Molecular targets and mechanisms of cortex mori for lung cancer treatment: a network pharmacology study, molecular docking and in vitro and in vivo experimental validation

Abstract

Introduction: The cortex mori comes from the white endothelium of the young root of Morus alba L., and its medical value was first described in Shen Nong Ben Cao Jing (Classic on Materia Medical of Shennong). It was originally intended to purge lung, relieve asthma and reduce swelling. More and more studies reported that its pharmacological effects include analgesic, anti-inflammatory, antitussive, antiasthmatic, hypoglycemic, hypolipidemic and anti-diabetic peripheral neuropathy. Accumulating clinical evidences exhibited that it can treat asthma, pneumonia and lung cancer. However, a comprehensive mechanism of cortex mori in the treatment of lung cancer needs to be further elucidated.To investigate the effect of cortex mori and its active components against lung cancer and explore its action and mechanism through network pharmacological analysis combined with biological experiments in vitro and In vivo.

Methods: GeneCards database was searched for the disease targets of lung cancer, and a Chinese medicine database, Traditional Chinese Medicine Systems Pharmacology (TCMSP), was used to screen cortex mori for its active components and targets. Targets related to lung cancer and action targets related to cortex mori were crossed. Protein-protein interactions (PPI) and gene ontologies (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were analyzed for intersection genes. In order to determine whether cortex mori affects lung cancer, MTS, wound healing, Western-blot, Hoechst assay, apoptosis assay and animal experiments were performed.

Results: 32 active ingredients and 434 targets of Chinese medicine cortex mori were obtained. Totally 2,3107 lung cancer related targets were collected, and 163 Chinese medicine-disease targets were derived from the intersection. The regulatory network of Chinese medicine-active ingredient-disease-targets showed that cortex mori acted on 163 disease targets of lung cancer mainly by cyclomolorusin, kuwanon D and Moracin A, etc. The core genes involving cortex mori treating lung cancer might consist of JUN, AKT1, etc. The core targets involved 162 biological processes, mainly including nuclear receptor activity, ligand-actived transcription factor activity, etc. The core study targeted 160 pathways, including AGE-RAGE signaling pathways associated with diabetes complications, fluid stress and atherosclerosis. Biologic cytological experiments showed that the effective active component cyclomorusin inhibited proliferation, inhibited migration and induced apoptosis of lung cancer through AKT-PI3K pathway. In vivo antitumor assay demonstrated that cyclomolorusin suppressed the tumor growth in mice.

Discussion: Cortex mori acts on AKT and other related disease targets of lung cancer cells through effective components such as cyclomolorusin, and plays a role in the treatment of lung cancer by inhibiting the signaling pathway associated with lung cancer occurrence and development.

Keywords: cortex mori; lung cancer; mechanism of action; network pharmacology; pathway; target.

Figure 1

The flowchart of the network pharmacology research and experiments of cortex mori against lung cancer.

Figure 2

Prediction of anti-lung cancer targets of active ingredients and drug-active ingredient-target gene network construction and analysis. (A) Venn diagram of TCM-disease target. 22944 target genes were identified and 163 drug-disease targets were identified according to intersections. (B) TCM-ingredients-disease-target regulatory network. Drug-component target network of active ingredients of cortex mori.

Figure 3

Analysis of target protein interaction network. (A) TCM-disease target protein interaction network. Anti-cancer protein target interaction network (PPI). (B) A bar chart of TCM-core disease targets. MAPK1, AKT1 and JUN were the three proteins with more than 30 connectivity degrees and may be key targets for anticancer therapies.

Figure 4

Enrichment analysis of the core targets from PPI analysis. (A) Histogram of GO functional enrichment analysis of anti-cancer targets gene from active ingredients of cortex mori. (B) Enrichment analysis histogram of KEGG pathway of anti-cancer targets gene from active ingredients of cortex mori.

Figure 5

Small cell lung cancer pathway including PI3K-Akt signaling pathway and p53 signaling pathway.

Figure 6

Non-small cell lung cancer pathway including PI3K-Akt signaling pathway and p53 signaling pathway.

Figure 7

Molecular docking analysis of molecular interactions of AKT and the active ingredient cyclomorusin.

Figure 8

Inhibitive effect of Cyclomorusin on the growth of human lung cancer cells in vitro. (A) Lung cancer cells (NCI-H1299 and A549) were treated with 50 μM and 100 μM cyclomorusin for 24 h Cell morphological changes were determined under phase contrast microscope; (B) Lung cancer cells (NCI-H1299 and A549) were treated with a series of cyclomorusin and cis-platinum concentrations (0-200µM) for 24 h or 48 h followed by MTS assay; Values were mean ± SD (n = 3). (C) Lung cancer cells (NCI-H1299 and A549) were treated with a series of concentrations of cyclomorusin combined with cis-platinum (0-200 μM) for 24 or 48 h and then detected by MTS assay. ZIP synergy score was all greater than -10 and less than 10, indicating that cyclomorusin and cis-platinum had additive effect; (D) Foci formation of NCI-H1299 and A549 cells was determined. After indicated treatments, lung cancer cells were trypsinized and plated in duplicates at low density. After 10 days, formed colonies were stained with crystal violet.

Figure 9

Effect of cyclomorusin on lung cancer cells death by restraining PI3K-AKT pathway. (A) Cells stained with Hoechst 33342 were detected and calculated by fluorescent photomicrographs; (B) A flow cytometric analysis of lung cancer cells (NCI-H1299) treated with different treatments was conducted using AnnexinV/PI. A cell death was observed in the right upper quadrant, as well as in the left upper quadrant. The values were the mean + SD (n=3); (C) After lung cancer cells were treated with cyclomorusin and cis-platinum for 24 h, protein levels of Bax, Bcl-2, PI3K, AKT, mTOR, p-AKT, p-PI3K, and p-mTOR were analyzed by western blot (*p < 0.05, **p < 0.01).

Figure 10

Metastasis and cell cycle inhibition by cyclomorusin in lung cancer cells. (A) The effect of cyclomorusin at various levels on the advancement of the cell cycle was examined using propidium iodide flow cytometry after 24 h; (B) Inhibitive effects of cyclomorusin and cis-platinum on lung cancer cell migratory ability. The smaller the scratch, the stronger the migration ability; (C) Wound closure rate of the lung cancer cells with different treatments. (D) The effect of cyclomorusin and cis-platinum on proteins related to metastasis. (p = ns (no significance), *p < 0.05, **p < 0.01).

Figure 11

The effect of cyclomorusin on zebrafish embryo development. (A) The growth of zebrafish embryos was observed at 24, 48, 72, and 96 h after various treatments using a microscope; (B) The zebrafish embryo hatching rate; (C) The zebrafish malformation rate (**p < 0.01).

Figure 12

The in vivo experiments of cyclomorusin. (A) Tumor images of the control group and the experimental group receiving cyclomorusin treatment; (B) The effect of cyclomorusin on tumor volume after resection and on mice body weight (p = ns (no significance), **p < 0.01); (C) Illustrative images of H&E staining of the heart, liver, spleen, lung, kidney, and tumor.