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Scientific Reports Apr 2020Clostridioides difficile is the most common cause of healthcare-associated diarrhea. Infection of the gastrointestinal tract with this Gram-positive, obligate anaerobe...
Clostridioides difficile is the most common cause of healthcare-associated diarrhea. Infection of the gastrointestinal tract with this Gram-positive, obligate anaerobe can lead to potentially life-threatening conditions in the antibiotic-treated populace. New therapeutics are urgently needed to treat this infection and prevent its recurrence. Here, we screened two libraries from the National Cancer Institute, namely, the natural product set III library (117 compounds) and the approved oncology drugs set V library (114 compounds), against C. difficile. In the two libraries screened, 17 compounds from the natural product set III library and 7 compounds from the approved oncology drugs set V library were found to exhibit anticlostridial activity. The most potent FDA-approved drugs (mitomycin C and mithramycin A) and a promising natural product (aureomycin) were further screened against 20 clinical isolates of C. difficile. The anticancer drugs, mitomycin C (MIC = 0.25 μg/ml) and mithramycin A (MIC = 0.015 μg/ml), and the naturally derived tetracycline derivative, aureomycin (MIC = 0.06 μg/ml), exhibited potent activity against C. difficile strains. Mithramycin A and aureomycin were further found to inhibit toxin production by this pathogen. Given their efficacy, these compounds can provide a quick supplement to current treatment to address the unmet needs in treating C. difficile infection and preventing its recurrence.
Topics: Anti-Bacterial Agents; Biological Products; Clostridioides difficile; Clostridium Infections; Diarrhea; Drug Approval; Drug Evaluation, Preclinical; Gastrointestinal Tract; Humans; Microbial Sensitivity Tests
PubMed: 32249833
DOI: 10.1038/s41598-020-63029-0 -
Journal of Medicinal Chemistry Nov 2020Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address...
Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2'-oxime (MTM) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTM analogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusion-independent/dependent selectivity indices for select 2'-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified (a Phe-Trp dipeptide-based 2'-conjugate) for in vivo testing. Relative to MTM, displayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.
Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Bone Neoplasms; Cell Proliferation; Female; Humans; Mice; Mice, SCID; Oximes; Plicamycin; Sarcoma, Ewing; Tissue Distribution; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 33191745
DOI: 10.1021/acs.jmedchem.0c01526 -
ACS Pharmacology & Translational Science Sep 2022Drug resistance is a leading cause for the failure of cancer treatments. Plasticity of cancer cells to acquire stem cell-like properties enables them to escape drug...
Drug resistance is a leading cause for the failure of cancer treatments. Plasticity of cancer cells to acquire stem cell-like properties enables them to escape drug toxicity through different adaptive mechanisms. Eliminating cancer stem cells (CSCs) can potentially improve treatment outcomes for patients. To determine the role of CSCs in resistance of colorectal cancer cells to targeted therapies and identify treatment strategies, we treated spheroids of BRAF and KRAS colorectal cancer cells with inhibitors of the mitogen-activated protein kinase pathway and studied resistance mechanisms through gene and protein expression analyses. We found that treatments activated several oncogenic pathways and expression of CSC markers CD166 and ALDH1A3. We identified a specific combination treatment using trametinib and mithramycin A to simultaneously inhibit the CSC phenotype and activities of several pathways in cancer cells. This study demonstrates the feasibility of therapeutic targeting of CSCs as a strategy to block tumorigenic activities of cancer cells.
PubMed: 36110381
DOI: 10.1021/acsptsci.1c00257 -
Biomedicine & Pharmacotherapy =... Jul 2022Glutathione S-transferases (GSTs) belong to one class of phase 2 detoxification enzymes which are important in metabolism and/or detoxification of various electrophilic...
Glutathione S-transferases (GSTs) belong to one class of phase 2 detoxification enzymes which are important in metabolism and/or detoxification of various electrophilic endogenous metabolites and xenobiotics. From the available database, we found that GSTM2 gene expression is lower in high stages of bladder urothelial carcinoma than in stage 1 and normal bladder tissue. GSTM2 overexpression retards invasion, migration and tumor sphere formation of bladder cancer cells. Analysis of GSTM2 promoter activity shows that one SP1 site located at - 48 to - 40 bp is important for GSTM2 gene expression in BFTC 905 cells. An SP1 inhibitor, mithramycin A, inhibits GSTM2 promoter activity and protein expression. SP1 overexpression also increases GSTM2 expression in BFTC 905 and 5637 cells. Eight potential phytochemicals were analyzed for GSTM2 promoter activation, and results indicated that baicalein, berberrubine, chalcone, curcumin, resveratrol, and wogonin can increase promoter activity. In endogenous GSTM2 expression, berberrubine and resveratrol activated GSTM2 mRNA and protein expression the most. A DNA methylation inhibitor, 5-aza-deoxycytidine, can decrease GSTM2 gene methylation level and then increase its gene expression; 50 μM berberrubine decreased the GSTM2 gene methylation level, providing a mechanism for activating GSTM2 gene expression. Berberrubine and resveratrol also increased SP1 protein expression as one of the mechanisms for GSTM2 gene expression. In summary, berberrubine and resveratrol activates GSTM2 expression which inhibits cell proliferation, migration, and invasion of bladder cancer cells. The GSTM2 expression mechanism is partially via SP1 activation, and the effect of berberrubine is also partly via DNA CpG demethylation.
Topics: Carcinoma, Transitional Cell; DNA Methylation; Glutathione Transferase; Humans; Phytochemicals; Resveratrol; Sp1 Transcription Factor; Urinary Bladder Neoplasms
PubMed: 35594716
DOI: 10.1016/j.biopha.2022.113102 -
Journal of Thoracic Oncology : Official... Jan 2021Ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) encodes a master regulator of DNA methylation that has emerged as an epigenetic driver in human...
INTRODUCTION
Ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) encodes a master regulator of DNA methylation that has emerged as an epigenetic driver in human cancers. To date, no studies have evaluated UHRF1 expression in malignant pleural mesothelioma (MPM). This study was undertaken to explore the therapeutic potential of targeting UHRF1 in MPM.
METHODS
Microarray, real-time quantitative reverse transcription-polymerase chain reaction, immunoblot, and immunohistochemistry techniques were used to evaluate UHRF1 expression in normal mesothelial cells (NMCs) cultured with or without asbestos, MPM lines, normal pleura, and primary MPM specimens. The impact of UHRF1 expression on MPM patient survival was evaluated using two independent databases. RNA-sequencing, proliferation, invasion, and colony formation assays, and murine xenograft experiments were performed to evaluate gene expression and growth of MPM cells after biochemical or pharmacologic inhibition of UHRF1 expression.
RESULTS
UHRF1 expression was significantly higher in MPM lines and specimens relative to NMC and normal pleura. Asbestos induced UHRF1 expression in NMC. The overexpression of UHRF1 was associated with decreased overall survival in patients with MPM. UHRF1 knockdown reversed genomewide DNA hypomethylation, and inhibited proliferation, invasion, and clonogenicity of MPM cells, and growth of MPM xenografts. These effects were phenocopied by the repurposed chemotherapeutic agent, mithramycin. Biochemical or pharmacologic up-regulation of p53 significantly reduced UHRF1 expression in MPM cells. RNA-sequencing experiments exhibited the pleiotropic effects of UHRF1 down-regulation and identified novel, clinically relevant biomarkers of UHRF1 expression in MPM.
CONCLUSIONS
UHRF1 is an epigenetic driver in MPM. These findings support the efforts to target UHRF1 expression or activity for mesothelioma therapy.
Topics: Animals; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; Cell Proliferation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Mice; Pleural Neoplasms; Ubiquitin-Protein Ligases
PubMed: 32927122
DOI: 10.1016/j.jtho.2020.08.024 -
Cancer Letters Sep 2020The dependency of prostate cancer (PCa) growth on androgen receptor (AR) signaling has been harnessed to develop first-line therapies for high-risk localized and...
The dependency of prostate cancer (PCa) growth on androgen receptor (AR) signaling has been harnessed to develop first-line therapies for high-risk localized and metastatic PCa treatment. However, the occurrence of aberrant expression, mutated or splice variants of AR confers resistance to androgen ablation therapy (ADT), radiotherapy or chemotherapy in AR-positive PCa. Therapeutic strategies that effectively inhibit the expression and/or transcriptional activity of full-length AR, mutated AR and AR splice variants have remained elusive. In this study, we report that mithramycin (MTM), an antineoplastic antibiotic, suppresses cell proliferation and exhibits dual inhibitory effects on expression and transcriptional activity of AR and AR splice variants. MTM blocks AR recruitment to its genomic targets by occupying AR enhancers and causes downregulation of AR target genes, which includes key DNA repair factors in DNA damage repair (DDR). We show that MTM significantly impairs DDR and enhances the effectiveness of ionizing radiation or the radiomimetic agent Bleomycin in PCa. Thus, the combination of MTM treatment with RT or radiomimetic agents, such as bleomycin, may present a novel effective therapeutic strategy for patients with high-risk, clinically localized PCa.
Topics: Antibiotics, Antineoplastic; Cell Line, Tumor; DNA Damage; DNA Repair; Humans; Male; Plicamycin; Prostatic Neoplasms, Castration-Resistant; Receptors, Androgen
PubMed: 32485222
DOI: 10.1016/j.canlet.2020.05.027 -
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