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PloS One 2013Aberrant epigenetic patterns are central in the pathogenesis of haematopoietic diseases such as myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML)....
BACKGROUND
Aberrant epigenetic patterns are central in the pathogenesis of haematopoietic diseases such as myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Vorinostat is a HDACi which has produced responses in these disorders. The purpose of this study was to address the functional effects of vorinostat in leukemic cell lines and primary AML and MDS myeloid cells and to dissect the genetic and molecular mechanisms by which it exerts its action.
METHODOLOGY/PRINCIPAL FINDINGS
Functional assays showed vorinostat promoted cell cycle arrest, inhibited growth, and induced apoptosis and differentiation of K562, HL60 and THP-1 and of CD33(+) cells from AML and MDS patients. To explore the genetic mechanism for these effects, we quantified gene expression modulation by vorinostat in these cells. Vorinostat increased expression of genes down-regulated in MDS and/or AML (cFOS, COX2, IER3, p15, RAI3) and suppressed expression of genes over-expressed in these malignancies (AXL, c-MYC, Cyclin D1) and modulated cell cycle and apoptosis genes in a manner which would favor cell cycle arrest, differentiation, and apoptosis of neoplastic cells, consistent with the functional assays. Reporter assays showed transcriptional effect of vorinostat on some of these genes was mediated by proximal promoter elements in GC-rich regions. Vorinostat-modulated expression of some genes was potentiated by mithramycin A, a compound that interferes with SP1 binding to GC-rich DNA sequences, and siRNA-mediated SP1 reduction. ChIP assays revealed vorinostat inhibited DNA binding of SP1 to the proximal promoter regions of these genes. These results suggest vorinostat transcriptional action in some genes is regulated by proximal promoter GC-rich DNA sequences and by SP1.
CONCLUSION
This study sheds light on the effects of vorinostat in AML and MDS and supports the implementation of clinical trials to explore the use of vorinostat in the treatment of these diseases.
Topics: Antineoplastic Agents; Apoptosis; Base Sequence; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; GC Rich Sequence; HL-60 Cells; Humans; Hydroxamic Acids; K562 Cells; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Vorinostat
PubMed: 23320102
DOI: 10.1371/journal.pone.0053766 -
PloS One 2011Despite the well known importance of apolipoprotein (Apo) E in cholesterol efflux, the effect of ApoE on the expression of ATP-binding cassette transporter A1 (ABCA1)...
Despite the well known importance of apolipoprotein (Apo) E in cholesterol efflux, the effect of ApoE on the expression of ATP-binding cassette transporter A1 (ABCA1) has never been investigated. The objective of this study was to determine the effect of ApoE on ApoB-carrying lipoprotein-induced expression of ABCA1, a protein that mediates cholesterol efflux. Our data demonstrate that ApoB-carrying lipoproteins obtained from both wild-type and ApoE knockout mice induced ApoAI-mediated cholesterol efflux in mouse macrophages, which was associated with an enhanced ABCA1 promoter activity, and an increased ABCA1 mRNA and protein expression. In addition, these lipoproteins increased the level of phosphorylated specificity protein 1 (Sp1) and the amount of Sp1 bound to the ABCA1 promoter. However, all these inductions were significantly diminished in cells treated with ApoE-free lipoproteins, when compared to those treated with wild-type lipoproteins. Enrichment with human ApoE3 reversed the reduced inducibility of ApoE-free lipoproteins. Moreover, we observed that inhibition of Sp1 DNA-binding by mithramycin A diminished ABCA1 expression and ApoAI-mediated cholesterol efflux induced by ApoB-carrying lipoproteins, and that mutation of the Sp1-binding motif in the ABCA1 promoter region diminished ApoB-carrying lipoprotein-induced ABCA1 promoter activity. Collectively, these data suggest that ApoE associated with ApoB-carrying lipoproteins has an upregulatory role on ABCA1 expression, and that induction of Sp1 phosphorylation is a mechanism by which ApoE upregulates ABCA1 expression.
Topics: ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Animals; Apolipoproteins B; Apolipoproteins E; Biological Transport; Blotting, Western; Cholesterol; Chromatin Immunoprecipitation; Lipoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Plicamycin; Promoter Regions, Genetic; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Sp1 Transcription Factor
PubMed: 21779326
DOI: 10.1371/journal.pone.0021453 -
Journal of Cell Communication and... Mar 2012It is becoming increasingly apparent that many of the basic mechanisms underlying cancers also underlie fibrotic diseases. For example, the Sp1 family of transcription...
It is becoming increasingly apparent that many of the basic mechanisms underlying cancers also underlie fibrotic diseases. For example, the Sp1 family of transcription factors plays an essential role in controlling the gene expression of proteins that promote both oncogenesis and fibrogenesis. The drug mithramycin, which prevents Sp1 binding to DNA, has been in use clinically for some cancers, but has side-effects. However, other drugs exist that affect Sp1 activity through promoting Sp1 protein degradation. Evidence has emerged that low levels of mithramycin can be combined with these drugs to result in potent antitumorigenic effects without resulting in obvious toxicity (Gao et al. Cancer Res 2011 Jun 20; Jia et al. Cancer Res 70:1111-1119, 2010). Given that Sp1 proteins also promote expression of profibrotic genes such as collagen type I and CCN2, it is possible that this combinatorial approach may be taken in the future to block not only cancer but also fibrosis.
PubMed: 21822787
DOI: 10.1007/s12079-011-0147-x -
Arthritis Research & Therapy 2005Interleukin-1 (IL-1), IL-17 and tumor necrosis factor alpha (TNF-alpha) are the main proinflammatory cytokines implicated in cartilage breakdown by matrix... (Comparative Study)
Comparative Study
Interleukin-1 (IL-1), IL-17 and tumor necrosis factor alpha (TNF-alpha) are the main proinflammatory cytokines implicated in cartilage breakdown by matrix metalloproteinase (MMPs) in arthritic joints. We studied the impact of an anti-neoplastic antibiotic, mithramycin, on the induction of MMPs in chondrocytes. MMP-3 and MMP-13 gene expression induced by IL-1beta, TNF-alpha and IL-17 was downregulated by mithramycin in human chondrosarcoma SW1353 cells and in primary human and bovine femoral head chondrocytes. Constitutive and IL-1-stimulated MMP-13 levels in bovine and human cartilage explants were also suppressed. Mithramycin did not significantly affect the phosphorylation of the mitogen-activated protein kinases, extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase. Despite effective inhibition of MMP expression by mithramycin and its potential to reduce cartilage degeneration, the agent might work through multiple unidentified mechanisms.
Topics: Animals; Cattle; Cells, Cultured; Chondrocytes; Cytokines; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Hip Joint; Humans; Joint Capsule; Knee Joint; Matrix Metalloproteinases; Plicamycin
PubMed: 15987479
DOI: 10.1186/ar1735 -
Frontiers in Immunology 2021The axis of Programmed cell death-1 receptor (PD-1) with its ligand (PD-L1) plays a critical role in colorectal cancer (CRC) in escaping immune surveillance, and...
The axis of Programmed cell death-1 receptor (PD-1) with its ligand (PD-L1) plays a critical role in colorectal cancer (CRC) in escaping immune surveillance, and blocking this axis has been found to be effective in a subset of patients. Although blocking PD-L1 has been shown to be effective in 5-10% of patients, the majority of the cohorts show resistance to this checkpoint blockade (CB) therapy. Multiple factors assist in the growth of resistance to CB, among which T cell exhaustion and immunosuppressive effects of immune cells in the tumor microenvironment (TME) play a critical role along with other tumor intrinsic factors. We have previously shown the polyketide antibiotic, Mithramycin-A (Mit-A), an effective agent in killing cancer stem cells (CSCs) and in a subcutaneous murine model. Since TME plays a pivotal role in CB therapy, we tested the immunomodulatory efficacy of Mit-A with anti-PD-L1 mAb (αPD-L1) combination therapy in an immunocompetent MC38 syngeneic orthotopic CRC mouse model. Tumors and spleens were analyzed by flow cytometry for the distinct immune cell populations affected by the treatment, in addition to RT-PCR for tumor samples. We demonstrated the combination treatment decreases tumor growth, thus increasing the effectiveness of the CB. Mit-A in the presence of αPD-L1 significantly increased CD8 T cell infiltration and decreased immunosuppressive granulocytic myeloid-derived suppressor cells and anti-inflammatory macrophages in the TME. Our results revealed Mit-A in combination with αPD-L1 has the potential for augmented CB therapy by turning an immunologically "cold" into "hot" TME in CRC.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Colorectal Neoplasms; Disease Models, Animal; Female; Immune Checkpoint Inhibitors; Mice; Mice, Inbred C57BL; Plicamycin
PubMed: 34381456
DOI: 10.3389/fimmu.2021.706133 -
Pharmaceuticals (Basel, Switzerland) Aug 2011Mithramycin A (MTM) and histone deacetylase inhibitors (HDACi) are effective therapeutic agents for cancer and neurodegenerative diseases. MTM is a FDA approved aureolic...
Mithramycin A (MTM) and histone deacetylase inhibitors (HDACi) are effective therapeutic agents for cancer and neurodegenerative diseases. MTM is a FDA approved aureolic acid-type antibiotic that binds to GC-rich DNA sequences and interferes with Sp1 transcription factor binding to its target sites (GC box). HDACi, on the other hand, modulate the activity of class I and II histone deacetylases. They mediate their protective function, in part, by regulating the acetylation status of histones or transcription factors, including Sp1, and in turn chromatin accessibility to the transcriptional machinery. Because these two classes of structurally and functionally diverse compounds mediate similar therapeutic functions, we investigated whether they act on redundant or synergistic pathways to protect neurons from oxidative death. Non-protective doses of each of the drugs do not synergize to create resistance to oxidative death suggesting that these distinct agents act via a similar pathway. Accordingly, we found that protection by MTM and HDACi is associated with diminished expression of the oncogene, Myc and enhanced expression of a tumor suppressor, p21(waf1/cip1). We also find that neuroprotection by MTM or Myc knockdown is associated with downregulation of class I HDAC levels. Our results support a model in which the established antitumor drug MTM or canonical HDACi act via distinct mechanisms to converge on the downregulation of HDAC levels or activity respectively. These findings support the conclusion that an imbalance in histone acetylase and HDAC activity in favor of HDACs is key not only for oncogenic transformation, but also neurodegeneration.
PubMed: 22582024
DOI: 10.3390/ph4081183 -
Cell Death & Disease Oct 2021Colorectal cancers (CRC) can be classified into four consensus molecular subtypes (CMS), among which CMS1 has the best prognosis, contrasting with CMS4 that has the...
Colorectal cancers (CRC) can be classified into four consensus molecular subtypes (CMS), among which CMS1 has the best prognosis, contrasting with CMS4 that has the worst outcome. CMS4 CRC is notoriously resistant against therapeutic interventions, as demonstrated by preclinical studies and retrospective clinical observations. Here, we report the finding that two clinically employed agents, everolimus (EVE) and plicamycin (PLI), efficiently target the prototypic CMS4 cell line MDST8. As compared to the prototypic CMS1 cell line LoVo, MDST8 cells treated with EVE or PLI demonstrated stronger cytostatic and cytotoxic effects, increased signs of apoptosis and autophagy, as well as a more pronounced inhibition of DNA-to-RNA transcription and RNA-to-protein translation. Moreover, nontoxic doses of EVE and PLI induced the shrinkage of MDST8 tumors in mice, yet had only minor tumor growth-reducing effects on LoVo tumors. Altogether, these results suggest that EVE and PLI should be evaluated for their clinical activity against CMS4 CRC.
Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Proliferation; Colorectal Neoplasms; Cytoskeletal Proteins; Everolimus; Humans; Mice; Plicamycin
PubMed: 34675191
DOI: 10.1038/s41419-021-04270-x -
BMC Cancer Nov 2011Tumor Necrosis Factor-α Related Apoptosis Inducing Ligand (TRAIL) and agonistic antibodies to death receptor 4 and 5 are promising candidates for cancer therapy due to...
BACKGROUND
Tumor Necrosis Factor-α Related Apoptosis Inducing Ligand (TRAIL) and agonistic antibodies to death receptor 4 and 5 are promising candidates for cancer therapy due to their ability to induce apoptosis selectively in a variety of human cancer cells, while demonstrating little cytotoxicity in normal cells. Although TRAIL and agonistic antibodies to DR4 and DR5 are considered safe and promising candidates in cancer therapy, many malignant cells are resistant to DR-mediated, TRAIL-induced apoptosis. In the current work, we screened a small library of fifty-five FDA and foreign-approved anti-neoplastic drugs in order to identify candidates that sensitized resistant prostate and pancreatic cancer cells to TRAIL-induced apoptosis.
METHODS
FDA-approved drugs were screened for their ability to sensitize TRAIL resistant prostate cancer cells to TRAIL using an MTT assay for cell viability. Analysis of variance was used to identify drugs that exhibited synergy with TRAIL. Drugs demonstrating the highest synergy were selected as leads and tested in different prostate and pancreatic cancer cell lines, and one immortalized human pancreatic epithelial cell line. Sequential and simultaneous dosing modalities were investigated and the annexin V/propidium iodide assay, in concert with fluorescence microscopy, was employed to visualize cells undergoing apoptosis.
RESULTS
Fourteen drugs were identified as having synergy with TRAIL, including those whose TRAIL sensitization activities were previously unknown in either prostate or pancreatic cancer cells or both. Five leads were tested in additional cancer cell lines of which, doxorubicin, mitoxantrone, and mithramycin demonstrated synergy in all lines. In particular, mitoxantrone and mithramycin demonstrated significant synergy with TRAIL and led to reduction of cancer cell viability at concentrations lower than 1 μM. At these low concentrations, mitoxantrone demonstrated selectivity toward malignant cells over normal pancreatic epithelial cells.
CONCLUSIONS
The identification of a number of FDA-approved drugs as TRAIL sensitizers can expand chemotherapeutic options for combination treatments in prostate and pancreatic cancer diseases.
Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Female; Humans; Male; Pancreatic Neoplasms; Prostatic Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured
PubMed: 22044796
DOI: 10.1186/1471-2407-11-470 -
Structure (London, England : 1993) May 2021Fusion products with the ETS family of transcription factors play critical roles in the etiology of several cancers. In this issue of Structure, Hou et al. (2020)...
Fusion products with the ETS family of transcription factors play critical roles in the etiology of several cancers. In this issue of Structure, Hou et al. (2020) provide insight into allosteric mechanisms by which mithramycin and its analogs perturb protein-DNA interactions in higher-order complexes at a DNA enhancer site.
Topics: Base Sequence; DNA; Plicamycin; Transcription Factors
PubMed: 33961789
DOI: 10.1016/j.str.2021.03.010 -
Cytometry Jan 1986Cells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with...
Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde.
Cells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide. With these DNA-fluorochromes, the fluorescence is quenched by formaldehyde less in mitotic nuclei than in interphase nuclei. Mitotic nuclei have a 20-40% increased mithramycin fluorescence and 30-60% decreased light scatter in comparison to those of G2 nuclei. There is a high correlation (r = 0.95; P less than 0.001) between microscope counts of mitotic figures in smear preparations of the initial cell suspension and the flow cytometrically estimated fraction of nuclei with increased mithramycin fluorescence. Flow sorting (FACS) demonstrates that the mitotic nuclei are confined to the peak of increased mithramycin fluorescence and decreased light scatter. The method has been applied to cultures of Yoshida ascites tumor cells, JB-1 reticulosarcoma cells, and PHA-stimulated human lymphocytes, incubated in the presence or absence of vinblastine for mitotic arrest. In a heteroploid mixture of fixed Yoshida (near-diploid) and JB-1 (hypotetraploid) nuclei, the mitotic fractions of the two cell lines could be estimated separately when analyzed with mithramycin fluorescence versus light scatter or with mithramycin fluorescence versus propidium iodide fluorescence.
Topics: Animals; Carcinoma, Hepatocellular; Detergents; Ethidium; Fixatives; Flow Cytometry; Fluorescent Dyes; Formaldehyde; Humans; Interphase; Liver Neoplasms; Liver Neoplasms, Experimental; Mice; Mitosis; Plicamycin; Propidium; Rats; Scattering, Radiation; Staining and Labeling; Vinblastine
PubMed: 2419056
DOI: 10.1002/cyto.990070108