FOXM1 (Forkhead box M1) [2]
EWS/FLI1 oncoprotein [3]

Thiostrepton (0.01-1000 μM; 48 hours) suppresses cell viability in A2780 and HEC-1A[2].

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Thiostrepton downregulates FOXM1 protein expression in A2780 and HEC-1A cancer cell lines (both wild-type and mutant TP53) [2].
Thiostrepton induces apoptosis in cancer cell lines as evidenced by caspase-3 and PARP1 cleavage, and increased Annexin V labeling [2].
Thiostrepton suppresses cell viability in A2780 (IC50 approximately 1.10 μM) and HEC-1A (IC50 approximately 2.22 μM) cells. For comparison, cisplatin IC50 is 7.16 μM in A2780 and 14.82 μM in HEC-1A [2].
Thiostrepton (2.5, 5, and 10 μM) shows synergistic drug interactions with 1 μM cisplatin in both A2780 and HEC-1A cell lines [2].
Thiostrepton inhibits proliferation of Ewing's sarcoma (EWS) cell lines A4573, SK-ES-1, and TC-71 (representing three major EWS/FLI1 translocation types) with ~50% decrease in viable cell numbers at 1 μM treatment for 48 hours [3].
Thiostrepton (1 μM, 48h) alters cell cycle progression in EWS cells, causing a decrease in S-phase population and accumulation of cells in G1 or G2-M phase [3].
Thiostrepton treatment (1 μM, 48h) causes morphological changes in EWS cells (round morphology, detachment from plates) and alters actin cytoskeleton organization (intense staining near nuclear periphery) [3].
Thiostrepton (1 μM, 48h) reduces FOXM1 protein and mRNA expression in EWS cells (A4573, SK-ES-1, TC-71). Downstream targets of FOXM1 (PLK-1, Cep55, cyclin D1) are also decreased. No significant change in Akt expression [3].
Thiostrepton (1 μM, 48h) reduces EWS/FLI1 oncoprotein and mRNA levels in EWS cells [3].
Thiostrepton (1 μM, 48h) induces a caspase-dependent apoptotic response in EWS cells, with 3- to 6-fold elevation in caspase 3/7 activity, PARP cleavage, decreased XIAP and survivin expression, and cleavage of caspase 7 (but not caspase 3) [3].

Thiostrepton (i.p. ; 17 mg/kg) decreases Ewing's sarcoma (EWS) cell carcinogenicity. The tumor volumes of control mice have increased approximately six times since the start of treatment, whereas the tumor volumes of mice treated with Thiostrepton have increased only approximately 1.7 times, showing a ~3.5-fold reduction, in comparison to controls[3].

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Thiostrepton (30 mg/kg, daily, 5 days/week) as monotherapy showed limited anti-tumor activity in HEC-1A xenograft model. However, combination with carboplatin (80 mg/kg weekly, then reduced to 20 mg/kg) showed a trend of tumor progression suppression despite suboptimal carboplatin dose [2].
Thiostrepton (17 mg/kg, intraperitoneal, every third day) significantly delays tumor growth in nude mouse xenografts of A4573 Ewing's sarcoma cells. At day 16 post-injection, thiostrepton-treated tumors increased only ~1.7-fold vs ~6-fold in controls (p≤0.0005). At day 22, thiostrepton-treated tumors increased ~4-fold vs ~19-fold in controls (~4.75-fold difference in volume) [3].
Thiostrepton treatment (17 mg/kg, i.p., every third day) reduces FOXM1, its downstream targets, and EWS/FLI1 protein levels in excised xenograft tumors (A4573) as shown by western blot and immunohistochemistry [3].
Thiostrepton treatment (17 mg/kg, i.p., every third day) induces caspase 7-dependent apoptosis in xenograft tumors (A4573), evidenced by PARP cleavage, decreased survivin, and cleaved caspase 7 [3].

Cell Line: A2780 and HEC-1A cells
Concentration: 0.01, 0.1, 1, 10, 100, 1000 μM
Incubation Time: 48 hours
Result: The IC50s are 1.10 μM in A2780 and 2.22 μM in HEC-1A, respectively.

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Cell viability assay (AlamarBlue): Cells were plated into 96-well plates (2×10³ cells/200 μl well) and cultured overnight. The next day, cells were treated with various concentrations of thiostrepton, cisplatin, or combinations. After 48 hours, cell viability was assessed using AlamarBlue reagent. IC50 values were determined using GraphPad Prism (version 6). Isobolograms for drug synergy were determined using CompuSyn software [2].
Apoptosis detection (Annexin V/PI staining): Cells were treated with thiostrepton, harvested, and stained with Annexin V and propidium iodide. Early and late apoptosis were quantified by flow cytometry [2].
Western blot analysis: Cells were collected after treatment, and total proteins were extracted using RIPA buffer containing protease/phosphatase inhibitor cocktail. Protein concentrations were determined by BCA assay. Equal amounts of protein were subjected to SDS-PAGE and electroblotted onto PVDF membranes. After blocking with 5% nonfat dry milk in TBS-Tween for 2 hours at room temperature, blots were incubated with primary antibodies overnight at 4°C, then with HRP-conjugated secondary antibodies for 1 hour. Protein bands were visualized using chemiluminescence kit. Blots were stripped and re-probed for β-actin for normalization [2].
Real-time quantitative RT-PCR (qPCR): Total RNA was extracted using Trizol reagent. cDNA was synthesized using iScript Reverse Transcription Supermix. The resulting cDNA was diluted 1:5 in sterile water, and 1 μl aliquots were used in qPCR reactions with primers designed with Primer3 plus. qPCR was carried out on a CFX384 Real-Time System. GAPDH was used for normalization. Each cDNA sample was run in triplicate [2].
Cell cycle analysis: EWS cells were harvested 48 hours after exposure to thiostrepton (1 μM), washed once in PBS, fixed in 70% ethanol in PBS, and stained with propidium iodide. Flow cytometric analysis was carried out on a FACScan instrument [3].
Reverse transcription-PCR (RT-PCR): RNA extraction, RT-PCR conditions, and PCR product analysis were carried out. Primer pairs were used to amplify EWS/FLI-1, FoxM1, and tubulin (internal control). The number of cycles for PCR was adjusted to ensure end-points remained within exponential phase of product amplification to get a semi-quantitative estimate of relative mRNA abundance [3].
Immunofluorescence analysis: Cells cultured on coverslips were fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature, permeabilized with 0.2% Triton X-100 for 10 minutes, and blocked with 10% normal goat serum in PBS for 30 minutes. Cells were incubated with FoxM1 antibody (1:500 dilution) for 1 hour at room temperature, then with Alexa fluor 488-conjugated anti-rabbit IgG for 1 hour. DNA was stained with DAPI for 5 minutes. Images were processed with ImageJ software [3].
Caspase 3/7 activity assay: Caspase 3/7 activity in treated cells was determined using a luminescent assay kit according to the manufacturer's protocol. Fold changes in activity were calculated relative to controls [3].

Animal Model: Athymic (BALB/c nu/nu) nude mice bearing A4573 cells[3]
Dosage: 17 mg/kg
Administration: Administered i.p.
Result: Treatment inhibited the growth of EWS-derived tumors in vivo.

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In vivo efficacy study (ovarian cancer model): Athymic Nu/Nu female mice (7-9 weeks old) were inoculated with luciferase-expressing HEC-1A cells (2×10⁶) by intraperitoneal (i.p.) injection. Three days after tumor inoculation, baseline tumor burden was determined by bioluminescence imaging. On day 4, treatment was initiated. Mice were administered either carboplatin (80 mg/kg weekly) or thiostrepton (30 mg/kg daily, five days per week) for monotherapy. A third group received combination of carboplatin (80 mg/kg weekly) and thiostrepton (10 mg/kg daily, five days per week). Control group received vehicle (water for carboplatin and DMSO for thiostrepton). After the first 3 weeks, carboplatin dose was reduced to 20 mg/kg. Mice were imaged weekly using IVIS spectrum imager. Region of interest (ROI) boxes were drawn around the entire body, and measurements expressed as flux (photons/second) [2].
In vivo xenograft study (Ewing's sarcoma model): Male immunodeficient athymic nude (BALB/c nu/nu) mice were injected subcutaneously with A4573 EWS cells (5×10⁶ cells in 100 μl serum-free medium mixed with 100 μl Matrigel) into the posterior flank. Once tumors reached a mean volume of about 150 mm³ (around day 8), mice were divided into control and treatment groups (10 animals per group). Treatment groups received thiostrepton (17 mg/kg, intraperitoneally) every third day. Control groups received vehicle (thiostrepton carrier solution) in equal volume by i.p. injection. Tumors were measured every alternate day. Primary tumor volumes were calculated by formula V = (1/2)×a×b² (a = longest axis, b = shortest axis). At specified times or when tumors reached maximum allowable volume, animals were sacrificed, and tumors were excised for analysis [3].

Evidence of toxicity from carboplatin treatment was observed as drastic weight loss in mice treated with 80 mg/kg carboplatin, leading to dose reduction to 20 mg/kg starting at Day 22 [2].

[1]. A combat with the YAP/TAZ-TEAD oncoproteins for cancer therapy. Theranostics. 2020 Feb 18;10(8):3622-3635.

[2]. Targeting of mutant p53-induced FoxM1 with Thiostrepton induces cytotoxicity and enhances carboplatin sensitivity in cancer cells. Oncotarget. 2014 Nov 30;5(22):11365-80.

[3]. The dual inhibitory effect of Thiostrepton on FoxM1 and EWS/FLI1 provides a novel therapeutic option for Ewing's sarcoma. Int J Oncol. 2013 Sep;43(3):803-12.

Thiostrepton is a natural cyclic oligopeptide antibiotic derived from various Streptomycete strains, including Streptomyces azureus and Streptomyces laurentii. Thiostrepton is a ribosome-synthesized and post-translational modified peptide natural product. Brymycin has also been reported in Streptomyces albidoflavus, Streptomyces aureus, and other microorganisms with relevant data. Thiostrepton is a naturally occurring sulfur-rich cyclic oligopeptide antibiotic belonging to the thiopeptide class. It is an irreversible inhibitor of mitochondrial thioredoxin-dependent peroxidases (peroxidase-3; PRX3; antioxidant protein 1; AOP-1) and possesses potential antitumor activity. After intrapleural administration, thioredoxin irreversibly binds to and inhibits PRX3 activity. This inhibits the peroxidase activity of the mitochondrial thioredoxin reductase 2 (TXNRD2)-thioredoxin 2 (TRX2)-PRX3 antioxidant signaling network, potentially leading to hydrogen peroxide accumulation and ultimately tumor cell death. PRX3 is upregulated in cancer cells and plays an important role in the regulation of oxidative stress pathways. It is one of the cyclic peptide compounds from Streptomyces, active against Gram-positive bacteria. In veterinary medicine, it has been used to treat mastitis and dermatitis caused by Gram-negative bacteria. See also: Thiostithrcin (note moved to).

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Thiostrepton is a naturally occurring small molecule (thiazole antibiotic) isolated from Streptomyces azureus that selectively inhibits FoxM1 expression in cancer cells [3].
Thiostrepton is known to inhibit the binding of FoxM1 to genomic target sites [3].
Thiostrepton downregulates FoxM1 expression in several ovarian cancer cell lines as well as in endometrial (HEC-1A) and lung (NCI-H23) cancer cell lines [2].
Thiostrepton enhances sensitivity to cisplatin in vitro and carboplatin in vivo [2].
Thiostrepton shows greater efficacy against Ewing's sarcoma than against other tumor types, as it is active on EWS cells and tumors at concentrations lower than those reported to have effective inhibitory activity on tumor cells derived from other cancers [3].
Thiostrepton exerts a dual inhibitory effect on EWS/FLI1 and FoxM1 in Ewing's sarcoma [3].

C72H85N19O18S5

1664.89

1663.49

C, 51.94; H, 5.15; N, 15.99; O, 17.30; S, 9.63

1393-48-2

16129666

White to off-white solid powder

1.64 g/cm3

248-257°C (dec.)

1.768

4.086

17

31

12

114

3940

0

S1C([H])=C2C(N([H])C([H])(C(N([H])C(=C([H])C([H])([H])[H])C3=NC([H])(C([H])([H])S3)C(N([H])C([H])(C3=NC(=C([H])S3)C(N([H])C3([H])C([H])(C([H])([H])[H])OC(C4C([H])=C(C([H])(C([H])([H])[H])O[H])C5C([H])=C([H])C([H])(C([H])(C=5N=4)O[H])N([H])C([H])(C(N([H])C([H])(C([H])([H])[H])C(N([H])C(=C([H])[H])C(N([H])C([H])(C([H])([H])[H])C(N([H])C4(C1=N2)C([H])([H])C([H])([H])C(C1=NC(C(N([H])C(=C([H])[H])C(N([H])C(=C([H])[H])C(N([H])[H])=O)=O)=O)=C([H])S1)=NC4([H])C1=C([H])SC3=N1)=O)=O)=O)=O)C([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])=O)=O)C(C([H])([H])[H])(C([H])(C([H])([H])[H])O[H])O[H])=O)=O)C([H])(C([H])([H])[H])O[H])=O

NSFFHOGKXHRQEW-DVRIZHICSA-N

InChI=1S/C72H85N19O18S5/c1-14-26(3)47-63(105)78-30(7)57(99)75-28(5)56(98)76-31(8)58(100)91-72-19-18-40(66-85-43(22-111-66)59(101)77-29(6)55(97)74-27(4)54(73)96)81-52(72)42-21-112-67(83-42)49(34(11)109-69(107)41-20-37(32(9)92)36-16-17-39(79-47)51(95)50(36)80-41)89-60(102)44-24-113-68(86-44)53(71(13,108)35(12)94)90-62(104)45-23-110-65(84-45)38(15-2)82-64(106)48(33(10)93)88-61(103)46-25-114-70(72)87-46/h15-17,20-22,24-26,30-35,39,45,47-49,51-53,79,92-95,108H,4-6,14,18-19,23H2,1-3,7-13H3,(H2,73,96)(H,74,97)(H,75,99)(H,76,98)(H,77,101)(H,78,105)(H,82,106)(H,88,103)(H,89,102)(H,90,104)(H,91,100)/b38-15+

N-[3-[(3-amino-3-oxoprop-1-en-2-yl)amino]-3-oxoprop-1-en-2-yl]-2-[(11Z)-37-butan-2-yl-18-(2,3-dihydroxybutan-2-yl)-11-ethylidene-59-hydroxy-8,31-bis(1-hydroxyethyl)-26,40,46-trimethyl-43-methylidene-6,9,16,23,28,38,41,44,47-nonaoxo-27-oxa-3,13,20,56-tetrathia-7,10,17,24,36,39,42,45,48,52,58,61,62,63,64-pentadecazanonacyclo[23.23.9.329,35.12,5.112,15.119,22.154,57.01,53.032,60]tetrahexaconta-2(64),4,12(63),19(62),21,29(61),30,32(60),33,51,54,57-dodecaen-51-yl]-1,3-thiazole-4-carboxamide

NSC-170365; NSC170365;NSC 170365;

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 100~125 mg/mL ( 60.06~75.08 mM )

Solubility in Formulation 1: 5 mg/mL (3.00 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 5 mg/mL (3.00 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 4.17 mg/mL (2.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 41.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5% DMSO+ 40% PEG300+ 5% Tween 80+ 50% ddH2O: 1.25mg/ml (0.75mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)