Network Pharmacology and Experimental Evidence Reveal Dioscin Suppresses Proliferation, Invasion, and EMT via AKT/GSK3b/mTOR Signaling in Lung Adenocarcinoma

Figures & data

Table 1 Predicted Lung Cancer-Related Targets of Dioscin

No.	Gene Symbol	No.	Gene Symbol	No.	Gene Symbol
1	CASP3	15	IL6	29	IL1B
2	CASP9	16	KRT18	30	KDR
3	MAPK1	17	AKT1	31	KEAP1
4	MAPK3	18	BCL2L1	32	MAPK8
5	PARP1	19	BIRC5	33	mTOR
6	TNF	20	CASP8	34	NFE2L2
7	ABCB1	21	CCNB1	35	NFKBIA
8	BAK1	22	CCND1	36	PTK2
9	BAX	23	CDK1	37	RELA
10	BCL2	24	CDKN1A	38	SRC
11	CTNNB1	25	CHEK2	39	TNFRSF10B
12	CYP2E1	26	FAS	40	TNFRSF1A
13	ESR1	27	FASLG	41	TP53
14	ESR2	28	HMOX1	42	VEGFA

Figure 1 Analysis of dioscin compound-target network. (A) The overlapped targets of lung cancer and dioscin-related targets were analyzed by STRING to generate the compound-target network. The diameter of the nodes was determined by the degree of freedom. The larger the diameter, the more critical the node is in the network. (B) GO functional annotation of potential targets of dioscin. Biological processes (BP), Cellular components (CC) and Molecular functions (MF) were ranked according to -logP values. (C) KEGG enrichment analysis for potential targets of dioscin. The top 20 with lower P-value was shown.

Figure 2 Modeling of dioscin with the potential targets. (A) Dioscin-AKT docking model with predicted binding energy of −8.91 kcal/mol. The Docking model of AKT and the crystal ligand has predicted binding energy of −10.89 kcal/mol. (B) Dioscin-mTOR docking model with predicted binding energy of −9.62 kcal/mol. The Docking model of AKT and the crystal ligand has predicted binding energy of −18.57 kcal/mol.

Figure 3 Dioscin inhibited the proliferation, migration, and invasion of A549 and H1299 cells. (A) The cell viability of A549 and H1299 cells treated with dioscin (1, 2, 4, and 8 µM) for 24 h and 48 h was determined using a Cell Counting Kit 8 assay. The width of scratches was measured in cells treated or not with dioscin, at 0 and 24 h in A549 (B) and H1299 (C) cells. Additionally, using a Transwell migration assay and Matrigel invasion assay, the number of migrated or invaded cells treated with dioscin per chamber was counted and compared with that in the control group from three independent experiments (*p < 0.05; **p< 0.01; ***p< 0.001).

Figure 4 Effect of dioscin on the expression of EMT markers in H1299 and A549 cells. (A) Cells were treated with dioscin (1 and 2 µM) for 24 h, and the EMT-associated biomarkers were detected using Western blotting. Typical graphs and a histogram (mean ± SD) are shown, n = 3. *p < 0.05; **p< 0.01; ***p< 0.001 compared with the control group. (B) After treatment with dioscin (1 and 2 µM) for 24 h, immunofluorescence staining was performed to determined Vimentin and E-cadherin expression in H1299 and A549 cells using confocal microscopy. Blue fluorescence indicates DAPI-labeled nuclei, and Green fluorescence indicates Vimentin or E-cadherin positive expression.

Figure 5 Effects of dioscin on the AKT/GSK3β/mTOR pathway in A549 and H1299 cells. (A) Cells were treated with dioscin (1 and 2 µM) for 24 h. The levels of AKT/GSK3/mTOR related pathway proteins, including phosphorylated (p)-AKT, AKT, p-GSK3, GSK3, p-mTOR, and mTOR, were examined using Western blotting analysis. (B, C) Cells were pre-treated with 10 µM LY294002 (a PI3K/AKT inhibitor, LY) for 2 h and then treated with dioscin (1 or 2 µM) for 24 h. Western blotting was then employed to determine the protein levels of Vimentin, E-cadherin, and N-cadherin (B) and p-AKT, AKT, p-GSK3, GSK3, p-mTOR, and mTOR (C). GAPDH was used as a loading control. Typical graphs and a histogram (mean±SD) are shown, n=3. *p < 0.05; **p< 0.01; ***p< 0.001 compared with the control group.