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Supplementary Materials Supplementary Materials A network pharmacology study on the molecular mechanisms of FDY003 for breast cancer Ho-Sung Lee1,2, In-Hee Lee1, Kyungrae Kang2, Sang-In Park3, Seung-Joon Moon2, Chol Hee Lee2, and Dae-Yeon Lee1,2,* 1The Fore, 87 Ogeum-ro, Songpa-gu, Seoul 05542, Republic of Korea. 2Forest Hospital, 129 Ogeum-ro, Songpa-gu, Seoul 05549, Republic of Korea. 3Forestheal Hospital, 173 Ogeum-ro, Songpa-gu, Seoul 05641, Republic of Korea. *Correspondence should be addressed to Dae-Yeon Lee; [email protected] Supplementary Figures Supplementary Figure S1. Effects of FDY003 on the viability of breast cancer cells. MCF-7, MDA-MB-453, and MDA-MB-231 human breast cancer cells were treated with indicated doses of FDY003 for 72 hours and their viability was measured. Data represent the mean ± S.D. of five replicates. p-values were calculated by comparison with untreated control cells. **, p < 0.01; ***, p < 0.001; two-tailed Student’s t-test. 2 Supplementary Figure S2. Contribution index analysis of active compounds of FDY003. A graph depicting the analysis result of contribution index (CI) of active chemical compounds in FDY003. The sum of CIs for the top two compounds, quercetin and luteolin, was found to exceed 85%. 3 Category Term Count Percent [%] p-value Cellular aromatic compound metabolic process 82 1.38 7.89E-07 Cellular hormone metabolic process 16 12.31 8.26E-12 Cellular response to chemical stimulus 95 2.78 1.42E-33 Cellular response to chemical stress 28 7.82 4.45E-17 Cellular response to drug 22 4.91 1.01E-08 Cellular response to growth f actor stimulus 22 3.08 6.48E-05 Cellular response to hormone stimulus 31 4.28 9.71E-12 Cellular response to stimulus 115 1.48 2.16E-18 Cellular response to stress 53 2.52 1.85E-12 Cellular response to toxic substance 15 5.93 2.42E-06 Cellular response to v ascular endothelial growth f actor stimulus 7 10.00 2.33E-03 Drug metabolic process 29 4.83 3.78E-12 Estrogen metabolic process 11 34.38 9.27E-13 Hormone metabolic process 18 7.38 1.20E-09 Immune response 41 1.79 3.04E-04 Positiv e regulation of blood v essel endothelial cell migration 7 8.86 5.31E-03 Regulation of apoptotic process 53 3.30 1.18E-17 Regulation of apoptotic signaling pathway 19 4.59 1.02E-06 Regulation of cell cycle 27 2.13 2.71E-03 Regulation of cell death 56 3.19 3.03E-18 Regulation of cell migration 28 2.86 3.18E-06 Regulation of cell motility 30 2.88 6.45E-07 Regulation of cell population prolif eration 53 3.03 5.72E-16 Regulation of cellular metabolic process 92 1.44 4.53E-10 Gene ontology Biological process Regulation of cellular response to stress 28 3.66 1.08E-08 Regulation of def ense response 21 2.60 2.18E-03 Regulation of DNA-binding transcription factor activity 16 3.64 6.41E-04 Regulation of growth 22 3.17 3.71E-05 Regulation of hormone lev els 27 4.98 1.95E-11 Regulation of immune system process 40 2.37 1.39E-07 Regulation of kinase activity 36 3.95 4.77E-13 Regulation of metabolic process 96 1.37 1.16E-09 Regulation of mitotic cell cycle phase transition 14 3.13 2.09E-02 Regulation of programmed cell death 54 3.31 4.01E-18 Regulation of protein kinase activ ity 30 3.74 9.70E-10 Regulation of response to stimulus 80 1.81 2.54E-13 Regulation of response to stress 45 2.86 6.71E-12 Regulation of signal transduction 61 1.88 3.40E-09 Regulation of signaling 72 1.96 5.44E-13 Regulation of stress-activated protein kinase signaling cascade 10 4.15 3.78E-02 Regulation of v ascular endothelial growth f actor production 5 15.63 8.92E-03 Response to chemical 106 2.16 6.20E-30 Response to drug 49 4.48 1.17E-21 Response to estradiol 11 7.80 2.58E-05 Response to growth factor 22 2.96 1.27E-04 Response to hormone 40 3.91 1.00E-14 Response to radiation 21 4.70 7.37E-08 Response to toxic substance 28 5.09 3.35E-12 Signal transduction in response to DNA damage 8 5.97 1.97E-02 4 Category Term Count Percent [%] p-v alue Cell 24 2.15 7.02E-04 Chromosome 28 1.60 2.25E-02 Cy clin-dependent protein kinase holoenzy me complex 6 14.29 1.93E-04 Cytoplasm 113 0.97 2.77E-04 Cytosol 66 1.28 1.26E-04 Endomembrane sy stem 63 1.39 1.35E-05 Endoplasmic reticulum 33 1.82 2.71E-04 Cellular components Extracellular space 48 1.35 2.90E-03 Membrane 97 1.00 4.60E-03 Mitochondrion 26 1.59 4.70E-02 Nuclear chromosome 24 1.86 8.19E-03 Organelle 125 0.91 4.13E-04 Plasma membrane 63 1.12 2.72E-02 Protein kinase complex 8 7.84 2.81E-04 Vesicle 51 1.30 4.02E-03 Gene ontology Aromatase activ ity 7 25.93 7.64E-07 Catalytic activity 109 1.86 9.43E-26 Drug binding 52 2.95 1.36E-15 Enzy me binding 53 2.36 8.40E-12 Enzy me regulator activ ity 21 2.01 3.72E-02 Estrogen 16-alpha-hy droxy lase activ ity 5 62.50 9.77E-07 Estrogen 2-hy droxy lase activ ity 3 60.00 3.10E-03 Molecular f unction Kinase activ ity 37 4.49 7.76E-16 Ligand-activ ated transcription f actor activ ity 6 12.77 1.10E-03 Nuclear receptor activ ity 6 12.77 1.10E-03 Protein kinase activ ity 36 5.87 3.72E-19 Signaling receptor binding 32 1.95 4.92E-04 Steroid hormone receptor activ ity 5 8.93 4.95E-02 Steroid hy droxy lase activ ity 10 26.32 1.49E-10 Transcription f actor binding 21 3.03 5.60E-05 Apoptosis 11 8.09 9.38E-04 Breast cancer 11 7.48 1.96E-03 Cell cy cle 10 8.06 2.45E-03 Cellular senescence 13 8.23 1.18E-04 EGFR ty rosine kinase inhibitor resistance 12 15.19 3.23E-07 Endocrine resistance 17 17.71 1.06E-11 ErbB signaling pathway 10 11.90 7.17E-05 Estrogen signaling pathway 12 8.76 1.59E-04 Focal adhesion 14 7.07 2.80E-04 FoxO signaling pathway 13 10.16 1.02E-05 KEGG HIF-1 signaling pathway 11 10.09 1.07E-04 Pathway MAPK signaling pathway 15 5.08 6.38E-03 p53 signaling pathway 9 12.50 1.71E-04 Pathway s in cancer 36 6.81 3.21E-12 PD-L1 expression and PD-1 checkpoint pathway in cancer 11 12.36 1.34E-05 PI3K-Akt signaling pathway 24 6.82 1.18E-07 Platinum drug resistance 7 9.72 1.26E-02 Ras signaling pathway 12 5.19 2.84E-02 Steroid hormone biosy nthesis 12 20.00 1.14E-08 TNF signaling pathway 13 11.61 2.03E-06 VEGF signaling pathway 9 15.25 3.01E-05 Wnt signaling pathway 10 6.25 2.10E-02 Supplementary Figure S3. Functional enrichment analysis for the breast cancer-associated targets of FDY003. Tables showing the result of gene ontology and pathway enrichment analysis for the breast cancer-related targets of FDY003. 5 Supplementary Figure S4. Functional interaction analysis for the breast cancer-associated targets of FDY003. Black nodes, breast cancer-related targets; colored edges, distinct mechanisms underlying functional interactions between the targets. 6 Supplementary Tables Supplementary Table S1. List of the chemical compounds in FDY003. Herbal medicines Compound OB Caco-2 DL Hdon Hacc MW AlogP RBN LjT (-)-(3R,8S,9R,9aS,10aS)-9-ethenyl-8-(beta-D- 3.22 -1.72 0.80 5 12 443.45 -2.71 5 glucopyranosyloxy)-2,3,9,9a,10,10a-hexahydro-5- oxo-5H,8H-pyrano[4,3-d]oxazolo[3,2-a]pyridine- 3-carboxylic acid LjT (-)-(3R,8S,9R,9aS,10aS)-9-ethenyl-8-(beta-D- 87.47 -0.55 0.23 2 7 281.29 -0.96 2 glucopyranosyloxy)-2,3,9,9a,10,10a-hexahydro-5- oxo-5H,8H-pyrano[4,3-d]oxazolo[3,2-a]pyridine- 3-carboxylic acid_qt LjT (-)-Caryophyllene oxide 32.67 1.58 0.13 0 1 220.39 3.52 0 LjT ()-Menthol 59.33 1.27 0.03 1 1 156.30 2.78 1 LjT (-)-α-Pinene 46.25 1.85 0.05 0 0 136.26 2.87 0 LjT ()-α-Terpineol 46.30 1.28 0.03 1 1 154.28 2.42 1 LjT (+)-Ledol 16.96 1.43 0.12 1 1 222.41 3.20 0 LjT (1alpha,3R,4alpha,5R)-3,4,5-Tris[[(2E)-3-(3,4- 3.01 -1.46 0.45 7 15 677.63 2.86 13 dihydroxyphenyl)-1-oxo-2-propen-1-yl]oxy]-1- hydroxycyclohexanecarboxylic acid LjT (1R,2R,4R)-Dihydrocarveol 51.17 1.32 0.03 1 1 154.28 2.58 1 LjT (1R,4S,4aR)-1-isopropyl-4-methyl-7-methylene- 19.50 1.84 0.08 0 0 204.39 4.80 1 2,3,4,4a,5,6-hexahydro-1H-naphthalene LjT (1S,2S)-2-isopropenyl-4-isopropylidene-1- 34.47 1.87 0.06 0 0 204.39 4.93 2 methyl-1-vinylcyclohexane LjT (1S,4E,8E,10R)-4,8,11,11- 21.69 1.86 0.08 0 0 204.39 4.70 0 tetramethylbicyclo[8.1.0]undeca-4,8-diene LjT (2S)-2-methylbutan-1-ol 81.23 1.06 0.00 1 1 88.17 1.29 2 LjT (3R,4aS,5R,6R)-6-hydroxy-3-methoxy-5-vinyl- 60.89 0.09 0.10 1 5 226.25 -0.04 2 4,4a,5,6-tetrahydro-3H-pyrano[5,4-c]pyran-1-one LjT (5Z,9Z)-6,10,14-trimethylpentadeca-5,9,13-trien- 37.84 1.58 0.10 0 1 262.48 5.45 9 2-one LjT (E,E,E)-3,7-11,16-tetramethyl hexadeca- 42.48 1.42 0.14 1 1 290.54 6.60 11 2,6,10,14-tetraen-1-ol LjT (E,Z)-farnesol 36.73 1.32 0.06 1 1 222.41 4.76 7 LjT (S)-phenethyl 2-bromopropanoate 9.10 1.27 0.05 0 2 257.14 3.19 5 LjT (Z,E)-farnesol 41.14 1.34 0.06 1 1 222.41 4.76 6 LjT [(1S)-endo]-(-)-Borneol 83.54 1.22 0.05 1 1 154.28 1.98 0 LjT 1,6-Dicyclohexylhexane 15.40 1.81 0.11 0 0 250.52 7.43 7 8 LjT 1H,3H-Pyrano(3,4-c)pyran-1-one, 5-ethenyl-6- 4.96 -1.08 0.38 4 9 358.38 -1.59 4 (beta-D-glucopyranosyloxy)-4,4a,5,6-tetrahydro-, (4aS-(4aalpha,5beta,6alpha))- LjT 2-(2,4-dimethoxyphenyl)-3-hydroxy-7-methoxy- 12.94 0.72 0.33 1 6 328.34 2.52 4 chromone LjT 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxy- 29.24 0.90 0.34 1 6 328.34 2.82 3 chromone LjT 2,3-DIMETHYLPYRAZINE 30.82 1.07 0.02 0 2 108.16 0.09 0 LjT 2-[(1R,3S,4S)-3-isopropenyl-4-methyl-4- 19.03 1.37 0.07 1 1 222.41 3.70 3 vinylcyclohexyl]propan-2-ol LjT 2-[(2S,5R)-5-ethenyl-5-methyloxolan-2- 68.08 0.99 0.04 1 2 170.28 1.43 2 yl]propan-2-ol LjT 2-bromododecane 17.60 1.82 0.03 0 0 249.27 5.89 9 LjT 2H-Pyran-5-carboxylic acid, 4-(2,2- 22.59 -1.25 0.45 4 11 434.49 -1.73 10 dimethoxyethyl)-3-ethenyl-2-(beta-D- glucopyranosyloxy)-3,4-dihydro-, methyl ester, (2S,3R,4S)- LjT 2-isopropenyl-5-methylhex-4-enal 27.69 1.33 0.02 0 1 152.26 2.83 4 LjT 2-METHYLPENTADECANE 4.35 1.82 0.06 0 0 226.50 7.47 12 LjT 3,4-Dicaffeoylquinic acid 1.71 -1.40 0.69 6 12
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