Ginger components and candidate genes associated with colon cancer
After searching, filtering, and removal of the duplicates, 6 putative components (MOL002464, MOL002501, MOL002514, MOL000358, MOL000359, and MOL002467) with OB ≥ 30% and DL ≥ 0.18 were selected from TCMSP database (listed in Table 1). Besides, the representative component 6-gingerol with pharmacological activities and high contents was also recruited in this study, although the DL value is 0.16.
Table 1 The active compounds and their properties and structures
Meanwhile, 285 target genes in total interacting with those potential components were collected, among which 251 were obtained from the Swiss TargetPrediction and 34 from TCMSP database. In addition, there remained 1356 colon cancer-associated genes, in which 1165 genes were from the GeneCards database, 156 from the OMIM, and 35 from the Drugbank. Finally, a total of 118 intersection genes, also the candidate genes, were collected for further mechanisms study of ginger on treatment of colon cancer (Figure 2A).
PPI network analysis
The 118 candidate genes were connected to establish an initial PPI network that included 104 nodes and 517 edges, and 14 isolated targets genes were removed (Figure 2B). In addition, the top two protein-protein interacting clusters were constructed (Figure 2C and 2D). Genes PIK3R1, CCNA2, and TP53 were as the core targets for one cluster (37 nodes and 179 edges), while another cluster (17 nodes and 42 edges) with STAT 3 and JAK2 as the core targets. Based on the analysis of this network, only the genes with higher values of “degree”, “betweenness” and “closeness” (above the median value) were collected as the key targets of ginger for colon cancer. Ultimately, 34 key targets, with PIK3CA, SRC, and TP53 as the top ones, were collected for pathway enrichment analysis (Table 2).
Table 2 The major targets of ginger for colon cancer treatment and the topological parameters
Uniprot ID
|
Gene symbol
|
Target name
|
Degree
|
Betweenness
|
Closeness
|
P42336
|
PIK3CA
|
Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform
|
42
|
0.187322
|
0.572254
|
P12931
|
SRC
|
Proto-oncogene tyrosine-protein kinase
|
38
|
0.107991
|
0.568966
|
P27986
|
PIK3R1
|
Phosphatidylinositol 3-kinase regulatory subunit alpha
|
38
|
0.106891
|
0.568966
|
P04637
|
TP53
|
Cellular tumor antigen p53
|
35
|
0.103695
|
0.546961
|
P27361
|
MAPK3
|
Mitogen-activated protein kinase
|
33
|
0.084312
|
0.543956
|
P40763
|
STAT3
|
Signal transducer and activator of transcription 3
|
31
|
0.080670
|
0.535135
|
P31749
|
AKT1
|
RAC-alpha serine/threonine-protein kinase
|
29
|
0.068649
|
0.532258
|
P07900
|
HSP90AA1
|
Heat shock protein HSP 90-alpha
|
27
|
0.066522
|
0.529412
|
P45983
|
MAPK8
|
Mitogen-activated protein kinase 8
|
23
|
0.065784
|
0.502538
|
O60674
|
JAK2
|
Tyrosine-protein kinase JAK2
|
20
|
0.056021
|
0.497487
|
P42338
|
PIK3CB
|
Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform
|
19
|
0.036367
|
0.492537
|
P00533
|
EGFR
|
Epidermal growth factor receptor
|
19
|
0.028652
|
0.485294
|
Q05513
|
PRKCZ
|
Protein kinase C zeta type
|
18
|
0.021649
|
0.480583
|
Q05655
|
PRKCD
|
Protein kinase C delta type
|
18
|
0.021286
|
0.478261
|
P23458
|
JAK1
|
Tyrosine-protein kinase JAK1
|
18
|
0.017349
|
0.478261
|
P03372
|
ESR1
|
Estrogen receptor
|
18
|
0.016778
|
0.473684
|
Q04206
|
RELA
|
Transcription factor p65
|
17
|
0.014046
|
0.469194
|
Q16539
|
MAPK14
|
Mitogen-activated protein kinase 14
|
17
|
0.011251
|
0.469194
|
Q05397
|
PTK2
|
Focal adhesion kinase 1
|
15
|
0.009972
|
0.464789
|
Q02750
|
MAP2K1
|
Dual specificity mitogen-activated protein kinase kinase 1
|
15
|
0.009205
|
0.464789
|
P04626
|
ERBB2
|
Receptor tyrosine-protein kinase erbB-2
|
15
|
0.008965
|
0.464789
|
P42574
|
CASP3
|
Caspase-3
|
15
|
0.008693
|
0.462617
|
P04150
|
NR3C1
|
Glucocorticoid receptor
|
14
|
0.00863
|
0.460465
|
P05412
|
JUN
|
Transcription factor AP-1
|
14
|
0.008501
|
0.458333
|
P52333
|
JAK3
|
Tyrosine-protein kinase JAK3
|
14
|
0.008096
|
0.450000
|
P06493
|
CDK1
|
Cyclin-dependent kinase 1
|
14
|
0.007042
|
0.450000
|
P17252
|
PRKCA
|
Protein kinase C alpha type
|
13
|
0.006885
|
0.447964
|
P10275
|
AR
|
Androgen receptor
|
13
|
0.005944
|
0.445946
|
P08069
|
IGF1R
|
Insulin-like growth factor I receptor
|
11
|
0.005841
|
0.443946
|
O14965
|
AURKA
|
Aurora kinase A
|
11
|
0.005668
|
0.440000
|
P00519
|
ABL1
|
Tyrosine-protein kinase ABL1
|
11
|
0.005616
|
0.432314
|
Q00987
|
MDM2
|
E3 ubiquitin-protein ligase Mdm2
|
10
|
0.00552
|
0.415966
|
P24941
|
CDK2
|
Cyclin-dependent kinase 2
|
10
|
0.004538
|
0.415966
|
P49841
|
GSK3B
|
Glycogen synthase kinase-3 beta
|
8
|
0.004458
|
0.415966
|
GO enrichment analysis
To investigate the biological function of target genes of ginger for colon cancer, the biological process was conducted by the Metascape database. As shown in Figure 3A, 20 markedly enriched BPs terms were shown (p < 0.01), with transmembrane receptor protein tyrosine kinase GO: 0007169, cellular response to nitrogen compound GO: 1901699, and peptidyl-serine phosphorylation GO: 0018105 as the top ones. The size of the dot in bubble chart indicates the number of target genes in the corresponding function pathway, and the enrichment expresses the ratio of the number of target genes belonging to the number of all the annotated genes located in the pathway. Besides, further analysis of the network was performed using Cytoscape plugin ClueGO, and we found that these BPs terms were mainly associated with the phosphatidylinositol 3-kinase signaling, cellular response to oxidative stress, cellular response to peptide hormone stimulus, and peptidyl-serine phosphorylation (Figure 3B).
For CCs, the items with significant enrichment were in perinuclear region of cytoplasm GO: 0048471, transferase complex, transferring phosphorus-containing groups GO: 0061695, and microtubule organizing center GO: 0005815; and for MFs in phosphotransferase activity, alcohol group receptor GO: 0016773, kinase binding GO: 0019900, and protein tyrosine kinase activity GO: 0004713.
KEGG pathway enrichment analysis
The results of pathway enrichment showed how ginger acts on the pathway, thereby playing a therapeutic role in colon cancer. Here, based on the 34 core target genes, 20 significant signaling pathways with p < 0.01 were picked out for further analysis, including pathways in cancer (hsa05200), endocrine resistance (hsa01522), EGFR tyrosine kinase inhibitor resistance (hsa01521), and PI3K-Akt signaling pathway (hsa04151) as the top ones. Among these pathways, pathways in cancer was identified as a set of important key pathway with the most target enrichment and lowest p value.
Drug-compound-disease-target-pathway network
The drug-compound-disease-target-pathway network was shown in Figure 5, which included 62 nodes (6 compounds, 34 targets, and 20 pathways) and 311 edges. The pink rectangle node is the Chinese herbal medicine ginger; yellow nodes is colon cancer, V nodes is 20 significant signaling pathway; purple rectangle nodes are 34 key targets; blue ellipse node represents 6 potential active components, while lines represent the interactions between them. According to the network analysis, multiple components from ginger acts on at least one target genes, and 6-gingerol was regarded as the most effective compound that interacts with 17 target genes. Besides, most of target genes were regulated by at least 2 active components, and at least 10 genes potentially involved in each pathway related to colon cancer. This network analysis indicated the characteristics of multiple components and multiple targets of ginger in the treatment of colon cancer.
Molecular docking result and analysis
A total of 8 target genes which have more interactions with other targets, pathway and potential components were selected to binding with the 6 putative components. The binding affinities of the testing components, indicating as LibDock Scores, were compared to the original ligands of target protein. As LibDock Scores displayed in Table 3 and heat map of the docking score exhibited in Figure 6, all the testing components had strong interactions than the prototype ligands, or similar effects to the ligands, with TP53, HSP90AA1, MAPK8, CASP3, JAK2, and ERBB2. Among these compounds, 1-monolinolein (MOL002464) and [(1S)-3-[(E)-but-2-enyl]-2-methyl-4-oxo-1-cyclopent-2-enyl](1R,3R)-3-[(E)-3-methoxy-2-methyl-3-oxoprop-1-enyl]-2,2-dimethylcyclopropane-1-carboxylate (MOL002501) always displayed the highest binding affinities, especially for the target HSP90AA1. These findings revealed that six components including MOL002464, MOL002501, MOL002467, MOL000358, MOL002514, and MOL000359 were forecasted as the active components of ginger for colon cancer and TP53, HSP90AA1, MAPK8, CASP3, JAK2, and ERBB2 were the major target for reaching this effect. The representative molecular docking results of the major targets and active components of ginger were exhibited in Figure 7.
Table 3 The LibDock Scores of 8 core targets and their interacting compounds
Target
|
PDB ID
|
Components
|
LibDock Score
|
SRC
|
2BDF
|
Ligand 1
|
141.019
|
MOL002464
|
126.807
|
MOL002501
|
108.312
|
MOL002467
|
107.09
|
MOL000358
|
113.863
|
MOL002514
|
94.4539
|
MOL000359
|
113.863
|
PIK3R1
|
4ZOP
|
Ligand 2
|
124.549
|
MOL002464
|
122.846
|
MOL002501
|
116.663
|
MOL002467
|
118.603
|
MOL000358
|
122.444
|
MOL002514
|
96.8916
|
MOL000359
|
120.734
|
TP53
|
5O1F
|
Ligand 3
|
105.7
|
MOL002464
|
135.803
|
MOL002501
|
135.516
|
MOL002467
|
122.954
|
MOL000358
|
115.344
|
MOL002514
|
112.737
|
MOL000359
|
115.344
|
HSP90AA1
|
4BQG
|
Ligand 4
|
89.7644
|
MOL002464
|
130.891
|
MOL002501
|
142.366
|
MOL002467
|
119.565
|
MOL000358
|
116.638
|
MOL002514
|
110.012
|
MOL000359
|
116.638
|
MAPK8
|
4E73
|
Ligand 5
|
91.9872
|
MOL002464
|
98.8619
|
MOL002501
|
99.3929
|
MOL002467
|
99.8414
|
MOL000358
|
99.7914
|
MOL002514
|
92.4867
|
MOL000359
|
99.7914
|
JAK2
|
3KCK
|
Ligand 6
|
106.149
|
MOL002464
|
116.428
|
MOL002501
|
112.673
|
MOL002467
|
103.286
|
MOL000358
|
101.945
|
MOL002514
|
93.3826
|
MOL000359
|
102.536
|
CASP3
|
1RE1
|
Ligand 7
|
85.4841
|
MOL002464
|
111.344
|
MOL002501
|
106.137
|
MOL002467
|
104.814
|
MOL000358
|
87.2257
|
MOL002514
|
85.8912
|
MOL000359
|
85.7436
|
ERBB2
|
3PPO
|
Ligand 8
|
103.488
|
MOL002464
|
139.158
|
MOL002501
|
149.566
|
MOL002467
|
128.035
|
MOL000358
|
136.813
|
MOL002514
|
103.498
|
MOL000359
|
136.762
|