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Toxicology in Vitro : An International... Oct 2015DIG-MSK (demycarosil-3D-β-D-digitoxosyl mithramycin SK; EC-8042), a novel analogue of mithramycin A, induced autophagy in HCT116 human colon carcinoma and, to a lesser...
DIG-MSK (demycarosil-3D-β-D-digitoxosyl mithramycin SK; EC-8042), a novel analogue of mithramycin A, induced autophagy in HCT116 human colon carcinoma and, to a lesser extent, in A2780 human ovarian carcinoma cell lines, which was followed by apoptosis and/or necrotic cell death in a time-dependent way. The effects of DIG-MSK included changes in the expression of a set of genes involved in autophagy, the progression of cells through the different phases of cell cycle, and their halting at the checkpoints. Cells treated with the glucose analogue 2-DG (2-deoxy-D-glucose), which induces autophagy because it impairs cell metabolism, or co-treated with 2-DG plus DIG-MSK, also showed altered gene expression and autophagy. In A2780 cells, some genes involved in autophagy were down-regulated by the different treatments, yet the levels of the proteins they encode could be enough to ensure autophagic flux. In HCT116 cells, up-regulation of several pro-autophagic genes resulted in strong autophagic response. Acidic cell organelles and autophagic flux were more evident in HCT116 than in A2780 cells. DIG-MSK was still cytotoxic in cells that underwent autophagy induced by 2-DG. Therefore, we verified that autophagy resulting from a stress response did not protect cells against DIG-MSK, but, instead, autophagy promoted by either 2-DG or the novel mithralogue can enhance the antitumour activity, which depended on the cell type.
Topics: Antineoplastic Agents; Apoptosis; Autophagy; Cell Line, Tumor; Colonic Neoplasms; Deoxyglucose; Female; Gene Expression Regulation, Neoplastic; Humans; Ovarian Neoplasms; Plicamycin
PubMed: 26079942
DOI: 10.1016/j.tiv.2015.06.008 -
Journal of Inorganic Biochemistry Mar 2016The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancer-related hypercalcemia and, experimentally, against Ewing sarcoma and lung...
The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancer-related hypercalcemia and, experimentally, against Ewing sarcoma and lung cancers. MTM exerts its cytotoxic effect by binding DNA as a divalent metal ion (Me(2+))-coordinated dimer and disrupting the function of transcription factors. A precise molecular mechanism of action of MTM, needed to develop MTM analogues selective against desired transcription factors, is lacking. Although it is known that MTM binds G/C-rich DNA, the exact DNA recognition rules that would allow one to map MTM binding sites remain incompletely understood. Towards this goal, we quantitatively investigated dimerization of MTM and several of its analogues, MTM SDK (for Short side chain, DiKeto), MTM SA-Trp (for Short side chain and Acid), MTM SA-Ala, and a biosynthetic precursor premithramycin B (PreMTM B), and measured the binding affinities of these molecules to DNA oligomers of different sequences and structural forms at physiological salt concentrations. We show that MTM and its analogues form stable dimers even in the absence of DNA. All molecules, except for PreMTM B, can bind DNA with the following rank order of affinities (strong to weak): MTM=MTM SDK>MTM SA-Trp>MTM SA-Ala. An X(G/C)(G/C)X motif, where X is any base, is necessary and sufficient for MTM binding to DNA, without a strong dependence on DNA conformation. These recognition rules will aid in mapping MTM sites across different promoters towards development of MTM analogues as useful anticancer agents.
Topics: Antibiotics, Antineoplastic; DNA; Dimerization; Plicamycin
PubMed: 26760230
DOI: 10.1016/j.jinorgbio.2015.12.011 -
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 -
Lancet (London, England) Mar 1975The treatment of rapidly progressive skeletal demineralisation in myelomatosis has been studied with the help of metabolic calcium balance in two patients; In one,...
The treatment of rapidly progressive skeletal demineralisation in myelomatosis has been studied with the help of metabolic calcium balance in two patients; In one, osteoporosis accelerated during treatment with melphalan and prednisolone, although he remained normocalcaemic throughout, suggesting that osteoporosis was aggravated by corticosteroid therapy. In the other patient, who was initially hypercalcaemic, conventional treatment produced clinical remission before eventual relapse with more hypercalcaemia and skeletal dissolution. Both patients were then treated with mithramycin alone, and, although neither obtained haematological remission, bone pain was relieved, hypercalciuria and hypercalcaemia were abolished, and calcium balances proved that mithramycin was effective in restoring calcium equilibrium. The results indicate that mithramycin may abolish excessive bone resorption in myelomatosis and that severe bone dissolution may occur in the absence of hypercalcaemia. Regular determination of 24-hour urinary calcium excretion as well as of plasma-calcium is important in monitoring process. Mithramycin should be considered in the early treatment not only of hypercalcaemia but also of severe hypercalciuria, if these complications do not rapidly remit during the first course of conventional myeloma therapy, with or without steroids. Finally, these results add to evidence that a humoral factor may be responsible for osteoclast stimulation in myelomatosis.
Topics: Aged; Calcium; Cyclophosphamide; Drug Therapy, Combination; Female; Humans; Hypercalcemia; Injections, Intravenous; Male; Melphalan; Middle Aged; Multiple Myeloma; Osteolysis; Phosphorus; Plicamycin; Prednisolone; Water-Electrolyte Balance
PubMed: 47484
DOI: 10.1016/s0140-6736(75)91631-1 -
Oncology 1982Some tumors release factors able to activate host osteoclasts. Mithramycin at sub-tumoricidal doses inhibits the release of calcium mediated by osteoclasts. If invasion...
Some tumors release factors able to activate host osteoclasts. Mithramycin at sub-tumoricidal doses inhibits the release of calcium mediated by osteoclasts. If invasion of bone by a cancer requires activation of these cells, their intermittent "blockade' might impede the development of metastases to bone or their local extension. Fetal rat bones prelabelled with 45Ca were cultured in the presence of 10(-7) M prostaglandin E2, sera from normal individuals, or from patients with multiple myeloma. Additional samples preincubated for 3 h with 1 microgram/ml of mithramycin, were washed before culture. Compared with controls, prostaglandin E2 stimulated the release of 45Ca by 28% (5 experiments) and mithramycin inhibited release by 15% (3 experiments). Preexposure to this cytotoxic antibiotic before culture with PGE2 reduced the augmented release. Sera from 4 patients with multiple myeloma were incubated with 45Ca-labelled bones, some pretreated with mithramycin. An additional 29% release of 45Ca (4 experiments) was prevented by mithramycin. These results are consistent with the hypothesis that augmented release of 45Ca due to stimulatory factors such as prostagladins or factors in sera from patients with multiple myeloma can be partially inhibited by pretreatment with mithramycin. Possibly, intermittent blockade of host osteoclasts can impair formation of metastases to bone by cancers dependent upon their activation for this event, or reduce the extent of local invasion by established metastases. Modifying the behavior of a cancer by altering the host-response to factors which it releases represents a potential alternative to cytotoxic chemotherapy.
Topics: Animals; Bone and Bones; Calcium; Humans; In Vitro Techniques; Multiple Myeloma; Osteoclasts; Plicamycin; Prostaglandins E; Rats
PubMed: 6460955
DOI: 10.1159/000225610 -
La Nouvelle Presse Medicale Apr 1975
Topics: Anemia; Blood Platelet Disorders; Blood Urea Nitrogen; Fever; Humans; Hypercalcemia; Plicamycin; Vomiting
PubMed: 124049
DOI: No ID Found -
Microbial Cell Factories May 2020Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized,...
BACKGROUND
Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis. To further study these compounds as potential new anticancer drugs requires their production yields to be improved significantly. The biosynthesis of mithramycin proceeds through the formation of the key intermediate 4-demethyl-premithramycinone. Extensive studies have characterized the biosynthesis pathway from this intermediate to mithramycin. However, the biosynthesis pathway for 4-demethyl-premithramycinone remains unclear.
RESULTS
Expression of cosmid cosAR7, containing a set of mithramycin biosynthesis genes, in Streptomyces albus resulted in the production of 4-demethyl-premithramycinone, delimiting genes required for its biosynthesis. Inactivation of mtmL, encoding an ATP-dependent acyl-CoA ligase, led to the accumulation of the tricyclic intermediate 2-hydroxy-nogalonic acid, proving its essential role in the formation of the fourth ring of 4-demethyl-premithramycinone. Expression of different sets of mithramycin biosynthesis genes as cassettes in S. albus and analysis of the resulting metabolites, allowed the reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, assigning gene functions and establishing the order of biosynthetic steps.
CONCLUSIONS
We established the biosynthesis pathway for 4-demethyl-premithramycinone, and identified the minimal set of genes required for its assembly. We propose that the biosynthesis starts with the formation of a linear decaketide by the minimal polyketide synthase MtmPKS. Then, the cyclase/aromatase MtmQ catalyzes the cyclization of the first ring (C7-C12), followed by formation of the second and third rings (C5-C14; C3-C16) catalyzed by the cyclase MtmY. Formation of the fourth ring (C1-C18) requires MtmL and MtmX. Finally, further oxygenation and reduction is catalyzed by MtmOII and MtmTI/MtmTII respectively, to generate the final stable tetracyclic intermediate 4-demethyl-premithramycinone. Understanding the biosynthesis of this compound affords enhanced possibilities to generate new mithramycin analogs and improve their production titers for bioactivity investigation.
Topics: Antibiotics, Antineoplastic; Bacterial Proteins; Plicamycin; Polyketides; Streptomyces
PubMed: 32448325
DOI: 10.1186/s12934-020-01368-3 -
Biomedicine & Pharmacotherapy =... Jun 2023Osteosarcomas are frequently associated to a poor prognosis and a modest response to current treatments. EC-8042 is a well-tolerated mithramycin analog that has...
Osteosarcomas are frequently associated to a poor prognosis and a modest response to current treatments. EC-8042 is a well-tolerated mithramycin analog that has demonstrated an efficient ability to eliminate tumor cells, including cancer stem cell subpopulations (CSC), in sarcomas. In transcriptomic and protein expression analyses, we identified NOTCH1 signaling as one of the main pro-stemness pathways repressed by EC-8042 in osteosarcomas. Overexpression of NOTCH-1 resulted in a reduced anti-tumor effect of EC-8042 in CSC-enriched 3D tumorspheres cultures. On the other hand, the depletion of the NOTCH-1 downstream target HES-1 was able to enhance the action of EC-8042 on CSCs. Moreover, HES1 depleted cells failed to recover after treatment withdrawal and showed reduced tumor growth potential in vivo. In contrast, mice xenografted with NOTCH1-overexpressing cells responded worse than parental cells to EC-8042. Finally, we found that active NOTCH1 levels in sarcoma patients was associated to advanced disease and lower survival. Overall, these data highlight the relevant role that NOTCH1 signaling plays in mediating stemness in osteosarcoma. Moreover, we demonstrate that EC-8042 is powerful inhibitor of NOTCH signaling and that the anti-CSC activity of this mithramycin analog highly rely on its ability to repress this pathway.
Topics: Animals; Mice; Bone Neoplasms; Cell Line, Tumor; Neoplastic Stem Cells; Osteosarcoma; Plicamycin; Receptor, Notch1; Receptors, Notch
PubMed: 37018985
DOI: 10.1016/j.biopha.2023.114627 -
Molecular Pharmacology Jan 2013In several human malignancies, overexpression of myeloid cell leukemia-1 (Mcl-1) confers resistance to induction of apoptosis; however, Mcl-1-mediated inhibition of...
In several human malignancies, overexpression of myeloid cell leukemia-1 (Mcl-1) confers resistance to induction of apoptosis; however, Mcl-1-mediated inhibition of apoptosis in oral squamous cell carcinoma (OSCC) is not fully understood and has been investigated in this study. The Mcl-1 promoter activators (TPA) and epidermal growth factor (EGF) enhanced neoplastic transformation of JB6 cells and this response was accompanied by enhanced expression of Mcl-1, and knockdown of Mcl-1 by RNA interference (RNAi) decreased JB6 cell transformation. In the same cell line, we also demonstrated that mithramycin A (Mith) decreased TPA-induced JB6 cell transformation and Mcl-1 expression. Mcl-1 was overexpressed in human oral tumors compared with normal oral mucosa and also in several OSCC cell lines including HN22 and HSC-4 cells. Treatment of these cells with Mith also decreased Mcl-1 expression and neoplastic cell transformation, and this was accompanied by induction of several markers of apoptosis. Knockdown of Mcl-1 by RNAi also induced apoptotic cell death. The downregulation of Mcl-1 by Mith and RNAi increased pro-apoptotic protein Bax, resulting in the Bax translocation into mitochondria and its oligomerization. Mith also suppressed tumor growth in vivo and induced apoptosis in tumor by also regulating expression of Mcl-1 and Bax proteins. These indicate a critical role for Mcl-1 in the growth and survival of OSCC and demonstrate that Mith may be a potential anticancer drug candidate for clinical treatment of OSCC.
Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Transformation, Neoplastic; Gene Knockdown Techniques; Humans; Mice; Mice, Nude; Mitochondria; Mouth Mucosa; Mouth Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Transplantation; Plicamycin; Polymerization; Protein Transport; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Transplantation, Heterologous; bcl-2-Associated X Protein
PubMed: 23019217
DOI: 10.1124/mol.112.081364 -
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