The DOI matches its Crossref record; 55 of the 55 references it cites were found in a registry or an index; 3 of the 30 sources its claims cite were checked for support.
Echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity.
Open accessPublished version of a preprint
Abstract
The identification of small molecules that inhibit the sequence-specific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1alpha and HIF-1beta proteins containing the basic-helix-loop-helix and PAS domains. Expressed recombinant HIF-1alpha and HIF-1beta proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1-targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a small-molecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1alpha and HIF-1beta proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-kappaB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters.
Overview
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Written from the paper’s full text.
TL;DR
Echinomycin (NSC-13502) was identified from a HIF-1-targeted compound screen as a sequence-specific DNA-binding inhibitor that blocks HIF-1 binding to hypoxia-responsive elements and reduces HIF-1-dependent transcription in U251 cells. It appears to act by binding DNA rather than by lowering HIF-1α protein, with selectivity over AP-1 and NF-κB in the assays used.
Key findings
- Of 128 active compounds from an NCI 140,000-compound HIF-1 cell-based screen, 20 inhibited HIF-1 DNA-binding activity by more than 50% at 10 µM; 12 of those also inhibited AP-1 and/or NF-κB binding, leaving 8 with relative HIF-1 selectivity.
- NSC-13502 dose-dependently inhibited recombinant HIF-1α/HIF-1β DNA binding in ELISA, with nearly complete inhibition at 320 nM, and in EMSA it did not inhibit AP-1 or NF-κB binding up to 10 µM.
- In ChIP assays, 0.5 µM NSC-13502 abolished HIF-1 binding to the VEGF promoter HRE but did not affect c-Fos binding to the cyclin D1 promoter or p65 binding to the ICAM-1 promoter; HIF-1α protein levels were not reduced.
- NSC-13502 inhibited hypoxic luciferase induction in U251-HRE cells with an EC50 of about 1.2 nM, blocked the roughly 5-fold hypoxic increase in VEGF mRNA (P < 0.01), and in microarray profiling 14 of 17 hypoxia-induced HIF-1 target genes were inhibited more than 3-fold.
Methods
- Recombinant truncated HIF-1α bHLH-PAS (amino acids 1–389) and HIF-1β bHLH-PAS (amino acids 1–475) proteins were expressed and used in a 96-well DNA-binding ELISA and EMSA with HRE, mutant HRE, AP-1, NF-κB, and E-box oligonucleotides; 128 compounds were screened at 10 µM.
- U251, U251-HRE, U251-pGL3, and MCF-7 cells were cultured under normoxia or 1% O2 hypoxia; assays included ChIP for VEGF, cyclin D1, and ICAM-1 promoters, Western blotting, luciferase reporter measurement, real-time PCR for VEGF, DNA thermal melting and ethidium bromide displacement, SRB viability, and oligonucleotide microarray analysis of about 11,000 genes.
- Main analyses compared dose-dependent inhibition, competition and supershift controls, promoter binding specificity, reporter activity, mRNA expression, and global gene-expression changes.
Limitations
- The paper states that NSC-13502’s sequence specificity may not be limited to HIF-1; it also inhibited nuclear protein binding to an E-box oligonucleotide, the binding site for Myc/Max.
- The paper states that in vivo feasibility and whether alternative dosing schedules could improve therapeutic potential remain to be established, and notes that earlier phase II clinical trials found minimal or no antitumor activity.
- Evident from the text: most mechanistic evidence comes from cell-free DNA-binding assays and cultured U251/MCF-7 cells, with no in vivo tumor efficacy experiment reported here; ChIP used 0.5 µM while the luciferase EC50 was about 1.2 nM, indicating different potency across assay contexts.
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Claims the paper makes
43 Own result · 16 No full text of the sources · 11 Own interpretation · 2 Not checked yet · 1 Supported · 1 Supported so far
The sources with a verdict support this claim; the rest weren’t checked yet, have no full text we could read, or their verdicts aren’t rated yet.
In the paper
“Notably, HIF-1a is overexpressed in human cancers due to hypoxia-dependent and/or hypoxia-independent pathways (6,18,19)”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Discussion
What the sources say
- Supportedcites [6] Karni R, Dor Y, Keshet E, Meyuhas O, Levitzki A. Activated pp60c-Src leads to elevated hypoxia-inducible factor (HIF)-1α expression under normoxia. J Biol Chem 2002; 277: 42919–25. source
The introduction says HIF-1α is stabilized under hypoxia and that many malignant tumors have high HIF-1α levels under normoxia. The Discussion identifies enhanced HIF-1α translation as a mechanism for its strong expression in many advanced tumors under normoxia. Together, these passages support hypoxia-dependent and hypoxia-independent pathways. (LLM, conf=0.90)
- Not checked — no full text of the sourcecites [18] Semenza G. Signal transduction to hypoxia-inducible factor 1. Biochem Pharmacol 2002; 64: 993–8. source
- Supportedcites [19] Laughner E, Taghavi P, Chiles K, Mahon PC, Semenza GL. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1α (HIF-1α) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol 2001; 21: 3995–4004. source
The Abstract states that HIF-1α is overexpressed in primary and metastatic human tumors and that HIF-1 activity increases through both intratumoral hypoxia and genetic alterations. The Discussion further describes hypoxia and altered signaling as mechanisms that increase HIF-1α expression. (LLM, conf=0.93)
plaindirect- Asserted: passes — The authors put it forward as true.
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- Supported
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We report here that echinomycin (NSC-13502) inhibits HIF-1 DNA-binding activity.”
- Also stated in: § Results · plain
“ELISA experiments (Fig. 2B) showed that NSC-13502 potently inhibited HIF-1a-389 and HIF-1h-475 DNA-binding activity in a dosedependent fashion, with almost complete inhibition at 320 nmol/L.”
- Also stated in: § Abstract · plain
“Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1A and HIF-1B proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-KB (NF-KB) consensus sequences.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Introduction
plaindirect- Asserted: passes — The authors put it forward as true.
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- Also stated in: § Results · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In contrast, HIF-1a mRNA expression, which was slightly decreased under hypoxic conditions relative to normoxia, was not affected by NSC-13502 (data not shown).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plain- Asserted: passes — The authors put it forward as true.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Treatment of U251 cells with NSC-13502 completely abrogated HIF-1 DNA-binding activity to the VEGF promoter.”
- Also stated in: § Abstract · plain
“Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-KB to promoter regions of corresponding target genes.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plaindirect- Asserted: passes — The authors put it forward as true.
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- Based: passes — The paper gives a reason why it holds.
- Also stated in: § Abstract · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“More importantly, NSC-13502 up to 10 Amol/L did not inhibit DNA-binding activities induced by c-Fos and JunD to the corres-ponding AP-1 DNA-binding sequence (Fig. 2C-b)”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plain- Asserted: passes — The authors put it forward as true.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In addition, cyclin D1 protein levels were not affected by treatment under hypoxia or with NSC-13502, consistent with data obtained in the ChIP assay.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plain- Asserted: passes — The authors put it forward as true.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Treatment of hypoxic U251 cells with NSC-13502 had a minimal effect on both constitutive and inducible HIF-1 DNA-binding activities detected in nuclear extracts (Fig. 2D-b, lane 3).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“In contrast, NSC-13502 did not significantly affect luciferase expression in the U251-pGL3 control cell line under either normoxic or hypoxic conditions (Fig. 4B).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“As shown in Fig. 4A, NSC-13502 very potently inhibited hypoxic induction of luciferase expression in U251-HRE in a dose-dependent fashion with an EC 50 of f1.2 nmol/L.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“More importantly, NSC-13502 up to 10 Amol/L did not inhibit DNA-binding activities induced by c-Fos and JunD to the corres-ponding AP-1 DNA-binding sequence (Fig. 2C-b) or the binding of p65 to the NF-nB consensus sequence (Fig. 2C-c).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plain- Asserted: passes — The authors put it forward as true.
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- Probably not a substantial claim. 9
- It may state two separate claims as one. 17
Every source the paper cites for this claim was checked and supports it.
In the paper
“Interestingly, a synthetic pyrroleimidazole polyamide that inhibits in a sequence-specific fashion HIF binding to the HRE of the VEGF promoter (44) caused 264 and 73 genes to be down-regulated and up-regulated by 2-fold, respectively, at a concentration of 1 Amol/L.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Discussion
What the sources say
- Supportedcites [44] Olenyuk BZ, Zhang GJ, Klco JM, et al. Inhibition of vascular endothelial growth factor with a sequence-specific hypoxia response element antagonist. Proc Natl Acad Sci U S A 2004; 101: 16768–73. source
Split into 2 atoms — 2 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
- Supported
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“NSC-13502 caused f50% displacement of ethidium bromide on HRE oligonucleotide at 3 Amol/L, whereas no significant influence on AP-1 oligonucleotide was observed even at a concentration of 10 Amol/L (data not shown).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plaindirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“These results clearly show that NSC-13502 specifically inhibits HIF-1, but not AP-1 or NF-nB, binding activity to endogenous promoters in cultured cells.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
boosteddirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“451 versus 276 by z2-fold and 65 versus 36 by z4-fold”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plaindirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“More importantly, NSC-13502 under the same experimental conditions did not block the binding of c-Fos to the AP-1 site of the cyclin D1 promoter or the binding of p65 to the NF-nB site of the ICAM-1 promoter.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Discussion
plain- Asserted: passes — The authors put it forward as true.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“only 186 genes were up-regulated under normoxia and 197 under hypoxia.”
- Also stated in: § Discussion · plain
“addition of NSC-13502 to U251 cells cultured under normoxic conditions only down-regulated or up-regulated 276 and 186 genes, respectively, by z2-fold.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plaindirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
- Also stated in: § Discussion · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Figure 2A shows the chemical structure of NSC-13502, one of the most potent compounds identified in the HIF-1 cell-based high-throughput screen and further validated using DNA-binding assays.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plain- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In contrast, HIF-1a mRNA expression, which was slightly decreased under hypoxic conditions relative to normoxia, was not affected by NSC-13502 (data not shown).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“276 and 451 genes were down-regulated by z2-fold under normoxic or hypoxic conditions, respectively, in the presence of NSC-13502 (Fig. 5B).”
- Also stated in: § Discussion · plain
“addition of NSC-13502 to U251 cells cultured under normoxic conditions only down-regulated or up-regulated 276 and 186 genes, respectively, by z2-fold.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
plaindirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
- Also stated in: § Discussion · plain
In the paper
“HIF-1a and HIF-1h belong to the basic-helix-loop-helix (bHLH)-PAS family of transcription factors.The basic domain is responsible for sequence-specific DNA binding, whereas the HLH and the PAS domains mediate dimerization and stabilization of the complex on the DNA (12).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Introduction
What the sources say
- Not checked — no full text of the sourcecites [12] Jiang BH, Rue E, Wang GL, Roe R, Semenza GL. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem 1996; 271: 17771–8. source
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
In the paper
“HIF-1 activates the transcription of genes whose products are involved in crucial aspects of cancer biology, including cell survival, glycolysis, angiogenesis, migration, and invasion (5).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Introduction
What the sources say
- Not checked — no full text of the sourcecites [5] Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003; 3: 721–32. source
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Results shown thus far indicate that NSC-13502 inhibits HIF-1, but not AP-1 or NF-nB, DNA-binding activity in vitro by binding DNA in a sequence-specific fashion.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
hedgeddirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A claim is checked only against the cited paper’s full text, never its abstract, and none is available for its sources.
In the paper
“Interestingly, NSC-13502 was originally discovered as a sequence-specific DNA-binding agent (15), whose sequence specificity encompasses the core sequence (5V-CGTG-3V) of HRE consensus sequence 5V-R(A/ G)CGTG-3V (12).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
What the sources say
- Not checked — no full text of the sourcecites [15] Van Dyke MM, Dervan PB. Echinomycin binding sites on DNA. Science 1984; 225: 1122–7. source
- Not checked — no full text of the sourcecites [12] Jiang BH, Rue E, Wang GL, Roe R, Semenza GL. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem 1996; 271: 17771–8. source
plain- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“Hypoxia-inducible factor-1 (HIF-1), a heterodimer composed of two subunits, HIF-1a and HIF-1h (also known as aryl hydrocarbon receptor nuclear translocator), mediates the transcriptional program to oxygen deprivation (3).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Introduction
What the sources say
- Not checked — no full text of the sourcecites [3] Semenza GL. Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology. Trends Mol Med 2001; 7: 345–50. source
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. It may state two separate claims as one.
In the paper
“More importantly, we have shown that topotecan administered on a chronic schedule inhibits HIF-1a protein accumulation, angiogenesis, and tumor growth in U251-HRE human glioblastoma xenografts (38).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Discussion
What the sources say
- Not checked — no full text of the sourcecites [38] Rapisarda A, Zalek J, Hollingshead M, et al. Schedule-dependent inhibition of hypoxia-inducible factor-1α protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts. Cancer Res 2004; 64: 6845–8. source
boosteddirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
In the paper
“The strong binding sites for NSC-13502 contain the central 2-bp sequence 5V-CG-3V, and the key recognition elements for NSC-13502 are contained in the sequences 5V-ACGT-3V and 5V-TCGT-3V (15).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Discussion
What the sources say
- Not checked — no full text of the sourcecites [15] Van Dyke MM, Dervan PB. Echinomycin binding sites on DNA. Science 1984; 225: 1122–7. source
plaincorrelational- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
In the paper
“One example is the Myc/ Max family of transcription factors, whose DNA-binding site (E-box, 5V-CACGTG-3V; ref. 46) encompasses the core sequence 5V-CGTG-3V, which is also present in the HIF-1 consensus sequence and is part of the sequence specificity of NSC-13502.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Discussion
What the sources say
- Not checked — no full text of the sourcecites [46] Nair SK, Burley SK. X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors. Cell 2003; 112: 193–205. source
plaincorrelational- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
In the paper
“NSC-13502 was brought into clinical trials by the NCI 20 years ago based on its antitumor activity against two i.p. implanted murine tumors, the B16 melanoma and the P388 leukemia (47).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Discussion
What the sources say
- Not checked — no full text of the sourcecites [47] Foster BJ, Clagett-Carr K, Shoemaker DD, et al. Echinomycin: the first bifunctional intercalating agent in clinical trials. Invest New Drugs 1985; 3: 403–10. source
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“Our group has recently shown that topotecan, a topoisomerase I poison, inhibits HIF-1a protein accumulation independently of DNA replication-mediated DNA damage, suggesting a distinct mechanism of action from the cytotoxic effects (35).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Discussion
What the sources say
- Not checked — no full text of the sourcecites [35] Rapisarda A, Uranchimeg B, Sordet O, et al. Topoisomerase I-mediated inhibition of hypoxia-inducible factor 1: mechanism and therapeutic implications. Cancer Res 2004; 64: 1475–82. source
hedgeddirect- Asserted: passes — The authors put it forward as true.
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- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“consistent with the notion that NSC-13502 binds DNA and not HIF-1a and/or HIF-1h proteins”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Discussion
hedged- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Most of the agents described above affect targets that are also involved in other biological functions, which will ultimately determine their therapeutic activity, and they can be defined nonselective inhibitors of HIF-1.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Discussion
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
In the paper
“HIF-1a is the downstream mediator of hypoxic and nonhypoxic signaling pathways, which play a crucial role in cancer progression.”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Discussion
What the sources say
- Not checked — no full text of the sourcecites [5] Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003; 3: 721–32. source
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
The authors state this as an assumption.
NSC-13502 inhibits HIF-1 DNA-binding activity in vitro by binding DNA in a sequence-specific fashion.
“Results shown thus far indicate that NSC-13502 inhibits HIF-1, but not AP-1 or NF-nB, DNA-binding activity in vitro by binding DNA in a sequence-specific fashion.If this assumption were correct, a ChIP assay would show whether NSC-13502 inhibited endogenous HIF-1 DNA-binding activity in cultured cells.”
The paper relies on this without support.
The core sequence of the HRE (5'-CGTG-3') is also present in the E-box (5'-CACGTG-3'), the DNA-binding site of the Myc/Max complex.
“The core sequence of the HRE (5V -CGTG-3V) is also present in the E-box (5V-CACGTG-3V), which is the DNA-binding site of the Myc/Max complex, raising the possibility that NSC-13502 might also inhibit the binding of nuclear proteins to the E-box.”
The paper relies on this without support.
The HRE recognized by HIF-1 contains the core sequence 5'-R(A/G)CGTG-3' and is present in promoters of HIF-1 target genes.
“The DNA-binding site recognized by HIF-1, known as hypoxia-responsive element (HRE), contains the core sequence 5V -R(A/G)CGTG-3V , which is present in promoters of HIF-1 target genes.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Echinomycin preferentially intercalates into DNA at sites containing the central 5′-CG-3′ dinucleotide, a critical recognition element within the HIF consensus HRE sequence (5′-ACGT-3′), thereby preventing binding of both HIF-1α and HIF-2α to their cognate response elements [383,384].”
From HIF inhibition: Current strategies and clinical challenges (2026) · cites this paper as [383]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“As a result, despite its potent ability to block HIF–DNA interactions, echinomycin exhibits limited specificity and a high propensity for off-target transcriptional effects.”
From HIF inhibition: Current strategies and clinical challenges (2026) · cites this paper as [383]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Echinomycin also binds DNA motifs recognized by other transcription factors, such as AP-1, NF-κB, or members of the Myc/Max family, whose E-box binding site (5′-CACGTG-3′) overlaps substantially with the HIF HRE core [383,385].”
From HIF inhibition: Current strategies and clinical challenges (2026) · cites this paper as [383]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Echinomycin is a potent small-molecule inhibitor of HIF-1α, which functions through sequence-specific binding to HRE to competitively inhibit HIF-1α binding to its target genes9.”
From Liposomal Formulation of HIF-1α Inhibitor Echinomycin Eliminates Established Metastases of Triple-negative Breast Cancer (2020) · cites this paper as [9]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“It binds specifically to the core of the HIF-1 recognition sequence 5′–CGTG-3′ in a sequence-dependent fashion precluding HIF-1 and DNA binding.”
From HIF-1α pathway: role, regulation and intervention for cancer therapy (2015) · cites this paper as [49]
In the citing paper
“The use of echinomycin, an inhibitor that has been characterized to inhibit HIF activity without modulating HIF-α subunit protein levels (23), led also to partial restoration of insulin-induced tyrosine phosphorylation of the insulin receptor in hypoxia (data not shown).”
From Hypoxia decreases insulin signaling pathways in adipocytes. (2009) · cites this paper as [23]
In the citing paper
“the HIF complex-induced binding to HRE regions in promoters of various genes can be achieved by echinomycin [87].”
From Hypoxia Signaling in Cancer: From Basics to Clinical Practice (2021) · cites this paper as [87]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
“Twenty of 128 compounds inhibited HIF-1h-389 and HIF-1h-475 DNA-binding activity by >50% relative to untreated control at the 10 Amol/L concentration (data not shown).”
§ Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity > Results
“we expressed recombinant HIF-1a-bHLH-PAS-His (HIF-1a-389) and Flag-HIF-1h-bHLH-PAS (HIF-1h-475) proteins”
Citations
Cites
Targeting HIF-1 for cancer therapy
Semenza, G. L. (2003) · Nature Reviews Cancer · doi:10.1038/nrc1187 · Cited by 6,717
Wang, G. L., Jiang, B. H., Rue, E. A., & Semenza, G. L. (1995) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.92.12.5510 · Cited by 6,212
The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis
Maxwell, P. H., Wiesener, M. S., Chang, G.-W., Clifford, S. C., Vaux, E. C., Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R., & Ratcliffe, P. J. (1999) · Nature · doi:10.1038/20459 · Cited by 5,180
Monks, A. P., Scudiero, D. A., Skehan, P., Shoemaker, R. H., Paull, K. D., Vistica, D. T., Hose, C. D., Langley, J. W., Cronise, P., Vaigro-Wolff, A., et al. (1991) · JNCI Journal of the National Cancer Institute · doi:10.1093/jnci/83.11.757 · Cited by 3,305
Self-citation(1999) · PubMed · Cited by 2,204
Small-molecule inhibitors of protein–protein interactions: progressing towards the dream
Arkin, M. R., & Wells, J. A. (2004) · Nature Reviews Drug Discovery · doi:10.1038/nrd1343 · Cited by 1,705
(2000) · PubMed · Cited by 1,445
Laughner, E., Taghavi, P., Chiles, K., Mahon, P. C., & Semenza, G. L. (2001) · Molecular and Cellular Biology · doi:10.1128/mcb.21.12.3995-4004.2001 · Cited by 1,256 · cited 1×
“Notably, HIF-1a is overexpressed in human cancers due to hypoxia-dependent and/or hypoxia-independent pathways (6, 18, 19), and overexpression of HIF-1a has been implicated as a poor prognostic indicator in a variety of tumors (20–23).”
BackgroundTranscription factors as targets for cancer therapy
Darnell, J. E. (2002) · Nature Reviews Cancer · doi:10.1038/nrc906 · Cited by 1,194
Hudson, C. C., Liu, M., Chiang, G. G., Otterness, D. M., Loomis, D. C., Kaper, F., Giaccia, A. J., & Abraham, R. T. (2002) · Molecular and Cellular Biology · doi:10.1128/mcb.22.20.7004-7014.2002 · Cited by 1,186
Cited by
Sorted by citations
Tumor cell metabolism: cancer's Achilles' heel
Kroemer, G., & Pouyssegur, J. (2008) · Cancer Cell · doi:10.1016/j.ccr.2008.05.005 · Cited by 2,337
HIF-1α pathway: role, regulation and intervention for cancer therapy
Masoud, G. N., & Li, W. (2015) · Acta Pharmaceutica Sinica B · doi:10.1016/j.apsb.2015.05.007 · Cited by 1,871
Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics
Semenza, G. L. (2010) · Oncogene · doi:10.1038/onc.2009.441 · Cited by 1,712
“intercalating drugs, including echinomycin (Kong et al., 2005) and anthracyclines, such as doxorubicin (Adriamycin) and daunorubicin (Lee et al.”
Hypoxia signalling in cancer and approaches to enforce tumour regression
Pouysségur, J., Dayan, F., & Mazure, N. M. (2006) · Nature · doi:10.1038/nature04871 · Cited by 1,676
Hypoxia-Induced Angiogenesis: Good and Evil
Krock, B. L., Skuli, N., & Simon, M. C. (2011) · Genes & Cancer · doi:10.1177/1947601911423654 · Cited by 1,236
Energy metabolism in tumor cells
Moreno‐Sánchez, R., Rodríguez‐Enríquez, S., Marín‐Hernández, Á., & Saavedra, E. (2007) · FEBS Journal · doi:10.1111/j.1742-4658.2007.05686.x · Cited by 1,121
Therapeutic targeting of hypoxia and hypoxia-inducible factors in cancer
Wigerup, C., Påhlman, S., & Bexell, D. (2016) · Pharmacology & Therapeutics · doi:10.1016/j.pharmthera.2016.04.009 · Cited by 687
“Echinomycin is a small antibiotic molecule isolated from Streptomyces echinatus that binds DNA in a sequence-specific manner.”
BackgroundInfluentialTriterpenoids and rexinoids as multifunctional agents for the prevention and treatment of cancer
Liby, K. T., Yore, M. M., & Sporn, M. B. (2007) · Nature Reviews Cancer · doi:10.1038/nrc2129 · Cited by 637
Rohwer, N., & Cramer, T. (2011) · Drug Resistance Updates · doi:10.1016/j.drup.2011.03.001 · Cited by 632
Hypoxia-inducible factors: cancer progression and clinical translation
Wicks, E. E., & Semenza, G. L. (2022) · Journal of Clinical Investigation · doi:10.1172/jci159839 · Cited by 626
“HIF inhibitors that have shown antitumor activity in mouse tumor models include acriflavine (47), digoxin (106), echinomycin (156), the HSP90 inhibitors 17-allylaminogeldanamycin (157) and ganetespib (158), 2-methoxyestradiol (159), PX-478 (160), and YC-1 (161).”
BackgroundCancer Cell Metabolism in Hypoxia: Role of HIF-1 as Key Regulator and Therapeutic Target
Infantino, V., Santarsiero, A., Convertini, P., Todisco, S., & Iacobazzi, V. (2021) · International Journal of Molecular Sciences · doi:10.3390/ijms22115703 · Cited by 541
“doxorubicin, impair binding of HIF to chromatin, resulting in blocking of its transcriptional activity [146,147].”
BackgroundN‐Methylation of Peptides and Proteins: An Important Element for Modulating Biological Functions
Chatterjee, J., Rechenmacher, F., & Kessler, H. (2012) · Angewandte Chemie International Edition · doi:10.1002/anie.201205674 · Cited by 509
Angiogenesis Inhibitors: Current Strategies and Future Prospects
Cook, K. M., & Figg, W. D. (2010) · CA A Cancer Journal for Clinicians · doi:10.3322/caac.20075 · Cited by 487
Hypoxia-inducible factors: central regulators of the tumor phenotype
Gordan, J. D., & Simon, M. C. (2007) · Current Opinion in Genetics & Development · doi:10.1016/j.gde.2006.12.006 · Cited by 458
Autophagy in cancer associated fibroblasts promotes tumor cell survival
Martinez‐Outschoorn, U., Trimmer, C., Zhao, L., Whitaker‐Menezes, D., Chiavarina, B., Zhou, J., Wang, C., Pavlides, S., Cantarin, M. P. M., Capozza, F., et al. (2010) · Cell Cycle · doi:10.4161/cc.9.17.12928 · Cited by 413
Targeting HIF1α Eliminates Cancer Stem Cells in Hematological Malignancies
Wang, Y., Liu, Y., Malek, S. N., Zheng, P., & Liu, Y. (2011) · Cell stem cell · doi:10.1016/j.stem.2011.02.006 · Cited by 410
Endoplasmic Reticulum Stress and Cancer
Yadav, R. K., Chae, S., Kim, H., & Chae, H. (2014) · Journal of Cancer Prevention · doi:10.15430/jcp.2014.19.2.75 · Cited by 402
“Therefore, blocking the HIF1α signal might be a novel and promising therapeutic target for the treatment of hypoxic tumors.(21)”
BackgroundGLUT1 Expression Is Increased in Hepatocellular Carcinoma and Promotes Tumorigenesis
Amann, T., Maegdefrau, U., Hartmann, A., Agaimy, A., Marienhagen, J., Weiß, T. S., Stoeltzing, O., Warnecke, C., Schölmerich, J., Oefner, P. J., et al. (2009) · American Journal Of Pathology · doi:10.2353/ajpath.2009.080596 · Cited by 370
Targeting Transcription Factors for Cancer Treatment
Lambert, M., Jambon, S., Depauw, S., & David‐Cordonnier, M. (2018) · Molecules · doi:10.3390/molecules23061479 · Cited by 359
“In terms of transcription factor inhibition, echinomycin was identified as a HIF1α and HIF1β/DNA binding inhibitor as evidenced using EMSA, luciferase activity and ChIP experiments on HIF-1 response elements (HRE) of the vascular endothelial growth factor promoter (VEGF, associated with tumor vascularization) [277].”
MethodTargeting hypoxia signalling for the treatment of ischaemic and inflammatory diseases
Eltzschig, H. K., Bratton, D. L., & Colgan, S. P. (2014) · Nature Reviews Drug Discovery · doi:10.1038/nrd4422 · Cited by 364
Cairns, R. A., Papandreou, I., & Denko, N. C. (2006) · Molecular Cancer Research · doi:10.1158/1541-7786.mcr-06-0002 · Cited by 375
“Several small-molecule inhibitors of HIF have been identified (119-122) and are now being tested for antitumor activity in preclinical trials.”
BackgroundEvaluation of HIF-1 inhibitors as anticancer agents
Semenza, G. L. (2007) · Drug Discovery Today · doi:10.1016/j.drudis.2007.08.006 · Cited by 363
Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis
Chourasia, A. H., Tracy, K., Frankenberger, C. A., Boland, M. L., Sharifi, M. N., Drake, L. E., Sachleben, J. R., Asara, J. M., Locasale, J. W., Karczmar, G. S., et al. (2015) · EMBO Reports · doi:10.15252/embr.201540759 · Cited by 344
“To assess to what extent elevated Hif levels contributed to the observed hyper-proliferative phenotype (Fig 1H), we treated cells with 10 ng/ml echinomycin (a small molecule inhibitor of Hif DNA binding activity) [25] and confirmed that echinomycin reduced expression of key Hif target genes (Fig 5E).”
Up-down regulation of HIF-1α in cancer progression
Rashid, M., Zadeh, L. R., Baradaran, B., Molavi, O., Ghesmati, Z., Sabzichi, M., & Ramezani, F. (2021) · Gene · doi:10.1016/j.gene.2021.145796 · Cited by 302
Jensen, R. L. (2009) · Journal of Neuro-Oncology · doi:10.1007/s11060-009-9827-2 · Cited by 294
“Using similar methods, we have been working on developing HRE-controlled expression of short hairpin vector-expressing inhibitory RNA for specific posttran- scriptional gene silencing (Fig.”
BackgroundInfluentialPharmacologic Targeting of Hypoxia-Inducible Factors
Semenza, G. L. (2019) · Annual Review of Pharmacology and Toxicology · doi:10.1146/annurev-pharmtox-010818-021637 · Cited by 288
Hypoxia decreases insulin signaling pathways in adipocytes.
Regazzetti, C., Peraldi, P., Grémeaux, T., Najem-Lendom, R., Ben-Sahra, I., Cormont, M., Bost, F., Le Marchand-Brustel, Y., Tanti, J.-F., & Giorgetti-Peraldi, S. (2009) · Diabetes · doi:10.2337/db08-0457 · Cited by 281
“The use of echinomycin, an inhibitor that has been characterized to inhibit HIF activity without modulating HIF- subunit protein levels (23), led also to partial nm ol D O G /m g pr ot /3 m in”
Lactate Activates HIF-1 in Oxidative but Not in Warburg-Phenotype Human Tumor Cells
Saedeleer, C. J. D., Copetti, T., Porporato, P. E., Verrax, J., Féron, O., & Sonveaux, P. (2012) · PLoS ONE · doi:10.1371/journal.pone.0046571 · Cited by 283
Recent advances in hypoxia-inducible factor (HIF)-1 inhibitors
Xia, Y., Choi, H.-K., & Lee, K. (2012) · European Journal of Medicinal Chemistry · doi:10.1016/j.ejmech.2012.01.033 · Cited by 278
Targeting Protein–Protein Interactions for Therapeutic Intervention: A Challenge for the Future
Zinzalla, G., & Thurston, D. E. (2009) · Future Medicinal Chemistry · doi:10.4155/fmc.09.12 · Cited by 266
Qiu, G., Jin, M., Dai, J., Sun, W., Feng, J., & Jin, W. (2017) · Trends in Pharmacological Sciences · doi:10.1016/j.tips.2017.05.002 · Cited by 250
Development of HIF‐1 inhibitors for cancer therapy
Onnis, B., Rapisarda, A., & Melillo, G. (2009) · Journal of Cellular and Molecular Medicine · doi:10.1111/j.1582-4934.2009.00876.x · Cited by 268
Self-citationPathania, D., Millard, M., & Neamati, N. (2009) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2009.05.010 · Cited by 269
Broekgaarden, M., Weijer, R., van Gulik, T. M., Hamblin, M. R., & Heger, M. (2015) · Cancer and Metastasis Reviews · doi:10.1007/s10555-015-9588-7 · Cited by 266
Inhibition of mitophagy drives macrophage activation and antibacterial defense during sepsis
Patoli, D., Mignotte, F., Deckert, V., Dusuel, A., Dumont, A., Rieu, A., Jalil, A., Dongen, K. V., Bourgeois, T., Gautier, T., et al. (2020) · Journal of Clinical Investigation · doi:10.1172/jci130996 · Cited by 251
“Cheng SC, et al. mTOR- and HIF-1α-mediated aerobic glycolysis as metabolic basis for trained immunity.”
BackgroundInfluentialTargeting hypoxia cell signaling for cancer therapy
Melillo, G. (2007) · Cancer and Metastasis Reviews · doi:10.1007/s10555-007-9059-x · Cited by 230
“Echinomycin was found to potently inhibit HIF-1 DNA binding activities both in ELISA and EMSA [ 91 ].”
BackgroundSelf-citationMolecular mechanisms mediating metastasis of hypoxic breast cancer cells
Semenza, G. L. (2012) · Trends in Molecular Medicine · doi:10.1016/j.molmed.2012.08.001 · Cited by 225
Metabolic control of the Treg/Th17 axis
Barbi, J. J., Pardoll, D. M., & Pan, F. (2013) · Immunological Reviews · doi:10.1111/imr.12029 · Cited by 227
“For instance, the well-characterized HIF-1 inhibiting compounds digoxin, acriflavine, and echinomycin suppress HIF-1 activity by reducing its expression (55), preventing HIF-1a/HIF-1b dimer formation (56), and interfering with HIF-1 DNA binding (58, 59), respectively.”
BackgroundTargeting the hypoxia-inducible factor (HIF) pathway in cancer
Poon, E., Harris, A. L., & Ashcroft, M. A. (2009) · Expert Reviews in Molecular Medicine · doi:10.1017/s1462399409001173 · Cited by 215
The hypoxic microenvironment: A determinant of cancer stem cell evolution
Carnero, A., & LLeonart, M. E. (2016) · BioEssays · doi:10.1002/bies.201670911 · Cited by 204
Kang, J., Kim, E., Kim, W., Seong, K. M., Youn, H., Kim, J. W., Kim, J., & Youn, B. (2013) · Journal of Biological Chemistry · doi:10.1074/jbc.m113.490482 · Cited by 189
Development of Inhibitors Targeting Hypoxia-Inducible Factor 1 and 2 for Cancer Therapy
Yu, T., Tang, B., & Sun, X. (2017) · Yonsei Medical Journal · doi:10.3349/ymj.2017.58.3.489 · Cited by 202
“Echinomycin (quinomycin A), a small molecule known to bind DNA in a sequence-specific fashion, was shown to inhibit the binding of HIF-1 to the core HRE sequences (5’-CGTG-3’) contained in the VEGF promoter.(54,55)”
HIFs, angiogenesis, and cancer
Yang, Y., Sun, M., Wang, L., & Jiao, B. (2012) · Journal of Cellular Biochemistry · doi:10.1002/jcb.24438 · Cited by 193
A complex task? Direct modulation of transcription factors with small molecules
Koehler, A. N. (2010) · Current Opinion in Chemical Biology · doi:10.1016/j.cbpa.2010.03.022 · Cited by 189
“The compounds include the natural product echinomycin [66], a potent yet nonspecific intercalator that blocks HIF-1 DNA binding and transcription, and NSC 50253 [67], a modest inhibitor of PAS-A domain interactions involving HIF-1α and HIF-1β.”
BackgroundInterfering with Tumor Hypoxia for Radiotherapy Optimization
Telarović, I., Wenger, R. H., & Pruschy, M. N. (2021) · Journal of Experimental & Clinical Cancer Research · doi:10.1186/s13046-021-02000-x · Cited by 208
Metabolic targeting of hypoxia and HIF1 in solid tumors can enhance cytotoxic chemotherapy
Cairns, R. A., Papandreou, I., Sutphin, P. D., & Denko, N. C. (2007) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0611662104 · Cited by 183
Yang, L., Lin, C., Wang, L., Guo, H., & Wang, X. (2012) · Experimental Cell Research · doi:10.1016/j.yexcr.2012.07.017 · Cited by 190
HIF1A Reduces Acute Lung Injury by Optimizing Carbohydrate Metabolism in the Alveolar Epithelium
Eckle, T. D. L. G., Brodsky, K. S., Bonney, M., Packard, T. A., Han, J., Borchers, C. H., Mariani, T. J., Kominsky, D. J., Mittelbronn, M. G. A., & Eltzschig, H. K. (2013) · PLoS Biology · doi:10.1371/journal.pbio.1001665 · Cited by 166
Inhibiting Hypoxia-Inducible Factor 1 for Cancer Therapy
Melillo, G. (2006) · Molecular Cancer Research · doi:10.1158/1541-7786.mcr-06-0235 · Cited by 168
“Perhaps not unexpectedly, only a few examples of HIF inhibitors that potentially target selective pathways associated with HIF activation have been described, among which echinomycin (29) and synthetic polyamides (30) that inhibit HIF-1 DNA binding, and chetomin (31), which blocks recruitment of coactivator p300/CBP (…”
BackgroundSelf-citationHypoxia-inducible factor pathway and diseases of the vascular wall
Lim, C. S., Kiriakidis, S., Sandison, A., Paleolog, E. M., & Davies, A. H. (2013) · Journal of Vascular Surgery · doi:10.1016/j.jvs.2013.02.240 · Cited by 154
“Examples of HIF inhibitors include EZN-2968,(132) sorafenib,(131) and echinomycin.(133) On the other hand, drugs that increase the activity of HIF-a are being developed for diseases such as ischemic heart diseases, stroke, peripheral arterial diseases, and chronic anemia.”
BackgroundMahabeleshwar, G. H., Kawanami, D., Sharma, N., Takami, Y., Zhou, G., Shi, H., Nayak, L. V., Jeyaraj, D., Grealy, R., White, M., et al. (2011) · Immunity · doi:10.1016/j.immuni.2011.04.014 · Cited by 149
“Echinomycin is a well-established inhibitor of HIF-1α (Kong et al., 2005).”
Maybin, J. A., Murray, A. A., Saunders, P. T. K., Hirani, N., Carmeliet, P., & Critchley, H. (2018) · Nature Communications · doi:10.1038/s41467-017-02375-6 · Cited by 159
Vadlapatla, R. K., Vadlapudi, A. D., & Mitra, A. K. (2013) · Current Drug Targets · doi:10.2174/13894501113149990015 · Cited by 148
ROS-activated Anticancer Prodrugs: a New Strategy for tumor-specific Damage
Peng, X., & Gandhi, V. V. (2012) · Therapeutic Delivery · doi:10.4155/tde.12.61 · Cited by 148
Hypoxia-induced alternative splicing: the 11th Hallmark of Cancer
Farina, A. R., Cappabianca, L. A., Sebastiano, M., Zelli, V., Guadagni, S., & Mackay, A. R. (2020) · Journal of Experimental & Clinical Cancer Research · doi:10.1186/s13046-020-01616-9 · Cited by 136
Hypoxia Contributes to Melanoma Heterogeneity by Triggering HIF1α-Dependent Phenotype Switching
Widmer, D. S., Hoek, K. S., Cheng, P. F., Eichhoff, O. M., Biedermann, T., Raaijmakers, M. I. G., Hemmi, S., Dummer, R., & Levesque, M. P. (2013) · Journal of Investigative Dermatology · doi:10.1038/jid.2013.115 · Cited by 147
“To inhibit HIF1a transcriptional activity, we treated the cells with Echinomycin during exposure to hypoxia (1% O2 for 72 hours) (Kong et al., 2005).”
MethodHypoxia-Inducible Factor-1: A Critical Player in the Survival Strategy of Stressed Cells
Chen, S., & Sang, N. (2015) · Journal of Cellular Biochemistry · doi:10.1002/jcb.25283 · Cited by 137
“, 2005]; PX478 and 103D5R showed the ability to inhibit HIF-1 function through unkown mechanisms [Welsh et al., 2004; Kong et al., 2005; Tan et al., 2005].”
BackgroundInfluentialPacary, E., Legros, H., Valable, S., Duchatelle, P., Lecocq, M., Petit, E., Nicole, O., & Bernaudin, M. (2006) · Journal of Cell Science · doi:10.1242/jcs.03004 · Cited by 145
“In addition, we used echinomycin, a small molecule previously reported by Kong et al. (Kong et al., 2005) which inhibits HIF- 1 DNA-binding activity.”
BackgroundMethodRole of the hypoxic tumor microenvironment in the resistance to anti-angiogenic therapies
Rapisarda, A., & Melillo, G. (2009) · Drug Resistance Updates · doi:10.1016/j.drup.2009.03.002 · Cited by 142
“, 2008) or the AhR ligand aminoflavone (Terzuoli and Melillo, submitted for publication) and inhibition of HIF-1 DNA binding by echinomycin (Kong et al., 2005) and doxorubicin (Lee et al.”
BackgroundSelf-citationHypoxia Inducible Factor Pathway Inhibitors as Anticancer Therapeutics
Burroughs, S. K., Kaluz, S., Wang, D., Wang, K., Van Meir, E. G., & Wang, B. (2013) · Future Medicinal Chemistry · doi:10.4155/fmc.13.17 · Cited by 140
Role and potential for therapeutic targeting of MYB in leukemia
Pattabiraman, D. R., & Gonda, T. J. (2012) · Leukemia · doi:10.1038/leu.2012.225 · Cited by 138
Hypoxia and Mucosal Inflammation
Colgan, S. P., Campbell, E. L., & Kominsky, D. J. (2016) · Annual Review of Pathology Mechanisms of Disease · doi:10.1146/annurev-pathol-012615-044231 · Cited by 137
Signaling by Hypoxia-Inducible Factors Is Critical for Ovulation In Mice
Kim, J., Bagchi, I. C., & Bagchi, M. K. (2009) · Endocrinology · doi:10.1210/en.2008-0948 · Cited by 137
“Echinomycin is a sequence-specific DNA-binding agent that binds the sequence 5 -CGTG-3 , which forms the core of the HRE [5 -(A/G)CGTG-3 ], which is found at the promoters of HIF-target genes (22).”
BackgroundInfluentialHai, Y., Wei, M., Wang, C., Gu, Y., & Shao, C. (2021) · Marine Life Science & Technology · doi:10.1007/s42995-021-00101-2 · Cited by 137
Wu, D., Zhau, H. E., Huang, W.-C., Iqbal, S. A., Habib, F. K., Sartor, O. A., Cvitanovic, L., Marshall, F. F., Xu, Z., & Chung, L. W. K. (2007) · Oncogene · doi:10.1038/sj.onc.1210316 · Cited by 134
“From the isolated DNA, a 135-bp region was amplified by PCR (Kong et al., 2005).”
BackgroundMethodS-Adenosylmethionine: more than just a methyl donor
Lee, Y., Ren, D., Jeon, B., & Liu, H. (2023) · Natural Product Reports · doi:10.1039/d2np00086e · Cited by 128
Marine antifungal theonellamides target 3β-hydroxysterol to activate Rho1 signaling
Nishimura, S., Arita, Y., Honda, M., Iwamoto, K., Matsuyama, A., Shirai, A., Kawasaki, H., Kakeya, H., Kobayashi, T., Matsunaga, S., et al. (2010) · Nature Chemical Biology · doi:10.1038/nchembio.387 · Cited by 125
Mitochondria as targets for chemotherapy
Gogvadze, V. G., Orrenius, S., & Zhivotovsky, B. (2009) · APOPTOSIS · doi:10.1007/s10495-009-0323-0 · Cited by 127
“Echinomycin HIF1 Inhibition achieved by DNA binding [ 119 ]”
MethodModulating Hypoxia-Inducible Transcription by Disrupting the HIF-1–DNA Interface
Nickols, N. G., Jacobs, C. S., Farkas, M. E., & Dervan, P. B. (2007) · ACS Chemical Biology · doi:10.1021/cb700110z · Cited by 128
“As previously reported, 100 nM echinomycin potently inhibits VEGF expression to levels below the non-induced control (21).”
BackgroundInfluentialHypoxia-inducible factors in cancer stem cells and inflammation
Peng, G., & Liu, Y. (2015) · Trends in Pharmacological Sciences · doi:10.1016/j.tips.2015.03.003 · Cited by 127
“This HRE sequence was specifically targeted by echinomycin [9, 42].”
The potential role of HIF on tumour progression and dissemination
Unwith, S., Zhao, H., Hennah, L., & Ma, D. (2014) · International Journal of Cancer · doi:10.1002/ijc.28889 · Cited by 130
Zou, G.-M., & Maitra, A. (2008) · Molecular Cancer Therapeutics · doi:10.1158/1535-7163.mct-08-0113 · Cited by 124
Zhang, J., Yao, M., Xia, S., Zeng, F., & Liu, Q. (2025) · Cellular & Molecular Biology Letters · doi:10.1186/s11658-024-00682-7 · Cited by 150
Hsu, S.-H., Chen, C.-T., & Wei, Y. (2013) · Stem Cells · doi:10.1002/stem.1441 · Cited by 122
“To further confirm the involvement of HIF-1 , we treated hMSCs with echinomycin, a small-molecule inhibitor of HIF-1 transcription activity [19], during differentiation under hypoxia.”
MethodTsai, J. M., Sinha, R., Seita, J., Fernhoff, N. B., Christ, S., Koopmans, T., Krampitz, G. W., McKenna, K. M., Xing, L., Sandholzer, M., et al. (2018) · Science Translational Medicine · doi:10.1126/scitranslmed.aan6735 · Cited by 121
Real-Time Imaging of HIF-1α Stabilization and Degradation
Moroz, E., Carlin, S., Dyomina, K., Burke, S. A., Thaler, H. T., Blasberg, R., & Serganova, I. S. (2009) · PLoS ONE · doi:10.1371/journal.pone.0005077 · Cited by 127
The magic of the hypoxia-signaling cascade
Benizri, E. I., Ginouvès, A., & Berra, E. (2008) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-008-7472-0 · Cited by 112
Development of novel therapeutic strategies that target HIF-1
Semenza, G. L. (2006) · Expert Opinion on Therapeutic Targets · doi:10.1517/14728222.10.2.267 · Cited by 120
“In cancer cells, the transcription of many tumour suppressor genes is turned off through epigenetic regulation of histone acetylation and DNA methylation [115].”
BackgroundInfluentialGlioblastoma, Cancer Stem Cells and Hypoxia
Bar, E. E. (2010) · Brain Pathology · doi:10.1111/j.1750-3639.2010.00460.x · Cited by 125
Hypoxia, drug therapy and toxicity
Lee, K., Roth, R. A., & LaPres, J. J. (2006) · Pharmacology & Therapeutics · doi:10.1016/j.pharmthera.2006.08.001 · Cited by 118
Targeting Intrinsically Disordered Transcription Factors: Changing the Paradigm
Tsafou, K., Tiwari, P. B., Forman-Kay, J. D., Metallo, S. J., & Toretsky, J. A. (2018) · Journal of Molecular Biology · doi:10.1016/j.jmb.2018.04.008 · Cited by 113
Hypoxia signaling in cancer: Implications for therapeutic interventions
Zhuang, Y., Liu, K., He, Q., Gu, X., Jiang, C., & Wu, J. (2023) · MedComm · doi:10.1002/mco2.203 · Cited by 119
Hypoxia inducible factor-1: a novel target for cancer therapy
Belozerov, V. E., & Meir, E. G. V. (2005) · Anti-Cancer Drugs · doi:10.1097/01.cad.0000180116.85912.69 · Cited by 120
Inhibition of transcription factors with small organic molecules
Berg, T. (2008) · Current Opinion in Chemical Biology · doi:10.1016/j.cbpa.2008.07.023 · Cited by 120
Baumeister, J., Chatain, N., Hubrich, A., Maié, T., Costa, I. G., Denecke, B., Han, L., Küstermann, C., Sontag, S., Seré, K., et al. (2019) · Leukemia · doi:10.1038/s41375-019-0629-z · Cited by 91
“While HIF-2α protein was detectable and gradually increasing between 48 and 96 h of cultivation up to a level of shHIF-1α transduced cells, no increase by echinomycin was found (Supplementary Fig.”
BackgroundInfluentialA chemical genomics screen highlights the essential role of mitochondria in HIF-1 regulation
Lin, X., David, C. A., Donnelly, J. B., Michaelides, M. R., Chandel, N. S., Huang, X., Warrior, U., Weinberg, F. D., Tormos, K. V., Fesik, S. W., et al. (2008) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0706585104 · Cited by 115
Targeting cancer cells through autophagy for anticancer therapy
Turcotte, S., & Giaccia, A. J. (2010) · Current Opinion in Cell Biology · doi:10.1016/j.ceb.2009.12.007 · Cited by 116
Recent agents targeting HIF‐1α for cancer therapy
Hu, Y., Liu, J., & Huang, H. (2013) · Journal of Cellular Biochemistry · doi:10.1002/jcb.24390 · Cited by 113
“specifically binds to the core of the HIF-1a recognition sequence and inhibits binding of HIF-1 to the HRE sequence [Kong, 2005].”
BackgroundDuette, G., Gerber, P. P., Rubione, J., Pérez, P. S., LANDAY, A. L., Crowe, S. M., Liao, Z., Witwer, K. W., Holgado, M. P., Salido, J., et al. (2018) · mBio · doi:10.1128/mbio.00757-18 · Cited by 105
Terzuoli, E., Puppo, M., Rapisarda, A., Uranchimeg, B., Cao, L., Burger, A. M., Ziche, M., & Melillo, G. (2010) · Cancer Research · doi:10.1158/0008-5472.can-10-1075 · Cited by 111
“We and others have identified small molecule inhibitors of HIF-1α that act by distinct mechanisms of action, including, but not limited to, inhibition of HIF-1α translation (31,34,37,38), inhibition of HIF-1 DNA binding (39) or transcriptional activity (40–42), inhibition of protein-protein interaction (43), increased…”
Self-citationHypoxia Signaling in Cancer: From Basics to Clinical Practice
Sebestyén, A., Kopper, L., Dankó, T., & Tímár, J. (2021) · Pathology and Oncology Research · doi:10.3389/pore.2021.1609802 · Cited by 110
“Of note, the HIF complex-induced binding to HRE regions in promoters of various genes can be achieved by echinomycin [87].”
BackgroundTargeting HIF-2 α in clear cell renal cell carcinoma: A promising therapeutic strategy
Martínez‐Sáez, O., Borau, P. G., Alonso‐Gordoa, T., Molina‐Cerrillo, J., & Grande, E. (2017) · Critical Reviews in Oncology/Hematology · doi:10.1016/j.critrevonc.2017.01.013 · Cited by 111
Belaïdi, E., Morand, J., Gras, E., Pépin, J., & Godin‐Ribuot, D. (2016) · Pharmacology & Therapeutics · doi:10.1016/j.pharmthera.2016.07.010 · Cited by 116
“Echinomycin is an antibiotic that can bind DNA in a sequence-speci fi c fashion and that interferes with HIF-1-induced gene transcription by binding to its hypoxia response elements (Kong et al., 2005).”
Hatano, N., Itoh, Y., Suzuki, H., Muraki, Y., Hayashi, H., Onozaki, K., Wood, I. C., Beech, D. J., & Muraki, K. (2012) · Journal of Biological Chemistry · doi:10.1074/jbc.m112.361139 · Cited by 108
“over, Echi and YC-1, which reduceHIF1 activity as HIF inhibitors (32, 33), effectively inhibited the action of TNF .”
BackgroundZhang, J., Hu, Z., Horta, C. A., & Yang, J. (2022) · Seminars in Cancer Biology · doi:10.1016/j.semcancer.2022.12.002 · Cited by 106
Dynamic regulation of stem cell specification and maintenance by hypoxia-inducible factors
Semenza, G. L. (2015) · Molecular Aspects of Medicine · doi:10.1016/j.mam.2015.09.004 · Cited by 94
Hemin-induced necroptosis involves glutathione depletion in mouse astrocytes
Laird, M. D., Wakade, C. G., Alleyne, C. H., & Dhandapani, K. M. (2008) · Free Radical Biology and Medicine · doi:10.1016/j.freeradbiomed.2008.07.003 · Cited by 108
Kwon, T., Zhao, X., Yang, Q., Li, Y., Ge, C., Zhao, G., & Franceschi, R. T. (2011) · Journal of Cellular Biochemistry · doi:10.1002/jcb.23289 · Cited by 102
Jin, W., Zhao, J., Yang, E., Wang, Y., Wang, Q., Wu, Y., Tong, F., Tan, Y., Zhou, J., & Kang, C. (2022) · Theranostics · doi:10.7150/thno.71029 · Cited by 104
“Echinomycin is a potent small-molecule and cell-permeable inhibitor of HIF-1α DNA-binding activity [34].”
BackgroundAML1/ETO cooperates with HIF1α to promote leukemogenesis through DNMT3a transactivation
Gao, X., Yan, F., Lin, J., Gao, L., Lu, X., Wei, S., Shen, N., Pang, J. X., Ning, Q., Komeno, Y., et al. (2015) · Leukemia · doi:10.1038/leu.2015.56 · Cited by 92
Metadata
- Authors
- Kong, D., Park, E. J., Stephen, A. G., Calvani, M., Cardellina, J. H., Monks, A., Fisher, R. J., Shoemaker, R. H., & Melillo, G.
- Affiliations
- Science Applications International Corporation (United States) (US)
- National Cancer Institute (US)
- Venue
- Cancer Research
- Published
- 2005-10-01
- Volume
- 65
- Issue
- 19
- Pages
- 9047-9055
- ISSN
- 0008-5472 (ISSN-L)
- 1538-7445
- OpenAlex
- W2144670533
- Cited by
- 508
- References
- 53
- Citations by year
- 2026: 12 · 2025: 24 · 2024: 23 · 2023: 25 · 2022: 31 · 2021: 34 · 2020: 26 · 2019: 22 · 2018: 23 · 2017: 26 · 2016: 22 · 2015: 23 · 2014: 20 · 2013: 23 · 2012: 26
- FWCI
- 7.64
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Cancer Research (publishedVersion)
- PubMed (publishedVersion)
- Florence Research (University of Florence) (submittedVersion, other-oa)
- Funders
- National Institutes of Health (n01co12400-79-0-22, N01-CO-12400)
- National Cancer Institute (N01CO12400)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
- Physical Sciences › Chemistry › Organic Chemistry › Heterocyclic Compound Synthesis and Bioactivity
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Kruppel-like factors research
- Keywords
- echinomycin, HIF-1, hypoxia response element, transcription factor-DNA binding, small molecule inhibitors, VEGF promoter, angiogenesis, tumor progression, metastasis, high-throughput screening
- MeSH terms
- Antibiotics, Antineoplastic, Breast Neoplasms, DNA, Neoplasm, Echinomycin, Enzyme-Linked Immunosorbent Assay, Glioma, Humans, Luciferases, Promoter Regions, Genetic, Recombinant Proteins, RNA, Messenger, Vascular Endothelial Growth Factor A, Cell Line, Tumor, Hypoxia-Inducible Factor 1
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