Unveiling the Mechanism of the ChaiShao Shugan Formula Against Triple-Negative Breast Cancer

Figures & data

Figure 1 Compounds of CSSGF scanned by LC-MS/MS. (a) MRM of CSSGF (positive mode). (b) MRM chromatogram of CSSGF (negative mode).

Abbreviations: CSSGF, Chaishao Shugan Formula; LC-MS/MS, Liquid Chromatograph Mass Spectrometer/Mass Spectrometer; MRM, Meteorological chromatography.

Figure 2 Prediction of TNBC related targets. (a) Identification of deferentially expressed genes (DEGs) between breast cancer and breast normal tissues.(b)The volcano data of different gene between breast cancer and breast normal tissues. The blue part shows the downregulated genes. The red part indicates the upregulated genes. And the black part represents the stable genes. (c) Identification of deferentially expressed genes in GEO data set with GSM38959.(d) The volcano data of different gene in the GSM38959 dataset. (e)The intersecting genes in the GEO and TCGA databases. (f)The volcano data of different gene of intersecting genes in the GEO and TCGA databases.

Abbreviations: GEO, Gene Expression Omnibus; TCGA, The Cancer Genome Atlas.

Table 1 Information on Active Compounds of CSSGF Screened by TCMSP

Mol ID	Molecule Name	OB	DL	Source
MOL001645	Linoleyl acetate	42.1	0.2	Chaihu
MOL000098	Quercetin	46.43	0.28	Chaihu
MOL000354	Isorhamnetin	49.6	0.31	Chaihu
MOL000422	Kaempferol	41.88	0.24	Chaihu, Baishao
MOL000449	Stigmasterol	43.83	0.76	Chaihu
MOL000490	Petunidin	30.05	0.31	Chaihu
MOL002776	Baicalin	40.12	0.75	Chaihu
MOL004598	3,5,6,7-tetramethoxy-2-(3,4,5- trimethoxyphenyl) chromone	31.97	0.59	Chaihu
MOL004609	Areapillin	48.96	0.41	Chaihu
MOL004624	Longikaurin A	47.72	0.53	Chaihu
MOL000211	Mairin	55.38	0.78	Baishao
MOL000358	Beta-sitosterol	36.91	0.75	Baishao, Taizishen
MOL000359	Sitosterol	36.91	0.75	Baishao
MOL001918	Paeoniflorgenone	87.59	0.37	Baishao
MOL001919	(3S,5R,8R,9R,10S,14S)-3,17-dihydroxy-4,4,8,10,14-pentamethyl-2,3,5,6,7,9-hexahydro-1H-cyclopenta[a]phenanthrene-15,16-dione	43.56	0.53	Baishao
MOL001924	Paeoniflorin	53.87	0.79	Baishao
MOL000492	(+)-catechin	54.83	0.24	Baishao
MOL000273	(2R)-2-[(3S,5R,10S,13R,14R,16R,17R)-3,16-dihydroxy-4,4,10,13,14-pentamethyl-2,3,5,6,12,15,16,17-octahydro-1H-cyclopenta[a]phenanthren-17-yl]-6-methylhept-5-enoic acid	30.93	0.81	Fuling
MOL000275	Trametenolic acid	38.71	0.8	Fuling
MOL000279	Cerevisterol	37.96	0.77	Fuling
MOL000282	Ergosta-7,22E-dien-3beta-ol	43.51	0.72	Fuling
MOL000296	Hederagenin	36.91	0.75	Fuling
MOL000022	12-senecioyl-2E,8E,10E-atractylentriol	62.4	0.22	Baishu
MOL000033	(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R,5S)-5-propan-2-yloctan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol	36.23	0.78	Baishu
MOL000049	3β-acetoxyatractylone	54.07	0.22	Baishu
MOL000072	8β-ethoxy atractylenolide III	35.95	0.21	Baishu
MOL000006	Luteolin	36.16	0.25	Taizishen
MOL001689	Acacetin	34.97	0.24	Taizishen
MOL001790	Linarin	39.84	0.71	Taizishen
MOL002464	1-Monolinolein	37.18	0.3	Taizishen
MOL006756	Schottenol	37.42	0.75	Taizishen

Figure 3 CSSGF-ingredient-target-network. (a)Venn diagram of the target of CSSGF and the target of TNBC. (b) CSSGF ingredient-TNBC target. (c)The PPI network of CSSGF and TNBC targets. (d)The top 30 gene of CSSGF ingredient-TNBC.(e) Top 10 hub genes.

Abbreviations: TNBC, triple-negative breast cancer; PPI, Protein-Protein Interaction.

Table 2 Top ten Targets Information of PPI Network

Name	Degree	Betweenness Centrality
IL6	57	817.8404
MYC	51	341.9149
JUN	50	430.77734
CCND1	49	352.96796
CASP3	49	464.73227
ESR1	48	327.55377
EGFR	48	588.0265
MMP9	48	391.15576
EGF	45	285.4833
PTGS2	43	219.10057

Figure 4 GO and KEGG enrichment analysis of CSSGF -TNBC targets. (a) GO-BP enrichment analysis for potential targets of CSSGF-TNBC. (b). GO-CC enrichment analysis for potential targets of CSSGF-TNBC.(c). GO-MF enrichment analysis for potential targets of CSSGF-TNBC.(d) KEGG pathway enrichment analysis for potential targets of CSSGF-TNBC. (q value refers to -log10 P value). (e) “CSSGF -TNBC target–key pathways” network.

Abbreviations: CC, cellular component; MF, molecular function; BP, biological process; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5 The survival analysis via the Kaplan–Meier plotter. (a) The survival plot of JUN gene.(b) The survival plot of EGFR gene. (c) The survival plot of IL-6 gene.(d) The survival plot of ESR1 gene. (e) The survival plot of PTGS2 gene.(f) The survival plot of c-Myc gene.(g)The survival plot of MMP1 gene.(h)The survival plot of Apopain (Caspase-3) gene.

Abbreviation: HR, hazard risk.

Figure 6 CSSGF could down-regulate the expression of c-Myc and up-regulate the expression of EGFR both in MDA-MB-231 and MCF-7 cells. (a) WB results after drug treatetment in MDA-MB-231 cells.(b) Statistic results of a.**p<0.01,*: p<0.05, ns: no significant difference. (c) WB results after drug treatetment in MCF-7 cells.(d) Statistic results of c.**: p<0.01.*: p<0.05.

Abbreviation: ns: no significant difference.

Figure 7 Molecular docking results of core chemical components of CSSGF. (a) Quercetin- EGFR; (b) Quercetin- EGF; (c) Quercetin- c-Myc; (d) Beta-sitosterol-JUN; (e) Luteolin- CASP3; (f) Catechin- ESR1.

Figure 8 Quercetin could downregulate the expression of C-Myc both in MCF-7 cells and MDA-MB-231 cells. (a) CCK-8 assay in quercetin treated MDA-MB-231 cells.(b) CCK-8 assay in quercetin treated MCF-7 cells. (c)WB results of quercetin treated MDA-MB-231 cells.(d) Statistic results of c.**p<0.01.(e) WB results of quercetin treated MCF-7 cells.(f) Statistic results of e.**p<0.01.