RESUMO
Four new echinomycin congeners, quinomycins M-P (1-4) were isolated from the cultures of the soil-derived Streptomyces sp. CPCC205575. The planar structures were determined by comprehensive analyses of NMR and HRESIMS/MS data. The absolute configurations were elucidated by the advanced Marfey's method combined with biosynthetic gene analysis. Compounds 1-4 represent the first examples of quinomycin-type natural products with the sulfur atom at the N,S-dimethylcysteine residue oxidized as a sulfoxide group forming the unusual N-methyl-3-methylsulfinyl-alanine residue. Bioassay results revealed that the oxidation of the sulfur atom at the Cys or Cys' residues led to dramatic decrease of cytotoxicity and antimicrobial activity.
Assuntos
Streptomyces , Streptomyces/metabolismo , Streptomyces/química , Humanos , Testes de Sensibilidade Microbiana , Equinomicina/farmacologia , Equinomicina/química , Equinomicina/análogos & derivados , Espectroscopia de Ressonância Magnética , Microbiologia do Solo , Alanina/análogos & derivados , Alanina/química , Linhagem Celular Tumoral , Estrutura Molecular , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificaçãoRESUMO
Colorectal cancer is the third most commonly diagnosed cancer and the second leading cause of cancer-related death, thus a novel chemotherapeutic agent for colon cancer therapy is needed. In this study, analogues of echinomycin, a cyclic peptide natural product with potent toxicity to several human cancer cell lines, were synthesized, and their biological activities against human colon cancer cells were investigated. Analogue 3 as well as 1 inhibit HIF-1α-mediated transcription. Notably, transcriptome analysis indicated that the cell cycle and its regulation were involved in the effects on cells treated with 3. Analogue 3 exhibited superior in vivo efficacy to echinomycin without significant toxicity in mouse xenograft model. The low dose of 3 needed to be efficacious in vivo is also noteworthy and our data suggest that 3 is an attractive and potentially novel agent for the treatment of colon cancer.
Assuntos
Neoplasias do Colo , Equinomicina , Humanos , Animais , Camundongos , Equinomicina/farmacologia , Neoplasias do Colo/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por HipóxiaRESUMO
Current data suggest a hypothesis of vascular pathogenesis for the development and progression of Alzheimer's disease (AD). To investigate this, we studied the association of apolipoprotein E4 (APOE4) gene on microvessels in human autopsy-confirmed AD with and without APOE4, compared with age/sex-matched control (AC) hippocampal CA1 stratum radiatum. AD arterioles (without APOE4 gene) had mild oxidative stress and loss of vascular endothelial growth factor (VEGF) and endothelial cell density, reflecting aging progression. In AD + APOE4, an increase in strong oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG), VEGF, and endothelial cell density were associated with increased diameter of arterioles and perivascular space dilation. In cultured human brain microvascular cells (HBMECs), treatment of ApoE4 protein plus amyloid-ß (Aß) oligomers increased superoxide production and the apoptotic marker cleaved caspase 3, sustained hypoxia inducible factor-1α (HIF-1α) stability that was associated with an increase in MnSOD, VEGF, and cell density. This cell over-proliferation was inhibited with the antioxidants N-acetyl cysteine and MnTMPyP, the HIF-1α inhibitor echinomycin, the VEGFR-2 receptor blocker SU1498, the protein kinase C (PKC) ε knock-down (KD) and the extracellular signal-regulated kinase 1/2 (ERK) inhibitor FR180204. The PKCε KD and echinomycin decreased VEGF and/or ERK. In conclusion, AD capillaries and arterioles in hippocampal CA1 stratum radiatum of non-APOE4 carriers are related with aging, while those in APOE4 carriers with AD are related with pathogenesis of cerebrovascular disease.
Assuntos
Doença de Alzheimer , Equinomicina , Humanos , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Apolipoproteínas E , Região CA1 Hipocampal/patologia , Equinomicina/metabolismo , Hipocampo/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Echinomycin is a natural product DNA bisintercalator antibiotic. The echinomycin biosynthetic gene cluster in Streptomyces lasalocidi includes a gene encoding the self-resistance protein Ecm16. Here, we present the 2.0 Å resolution crystal structure of Ecm16 bound to adenosine diphosphate. The structure of Ecm16 closely resembles that of UvrA, the DNA damage sensor component of the prokaryotic nucleotide excision repair system, but Ecm16 lacks the UvrB-binding domain and its associated zinc-binding module found in UvrA. Mutagenesis study revealed that the insertion domain of Ecm16 is required for DNA binding. Furthermore, the specific amino acid sequence of the insertion domain allows Ecm16 to distinguish echinomycin-bound DNA from normal DNA and link substrate binding to ATP hydrolysis activity. Expression of ecm16 in the heterologous host Brevibacillus choshinensis conferred resistance against echinomycin and other quinomycin antibiotics, including thiocoraline, quinaldopeptin, and sandramycin. Our study provides new insight into how the producers of DNA bisintercalator antibiotics fend off the toxic compounds that they produce.
Assuntos
Equinomicina , Streptomyces , Equinomicina/farmacologia , Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Antibacterianos/química , Streptomyces/genética , Streptomyces/metabolismoRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several human cancers, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), which preferentially arise in immunocompromised patients while lack of effective therapeutic options. Oncoproteins Myc and hypoxia-inducible factor-1α (HIF1α) have been found closely related to KSHV infection, replication and oncogenesis. However, the strategies of dual targeting these two oncoproteins have never been developed and tested for treatments of KSHV-related malignancies. In the current study, we report that treatment of echinomycin dramatically regresses cell growth both in vitro-cultured KSHV + tumor cells and in vivo KS or PEL xenograft mice models, through simultaneously inhibiting Myc and HIF1α expression. Echinomycin treatment also induces viral lytic gene expression whereas not increasing infectious virions production from KSHV + tumor cells. Our comparative transcriptomic analysis has identified a bunch of new Echinomycin-regulated, Myc- and HIF1α-related genes contributed to KSHV pathogenesis, including KDM4B and Tau, which are required for the survival of KSHV + tumor cells with functional validation. These data together reveal that dual targeting Myc and HIF1α such as using Echinomycin may represent a new and promising option for treatments of these virus-associated malignancies.
Assuntos
Equinomicina , Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Animais , Camundongos , Herpesvirus Humano 8/genética , Equinomicina/farmacologia , Equinomicina/uso terapêutico , Latência Viral/genética , Sarcoma de Kaposi/tratamento farmacológico , Sarcoma de Kaposi/metabolismo , Ciclo Celular , Histona Desmetilases com o Domínio JumonjiRESUMO
Combination cancer chemotherapy is one of the most useful treatment methods to achieve a synergistic effect and reduce the toxicity of dosing with a single drug. Here, we use a combination of two well-established anticancer DNA intercalators, actinomycin D (ActD) and echinomycin (Echi), to screen their binding capabilities with DNA duplexes containing different mismatches embedded within Watson-Crick base-pairs. We have found that combining ActD and Echi preferentially stabilised thymine-related T:T mismatches. The enhanced stability of the DNA duplex-drug complexes is mainly due to the cooperative binding of the two drugs to the mismatch duplex, with many stacking interactions between the two different drug molecules. Since the repair of thymine-related mismatches is less efficient in mismatch repair (MMR)-deficient cancer cells, we have also demonstrated that the combination of ActD and Echi exhibits enhanced synergistic effects against MMR-deficient HCT116 cells and synergy is maintained in a MMR-related MLH1 gene knockdown in SW620 cells. We further accessed the clinical potential of the two-drug combination approach with a xenograft mouse model of a colorectal MMR-deficient cancer, which has resulted in a significant synergistic anti-tumour effect. The current study provides a novel approach for the development of combination chemotherapy for the treatment of cancers related to DNA-mismatches.
Assuntos
Neoplasias Colorretais , Equinomicina , Humanos , Animais , Camundongos , Dactinomicina/química , Equinomicina/química , Timina , Sequência de Bases , Sítios de Ligação , Conformação de Ácido Nucleico , DNA/químicaRESUMO
Mangrove actinomycetia have been proven to be one of the promising sources for discovering novel bioactive natural products. Quinomycins K (1) and L (2), two rare quinomycin-type octadepsipeptides without intra-peptide disulfide or thioacetal bridges, were investigated from the Maowei Sea mangrove-derived Streptomyces sp. B475. Their chemical structures, including the absolute configurations of their amino acids, were elucidated by a combination of NMR and tandem MS analysis, electronic circular dichroism (ECD) calculation, advanced Marfey's method, and further unequivocally confirmed by the first total synthesis. The two compounds displayed no potent antibacterial activity against 37 bacterial pathogens and had no significant cytotoxic activity against H460 lung cancer cells.
Assuntos
Equinomicina , Streptomyces , Streptomyces/metabolismo , Equinomicina/metabolismo , Antibacterianos/química , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
Chronic myeloid leukemia (CML) has a markedly improved prognosis with the use of breakpoint cluster region-abelson 1 (BCR-ABL1) tyrosine kinase inhibitors (BCR-ABL1 TKIs). However, approximately 40% of patients are resistant or intolerant to BCR-ABL1 TKIs. Hypoxia-inducible factor 1α (HIF-1α) is a hypoxia response factor that has been reported to be highly expressed in CML patients, making it a therapeutic target for BCR-ABL1 TKI-sensitive CML and BCR-ABL1 TKI-resistant CML. In this study, we examined whether HIF-1α inhibitors induce cell death in CML cells and BCR-ABL1 TKI-resistant CML cells. We found that echinomycin and PX-478 induced cell death in BCR-ABL1 TKIs sensitive and resistant CML cells at similar concentrations while the cell sensitivity was not affected with imatinib or dasatinib in BCR-ABL1 TKIs resistant CML cells. In addition, echinomycin and PX-478 inhibited the c-Jun N-terminal kinase (JNK), Akt, and extracellular-regulated protein kinase 1/2 (ERK1/2) activation via suppression of BCR-ABL1 and Met expression in BCR-ABL1 sensitive and resistant CML cells. Moreover, treatment with HIF-1α siRNA induced cell death by inhibiting BCR-ABL1 and Met expression and activation of JNK, Akt, and ERK1/2 in BCR-ABL1 TKIs sensitive and resistant CML cells. These results indicated that HIF-1α regulates BCR-ABL and Met expression and is involved in cell survival in CML cells, suggesting that HIF-1α inhibitors induce cell death in BCR-ABL1 TKIs sensitive and resistant CML cells and therefore HIF-1α inhibitors are potential candidates for CML treatment. [BMB Reports 2023; 56(2): 78-83].
Assuntos
Equinomicina , Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Equinomicina/uso terapêutico , Proteínas Proto-Oncogênicas c-akt , Resistencia a Medicamentos Antineoplásicos , Apoptose , Inibidores de Proteínas Quinases/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Morte CelularRESUMO
Objective: Sepsis Associated Encephalopathy (SAE) is a common complication in critically ill patients and perioperative period, but its pathogenesis is still unclear. This study aimed to explore the effect of the HIF-1α (hypoxia-inducible factor-1α)/BNIP3L (Bcl-2/adenovirus E1B 19-kDa interaction protein) signaling pathway on SAE. Methods: C57BL/6J male mice were divided into four groups, using a random number table method: control group, sham group, sepsis group, sepsis+HIF-1α activity inhibitor (echinomycin) group. Sepsis was induced by cecal ligation and puncture (CLP). At 24 h after surgery, brain tissue was sampled. HE was staining to observe changes in the hippocampus structure. Fluoroscopy observes changes in mitochondrial structure. Western blot, QT-PCR, and immunofluorescence were used to assess the amount of expression of HIF-1α and BNIP3L in the hippocampus and mitochondrion of hippocampus neurons. Observation of neuronal apoptosis by TUNEL staining. Seven days after surgery, mice were tested in a Morris water maze test to assess cognitive function after CLP. Results: Our results show that CLP-induced hippocampus-dependent cognitive deficits were accompanied with increased HIF 1a and decreased BNIP3L, increased protein levels of TNF-α, IL-6, and IL-ß, and damage to mitochondrial structures and neuronal apoptosis in the hippocampus. In addition, administration of echinomycin rescues cognitive deficits, ameliorates HIF-1α and BNIP3L-mediated neuronal pyroptosis and damaged mitochondrial structures, and decreases the expression of TNF-α and IL-6 in the hippocampus. Conclusions: HIF-1α and the BNIP3L promote mitochondrial damage, and neuronal apoptosis and the expression of inflammatory factors may be the mechanism of SAE in critically ill patients and perioperative period.
Assuntos
Equinomicina , Encefalopatia Associada a Sepse , Sepse , Camundongos , Masculino , Animais , Fator de Necrose Tumoral alfa , Estado Terminal , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Interleucina-6 , Camundongos Endogâmicos C57BL , Sepse/complicações , CogniçãoRESUMO
OBJECTIVE: To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms. METHODS: EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, ß-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment. RESULTS: The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of ß-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, ß-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05). CONCLUSION: Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/ß-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.
Assuntos
Equinomicina , Células-Tronco Pluripotentes Induzidas , Equinomicina/metabolismo , Corpos Embrioides/metabolismo , Humanos , Hipóxia/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , beta Catenina/metabolismoRESUMO
Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic-producing bacteria. In this work, we characterize the self-resistance protein Ecm16 encoded by echinomycin-producing bacteria. Ecm16 is a structural homolog of the nucleotide excision repair protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 binds DNA (double-stranded and single-stranded) using a nucleotide-independent binding mode. Ecm16's binding affinity for DNA increased by 1.7-fold when the DNA is intercalated with echinomycin. Ecm16 can render resistance against echinomycin toxicity independently of the nucleotide excision repair system. Similar to UvrA, Ecm16 has ATPase activity, and this activity is essential for Ecm16's ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each other's function. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin.
Assuntos
Equinomicina , Proteínas de Escherichia coli , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Equinomicina/química , Equinomicina/metabolismo , Equinomicina/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
OBJECTIVE: To investigate the antitumor effects of the selective hypoxia-inducible factor-1 (HIF-1) inhibitors echinomycin and PX-478 on uterine fibroids. DESIGN: Experimental study using in vitro primary culture systems and an in vivo mouse xenograft model. SETTING: Academic university center. PATIENT(S): Women with uterine fibroids who underwent hysterectomy or myomectomy. INTERVENTION(S): Administration of the selective HIF-1 inhibitors echinomycin and PX-478 to the media of the primary cultured uterine fibroid cells and to nonobese diabetic/severe combined immunodeficient mice bearing fibroid xenografts consisting of the primary cultured fibroid cells and type â collagen gels beneath the kidney capsule. MAIN OUTCOME MEASURE(S): Cell proliferation was measured by Cell Counting Kit-8 assay. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and by measuring caspase 3 and 7 activities. The xenografts were evaluated by gross appearance, surface area, and histology. The Ki-67 index was measured to evaluate proliferation of the xenografts. RESULT(S): Both echinomycin and PX-478 inhibited cell proliferation and induced apoptosis in fibroid cells cultured under hypoxia and normoxia. Enlargement of the fibroid xenografts was significantly attenuated. The Ki-67 index significantly decreased after the administration of the HIF-1 inhibitors in the xenograft model. Eight of 27 xenografts treated with the HIF-1 inhibitors contained calcification and hyalinizing components from 3 days after the grafting to 2 weeks, suggesting that the HIF-inhibitors induce degeneration of the fibroid xenografts. CONCLUSION(S): The selective HIF-1 inhibitors echinomycin and PX-478 show antitumor effects against uterine fibroids both in vitro and in vivo. These findings support the potential use of HIF-1 inhibitors for the treatment of uterine fibroids.
Assuntos
Equinomicina , Leiomioma , Animais , Equinomicina/farmacologia , Feminino , Humanos , Hipóxia , Fator 1 Induzível por Hipóxia , Antígeno Ki-67 , Leiomioma/tratamento farmacológico , Camundongos , Compostos de Mostarda , FenilpropionatosRESUMO
A combination of anti-CTLA-4 plus anti-PD-1/PD-L1 is the most effective cancer immunotherapy but causes high incidence of immune-related adverse events (irAEs). Here we report that targeting of HIF-1α suppressed PD-L1 expression on tumor cells and tumor-infiltrating myeloid cells, but unexpectedly induced PD-L1 in normal tissues by an IFN-γ-dependent mechanism. Targeting the HIF-1α/PD-L1 axis in tumor cells reactivated tumor-infiltrating lymphocytes and caused tumor rejection. The HIF-1α inhibitor echinomycin potentiated the cancer immunotherapeutic effects of anti-CTLA-4 therapy, with efficacy comparable to that of anti-CTLA-4 plus anti-PD-1 antibodies. However, while anti-PD-1 exacerbated irAEs triggered by ipilimumab, echinomycin protected mice against irAEs by increasing PD-L1 levels in normal tissues. Our data suggest that targeting HIF-1α fortifies the immune tolerance function of the PD-1/PD-L1 checkpoint in normal tissues but abrogates its immune evasion function in the tumor microenvironment to achieve safer and more effective immunotherapy.
Assuntos
Equinomicina , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias , Animais , Antígeno B7-H1 , Equinomicina/farmacologia , Evasão da Resposta Imune , Tolerância Imunológica , Linfócitos do Interstício Tumoral , Camundongos , Neoplasias/terapia , Microambiente TumoralRESUMO
Oncogenic Ras mutations are highly prevalent in hematopoietic malignancies. However, it is difficult to directly target oncogenic RAS proteins for therapeutic intervention. We have developed a Drosophila acute myeloid leukemia model induced by human KRASG12V, which exhibits a dramatic increase in myeloid-like leukemia cells. We performed both genetic and drug screens using this model. The genetic screen identified 24 candidate genes able to attenuate the oncogenic RAS-induced phenotype, including two key hypoxia pathway genes HIF1A and ARNT (HIF1B). The drug screen revealed that echinomycin, an inhibitor of HIF1A, can effectively attenuate the leukemia phenotype caused by KRASG12V. Furthermore, we showed that echinomycin treatment can effectively suppress oncogenic RAS-driven leukemia cell proliferation, using both human leukemia cell lines and a mouse xenograft model. These data suggest that inhibiting the hypoxia pathway could be an effective treatment approach and that echinomycin is a promising targeted drug to attenuate oncogenic RAS-induced cancer phenotypes. This article has an associated First Person interview with the first author of the paper.
Assuntos
Equinomicina , Leucemia Mieloide Aguda , Animais , Equinomicina/farmacologia , Equinomicina/uso terapêutico , Genes ras , Humanos , Hipóxia/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Camundongos , FenótipoRESUMO
BACKGROUND: Glioblastoma multiforme (GBM), a lethal brain tumor, remains the most daunting challenge in cancer therapy. Overexpression and constitutive activation of PDGFs and PDGFRα are observed in most GBM; however, available inhibitors targeting isolated signaling pathways are minimally effective. Therefore, better understanding of crucial mechanisms underlying GBM is needed for developing more effective targeted therapies. METHODS: Target genes controlled by HIF1α in GBM were identified by analysis of TCGA database and by RNA-sequencing of GBM cells with HIF1α knockout by sgRNA-Cas9 method. Functional roles of HIF1α, PDGFs and PDGFRs were elucidated by loss- or gain-of-function assays or chemical inhibitors, and compared in response to oxygen tension. Pharmacological efficacy and gene expression in mice with intracranial xenografts of primary GBM were analyzed by bioluminescence imaging and immunofluorescence. RESULTS: HIF1α binds the PDGFD proximal promoter and PDGFRA intron enhancers in GBM cells under normoxia or mild-hypoxia to induce their expression and maintain constitutive activation of AKT signaling, which in turn increases HIF1α protein level and activity. Paradoxically, severe hypoxia abrogates PDGFRα expression despite enhancing HIF1α accumulation and corresponding PDGF-D expression. Knockout of HIF1A, PDGFD or PDGFRA in U251 cells inhibits cell growth and invasion in vitro and eradicates tumor growth in vivo. HIF1A knockdown in primary GBM extends survival of xenograft mice, whereas PDGFD overexpression in GL261 shortens survival. HIF1α inhibitor Echinomycin induces GBM cell apoptosis and effectively inhibits growth of GBM in vivo by simultaneously targeting HIF1α-PDGFD/PDGFRα-AKT feedforward pathway. CONCLUSIONS: HIF1α orchestrates expression of PDGF-D and PDGFRα for constitutive activation of AKT pathway and is crucial for GBM malignancy. Therefore, therapies targeting HIF1α should provide an effective treatment for GBM.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/patologia , Equinomicina/uso terapêutico , Glioblastoma/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Linfocinas/metabolismo , Oxigênio/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ativação Enzimática , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Linfocinas/genética , Camundongos , Camundongos Endogâmicos NOD , Invasividade Neoplásica , Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genéticaRESUMO
Polycystic kidney disease (PKD) is a genetic disorder characterized by aberrant renal epithelial cell proliferation and formation and progressive growth of numerous fluid-filled cysts within the kidneys. Previously, we showed that there is elevated Notch signaling compared to normal renal epithelial cells and that Notch signaling contributes to the proliferation of cystic cells. Quinomycin A, a bis-intercalator peptide, has previously been shown to target the Notch signaling pathway and inhibit tumor growth in cancer. Here, we show that Quinomycin A decreased cell proliferation and cyst growth of human ADPKD cyst epithelial cells cultured within a 3D collagen gel. Treatment with Quinomycin A reduced kidney weight to body weight ratio and decreased renal cystic area and fibrosis in Pkd1RC/RC ; Pkd2+/- mice, an orthologous PKD mouse model. This was accompanied by reduced expression of Notch pathway proteins, RBPjk and HeyL and cell proliferation in kidneys of PKD mice. Quinomycin A treatments also normalized cilia length of cyst epithelial cells derived from the collecting ducts. This is the first study to demonstrate that Quinomycin A effectively inhibits PKD progression and suggests that Quinomycin A has potential therapeutic value for PKD patients.
Assuntos
Antibacterianos/farmacologia , Cistos/tratamento farmacológico , Modelos Animais de Doenças , Equinomicina/farmacologia , Doenças Renais Policísticas/complicações , Canais de Cátion TRPP/fisiologia , Animais , Cistos/etiologia , Cistos/metabolismo , Cistos/patologia , Progressão da Doença , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND: Echinomycin (EKN), an inhibitor of hypoxia-inducible factor (HIF)-1 DNA-binding activity, has been implied as a possible therapeutic agent in ischemic diseases. Here, we assess EKN in hypoxia-driven responses in vitro using human primary adult retinal pigment epithelium cells (aRPE) and retinal endothelial cells (hREC), and in vivo using the laser-induced mouse choroidal neovascularization (CNV) model. METHODS: Effects of EKN on hypoxia-mediated pathways in aRPE were analyzed by Western blotting for HIF-1α protein, quantitative PCR of HIF-target genes, and proteome array for soluble angiogenic factors. In vitro inhibition of angiogenesis by EKN was determined in hREC. In vivo inhibition of angiogenesis by EKN was determined in the mouse laser-induced CNV, as a model of HIF-associated ocular neovascularization. CNV lesion area was determined by fundus fluorescein angiography. RESULTS: aRPE treated with EKN showed hypoxia-dependent significantly decreased cell recovery in the wound healing assay. These results were supported by lower levels of HIF-mediated transcripts detected in hypoxic aRPE cells treated with EKN compared with non-treated controls, and confirmed by proteome profiler for angiogenic factors. hREC exposed to aRPE EKN-conditioned medium displayed reduced sprouting angiogenesis. Mice with laser-induced CNV treated with intravitreally injected EKN showed significantly decreased vascular lesion area when compared with a mouse equivalent of aflibercept, or vehicle-treated controls. CONCLUSIONS: Our data proposes EKN as a potent inhibitor of HIF-mediated angiogenesis in retinal cells and in the mouse model of CNV, which could have future implications in the treatment of patients with neovascular age-related macular degeneration.
Assuntos
Neovascularização de Coroide/tratamento farmacológico , Equinomicina/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Adulto , Células Cultivadas , Neovascularização de Coroide/metabolismo , Angiofluoresceinografia , Fundo de Olho , Humanos , Epitélio Pigmentado da Retina/patologia , Transdução de SinaisRESUMO
Nonribosomal peptides (NRPs) are natural products that are biosynthesized by large multi-enzyme assembly lines called nonribosomal peptide synthetases (NRPSs). We have previously discovered that backbone or side chain methylation of NRP residues is carried out by an interrupted adenylation (A) domain that contains an internal methyltransferase (M) domain, while maintaining a monolithic AMA fold of the bifunctional enzyme. A key question that has remained unanswered is at which step of the assembly line mechanism the methylation by these embedded M domains takes place. Does the M domain methylate an amino acid residue tethered to a thiolation (T) domain on same NRPS module (in cis), or does it methylate this residue on a nascent peptide tethered to a T domain on another module (in trans)? In this study, we investigated the kinetics of methylation by wild-type AMAT tridomains from two NRPSs involved in biosynthesis of anticancer depsipeptides thiocoraline and echinomycin, and by mutants of these domains, for which methylation can occur only in trans. The analysis of the methylation kinetics unequivocally demonstrated that the wild-type AMATs methylate overwhelmingly in cis, strongly suggesting that this is also the case in the context of the entire NRPS assembly line process. The mechanistic insight gained in this study will facilitate rational genetic engineering of NRPS to generate unnaturally methylated NRPs.
Assuntos
Depsipeptídeos/metabolismo , Equinomicina/metabolismo , Metiltransferases/metabolismo , Microsporídios/enzimologia , Peptídeo Sintases/metabolismo , Streptomyces/enzimologia , Monofosfato de Adenosina/metabolismo , Depsipeptídeos/química , Equinomicina/química , Cinética , Metilação , Metiltransferases/química , Microsporídios/metabolismo , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeo Sintases/química , Domínios Proteicos , Streptomyces/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Hypoxia-inducible factor 1α (HIF-1α) is recognized as a prime molecular target for metastatic cancer. However, no specific HIF-1α inhibitor has been approved for clinical use. Here, we demonstrated that in vivo efficacy of echinomycin in solid tumors with HIF-1α overexpression is formulation-dependent. Compared to previously-used Cremophor-formulated echinomycin, which was toxic and ineffective in clinical trials, liposomal-echinomycin provides significantly more inhibition of primary tumor growth and only liposome-formulated echinomycin can eliminate established triple-negative breast cancer (TNBC) metastases, which are the leading cause of death from breast cancer, as available therapies remain minimally effective at this stage. Pharmacodynamic analyses reveal liposomal-echinomycin more potently inhibits HIF-1α transcriptional activity in primary and metastasized TNBC cells in vivo, the latter of which are HIF-1α enriched. The data suggest that nanoliposomal-echinomycin can provide safe and effective therapeutic HIF-1α inhibition and could represent the most potent HIF-1α inhibitor in prospective trials for metastatic cancer.
Assuntos
Equinomicina/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Lipossomos/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Equinomicina/química , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Lipossomos/química , Camundongos , Metástase Neoplásica , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Systemic chemotherapy is often the last resort of advanced cutaneous T-cell lymphoma (CTCL). Tumor recurrence and adverse effects of systemic chemotherapy are the main limitations. OBJECTIVE: We aim to investigate the metabolic alterations in tumor cells after CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin and prednisone) chemotherapy. METHODS AND RESULTS: In advanced CTCL, CHOP chemotherapy has no survival benefit and the duration of response is significantly inferior to other canonical treatments. HIF-1α is significantly elevated in lesions of advanced MF patients as well as tumor cell line Hut78 and tumor xenograft mice model. CHOP therapy also increased glycolytic activities in a HIF-1α-dependent manner. In CTCL xenograft tumor mice model, lesional cells showed a significant increase in IL-17F after chemotherapy, shifting toward a Th17 phenotype, which process is also regulated by HIF-1α. Echinomycin, HIF-1α inhibitor, was co-administered in xenograft tumor mouse models with CHOP and showed a significant reduction in tumor growth. CONCLUSION: CHOP chemotherapy promotes glycolysis and IL-17 pathways in a HIF-1α-dependent fashion. Furthermore, HIF-1α blockade is promising as an accompanying agent in systemic chemotherapy for patients with advanced CTCL.