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. 2021 Sep 9:9:685954.
doi: 10.3389/fcell.2021.685954. eCollection 2021.

Network Pharmacology and Experimental Validation Reveal the Effects of Chidamide Combined With Aspirin on Acute Myeloid Leukemia-Myelodysplastic Syndrome Cells Through PI3K/AKT Pathway

Simin Liang  1 , Xiaojia Zhou  2 , Duo Cai  1 , Fernando Rodrigues-Lima  3 , Jianxiang Chi  4 , Li Wang  1
Affiliations

Network Pharmacology and Experimental Validation Reveal the Effects of Chidamide Combined With Aspirin on Acute Myeloid Leukemia-Myelodysplastic Syndrome Cells Through PI3K/AKT Pathway

Simin Liang et al. Front Cell Dev Biol. .

Abstract

Chidamide (CDM), a novel histone deacetylase inhibitor, is currently used for patients with peripheral T-cell lymphoma. Aspirin (ASA), an anti-inflammatory drug, has been shown to exert anticancer activity. Herein, we investigated the effect of CDM combined with ASA on myelodysplastic syndromes-derived acute myeloid leukemia (AML-MDS) cells and explored the underlying mechanism. The putative targets of CDM and ASA were predicted by network pharmacology approach. GO functional and KEGG pathway enrichment analyses were performed by DAVID. Furthermore, experimental validation was conducted by Cell Counting Kit-8 assay, Flow cytometry and Western blotting. Network pharmacology analysis revealed 36 AML-MDS-related overlapping genes that were targets of CDM and ASA, while 10 hub genes were identified by the plug-in cytoHubba in Cytoscape. Pathway enrichment analysis indicated CDM and ASA significantly affected PI3K/AKT signaling pathway. Functional experiments demonstrated that the combination of CDM and ASA had a remarkable synergistic anti-proliferative effect by blocking the cell cycle in G0/G1 phase and inducing apoptosis. Mechanistically, the combination treatment significantly down-regulated the phosphorylation levels of PI3K and AKT. In addition, insulin-like growth factor 1 (IGF-1), an activator of PI3K/AKT pathway, reversed the effects of the combination treatment. Our findings suggested that CDM combined with ASA exerted a synergetic inhibitory effect on cell growth by inactivating PI3K/AKT pathway, which might pave the way for effective treatments of AML-MDS.

Keywords: PI3K/Akt pathway; aspirin; chidamide; myelodysplastic syndromes; network pharmacology.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Flowchart of the study design based on network pharmacology approaches for deciphering the mechanisms of CDM and ASA acting on AML-MDS. CDM, Chidamide; ASA, Aspirin; MDS, myelodysplastic syndrome; AML, acute myeloid leukemia; AML-MDS, myelodysplastic syndromes-derived acute myeloid leukemia.
FIGURE 2
FIGURE 2
The network pharmacology of CDM and ASA against AML-MDS and cluster analysis. (A) The Venn diagram of the potential targets of CDM and ASA against AML-MDS. (B) A PPI network of the overlapping targets of CDM and ASA against AML-MDS by Cytoscape software. (C) Clusters of interacted proteins by use of MCODE algorithm. (D) Hub targets of the PPI network by use of cytoHubba. The size of node and edge was mapped to the degree and edge betweenness, respectively. The color of the node represents the size of the degree value. The redder the color, the larger the node and the more important it is in the network. Conversely, the greener the color, the smaller the node and the less important it is in the network.
FIGURE 3
FIGURE 3
GO and KEGG pathway enrichment analyses for the hub genes of CDM and ASA against AML-MDS. (A) Biological process (BP); (B) cellular component (CC); (C) molecular function (MF); (D) the top 20 of KEGG enrichment analysis (left panel), alluvial plot of interaction among drugs, major hub genes and three main pathways (right panel).
FIGURE 4
FIGURE 4
CDM in combination with ASA inhibited the proliferation of AML-MDS cells. (A) Cell viability detected by Cell Titer-Glo luminescent cell viability assay in SKM-1 cells treated with 8 drugs. (B,C) SKM-1 and Molt-4 cells were exposed to CDM (0.5, 1, 2, 4, and 8 μM) or ASA (0.5, 1, 2, 5, and 10 mM) alone. (D) Combination index values were calculated with CompuSyn software. CI < 1 indicates synergy; CI = 1 is additive; and CI > 1 means antagonism. CI, combination index; Fa, effect levels. (E) SKM-1 and Molt-4 cells were treated with 0.5 μM CDM combined with 1 mM ASA for 24, 48, and 72 h. The cell viability was determined by CCK-8 assay. Data are mean ± SD of three independent experiments.
FIGURE 5
FIGURE 5
CDM in combination with ASA arrested the cell cycle at G0/G1 phase in AML-MDS cells. (A) Cell cycle distribution was detected by flow cytometry after treatment with 0.5 μM CDM and 1 mM ASA for 48 h. The red color areas on the left and right of the images represent the proportion of cells in the G0/G1 and G2/M phases, respectively. (B) Relative expression of P21 mRNA compared with the control group. (C) Expressions of cell cycle related protein (CDK2, CDK4, p21) were detected by western blot. β-actin was used as a loading control. Data are mean ± SD of three independent experiments. “∗” indicates a significant difference relative to the control group (∗p < 0.05), “#” indicates a significant difference relative to CDM-treated group (#p < 0.05), “&” indicates a significant difference relative to ASA-treated group (&p < 0.05).
FIGURE 6
FIGURE 6
CDM in combination with ASA induced cell apoptosis in AML-MDS cells. (A) Cell apoptotic rate was detected by flow cytometry after treatment of the combination of 0.5 μM CDM and 1 mM ASA for 48 h. (B) Relative mRNA expression of Bcl-2 and caspase-3 compared with the control group. (C) Expressions of the apoptosis-related protein (Bcl-2, caspase-3, cleaved caspase-3) were detected by western blot. β-actin was used as a loading control. Data are mean ± SD of three independent experiments. “∗” indicates a significant difference relative to the control group (∗p < 0.05), “#” indicates a significant difference relative to CDM-treated group (#p < 0.05), “&” indicates a significant difference relative to ASA-treated group (&p < 0.05), “NS” indicates no significant difference relative to ASA-treated group or CDM-treated group.
FIGURE 7
FIGURE 7
CDM combined with ASA inhibited the activation of PI3K/AKT pathway in AML-MDS cells. (A) Western blot analysis of PI3K, p-PI3K AKT, p-AKT. (B) Western blotting of PI3K, p-PI3K, AKT and p-AKT. (C) Western blotting of CDK2, CDK4 and p21. (D) Western blotting of Bcl-2, caspase-3 and cleaved caspase-3. IGF-1 reversed the effect of CDM combined with ASA on AML-MDS cells. β-actin served as a loading control. Data are mean ± SD of three independent experiments. “∗” indicates a significant difference relative to the control group (∗p < 0.05), “&” indicates a significant difference relative to ASA-treated group (&p < 0.05), “NS” indicates no significant difference relative to the control group.
FIGURE 8
FIGURE 8
A schematic representation of the proposed pathway responsible for CDM combined with ASA in AML-MDS cells. (A) The predictive pathways of CDM and ASA in AML-MDS through network pharmacology. (B) CDM combined with ASA could inhibited the activation of PI3K/AKT signaling pathways, and then affected the expression of cell cycle and apoptosis-related proteins to induce cell cycle arrest and apoptosis in AML-MDS cells through experimental validation.
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