most common and lethal brain tumour with high mortality rates in both paediatric and adult patients. The signal transducer and activator of transcription 3 (STAT3) is an important oncogenic driver of GBM. Although STAT3 reportedly plays a role in autophagy of some cells, its role in cancer cell autophagy remains unclear. In this study, we found Serine-727 and Tyrosine-705 phosphorylation of STAT3 was constitutive in GBM cell lines. Tyrosine phosphorylation of STAT3 in GBM cells suppresses autophagy, whereas knockout (KO) of STAT3 increases ULK1 gene expression, increases TSC2-AMPKα-ULK1 signalling, and increases lysosomal Cathepsin D processing, leading to the stimulation of autophagy. Rescue of STAT3-KO cells by the enforced expression of wild-type (WT) STAT3 reverses these pathways and inhibits autophagy. Conversely, expression of Y705F- and S727A-STAT3 phosphorylation deficient mutants in STAT3-KO cells did not suppress autophagy. Inhibition of ULK1 activity (by treatment with MRT68921) or its expression (by siRNA knockdown) in STAT3-KO cells inhibits autophagy and sensitizes cells to apoptosis. Taken together, our findings suggest that serine and tyrosine phosphorylation of STAT3 play critical roles in STAT3-dependent autophagy in GBM, and thus are potential targets to treat GBM.
Assuntos
Proteínas Quinases Ativadas por AMP , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Glioblastoma , Peptídeos e Proteínas de Sinalização Intracelular , Fator de Transcrição STAT3 , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Glioblastoma/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Fator de Transcrição STAT3/metabolismo , Serina/metabolismo , Tirosina/metabolismoRESUMO
Glioblastoma (GBM) is a highly aggressive cancer with a dismal prognosis. Constitutively active STAT3 has a causal role in GBM progression and is associated with poor patient survival. We rationally designed a novel small molecule, SS-4, by computational modeling to specifically interact with STAT3. SS-4 strongly and selectively inhibited STAT3 tyrosine (Y)-705 phosphorylation in MT330 and LN229 GBM cells and inhibited their proliferation and induced apoptosis with an IC50 of â¼100 nM. The antiproliferative and apoptotic actions of SS-4 were Y-705 phosphorylation dependent, as evidenced by its lack of effects on STAT3 knockout (STAT3KO) cells or STAT3KO cells that overexpressed a phospho-Y705 deficient (STAT3Y705F) mutant, and the recovery of effects when wild-type STAT3 or a phospho-serine (S)727 deficient mutant was expressed in STAT3KO cells. SS-4 increased the expression of STAT3 repressed genes, while decreasing the expression of STAT3 promoted genes. Importantly, SS-4 markedly reduced the growth of GBM intracranial tumor xenografts. These data together identify SS-4 as a potent STAT3 inhibitor that selectively blocks Y705-phosphorylation, induces apoptosis, and inhibits growth of human GBM models in vitro and in vivo.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Fosforilação , Fator de Transcrição STAT3/metabolismo , Tirosina/metabolismoRESUMO
Glioblastoma (GBM) is the most aggressive and treatment-refractory malignant adult brain cancer. After standard of care therapy, the overall median survival for GBM is only â¼6 months with a 5-year survival <10%. Although some patients initially respond to the DNA alkylating agent temozolomide (TMZ), unfortunately most patients become resistant to therapy and brain tumors eventually recur. We previously found that knockout of BRG1 or treatment with PFI-3, a small molecule inhibitor of the BRG1 bromodomain, enhances sensitivity of GBM cells to temozolomide in vitro and in vivo GBM animal models. Those results demonstrated that the BRG1 catalytic subunit of the SWI/SNF chromatin remodeling complex appears to play a critical role in regulating TMZ-sensitivity. In the present study we designed and synthesized Structurally Related Analogs of PFI-3 (SRAPs) and tested their bioactivity in vitro. Among of the SRAPs, 9f and 11d show better efficacy than PFI-3 in sensitizing GBM cells to the antiproliferative and cell death inducing effects of temozolomide in vitro, as well as enhancing the inhibitor effect of temozolomide on the growth of subcutaneous GBM tumors.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Compostos Azabicíclicos/farmacologia , DNA Helicases/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Piridinas/farmacologia , Temozolomida/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Animais , Antineoplásicos Alquilantes/química , Compostos Azabicíclicos/química , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA Helicases/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos NOD , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Nucleares/metabolismo , Piridinas/química , Relação Estrutura-Atividade , Temozolomida/química , Fatores de Transcrição/metabolismoRESUMO
Glioblastoma (GBM) is a deadly and incurable brain cancer with limited therapeutic options. PFI-3 is a small-molecule bromodomain (BRD) inhibitor of the BRM/BRG1 subunits of the SWI/SNF chromatin remodeling complex. The objective of this study is to determine the efficacy of PFI-3 as a potential GBM therapy. We report that PFI-3 binds to these BRDs when expressed in GBM cells. PFI-3 markedly enhanced the antiproliferative and cell death-inducing effects of temozolomide (TMZ) in TMZ-sensitive GBM cells as well as overcame the chemoresistance of highly TMZ-resistant GBM cells. PFI-3 also altered gene expression in GBM and enhanced the basal and interferon-induced expression of a subset of interferon-responsive genes. Besides the effects of PFI-3 on GBM cells in vitro, we found that PFI-3 markedly potentiated the anticancer effect of TMZ in an intracranial GBM animal model, resulting in a marked increase in survival of animals bearing GBM tumors. Taken together, we identified the BRG1 and BRM subunits of SWI/SNF as novel targets in GBM and revealed the therapeutic potential of applying small molecule inhibitors of SWI/SNF to improve the clinical outcome in GBM using standard-of-care chemotherapy.
RESUMO
The stability and processing capacity of membrane bioreactor can be improved with long sludge retention time. However, phosphorus removal will be markedly reduced under long sludge retention time and membrane fouling will be aggravated. Adding aluminum (Al) salt is a common way to achieve chemical phosphorus removal and membrane fouling reduction. But, accumulated Al will cause the decline of metabolic activity of activated sludge. In this study, biofilm-membrane flocculation reactor was proposed to enhance simultaneous nutrients removal and membrane fouling reduction. It showed that the removal efficiencies of chemical oxygen demand (COD), ammonia nitrogen (NH4+-N), total nitrogen (TN), and total phosphorus (TP) in biofilm-membrane flocculation reactor were 95.7%, 96.7%, 87.4%, and 97.2%, respectively. Compared with the control group, accumulated Al increased extracellular polymeric substances (EPS) secretion by 1.9%-35.4%, biofilm biomass by 12.4%-26.1%, and the activities of ammonia oxidation bacteria (AOB) and nitrite oxidation bacteria (NOB) in the biofilm increased by 42.9% and 65.9%, respectively. The relative abundance of Nitrospira, Dechloromonas, and Terrimonas in the biofilm increased by 1.78%, 3.01%, and 2.88%, respectively, which was conducive to facilitating the nitrification. Therefore, biofilm-membrane flocculation reactor is a promising way for enhancing simultaneous nutrients removal and membrane fouling reduction.
Assuntos
Reatores Biológicos , Nitrificação , Biofilmes , Floculação , Nitrogênio , Nutrientes , Fósforo , EsgotosRESUMO
Anaerobic ceramic membrane bioreactor (AnCMBR) is an attractive alternative for the treatment of high-strength phenol wastewater, but the effects of sludge retention time (SRT) on the performance and membrane fouling are still unclear. The results indicated that the AnCMBR was successfully employed to treat high-strength wastewater containing 5 g phenol L-1. The removal efficiencies of phenol and chemical oxygen demand (COD) reached over 99.5% and 99%, respectively, with long SRT and short SRT. SRT had no obvious effect on the performance of the AnCMBR treating high-strength phenol wastewater with long time operation. The strong performance robustness of AnCMBR benefited from the enrichment of hydrogenotrophic methanogens and syntrophic phenol-degrading bacteria. However, the decline of SRT led to a more severe membrane fouling in the AnCMBR, which was caused by the small size of sludge flocs and high concentration of protein in the biopolymers. Therefore, this work presented a comprehensive insight to the feasibility and robustness of the AnCMBR for treating high-strength phenol wastewater.
Assuntos
Reatores Biológicos , Cerâmica , Membranas Artificiais , Fenóis/química , Esgotos/química , Águas Residuárias/química , Anaerobiose , Fatores de Tempo , Eliminação de Resíduos Líquidos/instrumentação , Eliminação de Resíduos Líquidos/métodosRESUMO
Poly-aluminium chloride (PAC) is often used to enhance phosphorus removal and control membrane fouling in membrane bioreactors (MBRs). However, the influence of aluminium accumulation on the biological nitrification and phosphorus removal of MBRs has not been well assessed. In the present study, the effects of accumulated aluminium on sludge activity and morphology were investigated in a lab-scale anoxic-oxic membrane bioreactor. The reasonably high removal efficiencies of NH4+-N, TN, and COD, i.e. 94.9%, 84.8%, and 92.8%, respectively, were achieved in the reactor when the percentage of atomic aluminium on sludge surface increased to 14.2%. However, the decreases in the ammonia oxidation rate, nitrite oxidation rate, and specific oxygen uptake rate of sludge by 82.1%, 79.8%, and 46.4%, respectively, were observed. Meanwhile, the activity of phosphate-accumulating organisms was completely inhibited. Furthermore, the protein content in the extracellular polymeric substances of sludge decreased substantially, and the sludge became more dispersed due to the alum accumulation, compared with that of the initial phase. Therefore, long-term dosing of PAC in the MBR should be managed to avoid excessive aluminium accumulation in the sludge.
Assuntos
Alumínio/metabolismo , Reatores Biológicos , Nitrificação/fisiologia , Fósforo/metabolismo , Eliminação de Resíduos Líquidos , Membranas Artificiais , Nitrogênio , Oxirredução , EsgotosRESUMO
Ovarian cancer is one of the most common malignancies in women and has a high mortality rate due to metastatic progression and tumor recurrence. ASAP1 (ArfGAP with SH3 Domain, Ankyrin Repeat and PH Domain 1) is an ADP-ribosylation factor GTPase-activating protein, which is involved in tumor metastasis. However, the role of ASAP1 in ovarian cancer is completely unknown. The present study reported that ASAP1 was highly expressed in ovarian carcinoma, and expression positively-correlated with overall poor survival and prognosis of patients. Lentiviral vector mediated ASAP1 expression promoted cell migration and invasion in ovarian cancer cell lines SKOV3 and OVCAR3. In addition, ASAP1 promoted cell proliferation, survival and inhibited chemotherapy drug paclitaxel-induced cell apoptosis. Furthermore, ASAP1 expression promoted epithelial to mesenchymal transition (EMT) by upregulating the mesenchymal cell markers N-cadherin and vimentin, and downregulating epithelial cell marker E-cadherin in the ovarian cancer cell lines. The data indicate for the first time that ASAP1 exhibits an oncogenic role by promoting EMT in ovarian cancer cells.
RESUMO
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.
RESUMO
KLF4 is a transcriptional factor that can function either as a tumor suppressor or oncogene in cancer based on its cellular context. We recently demonstrated that KLF4 was a tumor suppressor in ovarian cancer cells by inhibiting the epithelial to mesenchymal transition. Here we report that KLF4 expression was downregulated in ovarian cancer tissue compared to normal ovarian tissue, and low KLF4 expression correlated with high risk ovarian carcinoma and poor patient survival. Enforced KLF4 expression by lentiviral transduction sensitized ovarian cancer cells to the effects of the chemotherapy drugs, paclitaxel and cisplatin. Treatment of ovarian cancer cells with APTO-253, a small molecule inducer of KLF4, enhanced the efficacy of both chemotherapy drugs. KLF4 expression mediated by lentiviral vector or induced by APTO-253 resulted in G1 phase arrest in ovarian cancer cells. Our results demonstrate that for the first time that inducing KLF4 expression with APTO-253 is a novel therapeutic strategy for treating ovarian cancer.
Assuntos
Antineoplásicos/administração & dosagem , Biomarcadores Tumorais/metabolismo , Imidazóis/administração & dosagem , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Fenantrolinas/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/agonistas , Neoplasias Ovarianas/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
The transcription factor Krüppel-like factor 4 (KLF4) has been implicated in regulating cell proliferation, migration and differentiation in a variety of human cells and is one of four factors required for the induction of pluripotent stem cell reprogramming. However, its role has not been addressed in ocular neovascular diseases. This study investigated the role of KLF4 in angiogenesis and underlying molecular mechanisms in human retinal microvascular endothelial cells (HRMECs). The functional role of KLF4 in HRMECs was determined following lentiviral vector mediated inducible expression and shRNA knockdown of KLF4. Inducible expression of KLF4 promotes cell proliferation, migration and tube formation. In contrast, silencing KLF4 inhibits cell proliferation, migration, tube formation and induces apoptosis in HRMECs. KLF4 promotes angiogenesis by transcriptionally activating VEGF expression, thus activating the VEGF signaling pathway in HRMECs.
Assuntos
Células Endoteliais/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Neovascularização Fisiológica/fisiologia , Vasos Retinianos/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Apoptose/genética , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Retina/citologia , Transdução de Sinais/fisiologia , Ativação Transcricional/genéticaRESUMO
PURPOSE: To evaluate the role of DiGeorge Critical Region 8 (DGCR8), a key component of miRNA biogenesis pathway in ovarian cancer. METHODS: The expression of DGCR8 in ovarian cancer was detected by immunostaining and DGCR8 knockdown in ovarian cancer cells was achieved using lentiviral shRNA. Differential expression of miRNAs was determined using Nanostring miRNA arrays and validated by real-time RT-PCR. RESULTS: DGCR8 was highly expressed in ovarian cancer. Knockdown of DGCR8 expression inhibits cell proliferation, migration, and invasion, as well as sensitizes cells to apoptosis induced by the chemotherapeutic drug cisplatin. Cellular survival pathways including ERK1/2 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT were attenuated in DGCR8 knockdown cells. DGCR8 knockdown results in dysregulated miRNA gene expression. miR-27b was identified as the most highly down-regulated miRNA in DGCR8 knockdown cells and promoted cell proliferation in ovarian cancer cells. CONCLUSIONS: DGCR8 functions as an oncogene in ovarian cancer, which is in part mediated by miR-27b.
Assuntos
Movimento Celular , Proliferação de Células , Terapia Genética/métodos , Oncogenes , Neoplasias Ovarianas/terapia , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: Ovarian cancer is a gynecological malignancy that has a high mortality rate in women due to metastatic progression and recurrence. miRNAs are small, endogenous, noncoding RNAs that function as tumor suppressors or oncogenes in various human cancers by selectively suppressing the expression of target genes. The objective of this study is to investigate the role of miR-203 in ovarian cancer. METHODS: miR-203 was expressed in ovarian cancer SKOV3 and OVCAR3 cells using lentiviral vector and cell proliferation, migration, invasion were examined using MTT, transwell and Matrigel assays, respectively. Tumor growth was examined using Xenograft mouse model. RESULTS: miR-203 expression was downregulated, whereas expression of its target gene Snai2 was upregulated in human ovarian serous carcinoma tissue as compared to normal ovaries. In addition, high miR-203 expression was associated with long-term survival rate of ovarian cancer patients. miR-203 overexpression inhibited cell proliferation, migration, and invasion of SKOV3 and OVCAR3 ovarian cancer cells. Furthermore, miR-203 overexpression inhibited the epithelial to mesenchymal transition (EMT) in ovarian cancer cells. Silencing Snai2 with lentiviral short hairpin (sh) RNA mimics miR-203-mediated inhibition of EMT and tumor cell invasion. Xenografts of miR-203-overexpressing ovarian cancer cells in immunodeficient mice exhibited a significantly reduced tumor growth. CONCLUSION: miR-203 functions as a tumor suppressor by down regulating Snai2 in ovarian cancer.
RESUMO
DiGeorge Critical Region 8 (DGCR8) is a double-stranded RNA-binding protein that interacts with Drosha and facilitates microRNA (miRNA) maturation. However, the role of DGCR8 in vascular smooth muscle cells (VSMCs) is not well understood. To investigate whether DGCR8 contributes to miRNA maturation in VSMCs, we generated DGCR8 conditional knockout (cKO) mice by crossing VSMC-specific Cre mice (SM22-Cre) with DGCR8(loxp/loxp) mice. We found that loss of DGCR8 in VSMCs resulted in extensive liver hemorrhage and embryonic mortality between embryonic days (E) 12.5 and E13.5. DGCR8 cKO embryos displayed dilated blood vessels and disarrayed vascular architecture. Blood vessels were absent in the yolk sac of DGCR8 KOs after E12.5. Disruption of DGCR8 in VSMCs reduced VSMC proliferation and promoted apoptosis in vitro and in vivo. In DGCR8 cKO embryos and knockout VSMCs, differentiation marker genes, including αSMA, SM22, and CNN1, were significantly down-regulated, and the survival pathways of ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated. Knockout of DGCR8 in VSMCs has led to down-regulation of the miR-17/92 and miR-143/145 clusters. We further demonstrated that the miR-17/92 cluster promotes VSMC proliferation and enhances VSMC marker gene expression, which may contribute to the defects of DGCR8 cKO mutants. Our results indicate that the DGCR8 gene is required for vascular development through the regulation of VSMC proliferation, apoptosis, and differentiation.