Identification and comparison and analysis of DEGs
To investigate the activation of ROS-KEAP1 in cancer progression, we identified the DEGs in GSE41328 and GSE4107. As shown in Fig. 1A & 1B, 1744 genes were identified as DEGs in GSE4107, and 1309 genes were identified as DEGs in GSE4107. The intersected genes in four genes sets were shown by Venn diagram, and 48 genes were finally identified as key genes (Fig. 1C).
By performing the KEGG analysis using the 48 key genes, it showed that cancer-related pathways such as “the pathways in cancer”, “proteoglycans in cancer”, and “PI3K-Akt signaling pathway” were significantly enriched (Fig. 1D&E).
GO and KEGG Analyses of DEGs in ROS /KEAP1 -related genes
By screening the genes with differential expression in GSE41328 or GSE4107, 15 up -regulated genes (IGFBP7, FN1, AQP8, HIGD1A, COX5A, MGST1, CES2, COL1A2, CAV1, CLU, CXCL12, VDR, IQGAP2, CASP7, PIGR), and 17 down -regulated genes (PDGFRB, NOTCH3, LY6E, MELTF9, NR1H4, TNFAIP6, DUSP10, PTGS, CP, IL1B, FSCN1, EIF5A2, FCGR3B, TWIST1, CASP6, SLC22A5, PLCE1, PLD1) were determined. Heatmaps were constructed to show up and down -regulated DEGs (Fig. 2A). The 17 up and 15 down-regulated ROS/KEAP1-related DEGs were used for GO function enrichment, respectively. Results suggested that Gene Ontology Classification by 17 up and 15 down-regulated DEGs were significantly enriched in pathways, such as biological adhesion, biological regulation and cell killing (Fig. 2B). The Gene Ontology Classification by up and down-regulated DEGs were significantly enriched in pathways such as the Cancer: overview, Signal transduction and so on (Fig. 2C, 2D). Furthermore, in the KEGG analysis showed that up and down-regulated DEGs were significantly enriched in cancer, apoptosis, signal transduction, metabolism and immune-related pathways (Fig. 2E-2H), pathways such as ECM-receptor interaction, Amoebiasis, and PI3K-Akt signaling pathway were significantly enriched (Enrichment score > 10).
Pearson correlation of DEGs in the PPI network and Identification of hub genes
Significant positive or negative correlations between the 48 key genes were constructed and 20 top correlated genes were found (Fig. 3A, 3B). Furthermore, the interactions of 48 genes ranked by degree were identified. as key genes (Fig. 3C, 3D). Furthermore, 17 top genes (MCODE Score > 1.5, Clustered Node) were identified in the PPI network and shown in the Table 1. Then, the Venn diagram of 20 top correlated genes in Pearson correlation and 17 top genes in the PPI network were constructed in Cytoscape, and 7 overlapping genes (COL1A1, COL1A2, CXCL12, CAV1, FN1, TIMP3, IGFBP7) were identified as hub genes (Fig. 3E). The expression heatmap of 7 hub genes were shown in Fig. 3F.
Table 1
The hub genes in the PPI network were identified by the MCODE plugin
MCODE::Clusters (1)
|
MCODE::Node Status (1)
|
MCODE::Score (1)
|
name
|
selected
|
shared name
|
Cluster 2
|
Clustered
|
5.153846
|
FN1
|
TRUE
|
FN1
|
Cluster 2
|
Clustered
|
4.761905
|
ANXA1
|
TRUE
|
ANXA1
|
Cluster 2
|
Seed
|
5.515152
|
IL1B
|
TRUE
|
IL1B
|
Cluster 2
|
Clustered
|
5.333333
|
CAV1
|
TRUE
|
CAV1
|
Cluster 2
|
Clustered
|
5.333333
|
PTGS2
|
TRUE
|
PTGS2
|
Cluster 2
|
Clustered
|
5
|
TNFAIP6
|
TRUE
|
TNFAIP6
|
Cluster 2
|
Clustered
|
5.384615
|
CXCL12
|
TRUE
|
CXCL12
|
Cluster 2
|
Clustered
|
5
|
ITGA2
|
TRUE
|
ITGA2
|
Cluster 1
|
Clustered
|
6.377778
|
COL1A1
|
TRUE
|
COL1A1
|
Cluster 1
|
Clustered
|
6.377778
|
COL1A2
|
TRUE
|
COL1A2
|
Cluster 1
|
Clustered
|
7
|
PDGFRB
|
TRUE
|
PDGFRB
|
Cluster 1
|
Clustered
|
6.377778
|
BGN
|
TRUE
|
BGN
|
Cluster 1
|
Clustered
|
7
|
TIMP3
|
TRUE
|
TIMP3
|
Cluster 1
|
Clustered
|
6
|
IGFBP7
|
TRUE
|
IGFBP7
|
Cluster 1
|
Clustered
|
6
|
FBN1
|
TRUE
|
FBN1
|
Cluster 1
|
Clustered
|
5.785714
|
SPP1
|
TRUE
|
SPP1
|
Cluster 1
|
Seed
|
7
|
THBS2
|
TRUE
|
THBS2
|
Development and validation of hub genes’ prognostic signature for COAD
Next, expressions of 7 hub genes in COAD and normal tissues were analyzed in the TCGA- COAD. It suggested that COL1A1 and COL1A2 were significantly increased while CXCL12 and CAV1 were significantly decreased in tumor compared to normal tissues (Fig. 4A). Additionally, analyses based on tumor stages revealed differences in gene expressions for COL1A2 and FN1 (Fig. 4B). KM survival curves further demonstrated that high-risk patients with COL1A1 and COL1A2 had lower OS compared to low-risk patients (Fig. 4C).In the training cohort, the area under the ROC curves (AUCs) for the OS of patients. The "ROC plotter" tool was used to validate the predictive value of hub genes. The AUCs of COL1A1 for treatment with 5-Fu, capecitabine, and fluoropyrimidines monotherapy were 0.61, 0.70, and 0.72, respectively (Fig. 4D). The AUC of COL1A2 for treatment with Bevacizumab was 0.63, while the AUC of CAV1 for treatment with fluoropyrimidines monotherapy was 0.63, and the AUC of CXCL12 for treatment with Bevacizumab was 0.62.
Correlation between the hub genes’ prognostic signature and clinical characteristics
To elucidate the underlying mechanism by which hub genes impact the prognosis of COAD patients, we utilized the UALCAN online database to analyze 374 COAD samples and 41 normal samples from TCGA. Both COL1A1 and COL1A2 showed significantly higher expression in COAD samples compared to normal samples (Fig. 4E). Moreover, distinct connections were observed between the significant differed expression of COL1A1 and COL1A2 with different stages. On the other hand, both CXCL12 and CAV1 exhibited significantly lower expression in COAD samples compared to normal samples, and there were no significant differences in their expression based on different stages (Fig. 4E). However, high expression of COL1A1 and COL1A2 was observed in Adenocarcinoma and Mucinous adenocarcinoma compared to normal tissues (Fig. 4F). In contrast, CXCL12 and CAV1 expression showed a decrease in Adenocarcinoma and Mucinous adenocarcinoma in a histological subtype-dependent manner.
Correlation Between the Prognosis-Related Genes and the TME
The TISCH database was used to analyze the expression of key hub genes in TME-related cells. In immune cells (CD4Tconv, Tprolif, DC) and epithelial cells, COL1A1 expression was low to moderate (Fig. 5A). Fibroblasts/Myofibroblasts showed the highest expression of COL1A1 and COL1A2, while DC, Endothelial, and Malignant cells had lower to moderate COL1A2 expression. Additionally, CXCL12 and CAV1 had the highest expression in Endothelial cells, Fibroblasts, and Myofibroblasts. Fibroblasts, Leydia cells, and Endothelial cells were the most abundant cell types analyzed in the HPA database. Furthermore, CXCL12 and COL1A1 had higher infiltration in TME-related cells compared to CAV1.
Furthermore, the distributions and expressions of COL1A1, COL1A2, CXCL12, and CAV1 in different cells at the single-cell level were analyzed using the HPA database (Fig. 7B-7I). The infiltrations of COL1A1 (Fig. 5B, 5C) and COL1A2 (Fig. 5D, 5E) in TME-related cells were higher compared to CXCL12 (Fig. 5F, 5G) and CAV1 (Fig. 5H, 5I), consistent with the findings presented in Fig. 5A. These results provide further evidence supporting the close association between COL1A1, COL1A2, and the tumor immune microenvironment (TIM) in CRC.
Correlation Between Immune Infiltrates and hub genes in COAD
To investigate the impact of hub genes on the TIM, we examined the correlations between the expression of COL1A1, COL1A2, CXCL12, and CAV1 and the infiltration levels of immune cells in TCGA-COAD. We observed strong associations between hub genes and immune cell infiltrations in COAD according to the TIMER database (Fig. 6A-6D). The accompanying linear regression analysis demonstrated that high expression of COL1A1, COL1A2, CXCL12, and CAV1 were associated with increased levels of immune cell infiltration. Notably, CD4+ T cells, macrophages, and dendritic cells exhibited the most significant coefficients in relation to COL1A1, COL1A2, CXCL12, and CAV1 in COAD. Furthermore, infiltration levels of immune cells in different copy number alternations (CNAs) were compared as well, which suggested that CNAs of hub genes could influence the infiltration levels of immune cells in COAD (Fig. 6E).
Target predicition and Molecular Interaction Analysis
Predicting ATC codes or targets of α-hederin is valuable for drug development (Table 2). NFE2L2 and KEAP1 were predicted targets, with a probability of 53.8% and 51%, respectively, and model accuracies of 96% and 82%. We generated an interaction network between α-hederin and predicted targets using Cytoscape (Fig. 7A). The chemical structure of α-hederin is illustrated in Fig. 7B.
Table 2
The predicted targets of α-hederin in SuperPred
Target Name
|
ChEMBL-ID
|
UniProt ID
|
PDB Visualization
|
TTD ID
|
Probability
|
Model accuracy
|
Mineralocorticoid receptor
|
CHEMBL1994
|
P08235
|
4PF3
|
Not Available
|
80%
|
100%
|
Lipoxin A4 receptor
|
CHEMBL4227
|
P25090
|
6OMM
|
Not Available
|
68%
|
100%
|
Sphingosine 1-phosphate receptor Edg-8
|
CHEMBL2274
|
Q9H228
|
Not Available
|
T50089
|
62%
|
100%
|
Neurotensin receptor 2
|
CHEMBL2514
|
O95665
|
Not Available
|
Not Available
|
60%
|
100%
|
Mitogen-activated protein kinase kinase kinase 14
|
CHEMBL5888
|
Q99558
|
6Z1T
|
Not Available
|
58%
|
100%
|
Lysosomal Pro-X carboxypeptidase
|
CHEMBL2335
|
P42785
|
3N2Z
|
Not Available
|
58%
|
100%
|
Casein kinase II alpha/beta
|
CHEMBL3038477
|
P67870
|
6TLS
|
T51565
|
76%
|
99%
|
Glycine transporter 2
|
CHEMBL3060
|
Q9Y345
|
Not Available
|
Not Available
|
80%
|
99%
|
Cathepsin D
|
CHEMBL2581
|
P07339
|
4OD9
|
T67102
|
80%
|
99%
|
Neurokinin 2 receptor
|
CHEMBL2327
|
P21452
|
Not Available
|
T52790
|
57%
|
99%
|
Voltage-gated T-type calcium channel alpha-1H subunit
|
CHEMBL1859
|
O95180
|
Not Available
|
T54644
|
59%
|
99%
|
Acetyl-CoA carboxylase 2
|
CHEMBL4829
|
O00763
|
3TDC
|
T08922
|
66%
|
98%
|
Cysteinyl leukotriene receptor 2
|
CHEMBL4330
|
Q9NS75
|
Not Available
|
T74238
|
64%
|
98%
|
Leukotriene A4 hydrolase
|
CHEMBL4618
|
P09960
|
3U9W
|
T03691
|
60%
|
98%
|
Cystinyl aminopeptidase
|
CHEMBL2693
|
Q9UIQ6
|
5MJ6
|
Not Available
|
53%
|
98%
|
ADAM10
|
CHEMBL5028
|
O14672
|
6BE6
|
T31902
|
83%
|
97.5%
|
Aurora kinase B/Inner centromere protein
|
CHEMBL3430907
|
Q96GD4
|
6YIH
|
T46781
|
64%
|
97.5%
|
Proteasome subunit beta type-9
|
CHEMBL1944495
|
P28065
|
6E5B
|
Not Available
|
63%
|
97.5%
|
G-protein coupled receptor 6
|
CHEMBL3714130
|
P46095
|
Not Available
|
Not Available
|
95%
|
97%
|
Tissue factor pathway inhibitor
|
CHEMBL3713062
|
P10646
|
5NMV
|
T78890
|
67%
|
97%
|
Cannabinoid CB2 receptor
|
CHEMBL253
|
P34972
|
6KPF
|
Not Available
|
66%
|
97%
|
Voltage-gated N-type calcium channel alpha-1B subunit
|
CHEMBL4478
|
Q00975
|
Not Available
|
T38338
|
71%
|
97%
|
LSD1/CoREST complex
|
CHEMBL3137262
|
O60341
|
5L3D
|
Not Available
|
90%
|
97%
|
Sodium channel protein type III alpha subunit
|
CHEMBL5163
|
Q9NY46
|
Not Available
|
T76937
|
78%
|
96.9%
|
Heat shock protein HSP 90-beta
|
CHEMBL4303
|
P08238
|
5FWK
|
Not Available
|
88%
|
97%
|
Androgen Receptor
|
CHEMBL1871
|
P10275
|
3V49
|
T11211
|
55%
|
96%
|
WD repeat-containing protein 5
|
CHEMBL1075317
|
P61964
|
2GNQ
|
Not Available
|
53.8%
|
96%
|
Toll-like receptor 8
|
CHEMBL5805
|
Q9NR97
|
3WN4
|
T48703
|
60%
|
96%
|
Nuclear factor NF-kappa-B p105 subunit
|
CHEMBL3251
|
P19838
|
1SVC
|
Not Available
|
91%
|
96%
|
Coagulation factor XIII
|
CHEMBL4530
|
P00488
|
4KTY
|
Not Available
|
62%
|
96%
|
Nuclear factor erythroid 2-related factor 2
|
CHEMBL1075094
|
Q16236
|
2FLU
|
Not Available
|
53.8%
|
96%
|
Adenosine A1 receptor
|
CHEMBL226
|
P30542
|
5N2S
|
T92072
|
65%
|
96%
|
NT-3 growth factor receptor
|
CHEMBL5608
|
Q16288
|
6KZD
|
Not Available
|
82%
|
96%
|
Glutamate NMDA receptor; GRIN1/GRIN2B
|
CHEMBL1907603
|
Q05586
|
5EWM
|
Not Available
|
70%
|
96%
|
Inhibitor of nuclear factor kappa B kinase alpha subunit
|
CHEMBL3476
|
O15111
|
5EBZ
|
Not Available
|
64%
|
96%
|
Cystic fibrosis transmembrane conductance regulator
|
CHEMBL4051
|
P13569
|
6MSM
|
T55654
|
68%
|
96%
|
Transcription intermediary factor 1-alpha
|
CHEMBL3108638
|
O15164
|
4YBM
|
Not Available
|
88%
|
96%
|
Sodium channel protein type II alpha subunit
|
CHEMBL4187
|
Q99250
|
6J8E
|
Not Available
|
75%
|
95.5%
|
Nuclear receptor ROR-beta
|
CHEMBL3091268
|
Q92753
|
Not Available
|
Not Available
|
54%
|
95.5%
|
NADPH oxidase 1
|
CHEMBL1287628
|
Q9Y5S8
|
Not Available
|
Not Available
|
51.7%
|
95%
|
Protein-tyrosine phosphatase 1B
|
CHEMBL335
|
P18031
|
5QGF
|
Not Available
|
96%
|
95%
|
Tyrosine-protein kinase ITK/TSK
|
CHEMBL2959
|
Q08881
|
4HCU
|
T91761
|
73%
|
95%
|
Solute carrier family 40 member 1
|
CHEMBL3392948
|
Q9NP59
|
6WBV
|
T86264
|
59%
|
95%
|
DCN1-like protein 1
|
CHEMBL4105838
|
Q96GG9
|
6BG3
|
Not Available
|
58.1%
|
95%
|
Histone deacetylase 2
|
CHEMBL1937
|
Q92769
|
7KBG
|
T51191
|
67%
|
95%
|
Pregnane X receptor
|
CHEMBL3401
|
O75469
|
6TFI
|
T82702
|
65%
|
95%
|
Excitatory amino acid transporter 1
|
CHEMBL3085
|
P43003
|
5LM4
|
Not Available
|
64%
|
95%
|
Matrix metalloproteinase-1
|
CHEMBL332
|
P03956
|
1SU3
|
Not Available
|
75%
|
94.5%
|
Dual specificity protein kinase CLK4
|
CHEMBL4203
|
Q9HAZ1
|
6FYV
|
Not Available
|
85%
|
94%
|
PI3-kinase p110-alpha/p85-alpha
|
CHEMBL2111367
|
P27986
|
4JPS
|
T80276
|
76%
|
94%
|
Dual specificity protein phosphatase 3
|
CHEMBL2635
|
P51452
|
3F81
|
Not Available
|
75%
|
94%
|
Kinesin-like protein 1
|
CHEMBL4581
|
P52732
|
6TIW
|
T28484
|
54%
|
93%
|
Acetyl-CoA carboxylase 1
|
CHEMBL3351
|
Q13085
|
6G2H
|
Not Available
|
57%
|
93%
|
Calpain 1
|
CHEMBL3891
|
P07384
|
1ZCM
|
Not Available
|
53.8%
|
93%
|
Cyclin-dependent kinase 5/CDK5 activator 1
|
CHEMBL1907600
|
Q00535
|
1UNL
|
T20973
|
58%
|
93%
|
T-cell protein-tyrosine phosphatase
|
CHEMBL3807
|
P17706
|
1L8K
|
Not Available
|
75%
|
93%
|
Phosphodiesterase 3A
|
CHEMBL241
|
Q14432
|
7LRC
|
T88975
|
75%
|
93%
|
Serine/threonine-protein kinase mTOR
|
CHEMBL2842
|
P42345
|
6BCX
|
T75243
|
79%
|
93%
|
GABA-A receptor; alpha-1/beta-2/gamma-2
|
CHEMBL2095172
|
P14867
|
6X3T
|
T51487
|
51%
|
93%
|
Neprilysin
|
CHEMBL1944
|
P08473
|
6SUK
|
T05409
|
51%
|
93%
|
C5a anaphylatoxin chemotactic receptor
|
CHEMBL2373
|
P21730
|
6C1R
|
T15439
|
74%
|
93%
|
Toll-like receptor 4
|
CHEMBL5255
|
O00206
|
4G8A
|
T81443
|
70%
|
92.5%
|
Sodium channel protein type IV alpha subunit
|
CHEMBL2072
|
P35499
|
6AGF
|
T02546
|
55%
|
92%
|
Acyl coenzyme A: cholesterol acyltransferase 1
|
CHEMBL2782
|
P35610
|
6P2J
|
Not Available
|
65%
|
92%
|
Cyclin-dependent kinase 1/cyclin B1
|
CHEMBL1907602
|
P06493
|
6GU2
|
T49898
|
53.4%
|
91%
|
Cytochrome P450 3A4
|
CHEMBL340
|
P08684
|
5VCC
|
T37848
|
75%
|
91%
|
DNA-(apurinic or apyrimidinic site) lyase
|
CHEMBL5619
|
P27695
|
6BOW
|
T13348
|
95%
|
91%
|
Platelet-derived growth factor receptor alpha
|
CHEMBL2007
|
P16234
|
7LBF
|
T53524
|
85%
|
91%
|
Glycine receptor subunit alpha-1
|
CHEMBL5845
|
P23415
|
4X5T
|
T50269
|
79%
|
91%
|
Sodium/hydrogen exchanger 1
|
CHEMBL2781
|
P19634
|
7DSX
|
T82028
|
65%
|
90%
|
Protein Mdm4
|
CHEMBL1255126
|
O15151
|
6Q9Y
|
T36741
|
57%
|
90.2%
|
Cyclooxygenase-1
|
CHEMBL221
|
P23219
|
6Y3C
|
Not Available
|
78%
|
90%
|
Proteasome component C5
|
CHEMBL4208
|
P20618
|
6KWY
|
Not Available
|
68%
|
90%
|
Telomerase reverse transcriptase
|
CHEMBL2916
|
O14746
|
7BG9
|
T86052
|
62%
|
90%
|
Histone deacetylase 5
|
CHEMBL2563
|
Q9UQL6
|
5UWI
|
Not Available
|
55.5%
|
90%
|
Histone deacetylase 7
|
CHEMBL2716
|
Q8WUI4
|
3C10
|
Not Available
|
71%
|
89%
|
Aldo-keto-reductase family 1 member C3
|
CHEMBL4681
|
P42330
|
1S1P
|
T60857
|
56%
|
89%
|
DNA topoisomerase II alpha
|
CHEMBL1806
|
P11388
|
6ZY5
|
T17048
|
87%
|
89%
|
Histone deacetylase 11
|
CHEMBL3310
|
Q96DB2
|
Not Available
|
Not Available
|
54%
|
89%
|
Ras-related protein Rab-9A
|
CHEMBL1293294
|
P51151
|
1WMS
|
T66350
|
66%
|
88%
|
Cysteine protease ATG4B
|
CHEMBL1741221
|
Q9Y4P1
|
2CY7
|
Not Available
|
63%
|
87.5%
|
Glutamate receptor ionotropic, AMPA 2
|
CHEMBL4016
|
P42262
|
2WJW
|
T42392
|
73%
|
87%
|
Kruppel-like factor 5
|
CHEMBL1293249
|
Q13887
|
Not Available
|
Not Available
|
90%
|
86%
|
Bcr/Abl fusion protein
|
CHEMBL2096618
|
P00519
|
5N7E
|
Not Available
|
63%
|
86%
|
Beta-glucocerebrosidase
|
CHEMBL2179
|
P04062
|
6TN1
|
T84173
|
57%
|
85%
|
Photoreceptor-specific nuclear receptor
|
CHEMBL4374
|
Q9Y5X4
|
4LOG
|
Not Available
|
66%
|
85%
|
Signal transducer and activator of transcription 3
|
CHEMBL4026
|
P40763
|
6QHD
|
T29130
|
53%
|
83%
|
Kelch-like ECH-associated protein 1
|
CHEMBL2069156
|
Q14145
|
6WCQ
|
Not Available
|
51%
|
82%
|
Dual specificity phosphatase Cdc25B
|
CHEMBL4804
|
P30305
|
1QB0
|
Not Available
|
69%
|
79.5%
|
G-protein coupled receptor 55
|
CHEMBL1075322
|
Q9Y2T6
|
Not Available
|
T87670
|
86%
|
78%
|
Nuclear receptor subfamily 4 group A member 1
|
CHEMBL1293229
|
P22736
|
4RZF
|
Not Available
|
63%
|
78%
|
Bloom syndrome protein
|
CHEMBL1293237
|
P54132
|
4O3M
|
Not Available
|
100%
|
70%
|
To study the binding interactions between α-hederin and the core molecular targets (KEAP1-NFE2L2 and COL1A1), we used AutoDock Maestro 12.8 software for molecular docking analysis. The PDB structures of KEAP1-NFE2L2 and COL1A1 were employed for representing the targets in the docking analysis. (Fig. 7C&7D).
Furthermore, we visualized the molecular docking results by displaying small-molecule drug binding (Fig. 7E, 7F). For instance, α-hederin potentially binds to KEAP1-NFE2L2, forming hydrogen bonds with GLY-527, GLY-528, GLY-530, and ARC 482 near the active site. This may contribute to its biological functions. To evaluate α-hederin's docking scores against the active sites of COL1A1, we used the Glide module in Schrodinger suite software. The docking score of α-hederin with COL1A1 was − 3.39, indicating a favorable interaction. The 2D and 3D interaction diagrams of α-hederin in the active site of COL1A1 showed the formation of two hydrogen bonds with CLN B:20 and CLY C:19. (Fig. 7F).
In Vitro Validation of Predicted Targets of α-hederin in CRC Cells
α-Hederin inhibits cell growth and promotes cell death in HCT116 cells
To assess the cytotoxic effect of α-hederin on CRC cell lines, we treated HCT116 CRC cell lines with different concentrations of α-hederin or Oxaliplatin for varying durations (12, 24, 36, 48 hours) and evaluated cell viability using the CCK-8 assay. Our findings demonstrated that α-hederin exerted a significant concentration- and time-dependent reduction in the viability of HCT116 CRC cell lines compared to Oxaliplatin or untreated cells (Fig. 8A, 8B).
Effects of α-hederin on CRC apoptosis were detected. Results revealed that α-hederin treatment for 24 or 48 hours induced apoptosis. Interestingly, after 12 hours of treatment with α-hederin in HCT116 cells, a fraction of the cells accumulated in the Annexin V-/PI + region (Fig. 8C). This observation suggests that α-hederin may induce a distinct form of cell death within 12 hours, different from apoptosis. Additional cell death assays were conducted to investigate further. Results showed that α-hederin-induced cell death within 48 hours is mainly apoptotic, indicating that apoptosis could become more prominent with longer treatments. (Fig. 8D).
α-hederin induces ROS generation by mediating KEAP1\Nrf2-HO-1 activation in CRC cells
Our findings demonstrated that α-hederin induced rapid and concentration-dependent production of ROS in HCT116 cells, as compared to Oxaliplatin or untreated cells (Fig. 9A, 9B). GSH, an essential antioxidant involved in regulating redox balance [13]. By investigating the mechanism underlying α-hederin-induced ROS generation, results revealed a decrease in GSH levels upon treatment with α-hederin in HCT116 cells (Fig. 9C).
To understand the mechanism of α-hederin-induced cell death, we performed RNA-sequencing analysis. We found that α-hederin induced oxidative stress, leading to ROS activation. Specifically, we examined Nrf2, an upstream regulator of ROS-responsive genes. In HCT116 cells treated with α-hederin, we observed a significant concentration-dependent decrease in gene expression of Nrf2 and HO-1, compared to cells treated with Oxaliplatin (Fig. 9D). Protein level analysis after 24 hours of α-hederin treatment also showed reduced levels of Nrf2 and HO-1 in HCT116 cells (Fig. 9E). These results suggest that α-hederin inhibits the Nrf2-HO-1 signaling pathway, contributing to cell death in HCT116 cells.
Moreover, Western blot analysis revealed that α-hederin treatment resulted in a decrease in KEAP1\Nrf2 activation within 24 hours in HCT116 cells (Fig. 9F).
α-hederin induces CAV1, COL1A1 activation in CRC cells
To investigate the involvement of CAV1 and COL1A1 in α-hederin-induced cell death, we treated HCT116 cells with α-hederin and examined their expression levels. Western blot analysis revealed that α-hederin treatment led to reduced activation of CAV1 and COL1A1 in HCT116 cells within 24 hours (Fig. 11G). This suggests that α-hederin may influence the expression of CAV1 and COL1A1, potentially contributing to its ability to induce cell death in HCT116 cells.