Separase (non-competitive inhibitor) [1]
Forkhead box protein M1 (FoxM1) (expression is inhibited) [1]
Raf kinase family members (A-Raf, B-Raf, C-Raf) (expression is inhibited) [1]

DNA damage is caused by sepin-1 (0, 20, 40 µM; 24 h), which also prevents cell migration and wound healing [1]. In a simulated varied mode of expression, Sepin-1 (0, 10, 20, 40 µM; 24 h) suppresses the protein levels of Plk1, Cdk1, pericentrin, Aurora, and Lamin B1[1]. Assay for cell proliferation. Lower the amounts of mRNA and the expression of the FoxM1 protein [1].

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In breast cancer cell lines BT-474, MCF7, MDA-MB-231, and MDA-MB-468, Sepin-1 inhibited cell growth in a dose-dependent manner. The half-maximal effective concentration (EC₅₀) values were approximately 18 μM for BT-474 and MCF7 (luminal subtypes), and approximately 28 μM for MDA-MB-231 and MDA-MB-468 (basal-like subtypes) [1].
Sepin-1 treatment reduced Separase protein levels in all four breast cancer cell lines, with a more significant reduction observed in BT-474 and MCF7 cells compared to the triple-negative lines [1].
In Transwell migration assays using MDA-MB-231 and MDA-MB-468 cells, Sepin-1 significantly inhibited cell migration in a dose-dependent manner [1].
In wound healing assays using MDA-MB-468 cells, Sepin-1 significantly reduced wound closure, observable as early as 24 hours post-treatment [1].
TUNEL assay showed that treatment with 40 μM Sepin-1 resulted in approximately 40% TUNEL-positive cells in BT-474 and MCF7 cell lines, significantly higher than the <10% seen in control or 20 μM treated groups. In contrast, TUNEL-positive cells were rarely found in MDA-MB-231 and MDA-MB-468 cells treated with 40 μM Sepin-1 [1].
While Sepin-1 treatment increased levels of pro-apoptotic regulators Bak, Bax, and Bid in a concentration-dependent manner (RPPA data), it did not lead to the activation of caspases 3 and 7 or the cleavage of PARP (generation of 85 kD fragment), unlike the positive control etoposide. This suggests that Sepin-1-induced growth inhibition is not via classical apoptosis [1].
Sepin-1 treatment reduced FoxM1 expression at both the mRNA (qPCR) and protein (RPPA, Western blot) levels in breast cancer cells [1].
Sepin-1 inhibited the expression of Raf kinase family members A-Raf, B-Raf, and C-Raf, as shown by qPCR (for A-Raf) and Western blot [1].
Sepin-1 reduced the protein levels of FoxM1 target genes involved in cell cycle progression and proliferation, including Plk1, Cdk1, Aurora A, pericentrin, and Lamin B1, as demonstrated by RPPA and Western blot analyses [1].

In mice, sepin-1 (10 mg/kg; intravenously administered; once daily, five days a week for three weeks) exhibits anti-tumor action [2].

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Cell proliferation assay. Reduce the expression of FoxM1 protein and mRNA levels [1]. [1]
Cell Types: BT-474, MCF7, MDA-MB-231, MDA-MB-468 Cell
Tested Concentrations: 0-64 µM
Incubation Duration: 3 days
Experimental Results: Inhibited cell growth, EC50 was 18.03, 17.66, 27.33, 27.92 µM is applicable to BT-474, MCF7, MDA-MB-231, and MDA-MB-468 cells respectively.

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Western Blot Analysis[1]
Cell Types: BT-474, MCF7, MDA-MB-231, MDA-MB-468 Cell
Tested Concentrations: 0, 10, 20, 40 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: FoxM1 protein expression diminished with dose Decreases the expression of A-Raf, B-Raf and C-Raf in a dependent manner.

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Cell Viability Assay: Cells (BT-474, MCF7, MDA-MB-231, MDA-MB-468) were seeded in 96-well plates (100 μL medium/well) and incubated for 24 hours. Then, 50 μL of medium containing various concentrations of Sepin-1 or medium alone was added to each well. After 3 days of incubation, 20 μL of CellTiter-Blue® Reagent was added to each well. Following a 6-hour incubation, fluorescence intensity (FI) was measured using a microplate reader (excitation 560 nm, emission 590 nm). Wells without cells served as background, and wells without Sepin-1 treatment served as positive control. Cell viability (%) was calculated as 100 × ((FISepin-1 - FIbackground) / (FIpositive - FIbackground)). Each treatment was performed in triplicate and repeated in three independent experiments [1].
Cell Migration Assay (Transwell): Cells (MDA-MB-231, MDA-MB-468) were detached and suspended in serum-free medium at 1 x 10⁵ cells/mL. 100 μL of this cell suspension was plated on top of the filter membrane in a 24-well Transwell® insert and incubated for 10 minutes at 37°C to allow cells to settle. 600 μL of medium with or without Sepin-1 was added to the lower chamber. After 24 hours of incubation at 37°C, non-migrated cells on the top of the membrane were removed with cotton-tipped applicators. Cells on the opposite side of the membrane were fixed with 4% glutaraldehyde and stained with 0.2% crystal violet. Migrated cells were counted in five random fields per membrane using an inverted microscope. The assay was performed in triplicate [1].
Wound Healing Assay: MDA-MB-468 cells were seeded in 12-well plates at 2 x 10⁵ cells per well and incubated for 24 hours. A straight-line wound was made in each well using a 200 μL pipette tip. Medium was aspirated to remove debris and replaced with medium containing or lacking Sepin-1. Images were taken at 0, 24, 48, and 72 hours post-wounding. The wound gap distance was measured using a scale bar, and the percentage of wound closure was calculated. The assay was performed in triplicate [1].
TUNEL Assay: Cells were seeded in 6-well plates and treated with 0, 20, or 40 μM Sepin-1 for 24 hours. Cells were then detached and cytospun onto slides. The cells on slides were fixed, permeabilized, and labeled using the DeadEnd Fluorometric TUNEL System according to the manufacturer's protocol. Slides were examined under a fluorescence microscope. TUNEL-positive cells were counted in seven random fields per condition, and the percentage was calculated [1].
Reverse Phase Protein Array (RPPA): Cells were treated with Sepin-1 for 24 hours. Whole cell lysates were prepared and sent to the RPPA Core Facility at MD Anderson Cancer Center for analysis of protein expression levels [1].
Western Blot Analysis: Cells were treated with Sepin-1 for 24 hours. Protein lysates were prepared, separated by gel electrophoresis, and transferred to nitrocellulose membranes. Membranes were probed with specific primary antibodies (e.g., anti-Separase, anti-FoxM1, anti-Rafs, anti-Plk1, anti-Cdk1, anti-Aurora A, anti-pericentrin, anti-Lamin B1, anti-caspase 3, anti-caspase 7, anti-PARP) and appropriate secondary antibodies. Protein bands were visualized and analyzed [1].
Quantitative PCR (qPCR) Analysis: Cells were seeded in 10 cm plates and treated with different concentrations of Sepin-1 for 24 hours. Total RNA was extracted using an RNeasy Plus Mini Kit. cDNA was synthesized using an RT² First Strand Kit. qPCR was performed using RT² SYBR Green ROX qPCR Mastermix. Gene expression levels (e.g., FoxM1, A-Raf) were analyzed and normalized [1].

Animal/Disease Models: SCID beige mice (MCF7 xenograft) [2]
Doses: 10 mg/kg
Route of Administration: IV; one time/day, 5 days a week for 3 weeks
Experimental Results: Inhibition of tumor growth.

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[1]. Separase Inhibitor Sepin-1 Inhibits Foxm1 Expression and Breast Cancer Cell Growth. J Cancer Sci Ther. 2018;10(3):517.

[2]. Identification and Characterization of Separase Inhibitors (Sepins) for Cancer Therapy. J Biomol Screen. 2014 Jul;19(6):878-89.

Sepin-1 is a small molecule identified as a potent, non-competitive inhibitor of Separase, a protease that resolves sister chromatid cohesion and is overexpressed in many cancers [1].
The anti-proliferative effect of Sepin-1 in breast cancer cells is proposed to be mediated through inhibition of cell proliferation rather than apoptosis [1].
The proposed mechanism involves Sepin-1-induced downregulation of Raf kinase family members (A-Raf, B-Raf, C-Raf). This likely attenuates the Raf-Mek-Erk signaling cascade, which is responsible for phosphorylating and activating FoxM1. Reduced FoxM1 activity then diminishes the expression of its own gene (via a positive feedback loop) and its target genes (Plk1, Cdk1, Aurora A, Lamin B1, pericentrin), ultimately leading to cell cycle inhibition and growth arrest [1].
The differential sensitivity of breast cancer cell lines to Sepin-1 (luminal vs. basal-like) was noted, with luminal lines (BT-474, MCF7) showing lower EC₅₀ values and increased DNA damage (TUNEL positivity) compared to basal-like lines (MDA-MB-231, MDA-MB-468) [1].

C9H9N3O4

223.19

223.059

163126-81-6

2736269

Brown to reddish brown solid powder

1

0

5

0

16

338

0

CC1(N(C2=C([N+]1=O)C=CC(=C2)[N+](=O)[O-])[O-])C

YLVSVJDCTDBERH-UHFFFAOYSA-N

InChI=1S/C9H9N3O4/c1-9(2)10(13)7-4-3-6(12(15)16)5-8(7)11(9)14/h3-5H,1-2H3

2,2-dimethyl-5-nitro-3-oxidobenzimidazol-1-ium 1-oxide

Sepin-1 Sepin1 Sepin 1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~100 mg/mL (~448.05 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (11.20 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)