PI3K/AKT/β-catenin signaling pathway . [1]

In the 75, 100, and 200 µM groups, heterophyllin B (0-200 µM; 12-48 hours) decreased cell viability, while it had no effect on the 5, 10, 25, and 50 µM groups [1]. Heterophyllin B (0-50 µM; 6 hours) dose-dependently suppresses p-PI3K, p-AKT, and β-catenin expression levels in ECA-109 cells [1].

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- Heterophyllin B (10, 25, and 50 µM for 24 h) significantly suppressed the adhesion of ECA-109 human esophageal carcinoma cells in a dose-dependent manner. Adhesion rates were approximately 76.22%, 32.22%, and 21.34% of the control group at 10, 25, and 50 µM, respectively. [1]
- Heterophyllin B (10, 25, and 50 µM for 24 h) significantly inhibited the invasion of ECA-109 cells in a Transwell assay, with invasion rates of approximately 71.25%, 45.24%, and 21.25% of the control group, respectively. [1]
- Western blot analysis showed that Heterophyllin B (10, 25, and 50 µM for 6 h) dose-dependently decreased the protein expression levels of phosphorylated PI3K (p-PI3K), phosphorylated AKT (p-AKT), and β-catenin in ECA-109 cells. [1]
- RT-qPCR and western blot analyses revealed that Heterophyllin B (10, 25, and 50 µM) dose-dependently increased E-cadherin mRNA and protein expression, while decreasing the mRNA and protein expression of snail, vimentin, MMP2, and MMP9 in ECA-109 cells. [1]
- Treatment with PI3K-activating peptide (740Y-P, 500 µg/ml for 3 h) increased cell adhesion and invasion to 164.08±9.36% and 121.12±9.63% of control, respectively. Co-treatment with Heterophyllin B (25 µM for 24 h) after 740Y-P pretreatment significantly reduced adhesion and invasion to 29.26±2.30% and 62.50±7.09% of control, respectively. [1]
- Western blot analysis showed that 740Y-P (500 µg/ml for 6 h) increased p-AKT/AKT and β-catenin levels, upregulated snail, vimentin, MMP2, and MMP9, and decreased E-cadherin. Treatment with Heterophyllin B (25 µM for 6 h or 24 h) reversed these changes, reducing p-AKT/AKT, β-catenin, snail, vimentin, MMP2, MMP9, and increasing E-cadherin. [1]

Cell Viability Assay[1]
Cell Types: ECA-109 Cell
Tested Concentrations: 5 µM, 10 µM, 25 µM, 50 µM, 75 µM, 100 µM and 200 µM
Incubation Duration: 12, 24 and 48 hrs (hours)
Experimental Results: High reduction in cell viability concentration.

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Western Blot Analysis[1]
Cell Types: ECA-109 Cell
Tested Concentrations: 0 µM, 10 µM, 25 µM, 50 µM
Incubation Duration: 6 hrs (hours)
Experimental Results: diminished PI3K/AKT phosphorylation and β-catenin expression.

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- Cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8) assay. ECA-109 cells were seeded in 96-well plates at 2×10³ cells/well and cultured for 24 h. Then, Heterophyllin B (0, 5, 10, 20, 50, 100, 200 µM) with or without 740Y-P (500 µg/ml) was added, and cells were cultured for another 12, 24, and 48 h. CCK-8 solution (20 µl) was added to each well and incubated for 1 h at 37°C. Optical density was read at 450 nm. HB at 75, 100, and 200 µM significantly decreased cell proliferation (P<0.05), while lower concentrations (5, 10, 25, 50 µM) did not reduce cell viability. [1]
- Cell adhesion was measured by seeding ECA-109 cells (1×10⁵ cells/ml) into 12-well plates and incubating for 1 h at 37°C. After washing with PBS, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, stained with Giemsa for 30 min, and optical density was read at 570 nm. Adhesion rate (%) = (OD_treated / OD_control) × 100%. HB (10, 25, 50 µM for 24 h) dose-dependently suppressed adhesion. [1]
- Cell invasion was assessed using 24-well Transwell chambers (8 µm pore size) coated with Matrigel (50 µl, 1:2 dilution). ECA-109 cells (1×10⁵ cells in 100 µl serum-free medium) pretreated with 740Y-P (500 µg/ml) and/or HB for 24 h were added to the upper chamber; the lower chamber contained 10% FBS as chemoattractant. After 24 h incubation at 37°C, invaded cells on the filter were fixed, stained with 0.1% crystal violet, and counted in five random high-power fields under a microscope. HB (10, 25, 50 µM) dose-dependently reduced invasion. [1]
- Western blot analysis: Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors, incubated at 4°C for 10 min then 95°C for 10 min, and centrifuged at 12,000×g for 10 min at room temperature. Protein (20-30 µg) was separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies against PI3K, p-PI3K, AKT, p-AKT, β-catenin, E-cadherin, vimentin, snail, MMP2, MMP9, and GAPDH, followed by secondary antibodies. Band intensities were quantified. [1]
- Reverse transcription-quantitative polymerase chain reaction (RT-qPCR): Total RNA was isolated from HB-treated ECA-109 cells (10, 25, 50 µM for 6 h) using TRIzol. cDNA was synthesized from 2 µg RNA using a First Strand cDNA kit. PCR amplification was performed for 40 cycles (95°C for 15 sec, 60°C for 45 sec) after an initial 10 min at 95°C, using SYBR Premix Ex Taq. Specific primers for E-cadherin, vimentin, snail, MMP2, MMP9, and GAPDH were used. Data were analyzed using the 2⁻ΔΔCt method and normalized to GAPDH. [1]

[1]. Heterophyllin B inhibits the adhesion and invasion of ECA-109 human esophageal carcinoma cells by targeting PI3K/AKT/β-catenin signaling. Mol Med Rep. 2016 Feb;13(2):1097-104.

There have been reports of isophyllin B being found in Pseudostellaria heterophylla, and relevant data are available for reference.

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- Heterophyllin B is a cyclic octapeptide derived from Pseudostellaria heterophylla (Miq.) Pax, traditionally used to promote fluid production in Chinese medicine. The study demonstrated that HB effectively suppressed adhesion and invasion of human esophageal carcinoma cells (ECA-109) by mediating the PI3K/AKT/β-catenin pathway and regulating expression of adhesion- and invasion-associated genes (E-cadherin, vimentin, snail, MMP2, MMP9). These findings suggest potential relevance for esophageal carcinoma therapy. [1]

C40H58N8O8

778.9

778.437

145459-19-4

102022989

White to off-white solid

1.3±0.1 g/cm3

1159.7±65.0 °C at 760 mmHg

655.1±34.3 °C

0.0±0.3 mmHg at 25°C

1.604

-1.82

5

8

6

56

1480

7

CCC(C)C1C(=O)NC(CC2=CC=CC=C2)C(=O)NCC(=O)NCC(=O)NC(CC(C)C)C(=O)N3C(CCC3)C(=O)N4C(CCC4)C(=O)N5C(CCC5)C(=O)N1

KYAVLJJQNUHRMR-PPLFBOPFSA-N

InChI=1S/C40H58N8O8/c1-5-25(4)34-37(53)44-27(21-26-12-7-6-8-13-26)35(51)42-22-32(49)41-23-33(50)43-28(20-24(2)3)38(54)47-18-10-15-30(47)40(56)48-19-11-16-31(48)39(55)46-17-9-14-29(46)36(52)45-34/h6-8,12-13,24-25,27-31,34H,5,9-11,14-23H2,1-4H3,(H,41,49)(H,42,51)(H,43,50)(H,44,53)(H,45,52)/t25-,27-,28-,29-,30-,31-,34-/m0/s1

(3S,9S,15S,24S,27S,30S)-24-benzyl-27-[(2S)-butan-2-yl]-15-(2-methylpropyl)-1,7,13,16,19,22,25,28-octazatetracyclo[28.3.0.03,7.09,13]tritriacontane-2,8,14,17,20,23,26,29-octone

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 : ~100 mg/mL (~128.38 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.21 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 (3.21 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.21 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.)