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Basal and apical regulation of VEGF-A and placenta growth factor in the RPE/choroid and primary RPE.Molecular Vision 2015Members of the vascular endothelial growth factor (VEGF) family are strongly involved in pathological processes in the retina, such as age-related macular degeneration...
PURPOSE
Members of the vascular endothelial growth factor (VEGF) family are strongly involved in pathological processes in the retina, such as age-related macular degeneration and diabetic retinopathy. Cells of the retinal pigment epithelium (RPE) constitutively secrete VEGF-A, and the secretion of placental growth factor (PlGF) has also been described. RPE cells are strongly polarized cells with different secretome at the apical and basal side. In this study, we evaluated the basal and apical regulation of VEGF-A and PlGF secretion in RPE/choroid explants and primary RPE cells.
METHODS
RPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers, separating apical (RPE) and basal (choroid) supernatant. Primary RPE cells were also prepared from porcine eyes and cultivated on Transwell plates. Explants and cells were treated with inhibitors for VEGFR-2 (SU1498), p38 (SB203580), and the transcription factors nuclear factor-kappa B (NF-κB) and SP-1 (mithramycin), respectively. VEGF-A and PlGF content was evaluated with enzyme-linked immunosorbent assay (ELISA). In addition, western blots were performed.
RESULTS
In the RPE/choroid, VEGF-A can initially be found on the apical and basal sides with significantly more pronounced secretion on the basal side. VEGF-A secretion is differentially regulated on the apical and basal sides, with the inhibition of SP-1 and NF-κB showing strong effects apically and basally after 24 h and 48 h, the inhibition of p38 displaying its effect mainly on the basal side with some effect apically after 48 h, and the inhibition of VEGFR-2 reducing the secretion of VEGF only on the apical side at 24 h and 48 h. In the RPE cell culture, similar effects were found, with inhibition of NF-κB or SP-1 displaying a strong decrease in VEGF-A on both sides, and p38 inhibition displaying only an inhibitory effect on the basal side. In contrast, an apical effect of VEGFR-2 inhibition was not found. However, the western blot experiments exhibited a significant decrease in the VEGF-A protein under SU1498 treatment. In the RPE/choroid organ cultures, PlGF was initially found mainly on the basal site with only minute amounts of PlGF found apically. NF-κB and SP-1 were strongly involved in PlGF regulation apically and basally, while VEGFR2 and to a lesser degree p38 displayed some regulation at the basal site. In the primary RPE cell culture, PlGF was not found on the apical or basal side.
CONCLUSIONS
VEGF-A and PlGF were constitutively secreted and regulated by the RPE/choroid complex, with PlGF secreted mainly by the choroid. Although the transcription factors NF-κB and SP-1 were involved in apical and basal regulation of both growth factors, VEGFR-2 displayed a strong polarity, with regulation of apical VEGF-A and basal PlGF secretion.
Topics: Animals; Cells, Cultured; Choroid; NF-kappa B; Organ Culture Techniques; Placenta Growth Factor; Pregnancy Proteins; Retinal Pigment Epithelium; Sp1 Transcription Factor; Sus scrofa; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; p38 Mitogen-Activated Protein Kinases
PubMed: 26167115
DOI: No ID Found -
Journal of the American Chemical Society May 2003To gain initial structure-activity relationships regarding the highly functionalized pentyl side chain attached at C-3 of mithramycin (MTM), we focused on a...
Mithramycin SK, a novel antitumor drug with improved therapeutic index, mithramycin SA, and demycarosyl-mithramycin SK: three new products generated in the mithramycin producer Streptomyces argillaceus through combinatorial biosynthesis.
To gain initial structure-activity relationships regarding the highly functionalized pentyl side chain attached at C-3 of mithramycin (MTM), we focused on a post-polyketide synthase (post-PKS) tailoring step of the MTM biosynthesis by Streptomyces argillaceus ATCC 12956, which was proposed to be catalyzed by ketoreductase (KR) MtmW. In this last step of the MTM biosynthesis, a keto group of the pentyl side chain is reduced to a secondary alcohol, and we anticipated the generation of an MTM derivative with an additional keto group in the 3-side chain. Insertional inactivation of mtmW, a gene located ca. 8 kb downstream of the mithramycin-PKS genes, yielded an S. argillaceus mutant, which accumulated three new mithramycin analogues, namely mithramycin SA, demycarosyl-mithramycin SK, and mithramycin SK (MTM-SK). The structures of these three compounds confirmed indirectly the proposed role of MtmW in MTM biosynthesis. However, the new mithramycin derivatives bear unexpectedly shorter 3-side chains (ethyl or butyl) than MTM, presumably caused by nonenzymatic rearrangement or cleavage reactions of the initially formed pentyl side chain with a reactive beta-dicarbonyl functional group. The major product, MTM-SK, was tested in vitro against a variety of human cancer cell lines, as well as in an in vitro toxicity assay, and showed an improved therapeutic index, in comparison to the parent drug, MTM.
Topics: Antibiotics, Antineoplastic; Carbohydrate Sequence; Combinatorial Chemistry Techniques; Drug Screening Assays, Antitumor; Gene Silencing; Humans; Molecular Sequence Data; Mutagenesis, Insertional; Nuclear Magnetic Resonance, Biomolecular; Oxidoreductases; Plicamycin; Streptomyces; Trisaccharides; Tumor Cells, Cultured
PubMed: 12733914
DOI: 10.1021/ja034162h -
Gels (Basel, Switzerland) Sep 2022Gelatin is a popular biopolymer for biomedical applications due to its harmless impact with a negligible inflammatory response in the host organism. Gelatin interacts...
Gelatin is a popular biopolymer for biomedical applications due to its harmless impact with a negligible inflammatory response in the host organism. Gelatin interacts with soluble molecules in aqueous media as ionic counterparts such as ionic liquids (ILs) to be used as cosolvents to generate the so-called Ionogels. The perfluorinated IL (FIL), 1-ethyl-3-methylpyridinium perfluorobutanesulfonate, has been selected as co-hydrosolvent for fish gelatin due to its low cytotoxicity and hydrophobicity aprotic polar structure to improve the drug aqueous solubility. A series of FIL/water emulsions with different FIL content and their corresponding shark gelatin/FIL Ionogel has been designed to enhance the drug solubility whilst retaining the mechanical structure and their nanostructure was probed by simultaneous SAXS/WAXS, FTIR and Raman spectroscopy, DSC and rheological experiments. Likewise, the FIL assisted the solubility of the antitumoural Doxorubicin whilst retaining the performing mechanical properties of the drug delivery system network for the drug storage as well as the local administration by a syringe. In addition, the different controlled release mechanisms of two different antitumoral such as Doxorubicin and Mithramycin from two different Ionogels formulations were compared to previous gelatin hydrogels which proved the key structure correlation required to attain specific therapeutic dosages.
PubMed: 36135306
DOI: 10.3390/gels8090594 -
Microbiology (Reading, England) Feb 2015The mithramycin biosynthesis gene cluster of Streptomyces argillaceus ATCC 12956 contains 34 ORFs and includes two putative regulatory genes (mtmR and mtrY), which...
The mithramycin biosynthesis gene cluster of Streptomyces argillaceus ATCC 12956 contains 34 ORFs and includes two putative regulatory genes (mtmR and mtrY), which encode proteins of the SARP (Streptomyces antibiotic regulatory protein) and PadR transcriptional regulator families, respectively. MtmR was proposed to behave as a positive regulator of mithramycin biosynthesis. Inactivation and overexpression of mtrY indicated that it is also a positive regulator of mithramycin biosynthesis, being non-essential but required to maintain high levels of mithramycin production in the producer strain. Transcriptional analyses by reverse transcription PCR and quantitative real-time PCR of mithramycin genes, and promoter-probe assays in S. argillaceus polyketide synthase and regulatory mutants and the WT strain, and in the heterologous host Streptomyces albus, were carried out to analyse the role of MtmR and MtrY in the regulation of the mithramycin gene cluster. These experiments revealed that MtmR had a positive role, activating expression of at least six polycistronic units (mtmR-mtmE, mtmQ-mtmTII, mtmX-mtmY, mtmV-mtmTIII, mtmW-mtmMI and mtmGI-mtrB) and one monocistronic unit (mtmGII) in the mithramycin gene cluster. However, MtrY played a dual role in the mithramycin gene cluster: (i) repressing the expression of resistance genes and its coding gene itself by controlling the activity of the mtrYp promoter that directs expression of the regulator mtrY and resistance genes, with this repression being released in the presence of mithramycin; and (ii) enhancing the expression of mithramycin biosynthesis genes when mithramycin is present, by interacting with the mtmRp promoter that controls expression of the mtmR regulator, amongst others.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Gene Expression Regulation, Bacterial; Plicamycin; Promoter Regions, Genetic; Repressor Proteins; Streptomyces; Trans-Activators; Transcription, Genetic
PubMed: 25416691
DOI: 10.1099/mic.0.080895-0 -
Cell Reports Jan 2017Efforts to identify and target glioblastoma (GBM) drivers have primarily focused on receptor tyrosine kinases (RTKs). Clinical benefits, however, have been elusive....
Efforts to identify and target glioblastoma (GBM) drivers have primarily focused on receptor tyrosine kinases (RTKs). Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2) transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2) and zinc-finger E-box binding homeobox 1 (ZEB1), which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.
Topics: Animals; Astrocytes; Brain; Brain Neoplasms; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Down-Regulation; ErbB Receptors; Gefitinib; Glioblastoma; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Grading; Oligodendrocyte Transcription Factor 2; Plasmids; Plicamycin; Quinazolines; RNA Interference; Receptor, Platelet-Derived Growth Factor alpha; SOXB1 Transcription Factors; Zinc Finger E-box-Binding Homeobox 1
PubMed: 28122245
DOI: 10.1016/j.celrep.2016.12.064 -
Molecular Cancer Therapeutics May 2020There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor-targeted therapies. In this study, we exploit...
There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor-targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1-targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic "class" effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1-targeted combination therapy for Ewing sarcoma.
Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Bone Neoplasms; Cell Proliferation; Cyclin-Dependent Kinase 9; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Mice; Mice, Nude; Plicamycin; Sarcoma, Ewing; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 32127464
DOI: 10.1158/1535-7163.MCT-19-0775 -
Gynecologic Oncology Aug 2010Increased activity of Sp family of transcription factors is a frequent and critical event in cancer development and progression. Genes governing tumor growth, invasion...
OBJECTIVE
Increased activity of Sp family of transcription factors is a frequent and critical event in cancer development and progression. Genes governing tumor growth, invasion and angiogenesis are regulated by Sp factors, like Sp1, Sp3 or Sp4, and are frequently over-expressed in tumors. Targeting Sp factors has been explored as a therapeutic approach. Mithramycin (MTM) is a natural antibiotic that binds DNA and inhibit Sp1-dependent transcription. New analogues, named MTM-SDK and MTM-SK, were recently obtained by genetic engineering of the MTM biosynthetic pathway and have demonstrated improved transcriptional and antiproliferative activity in ovarian cancer cell lines in vitro. In the present study we evaluated the activity of the new compounds in human ovarian cancer xenografts.
METHODS
Expression of Sp1 and target proteins in ovarian cancer specimens and tumor xenografts was assessed by immunohistochemistry. Drug-induced silencing of Sp1-regulated genes in cells and tumor xenograft samples was assessed by quantitative RT-PCR. Toxicity and antitumor activity of the compounds were investigated in healthy and tumor-bearing immunocompromised mice, respectively.
RESULTS
Expression of Sp1 was frequently increased in human epithelial ovarian cancers. MTM-SDK and MTM-SK acted as potent inhibitors of Sp1-dependent transcription both in vitro and in tumor xenografts. Both compounds were well tolerated even after prolonged administration and delayed growth of ovarian tumor xenografts. MTM-SDK was particularly effective against orthotopic tumors leading to a significant increase of survival and delay of tumor progression.
CONCLUSIONS
MTM-SDK and MTM-SK show relevant activity in vivo and represent interesting candidates for treatment of ovarian cancers.
Topics: Animals; Antibiotics, Antineoplastic; Cell Growth Processes; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Ovarian Neoplasms; Plicamycin; Sp1 Transcription Factor; Xenograft Model Antitumor Assays
PubMed: 20452660
DOI: 10.1016/j.ygyno.2010.03.020 -
BMC Cancer Jun 2016It has been shown that epidermal growth factor receptor (EGFR) mutation status is associated with 5-fluorouracil (5-FU) sensitivity in non-small-cell lung cancer...
BACKGROUND
It has been shown that epidermal growth factor receptor (EGFR) mutation status is associated with 5-fluorouracil (5-FU) sensitivity in non-small-cell lung cancer (NSCLC). However, the relationship between EGFR mutation status and dihydropyrimidine dehydrogenase (DPD), a 5-FU degrading enzyme, is unknown.
METHODS
We elucidated the crosstalk among the EGFR signal cascade, the DPD gene (DPYD), and DPD protein expression via the transcription factor Sp1 and the effect of EGFR mutation status on the crosstalk.
RESULTS
In the PC9 (exon19 E746-A750) study, EGF treatment induced up-regulation of both Sp1 and DPD; gefitinib, an EGFR-tyrosine kinase inhibitor (EGFR-TKI), and mithramycin A, a specific Sp-1 inhibitor, suppressed them. Among EGFR-mutated (PC9, HCC827; exon19 E746-A750 and H1975; exon21 L858R, T790M, gefitinib resistant) and -non-mutated (H1437, H1299) cell lines, EGF administration increased DPYD mRNA expression only in mutated cells (p < 0.05). Accordingly, gefitinib inhibited DPD protein expression only in PC9 and HCC827 cells, and mithramycin A inhibited it in EGFR-mutated cell lines, but not in wild-type. FU treatment decreased the level of cell viability more in gefitinib-treated EGFR-TKI sensitive cell lines. Further, combination treatment of FU and mithramycin A suppressed cell viability even in a gefitinib resistant cell line.
CONCLUSIONS
The EGFR signal cascade regulates DPD expression via Sp1 in EGFR mutant cells. These results might be a step towards new therapies targeting Sp1 and DPD in NSCLC with different EGFR mutant status.
Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Dihydrouracil Dehydrogenase (NADP); Drug Resistance, Neoplasm; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Fluorouracil; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mutation; Plicamycin; Quinazolines; Signal Transduction; Sp1 Transcription Factor
PubMed: 27268079
DOI: 10.1186/s12885-016-2392-0 -
Journal of Chromatography. B,... Nov 2014Mithramycin is a neoplastic antibiotic synthesized by various Streptomyces bacteria. It is under investigation as a chemotherapeutic treatment for a wide variety of...
Mithramycin is a neoplastic antibiotic synthesized by various Streptomyces bacteria. It is under investigation as a chemotherapeutic treatment for a wide variety of cancers. Ongoing and forthcoming clinical trials will require pharmacokinetic analysis of mithramycin in humans, both to see if target concentrations are achieved and to optimize dosing and correlate outcomes (response/toxicity) with pharmacokinetics. Two published methods for mithramycin quantitation exist, but both are immunoassays that lack current bioanalytical standards of selectivity and sensitivity. To provide an upgraded and more widely applicable assay, a UPLC-MS/MS method for quantitation of mithramycin in human plasma was developed. Solid-phase extraction allowed for excellent recoveries (>90%) necessary for high throughput analyses on sensitive instrumentation. However, a ∼55% reduction in analyte signal was observed as a result of plasma matrix effects. Mithramycin and the internal standard chromomycin were separated on a Waters Acquity BEH C18 column (2.1×50 mm, 1.7 μm) and detected using electrospray ionization operated in the negative mode at mass transitions m/z 1083.5→268.9 and 1181.5→269.0, respectively, on an AB Sciex QTrap 5500. The assay range was 0.5-500 ng/mL and proved to be linear (r(2)>0.996), accurate (≤10% deviation), and precise (CV<15%). Mithramycin was stable in plasma at room temperature for 24 h, as well as through three freeze-thaw cycles. This method was subsequently used to quantitate mithramycin plasma concentrations from patients enrolled on two clinical trials at the NCI.
Topics: Blood Proteins; Chromatography, High Pressure Liquid; Humans; Linear Models; Plicamycin; Reproducibility of Results; Sensitivity and Specificity; Solid Phase Extraction; Tandem Mass Spectrometry
PubMed: 25247492
DOI: 10.1016/j.jchromb.2014.08.021 -
Journal of the American Heart... Jan 2020Background Activation of the YAP (Yes-associated protein) pathway has been demonstrated to be related to smooth muscle cells (SMCs) phenotypic modulation and vessel...
Background Activation of the YAP (Yes-associated protein) pathway has been demonstrated to be related to smooth muscle cells (SMCs) phenotypic modulation and vessel restenosis. The aim of this study was to illustrate the molecular mechanisms that regulate the expression of YAP during the process of SMCs phenotypic switch. Whether the molecular basis identified in the study could be a potential therapeutic target for drug-eluting stents is further tested. Methods and Results In cell culture and in rat carotid arterial injury models, Sp-1 (specificity protein 1) expression was significantly induced, and correlated with SMCs proliferative phenotype. Overexpression of Sp-1 promoted SMCs proliferation and migration. Conversely, siSp-1 transfection or Sp-1 inhibitor Mithramycin A treatment attenuates SMC proliferation and migration. Through gain- and loss-function assays, we demonstrated that YAP was involved in Sp-1-mediated SMC phenotypic switch. Mechanistically, activated Sp-1 regulated YAP transcriptional expression through binding to its promoter. Moreover, we fabricated a Sp-1 inhibitor Mithramycin A-eluting stent and further tested it. In the rabbit carotid model, Mithramycin A-eluting stent inhibited YAP transcription and attenuated in-stent restenosis through regulating YAP-mediated SMC phenotypic switch. Conclusions Sp-1 controls phenotypic modulation of SMC by regulating transcription factor YAP. Drug-eluting stent targeting Sp-1 might represent a novel therapeutic strategy to prevent in-stent restenosis.
Topics: Angioplasty, Balloon; Animals; Apoptosis Regulatory Proteins; Cardiovascular Agents; Carotid Arteries; Carotid Artery Injuries; Carotid Stenosis; Cell Movement; Cell Plasticity; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Drug-Eluting Stents; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phenotype; Plicamycin; Prosthesis Design; Rabbits; Rats; Signal Transduction; Sp1 Transcription Factor; YAP-Signaling Proteins
PubMed: 31880978
DOI: 10.1161/JAHA.119.014103