MMP9; NF-κB; p38 MAPK
Isoliquiritin Apioside suppresses PMA-induced activation of mitogen-activated protein kinase (MAPK) pathways including p38, ERK, and JNK. It also inhibits NF-κB activation by blocking nuclear translocation of the p65 subunit. Additionally, ISLA impairs the hypoxia-inducible factor-1α (HIF-1α) pathway, reducing HIF-1α accumulation and phosphorylation of Akt, mTOR, 4E-BP1, and p70S6K. No IC50, Ki, EC50, or DC50 values were reported for these targets. [1]

The gelatinolytic MMP-9 activity in HT1080 cells is efficiently inhibited by isoliquiritin apioside when exposed to PMA. Moreover, isoliquiritin apioside lessens the PMA-induced rise in MMP-9 synthesis in HT1080 cells. In malignant cancer cells and endothelial cells (EC), isoliquiritin apioside exhibits anti-metastatic and anti-angiogenic qualities without causing cytotoxicity [1].

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Isoliquiritin Apioside (up to 100 μM) did not affect cell proliferation of HT1080 cells; instead, 100 μM ISLA slightly increased cell viability by approximately 10% compared to untreated control cells (CCK-8 assay, 48 h treatment). [1]
ISLA suppressed PMA-induced increase in MMP-9 activity and production in HT1080 cells, as shown by gelatin zymography (PMA 20 nM stimulation; ISLA treatment reduced gelatinolytic MMP-9 activity). ISLA did not inhibit MMP-2 activity. Total MMPs activity in HT1080 conditioned media was reduced by ISLA in a dose-dependent manner (one-way ANOVA, F = 91.44, p < 0.0001). [1]
ISLA inhibited serum-induced migration of HT1080 cells in Transwell assays: at 100 μM, reductions of 56.8% (8 h) and 71.8% (12 h) compared to control (8 h: F = 259.7, p < 0.0001; 12 h: F = 312.0, p < 0.0001, one-way ANOVA). In scratch-wound migration assays, 100 μM ISLA inhibited wound closure by 60.6% at 24 h (24 h: F = 87.3, p < 0.0001, one-way ANOVA). [1]
ISLA suppressed invasion of HT1080 cells across Matrigel-coated Transwell membranes: 100 μM ISLA caused approximately 83.2% inhibition compared to untreated control (F = 272.5, p < 0.0001, one-way ANOVA). In scratch-wound invasion assays, 100 μM ISLA inhibited invasion by 63.6% at 24 h (24 h: F = 26.28, p < 0.0001, one-way ANOVA). In three-dimensional spheroid invasion assays, 100 μM ISLA reduced invasion from spheroids by 72.1% at 3 days and 87.3% at 5 days post-treatment (T3: F = 9.006, p = 0.0061; T5: F = 42.39, p < 0.0001, one-way ANOVA). [1]
ISLA blocked PMA-induced phosphorylation of p38, ERK, and JNK in HT1080 cells (western blot analysis; 100 μM ISLA pretreatment for 12 h followed by 20 nM PMA stimulation for indicated times). ISLA also inhibited PMA-induced nuclear translocation of NF-κB p65 subunit (50 μM ISLA pretreatment for 12 h, then 20 nM PMA for 30 min; cytosolic and nuclear fraction western blot). [1]
ISLA suppressed hypoxia-induced HIF-1α nuclear accumulation in HT1080 cells: at 100 μM ISLA, the proportion of cells with nuclear HIF-1α was reduced to approximately 5% of control cells under CoCl₂ stimulation (F = 184.3, p < 0.0001) and 1% O₂ (F = 646.4, p < 0.0001, one-way ANOVA) (fluorescence immunocytochemistry). ISLA also inhibited hypoxia-induced HIF-1α accumulation and phosphorylation of Akt, mTOR, 4E-BP1, and p70S6K in a dose-dependent manner (western blot). [1]
Under normoxic conditions, ISLA reduced production of pro-angiogenic factors MMP-9 and placental growth factor (PlGF) in HT1080 conditioned media. Under hypoxic conditions (1% O₂), MMP-9 and PlGF levels increased by 1.69-fold and 2.52-fold respectively compared to normoxia, and ISLA reduced these levels. VEGF levels were slightly decreased by ISLA under both normoxia and hypoxia (Proteome Profiler Antibody Array). [1]
ISLA suppressed EGM-2-induced migration of HUVECs across Transwell membranes: at 25, 50, and 100 μM, reductions of 51.9%, 81.9%, and 89.9% respectively compared to control (F = 471.4, p < 0.0001, one-way ANOVA). ISLA also inhibited tube formation of HUVECs on basement membrane extract in a dose-dependent manner (F = 577.1, p < 0.0001, one-way ANOVA). [1]

Isoliquiritin Apioside suppressed spontaneous angiogenesis in the chick chorioallantoic membrane (CAM) assay: topical application of ISLA (100 μg per disk) inhibited angiogenesis by approximately 37% compared to vehicle alone (ED day 9). ISLA also efficiently blocked VEGF-induced angiogenesis (VEGF 200 ng per disk), reducing vessel formation to approximately 46.8% of the vehicle control level (total vessel length measured by ImageJ, n=3 per group). [1]

To measure total MMPs activity, culture supernatants of ISLA-treated or untreated HT1080 cells were collected and assayed using a MMPs Activity Assay Kit. Green fluorescence intensity was measured using a multi-mode reader at Ex/Em 490/525 nm. [1]
For gelatin zymography, cells were pretreated with indicated concentrations of ISLA in serum-free medium for 12 h and then stimulated with 20 nM PMA for an additional 24 h. Culture supernatants were collected, centrifuged to remove cell debris, and electrophoresed on an 8% SDS-PAGE gel containing 0.1% gelatin. After washing with washing buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2.5% Triton X-100), gels were incubated in activation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl₂, 0.02% NaN₃, 1 μM ZnCl₂) at 37°C for 24-48 h. Gels were stained with Coomassie Brilliant Blue R-250 solution and destained. Gelatinolytic MMP-9 activity was detected at 92 kDa and MMP-2 at 72 and 64 kDa as transparent bands. [1]

Cell viability was assessed using the CCK-8 assay. HT1080 cells were treated with indicated concentrations of ISLA for 48 h, and viability was measured. [1]
For Transwell migration assay, HT1080 cells or HUVECs (1×10⁴ cells) suspended in serum-free medium were loaded onto the upper chamber of Transwell chambers (8 μm pore size). In lower chambers, 10% FBS/RPMI1640 or EGM-2 was added. After incubation, cells on the upper surface were removed, and migrated cells on the lower surface were stained with 0.2% crystal violet/20% methanol solution and counted under a phase contrast microscope. [1]
For scratch-wound migration assay, cells cultured to about 90% confluence were pre-treated with 25 μg/mL mitomycin C for 30 min. Wounds were made using a 96-pin wound maker. Cells were incubated with or without ISLA, and wound images were captured every 3 h. Relative wound migration was calculated based on wound width at 0 h. [1]
For Transwell invasion assay, the same procedure as migration assay was performed using Matrigel (diluted 1:4 with serum-free medium) as the invasive barrier. [1]
For three-dimensional (3D) spheroid invasion assay, cells (3×10⁵) suspended in 50 μL prechilled spheroid formation extracellular matrix (ECM) were added to a 96-well ultra-low attachment microplate. After centrifugation at 200×g for 3 min, cells were incubated to form spheroids for 3 days. Then 50 μL prechilled invasion matrix was added, and plates were incubated for 1 h at 37°C. Culture media with or without ISLA were added, and plates were incubated for 5 days. Cell invasion into surrounding matrix was observed and photographed every 24 h. [1]
For western blot analysis, whole cell lysates and nuclear/cytosolic fractions were prepared using protein extraction reagents. Protein concentrations were determined using a bicinchoninic acid (BCA) kit. Equal protein aliquots (25 μg) were resolved by SDS-PAGE and immunoblotted using specific antibodies. Protein levels were measured using a chemiluminescence imaging system. [1]
For fluorescence immunocytochemistry of HIF-1α nuclear translocation, cells grown on glass bottom dishes were treated with ISLA for 12 h and then stimulated with 200 μM CoCl₂ for 6 h. Cells were fixed with 4% paraformaldehyde, permeabilized and blocked, then stained with Alexa Fluor 488-conjugated rabbit anti-HIF-1α antibody (diluted 1:500). Nuclei were counterstained with DAPI, and cells were observed under a fluorescence microscope. [1]
For HUVEC tube formation assay, 50 μL ice-chilled basement membrane extract (BME) was added to a 96-well plate and solidified at 37°C for 30 min. HUVECs (5×10⁴) pretreated with or without ISLA for 12 h were suspended in 100 μL EGM-2 and added to each well. After 4 h, tube formation was visualized under a phase contrast inverted microscope. [1]

Fertilized chicken eggs were incubated at 37°C with 65% humidity. On embryonic development (ED) day 3, albumin was carefully removed, and a round window was made on the blunt end. Windows were covered with adhesive tape, and eggs were returned to the incubator. On ED day 6, 5 mm disks loaded with Isoliquiritin Apioside (100 μg) and/or VEGF (200 ng) in PBS (20 μL) were placed on the chorioallantoic membrane (CAM) of individual embryos. Eggs were further incubated for 3 days. On ED day 9, the vasculature was macroscopically observed and photographed, and total vessel length was measured using ImageJ software (three samples per group). [1]

Isoliquiritin Apioside up to 100 μM did not show cytotoxicity in HT1080 cells; instead, cell viability was slightly increased by approximately 10% at 100 μM compared to untreated control (CCK-8 assay, 48 h). No in vivo toxicity data (e.g., LD50, hepatotoxicity, nephrotoxicity) were reported. No information on plasma protein binding or drug-drug interactions was provided. [1]

[1]. Isoliquiritin Apioside Suppresses in vitro Invasiveness and Angiogenesis of Cancer Cells and Endothelial Cells.Front Pharmacol. 2018 Dec 10;9:1455.

Neo-matrine belongs to the flavonoid class of compounds and is a type of glycoside. It has been reported that licorice contains isoglycyrrhizin, and relevant data is available for reference.

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Isoliquiritin Apioside is a component isolated from Glycyrrhizae radix rhizome (the root of Glycyrrhiza uralensis). Previous studies have shown that ISLA exhibits inhibitory activity against H₂O₂ and 4NQO-induced DNA damage (SOS chromotest and Comet assay) and suppresses electrically induced tetanic contractions in rat gastrocnemius muscle (20 μmol/kg injection). This study is the first to report anti-metastatic and anti-angiogenic activities of ISLA in cancer cells and endothelial cells. ISLA suppressed migration, invasion, and angiogenesis without affecting cell proliferation or inducing cell death. ISLA did not affect adhesion of HT1080 cells to ECMs (fibronectin, type I collagen, vitronectin) nor expression of integrins or EMT-related proteins (β-catenin, E-cadherin, N-cadherin, snail, vimentin, ZO-1). The anti-metastatic mechanism involves blocking PMA-induced MAPK activation and NF-κB nuclear translocation, leading to decreased MMP activity. The anti-angiogenic mechanism involves suppression of the HIF-1α/Akt/mTOR pathway and reduction of pro-angiogenic factors (MMP-9, PlGF, VEGF). ISLA also directly suppressed angiogenic activities of HUVECs (migration, tube formation) and reduced vessel formation in CAM assay. The authors suggest that ISLA may be a safe and effective lead compound for anti-cancer drug development to limit primary tumor spread. [1]

C26H30O13

550.5086

550.168

120926-46-7

6442433

Yellow to orange solid

1.6±0.1 g/cm3

901.0±65.0 °C at 760 mmHg

301.9±27.8 °C

0.0±0.3 mmHg at 25°C

1.709

1.96

8

13

9

39

833

8

O([C@@]1([H])[C@@]([H])([C@@](C([H])([H])O[H])(C([H])([H])O1)O[H])O[H])[C@@]1([H])[C@]([H])(OC2C([H])=C([H])C(/C(/[H])=C(\[H])/C(C3C([H])=C([H])C(=C([H])C=3O[H])O[H])=O)=C([H])C=2[H])O[C@]([H])(C([H])([H])O[H])[C@]([H])([C@]1([H])O[H])O[H]

VMMVZVPAYFZNBM-KVFWHIKKSA-N

InChI=1S/C26H30O13/c27-10-19-20(32)21(33)22(39-25-23(34)26(35,11-28)12-36-25)24(38-19)37-15-5-1-13(2-6-15)3-8-17(30)16-7-4-14(29)9-18(16)31/h1-9,19-25,27-29,31-35H,10-12H2/b8-3+/t19-,20-,21+,22-,23+,24-,25+,26-/m1/s1

(E)-3-[4-[(2S,3R,4S,5S,6R)-3-[(2S,3R,4R)-3,4-dihydroxy-4-(hydroxymethyl)oxolan-2-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-1-(2,4-dihydroxyphenyl)prop-2-en-1-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~25 mg/mL (~45.41 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.54 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 (4.54 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.)