RESUMO
The antitumor antibiotic mithramycin A (MTA) binds to G/C-rich DNA sequences in the presence of dications. MTA inhibits transcription regulated by the Sp1 transcription factor, often enhanced during tumor development. It shows antitumor activity, but its clinical use was discontinued due to toxic side effects. However, recent observations have led to its use being reconsidered. The MTA biosynthetic pathways have been modified to produce mithramycin analogs (mithralogs) that encompass lower toxicity and improved pharmacological activity. Some mithralogs reduce gene expression in human ovarian and prostate tumors, among other types of cancer. They down-regulate gene expression in various cellular processes, including Sp1-responsive genes that control tumor development. Moreover, MTA and several mithralogs, such as EC-8042 (DIG-MSK) and EC-8105, effectively treat Ewing sarcoma by inhibiting transcription controlled by the oncogenic EWS-FLI1 transcription factor.
Assuntos
Neoplasias , Plicamicina , Humanos , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Plicamicina/uso terapêutico , Animais , Neoplasias/tratamento farmacológico , Neoplasias/genética , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Following acute human alphaherpesvirus 1 (HSV-1) infection of oral-facial mucosal surfaces, sensory neurons in trigeminal ganglia (TG) are important sites for life-long latency. Neurons in the central nervous system, including brainstem, also harbor viral genomes during latency. Periodically, certain cellular stressors trigger reactivation from latency, which can lead to recurrent HSV-1 disease: herpes labialis, herpes stromal keratitis, and encephalitis for example. Activation of the glucocorticoid receptor (GR) by stressful stimuli enhances HSV-1 gene expression, replication, and explant-induced reactivation. GR and certain stress-induced Krüppel like factors (KLF) cooperatively transactivate cis-regulatory modules (CRM) that drive expression of viral transcriptional regulatory proteins (ICP0, ICP4, and ICP27). These CRMs lack GR response elements (GRE); however, specificity protein 1 (Sp1) binding sites are crucial for GR and KLF15 or KLF4 mediated transactivation. Hence, we tested whether Sp1 or Sp3 regulate viral replication and transactivation of the ICP0 promoter. During early stages of explant-induced reactivation from latency, the number of Sp3+ TG neurons were significantly higher relative to TG from latently infected mice. Conversely, Sp1+ TG neurons were only increased in females, but not male mice, during explant-induced reactivation. Sp1 siRNA significantly reduced HSV-1 replication in cultured mouse (Neuro-2A) and monkey (CV-1) cells. Mithramycin A, an antibiotic that has anti-tumor activity preferentially interacts with GC-rich DNA, including Sp1 binding sites, significantly reduced HSV-1 replication indicating it has antiviral activity. GR and Sp1 or Sp3 transactivated the HSV-1 ICP0 promoter in Neuro-2A and CV-1 cells confirming these transcription factors enhance viral replication and gene expression.
Assuntos
Herpes Simples , Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Plicamicina/análogos & derivados , Feminino , Humanos , Camundongos , Animais , Herpesvirus Humano 1/genética , Receptores de Glucocorticoides/metabolismo , Ativação Viral , Latência Viral/genética , Proteínas Imediatamente Precoces/genética , Antibacterianos , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACKGROUND: Sarcomas comprise a group of aggressive malignancies with very little treatment options beyond standard chemotherapy. Reposition of approved drugs represents an attractive approach to identify effective therapeutic compounds. One example is mithramycin (MTM), a natural antibiotic which has demonstrated a strong antitumour activity in several tumour types, including sarcomas. However, its widespread use in the clinic was limited by its poor toxicity profile. RESULTS: In order to improve the therapeutic index of MTM, we have loaded MTM into newly developed nanocarrier formulations. First, polylactide (PLA) polymeric nanoparticles (NPs) were generated by nanoprecipitation. Also, liposomes (LIP) were prepared by ethanol injection and evaporation solvent method. Finally, MTM-loaded hydrogels (HG) were obtained by passive loading using a urea derivative non-peptidic hydrogelator. MTM-loaded NPs and LIP display optimal hydrodynamic radii between 80 and 105 nm with a very low polydispersity index (PdI) and encapsulation efficiencies (EE) of 92 and 30%, respectively. All formulations show a high stability and different release rates ranging from a fast release in HG (100% after 30 min) to more sustained release from NPs (100% after 24 h) and LIP (40% after 48 h). In vitro assays confirmed that all assayed MTM formulations retain the cytotoxic, anti-invasive and anti-stemness potential of free MTM in models of myxoid liposarcoma, undifferentiated pleomorphic sarcoma and chondrosarcoma. In addition, whole genome transcriptomic analysis evidenced the ability of MTM, both free and encapsulated, to act as a multi-repressor of several tumour-promoting pathways at once. Importantly, the treatment of mice bearing sarcoma xenografts showed that encapsulated MTM exhibited enhanced therapeutic effects and was better tolerated than free MTM. CONCLUSIONS: Overall, these novel formulations may represent an efficient and safer MTM-delivering alternative for sarcoma treatment.
Assuntos
Plicamicina/análogos & derivados , Plicamicina/farmacologia , Plicamicina/uso terapêutico , Sarcoma/patologia , Animais , Antibacterianos/uso terapêutico , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Condrossarcoma/tratamento farmacológico , Composição de Medicamentos , Feminino , Humanos , Hidrogéis/química , Hidrogéis/uso terapêutico , Lipossomos , Camundongos , Camundongos Nus , Nanopartículas/química , Nanopartículas/uso terapêutico , Poliésteres/química , Poliésteres/uso terapêutico , Sarcoma/tratamento farmacológicoRESUMO
The axis of Programmed cell death-1 receptor (PD-1) with its ligand (PD-L1) plays a critical role in colorectal cancer (CRC) in escaping immune surveillance, and blocking this axis has been found to be effective in a subset of patients. Although blocking PD-L1 has been shown to be effective in 5-10% of patients, the majority of the cohorts show resistance to this checkpoint blockade (CB) therapy. Multiple factors assist in the growth of resistance to CB, among which T cell exhaustion and immunosuppressive effects of immune cells in the tumor microenvironment (TME) play a critical role along with other tumor intrinsic factors. We have previously shown the polyketide antibiotic, Mithramycin-A (Mit-A), an effective agent in killing cancer stem cells (CSCs) in vitro and in vivo in a subcutaneous murine model. Since TME plays a pivotal role in CB therapy, we tested the immunomodulatory efficacy of Mit-A with anti-PD-L1 mAb (αPD-L1) combination therapy in an immunocompetent MC38 syngeneic orthotopic CRC mouse model. Tumors and spleens were analyzed by flow cytometry for the distinct immune cell populations affected by the treatment, in addition to RT-PCR for tumor samples. We demonstrated the combination treatment decreases tumor growth, thus increasing the effectiveness of the CB. Mit-A in the presence of αPD-L1 significantly increased CD8+ T cell infiltration and decreased immunosuppressive granulocytic myeloid-derived suppressor cells and anti-inflammatory macrophages in the TME. Our results revealed Mit-A in combination with αPD-L1 has the potential for augmented CB therapy by turning an immunologically "cold" into "hot" TME in CRC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Inibidores de Checkpoint Imunológico/farmacologia , Plicamicina/análogos & derivados , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Plicamicina/farmacologiaRESUMO
Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial-mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from -273 to -10 base pairs upstream of the transcription start site identified by 5'-RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB-downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression.
Assuntos
Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Sítios de Ligação , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Progressão da Doença , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HT29 , Humanos , Metástase Neoplásica , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb/genética , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica/genética , TransfecçãoRESUMO
Specific protein 1 (SP1) might act as a critical transcription regulator in myocardial infarction (MI), but little evidence about its function in regulating cardiac apoptosis, a major cause of MI development, has been revealed. This study tried to investigate the role of SP1 in MI and its interaction with poly-ADP-ribose polymerase (PARP)-1 by using SP1 inhibitor, mithramycin A (mithA). Primary mouse cardiomyocytes and commercial mouse cardiomyocytes were subjected to mithA treatment under hypoxia conditions, while cell viability, Nix promoter activity, and its expression were detected correspondingly. PARP overexpression and knockdown were conducted, respectively, in mithA-treated and SP1-overexpressing cells. Co-immunoprecipitation was used to verify the interaction between PARP and SP1. For in vivo experiments, mithA administration was performed after the injections of adenovirus for PARP overexpression, and then, MI introduction was carried out. Infarct size and lactate dehydrogenase level were measured to assess MI injury. SP1 inhibitor mithA attenuated hypoxia-induced decrease of cell viability and Nix transcriptional activation, which could be inhibited by PARP overexpression. Knockdown of PARP prevented SP1-induced transcription of Nix and cell viability change, and PARP showed direct interaction with SP1. Furthermore, mithA administration reduced MI injuries, while PARP overexpression could suppress the improvement. The cardioprotective role of SP1 inhibitor mithA was demonstrated here expanding the role of SP1 in MI development involving hypoxia-induced cardiac apoptosis. Moreover, PARP acted as a transcriptional coactivator in Nix transcription involving its interaction with SP1.
Assuntos
Cardiotônicos/farmacologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Plicamicina/análogos & derivados , Poli(ADP-Ribose) Polimerases/metabolismo , Fator de Transcrição Sp1/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Plicamicina/farmacologia , Ligação Proteica/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The oncogenic role of ErbB2 Receptor Tyrosine Kinase 2 (ERBB2) has been identified in several types of cancer, but less is known on its function and mechanism of action in cervical cancer cells. The present study employed a multipronged approach to investigate the role of ERBB2 in cervical cancer. ERBB2 and microRNA (miR)31845p expression was assessed in patientderived cervical cancer biopsy tissues, revealing that higher levels of ERBB2 and lower levels of miR31845p were associated with clinicopathological indicators of cervical cancer progression. Furthermore, ERBB2 stimulated proliferation, migration and sphereformation of cervical cancer cells in vitro. This effect was mediated by enhanced phosphatidylinositol4,5bisphosphate 3kinase catalytic subunit α activity. Additionally, it was revealed that miR31845p directly suppressed ERBB2 in cervical cancer cells. The p53 activator Mithramycin A stimulated p53 and miR31845p expression, thereby lowering the levels of ERBB2 and attenuating proliferation, migration and sphereformation of cervical cancer cells. In conclusion, the findings of the present study suggested ERBB2 as an oncogenic protein that may promote invasiveness in cervical cancer cells. Treatment of cervical cancer cells with the p53 activator Mithramycin A restored the levels of the endogenous ERBB2 inhibitor miR31845p and may represent a novel treatment strategy for cervical cancer.
Assuntos
MicroRNAs/metabolismo , Receptor ErbB-2/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/genética , Adulto , Idoso , Biópsia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Colo do Útero/patologia , Colo do Útero/cirurgia , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Histerectomia , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Proteína Supressora de Tumor p53/agonistas , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/cirurgiaRESUMO
PURPOSE: The oncogenic EWS:Fli1 fusion protein is a key transcriptional mediator of Ewing sarcoma initiation, progression, and therapeutic resistance. Mithramycin A (MithA) is a potent and specific inhibitor of transcription mediated by the EWS:Fli1. We tested the hypothesis that pretreatment with MithA could selectively radiosensitize EWS:Fli1+ tumor cells by altering the transcriptional response to radiation injury. METHODS AND MATERIALS: A panel of 4 EWS:Fli1+ and 3 EWS:Fli1- Ewing sarcoma cell lines and 1 nontumor cell line were subjected to MithA dose-response viability assays to determine the relative potency of MithA in cells possessing or lacking the EWS:Fli1 fusion. Radiosensitization by MithA was evaluated by clonogenic survival assays in vitro and in a murine xenograft model. DNA damage was evaluated by comet assay and γ-H2Ax flow cytometry. Immunoblotting, flow cytometry, and reverse-transcription, polymerase chain reaction were used to evaluate DNA damage-induced signaling and repair processes and apoptosis. RESULTS: We found that MithA alone could potently and selectively inhibit the growth of EWS:Fli1+ tumor cells, but not cells lacking this fusion. Pretreatment with MithA for 24 hours before irradiation significantly reduced clonogenic survival in vitro and delayed tumor regrowth in vivo, prolonging survival of EWS:Fli1+ tumor-bearing mice. Although MithA did not increase the level of DNA double-strand breaks, mechanistic studies revealed that MithA pretreatment selectively inhibited DNA double-strand break repair through downregulation of EWS:Fli1-mediated transcription, leading to tumor cell death by apoptosis. CONCLUSIONS: Our data indicate that MithA is an effective radiosensitizer of EWS:Fli1+ tumors and may achieve better local control at lower doses of radiation.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos dos fármacos , Proteínas de Fusão Oncogênica/metabolismo , Plicamicina/análogos & derivados , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Radiossensibilizantes/farmacologia , Sarcoma de Ewing/radioterapia , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Relação Dose-Resposta a Droga , Regulação para Baixo , Histonas/metabolismo , Camundongos , Plicamicina/farmacologia , Tolerância a Radiação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Ewing/química , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Prenyl diphosphate synthase subunit 2 (PDSS2) is the first key enzyme in the CoQ10 biosynthesis pathway, and contributes to various metabolic and nephritic diseases. It has been reported that PDSS2 is downregulated in several types of tumors and acts as a potential tumor suppressor gene to inhibit the proliferation and migration of cancer cells. However, the regulatory mechanism of PDSS2 expression remains elusive. In the present study, we first identified and characterized the PDSS2 promoter region. We established four different luciferase reporter constructs which mainly cover the 2 kb region upstream of the PDSS2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all four constructs have prominent promoter activity, and the core promoter of PDSS2 is mainly located within the 202 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the PDSS2 promoter contains binding sites for canonical transcription factors such as Sp1 and GATA-1. Overexpression of Sp1 significantly inhibited PDSS2 promoter activity, as well as its endogenous expression, at both mRNA and protein levels in lung cancer cells. Site-directed mutagenesis assay further confirmed that the Sp1 binding sites are essential for proximal prompter activity of PDSS2. Consistently, a selective Sp1 inhibitor, mithramycin A, treatment repressed the PDSS2 promoter activity, as well as its endogenous expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binds to the PDSS2 promoter in vivo. Of note, the expression of Sp1 and PDSS2 are negatively correlated, and higher Sp1 expression with low PDSS2 expression is significantly associated with poor prognosis in lung cancer. Taken together, our results strongly suggest the essential role of Sp1 in maintaining the basic constitutive expression of PDSS2, and the pathogenic implication of Sp1-mediated PDSS2 transcriptional repression in lung cancer cells.
Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Neoplasias Pulmonares/mortalidade , Fator de Transcrição Sp1/genética , Células A549 , Alquil e Aril Transferases/química , Alquil e Aril Transferases/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Prognóstico , Regiões Promotoras Genéticas/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , Análise de Sobrevida , Sítio de Iniciação de TranscriçãoRESUMO
B-cell receptor (BCR)-dependent signaling is central for leukemia B-cell homeostasis, as underscored by the promising clinical results obtained in patients with chronic lymphocytic leukemia (CLL) treated with novel agents targeting components of this pathway. Herein, we demonstrate that the mithralog EC-7072 displays high ex vivo cytotoxic activity against leukemia cells from CLL patients independently from high-risk prognostic markers and IGHV mutational status. EC-7072 was significantly less toxic against T cells and NK cells and did not alter the production of the immune effector molecules IFN-γ and perforin. EC-7072 directly triggered caspase-3-dependent CLL cell apoptosis, which was not abrogated by microenvironment-derived factors that sustain leukemia cell survival. RNA-sequencing analyses revealed a dramatic EC-7072-driven reprograming of the transcriptome of CLL cells, including a wide downregulation of multiple components and targets of the BCR signaling pathway. Accordingly, we found decreased levels of phosphorylated signaling nodes downstream of the BCR. Crosslinking-mediated BCR activation antagonized CLL cell death triggered by EC-7072, increased the phosphorylation levels of the abovementioned signaling nodes and upregulated BCL2 expression, suggesting that the mithralog disrupts CLL cell viability by targeting the BCR signaling axis at multiple levels. EC-7072 exerted similar or higher antileukemic activity than that of several available CLL therapies and displayed additive or synergistic interaction with these drugs in killing CLL cells. Overall, our findings provide rationale for future investigation to test whether EC-7072 may be a potential therapeutic option for patients with CLL and other B-cell malignancies.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Plicamicina/análogos & derivados , Receptores de Antígenos de Linfócitos B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Antibióticos Antineoplásicos/farmacologia , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Fosforilação/efeitos dos fármacos , Plicamicina/farmacologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacosRESUMO
The pivotal role of cancer initiating stem cells (CSCs) in tumor initiation, growth, metastasis and drug resistance has led to the postulation of a 'total cancer therapy' paradigm, which involves targeting both cancer cells and CSCs for effective therapy. However, the progress in identifying drugs for total cancer therapy has been limited. Herein, we show for the first time that mithramycin A (Mit-A) can successfully inhibit CSC proliferation, in addition to inhibiting bulk cancer cells in a model of colorectal cancer (CRC), the second leading cause of death among men and women in the United States. To this end, a polymeric nanofiber scaffold culture system was established to develop 3D tumor organoids (tumoroids) from CRC cell lines such as HT29, HCT116, KM12, CT26 and MC38 as well as ex vivo mouse tumors. These tumoroids possessed increased expression of CSC markers and transcription factors, expanded the number of CSCs in culture and increased CSC functional properties measured by aldehyde dehydrogenase activity. Screening of an NCI library of FDA approved drugs led to the identification of Mit-A as a potential total cancer therapy drug. In both sphere and tumoroid culture, Mit-A inhibits cancer growth by reducing the expression of cancer stemness markers. In addition, Mit-A inhibits the expression of SP1, a previously known target in CRCs. Moreover, Mit-A significantly reduces growth of tumoroids in ex vivo cultures and CRC tumor growth in vivo. Finally, a dose-dependent treatment on CRC cells indicate that Mit-A significantly induces the cell death and PARP-cleavage of both CSC and non-CSC cells. Taken together the results of these in vitro, ex vivo and in vivo studies lead to the inference that Mit-A is a promising drug candidate for total cancer therapy of CRCs.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Plicamicina/análogos & derivados , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Células HCT116 , Células HT29 , Humanos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/patologia , Plicamicina/farmacologia , Plicamicina/uso terapêuticoRESUMO
The restricted expression of Wilms tumor 1 (WT1) and cyclin A1 (CCNA1) in normal tissues, as opposed to their abnormal expression in leukemia demonstrates the applicability of WT1 and CCNA1 as cancer antigens for immunotherapy, and as markers for prognosis and relapse. In this study, the WT1 and CCNA1 mRNA levels were found to be elevated in bone marrow samples from pediatric acute promyelocytic leukemia (APL or AMLM3) patients, and to be quite varied in pediatric acute lymphocytic leukemia (ALL) patients, compared to nonleukemic bone marrow controls. Consistent with the observed upregulation of both WT1 and CCNA1 in APL, WT1 overexpression elevated the CCNA1 mRNA levels in K562 leukemia cells. Treatment with curcumin decreased the WT1 levels in K562 cells, and also decreased CCNA1 protein expression. The examination of the CCNA1 promoter identified potential canonical WT1 binding sites within the 3kb region upstream of the transcription start site. Chromatin immunoprecipitation and luciferase reporter assays confirmed WT1 binding and the activation of the CCNA1 promoter. Furthermore, the GCrich core CCNA1 promoter region provided additional noncanonical WT1 activation sites, as revealed by promoter assays. The importance of the GCrich core region of the CCNA1 promoter was confirmed by treating the K562 cells with mithramycin A, which blocks the binding of zinc finger transcription factors to GCrich sequences. Mithramycin A subsequently suppressed both CCNA1 promoter activity and protein expression in the K562 cells. Taken together, the data from the WT1 overexpression, and curcumin and mithramycin A treatment experiments, as well as those from chromatin binding assays, along with inferences from patient RNA analyses, establish a plausible link between WT1 and CCNA1, and support the functional significance of an elevated WT1 expression in leukemia, which may also affect CCNA1 expression.
Assuntos
Ciclina A1/genética , Leucemia Promielocítica Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas WT1/genética , Adolescente , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Criança , Pré-Escolar , Curcumina/farmacologia , Ciclina A1/química , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lactente , Células K562 , Leucemia Promielocítica Aguda/metabolismo , Masculino , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteínas WT1/química , Proteínas WT1/metabolismoRESUMO
BACKGROUND: The TMPRSS2-ERG gene fusion is the most frequent genetic rearrangement in prostate cancers and results in broad transcriptional reprogramming and major phenotypic changes. Interaction and cooperation of ERG and SP1 may be instrumental in sustaining the tumorigenic and metastatic phenotype and could represent a potential vulnerability in ERG fusion-positive tumors. OBJECTIVE: To test the activity of EC-8042, a compound able to block SP1, in cellular and mouse models of ERG-positive prostate cancer. DESIGN, SETTING, AND PARTICIPANTS: We evaluated the activity of EC-8042 in cell cultures and ERG/PTEN transgenic/knockout mice that provide reliable models for testing novel therapeutics in this specific disease context. Using a new protocol to generate tumor spheroids from ERG/PTEN mice, we also examined the effects of EC-8042 on tumor-propagating stem-like cancer cells with high self-renewal and tumorigenic capabilities. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The efficacy of EC-8042 was determined by measuring the proliferative capacity and target gene expression in cell cultures, invasive and metastatic capabilities in chick chorioallantoic membrane assays, and tumor development in mice. Significance was determined using statistical test. RESULTS AND LIMITATIONS: EC-8042 blocked transcription of ERG-regulated genes and reverted the invasive and metastatic phenotype of VCaP cells. EC-8042 blocked the expansion of stem-like tumor cells in tumor spheroids from VCaP cells and mouse-derived tumors. In ERG/PTEN mice, systemic treatment with EC-8042 inhibited ERG-regulated gene transcription, tumor progression, and tumor-propagating stem-like tumor cells. CONCLUSIONS: Our data support clinical testing of EC-8042 for the treatment of ERG-positive prostate cancer in precision medicine approaches. PATIENT SUMMARY: In this study, EC-8042, a novel compound with a favorable pharmacological and toxicological profile, exhibited relevant activity in cell cultures and in vivo in a genetically engineered mouse model that closely recapitulates the features of clinically aggressive ERG-positive prostate cancer. Our data indicate that further evaluation of EC-8042 in clinical trials is warranted.
Assuntos
Plicamicina/análogos & derivados , Neoplasias da Próstata/genética , Fator de Transcrição Sp1/antagonistas & inibidores , Regulador Transcricional ERG/genética , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos Transgênicos , Células-Tronco Neoplásicas , PTEN Fosfo-Hidrolase/genética , Plicamicina/farmacologia , Plicamicina/uso terapêutico , Neoplasias da Próstata/tratamento farmacológicoRESUMO
Mithramycin (MTM) has potent anticancer activity, but severe toxicities restrict its clinical use. Semi-synthetic approaches have yielded novel MTM analogs with potentially lower toxicity and similar efficacy. In an effort to transition these analogs into in vivo models, a bioanalytical method was developed for their quantification in mouse plasma. Here we present the validation of the method for the quantitation of mithramycin SA-tryptophan (MTMSA-Trp) as well as the applicability of the methodology for assaying additional analogs, including MTM, mithramycin SK (MTMSK) and mithramycin SA-phenylalanine (MTMSA-Phe) with run times of 6 min. Assay linearity ranged from 5 to 100 ng/mL. Accuracies of calibration standards and quality control samples were within 15% of nominal with precision variability of <20%. MTMSA-Trp was stable for 30 days at -80°C and for at least three freeze-thaw cycles. Methanol (-80°C) extraction afforded 92% of MTMSA-Trp from plasma. Calibration curves for MTM and analogs were also linear from ≤5 to 100 ng/mL. This versatile method was used to quantitate MTM analogs in plasma samples collected during preclinical pharmacokinetic studies.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Plicamicina/análogos & derivados , Plicamicina/sangue , Animais , Antibióticos Antineoplásicos , Estabilidade de Medicamentos , Feminino , Limite de Detecção , Modelos Lineares , Camundongos , Plicamicina/química , Plicamicina/farmacocinética , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: Melatonin, an endogenous neurohormone secreted predominately by the pineal gland, has a variety of physiological functions. However, its protective role in atherosclerosis is not clear. In this study, we sought to investigate the potential effects of melatonin in modulating atherosclerotic plaque stability in apolipoprotein E knockout (ApoE) mice. METHOD AND RESULTS: Smooth muscle cells were treated with melatonin, which significantly increased mRNA and protein levels of a key intracellular enzyme essential for collagen maturation and secretion, prolyl-4-hydroxylase α1 (P4Hα1). Mechanistically, melatonin increased Akt phosphorylation and transcriptional activation of specificity protein 1 (Sp1), which bound with the P4Hα1 promoter and then induced P4Hα1 expression. Pretreatment with either Akt inhibitor LY294002 or Sp1 inhibitor mithramycin A (MTM) could inhibit melatonin-induced P4Hα1 expression. Finally, atherosclerotic lesions were induced by placing a perivascular collar on the right common carotid artery of ApoE mice, which were received with or without different doses of melatonin or MTM. High-dose melatonin enhanced atherosclerotic plaque stability in ApoE mice in vivo by inducing the expression of P4Hα1, which was reversed by MTM. CONCLUSION: We propose that melatonin supplementation may provide a novel and promising approach to atherosclerosis treatment.
Assuntos
Apolipoproteínas E/genética , Depressores do Sistema Nervoso Central/farmacologia , Expressão Gênica/efeitos dos fármacos , Melatonina/farmacologia , Miócitos de Músculo Liso/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Animais , Linhagem Celular , Depressores do Sistema Nervoso Central/administração & dosagem , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Masculino , Melatonina/administração & dosagem , Camundongos , Camundongos Knockout , Morfolinas/farmacologia , Fosforilação/efeitos dos fármacos , Placa Aterosclerótica/tratamento farmacológico , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genéticaRESUMO
BACKGROUND: To deliver efficacious personalised cancer treatment, it is essential to characterise the cellular metabolism as well as the genetic stability of individual tumours. In this study, we describe a new axis between DNA repair and detoxification of aldehyde derivatives with important implications for patient prognosis and treatment. METHODS: Western blot and qPCR analyses were performed in relevant non-transformed and cancer cell lines from lung and liver tissue origin in combination with bioinformatics data mining of The Cancer Genome Atlas database from lung and hepatocellular cancer patients. RESULTS: Using both biochemical and bioinformatics approaches, we revealed an association between the levels of expression of the aldehyde detoxifying enzyme aldehyde dehydrogenase 2 (ALDH2) and the key DNA base excision repair protein XRCC1. Across cancer types, we found that if one of the corresponding genes exhibits a low expression level, the level of the other gene is increased. Surprisingly, we found that low ALDH2 expression levels associated with high XRCC1 expression levels are indicative for a poor overall survival, particularly in lung and liver cancer patients. In addition, we found that Mithramycin A, a XRCC1 expression inhibitor, efficiently kills cancer cells expressing low levels of ALDH2. CONCLUSIONS: Our data suggest that lung and liver cancers require efficient single-strand break repair for their growth in order to benefit from a low aldehyde detoxification metabolism. We also propose that the ratio of XRCC1 and ALDH2 levels may serve as a useful prognostic tool in these cancer types.
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Aldeído-Desidrogenase Mitocondrial/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Aldeído-Desidrogenase Mitocondrial/genética , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/genética , Dano ao DNA/fisiologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/genética , Plicamicina/análogos & derivados , Plicamicina/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/antagonistas & inibidores , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genéticaRESUMO
Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS transcription factor EWS-FLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic window, compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to generate analogues via peptide coupling and fragment-based drug development strategies. Cytotoxicity assays in ETS and non-ETS dependent cell lines identified two dipeptide analogues, 60 and 61, with 19.1- and 15.6-fold selectivity, respectively, compared to 1.5-fold for 1. Importantly, the cytotoxicity of 60 and 61 is <100 nM in ETS cells. Molecular assays demonstrated the inhibitory capacity of these analogues against EWS-FLI1 mediated transcription in Ewing sarcoma. Structural analysis shows that positioning the tryptophan residue in a distal position improves selectivity, presumably via interaction with the ETS transcription factor. Thus, these analogues may present new ways to target transcription factors for clinical use.
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Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Desenvolvimento de Medicamentos , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Plicamicina/análogos & derivados , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ets/antagonistas & inibidores , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Sarcoma de Ewing/tratamento farmacológico , Antibióticos Antineoplásicos/química , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Estrutura Molecular , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Células Tumorais CultivadasRESUMO
As the most malignant breast cancer subtype, triple-negative breast cancer (TNBC) does not have effective targeted therapies clinically to date. As a selective Sp1 inhibitor, Mithramycin A (MIT) has been reported to have anti-tumor activities in multiple cancers. However, the efficacy and the mechanism of MIT in breast cancer, especially TNBC, have not been studied. In this study, we demonstrated that MIT suppressed breast cancer cell survival in a dosage-dependent manner. Interestingly, TNBC cells were more sensitive to MIT than non-TNBC cells. MIT inhibited TNBC cell proliferation and promoted apoptosis in vitro in time- and dosage-dependent manners. MIT suppressed TNBC cell survival, at least partially, by transcriptionally down-regulating KLF5, an oncogenic transcription factor specifically expressed in basal TNBC. Finally, MIT suppressed TNBC cell growth in a xenograft mouse model. Taken together, our findings suggested that MIT inhibits basal TNBC via the Sp1/KLF5 axis and that MIT may be used for TNBC treatment.
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Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/genética , Plicamicina/análogos & derivados , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Plicamicina/farmacologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget's disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.
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Plicamicina/análogos & derivados , Policetídeos/metabolismo , Streptomyces lividans/metabolismo , Streptomyces/enzimologia , Biocatálise , Vias Biossintéticas , Clonagem Molecular , Fermentação , Família Multigênica , Plicamicina/biossíntese , Saccharomyces cerevisiae , Metabolismo Secundário , Streptomyces/genética , Streptomyces lividans/genéticaRESUMO
PURPOSE: Specificity protein 1 (SP1) is involved in the transcription of several genes implicated in tumor maintenance. We investigated the effects of mithramycin A (MTA), an inhibitor of SP1 DNA binding, on radiation response. METHODS AND MATERIALS: Clonogenic survival after irradiation was assessed in 2 tumor cell lines (A549, UM-UC-3) and 1 human fibroblast line (BJ) after SP1 knockdown or MTA treatment. DNA damage repair was evaluated using γH2AX foci formation, and mitotic catastrophe was assessed using nuclear morphology. Gene expression was evaluated using polymerase chain reaction arrays. In vivo tumor growth delay was used to evaluate the effects of MTA on radiosensitivity. RESULTS: Targeting of SP1 with small interfering RNA or MTA sensitized A549 and UM-UC-3 to irradiation, with no effect on the BJ radiation response. MTA did not alter γH2AX foci formation after irradiation in tumor cells but did enhance mitotic catastrophe. Treatment with MTA suppressed transcription of genes involved in cell death. MTA administration to mice bearing A549 and UM-UC-3 xenografts enhanced radiation-induced tumor growth delay. CONCLUSIONS: These results support SP1 as a target for radiation sensitization and confirm MTA as a radiation sensitizer in human tumor models.