Hypoxia-inducible factor-1 (HIF-1). [1]

SYP-5 prevents HIF-1 upregulation brought on by hypoxia. In Hep3B and Bcap37 cells, SYP-5 inhibits HIF-1 and downstream gene expression. SYP-5 blocks the PI3K/AKT and MAPK/ERK-dependent HIF-1 pathways, which in turn prevents tumor cell migration, invasion, and angiogenesis. SYP-5 downregulates two proteins that are targets of HIF-1: matrix metalloproteinase (MMP)-2 and vascular endothelial growth factor (VEGF). In both cell lines, hypoxia-induced overexpression of VEGF and MMP2 was significantly inhibited by SYP-5. SYP-5 inhibited hypoxia- and VEGF-induced angiogenesis in vitro in tube formation experiments. Additionally, SYP-5 can postpone the invasion and migration of Bcap37 and Hep3B cells brought on by FBS and hypoxia. The hypoxic induction of luciferase expression in U251-HRE is specifically inhibited by SYP-5, whereas U251-pGL3 is not affected [1].

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SYP-5 specifically inhibited hypoxia-induced luciferase expression in U251-HRE reporter cells (a model for HIF-1 activity) in a concentration-dependent manner, while it did not affect the control U251-pGL3 cells. [1]
In Hep3B (hepatocellular carcinoma) and Beap37 (breast cancer) cells under hypoxic conditions, SYP-5 (2, 10, 50 μM) treatment for 48 hours downregulated the protein levels of HIF-1α and HIF-1β, as well as the downstream HIF-1 target proteins vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2), in a concentration-dependent manner. [1]
In human umbilical vein endothelial cells (HUVECs), SYP-5 (2, 10, 50 μM) significantly inhibited capillary-like tube formation induced by either hypoxia or exogenous VEGF (20 ng/ml) in a concentration-dependent manner. [1]
SYP-5 (2, 10, 50 μM) suppressed the migration and invasion of HUVECs induced by VEGF (20 ng/ml), as measured by Real-Time Cell Analysis (RTCA) and Transwell assays. [1]
SYP-5 (2, 10, 50 μM) inhibited hypoxia-induced migration and invasion of Hep3B and Beap37 cells in Transwell assays. It also suppressed fetal bovine serum (FBS)-induced migration (RTCA) and invasion (Transwell) of these cancer cell lines. [1]
In Beap37 cells under hypoxia, SYP-5 (2, 10, 50 μM) treatment for 48 hours inhibited the activation (phosphorylation) of epidermal growth factor receptor (EGFR), protein kinase B (AKT), extracellular signal-regulated kinase (ERK1/2), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). [1]
Combination of SYP-5 (10 μM) with the PI3K/AKT pathway inhibitor LY294002 (10 μM) resulted in a dramatic synergistic suppression of hypoxia-induced invasion in Beap37 cells, greater than the effect of either agent alone or the combination with the MAPK/ERK inhibitor PD98059 (10 μM). Western blot analysis confirmed that the combination of SYP-5 with either LY294002 or PD98059 enhanced the inhibition of MMP-2 expression compared to single agent treatments. [1]
MTT assays showed that at concentrations up to 50 μM, SYP-5 did not significantly reduce cell viability (<80% viability) in HUVEC, Hep3B, and Beap37 cells after 24-hour treatment, indicating that the observed anti-angiogenic and anti-invasive effects at these concentrations were not due to general cytotoxicity. [1]

Luciferase Reporter Assay: U251-HRE cells (stably expressing a hypoxia-responsive element-luciferase construct) and control U251-pGL3 cells were seeded in 96-well plates. After overnight incubation, cells were pretreated with various concentrations of SYP-5 for 1 hour and then exposed to hypoxic conditions (1% O2) for 16 hours. Luciferase activity was measured using a commercial luciferase assay reagent. [1]
Cell Viability Assay (MTT): Cells (HUVEC, Hep3B, Beap37) were seeded in 96-well plates. After overnight incubation, cells were treated with various concentrations of SYP-5 for 24 hours. MTT solution was added to each well and incubated for 4 hours. The formed formazan crystals were dissolved in dimethyl sulfoxide, and the optical density was measured at 570 nm. [1]
Western Blot Analysis: Hep3B and Beap37 cells were treated with SYP-5 under hypoxic conditions. For inhibitor studies, cells were pretreated with pathway inhibitors for 1 hour before SYP-5 addition. Cells were lysed, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with specific primary antibodies against target proteins (e.g., HIF-1α, VEGF, MMP-2, p-EGFR, p-AKT, p-ERK) and corresponding secondary antibodies conjugated to horseradish peroxidase. Protein bands were visualized using enhanced chemiluminescence detection reagents. β-actin was used as a loading control. [1]
Tube Formation Assay: Matrigel was polymerized in 96-well plates. HUVECs were seeded onto the Matrigel layer with or without SYP-5 and then exposed to hypoxia or treated with VEGF (20 ng/ml). After 12 hours, tubular structures were stained with Calcein-AM and imaged. The number of branches per field was quantified. [1]
RTCA Migration Assay: Cell migration was monitored in real-time using a system with modified Boyden chambers. Cells treated with SYP-5 were placed in serum-free medium in the upper chamber. The lower chamber contained a chemoattractant (15% FBS or 20 ng/ml VEGF). Electrical impedance (Cell Index) across microelectrodes on the underside of the membrane, which changes as cells migrate through pores and adhere, was monitored every 5 minutes. [1]
Transwell Migration/Invasion Assay: For migration assay, cells were plated in the upper chamber of a Transwell insert (8 μm pore). For invasion assay, the insert membrane was pre-coated with Matrigel. SYP-5 was added to the lower chamber. Cells were incubated under normoxia or hypoxia for 16 hours. Non-migrating/invading cells on the upper surface were removed. Cells that migrated/invaded to the lower surface were stained with Calcein-AM, photographed, and counted. [1]

[1]. SYP-5, a novel HIF-1 inhibitor, suppresses tumor cells invasion and angiogenesis. Eur J Pharmacol. 2016 Nov 15;791:560-568.

SYP-5 is a novel chalcone compound that has been identified as a potential inhibitor of HIF-1. HIF-1 is a key transcription factor that is overexpressed in many solid tumors under hypoxic conditions and promotes tumor progression, angiogenesis, and metastasis. [1] Its mechanism of action is thought to be that SYP-5 inhibits the upregulation of hypoxia-induced HIF-1α and HIF-1β protein levels. This leads to the downregulation of HIF-1 target genes such as VEGF and MMP-2. The anti-angiogenic (inhibition of tubular formation) and anti-invasive/migration effects observed in vitro are attributed to the inhibition of the HIF-1 pathway. [1] Further mechanistic studies have shown that SYP-5 works by inhibiting the activation of EGFR and its downstream PI3K/AKT and MAPK/ERK signaling pathways, which are known to regulate HIF-1α expression. The PI3K/AKT pathway appears to play a particularly important synergistic role in the anti-invasive effects of SYP-5. [1] This study suggests that SYP-5 may be a potential anticancer drug targeting the HIF-1 pathway. [1]

C18H16O3S

312.3828

312.082

C, 69.21; H, 5.16; O, 15.36; S, 10.26

1384268-04-5

1384268-04-5;

123131800

Yellow to orange solid powder

1.3±0.1 g/cm3

502.0±50.0 °C at 760 mmHg

257.4±30.1 °C

0.0±1.3 mmHg at 25°C

1.649

6.34

1

4

3

22

483

0

S1C([H])=C([H])C([H])=C1C([H])=C([H])C(C1C([H])=C([H])C2=C(C=1O[H])C([H])=C([H])C(C([H])([H])[H])(C([H])([H])[H])O2)=O

OMVURDVBYIJCOI-FNORWQNLSA-N

InChI=1S/C18H16O3S/c1-18(2)10-9-14-16(21-18)8-6-13(17(14)20)15(19)7-5-12-4-3-11-22-12/h3-11,20H,1-2H3/b7-5+

(E)-1-(5-hydroxy-2,2-dimethylchromen-6-yl)-3-thiophen-2-ylprop-2-en-1-one

SYP-5; SYP5; SYP 5;

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 : ~33.33 mg/mL (~106.70 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.00 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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: ≥ 2.5 mg/mL (8.00 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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