Study on the mechanism of Cortex Lycii on lung cancer based on network pharmacology combined with experimental validation.

Xie Bai San is a Chinese medicine prescription that has been used to treat lung cancer in China for a long time. It has been proven to alleviate the symptoms and extend the survival time of lung cancer patients. Xie Bai San comprises Cortex Lycii, Cortex Mori, and Radix Glycyrrhizae Preparata. The effects and mechanisms of Cortex Mori and Glycyrrhizae on lung cancer have been reported, whereas the underlying mechanism of Cortex Lycii remains unknown. Network pharmacology was used to explore the unknown mechanisms underlying the effect of Cortex Lycii on lung cancer. Molecular docking was used to predict the binding of a compound to the protein. The fingerprint of Cortex Lycii was obtained by HPLC. Cell counting Kit-8 assay was used to determine the appropriate concentration of Cortex Lycii extract for human lung adenocarcinoma cells, A549 and H1299. Wound healing assay and Matrigel invasion assay were used to detect the influence of Cortex Lycii extract on the migration and invasion ability of A549 and H1299. The protein expression level was detected by western blot and immunohistochemical staining. Using network pharmacology, 38 components of Cortex Lycii and 79 possible lung cancer-related target genes of Cortex Lycii were obtained. The targets were assigned to 35 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and the PI3K-AKT signaling pathway contained the most targets and had the second-lowest P-value. The molecular docking showed the components of Cortex Lycii bound to HSP90AB1. Among them, 6 components bound to HSP90AB1 in which HSP90AB1 binds to and phosphorylates AKT. The functional experiments showed that Cortex Lycii suppressed the migration and invasion of human lung cancer cells in a dose-dependent manner. Cortex Lycii up-regulated E-Cadherin and down-regulated N-Cadherin, Vimentin, and MMP2. Furthermore, Cortex Lycii made no change in the total AKT and mTOR protein levels, but caused the down-regulation of p-AKT and p-mTOR in human lung cancer cells, which was reversed by Terazosin, an agonist of HSP90. Moreover, acacetin and apigenin, two components of Cortex Lycii, reduced the protein level of p-AKT and p-mTOR, and the reduction was also inhibited by Terazosin. Cortex Lycii suppressed epithelial-mesenchymal transition (EMT) in lung cancer cells through the PI3K-AKT-mTOR signaling pathway, possibly by targeting HSP90AB1 and inhibiting HSP90AB1-AKT binding.

Guo Z ，Yin H ，Wu T ，Wu S ，Liu L ，Zhang L ，He Y ，Zhang R ，Liu N ... -  《-》

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

Dioscin, a natural glycoside derived from many plants, has been proved to exert anti-cancer activity. Several studies have found that it reverses TGF-β1-induced epithelial-mesenchymal transition (EMT). Whether dioscin can reverse EMT by pathways other than TGF-β is still unknown. We used network-based pharmacological methods to systematically explore the potential mechanisms by which dioscin acts on lung cancer. Cell Counting Kit-8 assay, scratch healing, Transwell assay, Matrigel invasion assay, immunofluorescence assay, and Western blotting were employed to confirm the prediction of key targets and the effects of dioscin on EMT. Here, using network-based pharmacological methods, we found 42 possible lung cancer-related targets of dioscin, which were assigned to 98 KEGG pathways. Among the 20 with the lowest p-values, the PI3K-AKT signaling pathway is involved and significantly related to EMT. AKT1 and mTOR, with high degrees (reflecting higher connectivity) in the compound-target analysis, participate in the PI3K-AKT signaling pathway. Molecular docking indicated the occurrence of dioscin-AKT1 and dioscin-mTOR binding. Functional experiments demonstrated that dioscin suppressed the proliferation, migration, invasion, and EMT of human lung adenocarcinoma cells in a dose-dependent manner, without TGF-β stimulation. Furthermore, we determined that dioscin downregulated p-AKT, p-mTOR and p-GSK3β in human lung adenocarcinoma cells without affecting their total protein levels. The PI3K inhibitor LY294002 augmented these changes. Dioscin suppressed proliferation, invasion and EMT of lung adenocarcinoma cells via the inactivation of AKT/mTOR/GSK3β signaling, probably by binding to AKT and mTOR, and inhibiting their phosphorylation.

Mao W ，Yin H ，Chen W ，Zhao T ，Wu S ，Jin H ，Du B ，Tan Y ，Zhang R ，He Y ... -  《Drug Design Development and Therapy》

Hydroxysafflor yellow A inhibited lipopolysaccharide-induced non-small cell lung cancer cell proliferation, migration, and invasion by suppressing the PI3K/AKT/mTOR and ERK/MAPK signaling pathways.

Chronic inflammation plays a significant role in the occurrence and development of non-small cell lung cancer (NSCLC). Hydroxysafflor yellow A (HSYA), a chemical compound of the yellow color pigments extracted from the safflower, has been widely used in clinical treatment with positive antioxidation, anti-inflammation, and antitumor effects. However, the role and underlying mechanisms of HYSA on development and progress in inflammation-mediated NSCLC are unknown. Cell counting kit-8, colony formation, EdU, cell apoptosis, wound healing, Transwell migration and invasion, and enzyme-linked immunosorbent assays; flow cytometry; and Western blotting were conducted using human NSCLC cell lines A549 and H1299. Lipopolysaccharide (LPS) significantly promoted the proliferation and enhanced colony formation of A549 and H1299 cells, while HYSA notably reversed the effects of LPS. HYSA induced apoptosis of LPS-mediated A549 and H1299 cells in a dose dependent manner; and remarkably suppressed migration, invasion, and epithelial-mesenchymal transition (EMT), significantly regulated production of LPS-induced inflammation cytokines, and downregulated protein expression of PI3K/Akt/mTOR and ERK/MAPK signaling pathways in LPS-induced A549 and H1299 cells. Furthermore, PI3K (LY294002) and ERK (SCH772984) inhibitors remarkably inhibited proliferation, migration, invasion, and EMT, and induced apoptosis in LPS-mediated A549 and H1299 cells. These effects were even more obvious in the presence of HYSA and LY294002 or SCH772984 compared to those of either agent alone. HYSA suppressed LPS-mediated proliferation, migration, invasion, and EMT in A549 and H1299 cells by inhibiting the PI3K/Akt/mTOR and ERK/MAPK signaling pathways, indicating that HYSA may be a potential candidate to treat inflammation-mediated NSCLC.

Jiang M ，Zhou LY ，Xu N ，An Q ... -  《-》

Ginkgolic Acid Inhibits Invasion and Migration and TGF-β-Induced EMT of Lung Cancer Cells Through PI3K/Akt/mTOR Inactivation.

Epithelial-to-mesenchymal transition (EMT) is a critical cellular phenomenon regulating tumor metastases. In the present study, we investigated whether ginkgolic acid can affect EMT in lung cancer cells and the related underlying mechanism(s) of its actions. We found that ginkgolic acid C15:1 (GA C15:1) inhibited cell proliferation, invasion, and migration in both A549 and H1299 lung cancer cells. GA C15:1 also suppressed the expression of EMT related genes (Fibronectin, Vimentin, N-cadherin, MMP-9, MMP-2, Twist and Snail) and suppressed TGF-β-induced EMT as assessed by reduced expression of mesenchymal markers (Fibronectin, Vimentin, N-cadherin), MMP-9, MMP-2, Twist and Snail. However, GA C15:1 did not affect the expression of various epithelial marker proteins (Occludin and E-cadherin) in both A549 and H1299 cells. TGF-β-induced morphologic changes from epithelial to mesenchymal cells and induction of invasion and migration were reversed by GA C15:1. Finally, GA C15:1 not only abrogated basal PI3K/Akt/mTOR signaling cascade, but also reduced TGF-β-induced phosphorylation of PI3K/Akt/mTOR pathway in lung cancer cells. Overall, these findings suggest that GA C15:1 suppresses lung cancer invasion and migration through the inhibition of PI3K/Akt/mTOR signaling pathway and provide a source of potential therapeutic compounds to control the metastatic dissemination of tumor cells. J. Cell. Physiol. 232: 346-354, 2017. © 2016 Wiley Periodicals, Inc.

Baek SH ，Ko JH ，Lee JH ，Kim C ，Lee H ，Nam D ，Lee J ，Lee SG ，Yang WM ，Um JY ，Sethi G ，Ahn KS ... -  《-》

A network pharmacology approach and experimental validation to investigate the anticancer mechanism and potential active targets of ethanol extract of Wei-Tong-Xin against colorectal cancer through induction of apoptosis via PI3K/AKT signaling pathway.

Wei-Tong-Xin (WTX), derives from the Chinese herbal decoction (CHD) of Wan-Ying-Yuan in ancient China, has been shown to be effective therapeutic herbal decoction for treating gastrointestinal diseases. Present studies have demonstrated that WTX had potential to alleviate the symptoms of gastrointestinal inflammation, gastric ulcer and improve gastric motility. The study primarily focused on exploring the therapeutic effect and possible pharmacological mechanism of WTX on colorectal cancer (CRC) based on network pharmacology, in vitro and in vivo experiments. Firstly, colorectal cancer and WTX associated with targets were searched from GeneCards database and TCM Systems Pharmacology Database and Analysis Platform (TCMSP) respectively. The protein-protein interaction (PPI) network also was constructed to screening key targets. In addition, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to predict the underlying biological function and mechanism involving in the anti-colorectal cancer effect of WTX. Next, CCK-8, colony formation and transwell assays were performed to verify the influence of proliferation and metastasizing ability of HCT116 cells after treated with WTX. Cell cycle, apoptosis and reactive oxygen species (ROS) were analysis by flow cytometry. Hoechst 33258 staining was conducted to observe nuclear morphology changes. Protein expression of apoptosis and PI3K/AKT signaling as well as mRNA expression of ferroptosis and apoptosis were determined by Western Blotting and RT-qPCR. The effects of WTX and LY294002 combination on the PI3K/Akt/mTOR signaling pathway were measured by Western Blotting. Finally, the xenograft tumor mouse model was established by subcutaneous injection of CT26 cells to measure tumors volume and weight. Hematoxylin and eosin (HE) staining and immunohistochemical analysis were used to observe the pathological changes and the protein expression in tumor tissues. There were 286 potential treatment targets from 130 bioactive compounds in WTX, 1349 CRC-related targets were identified. Eleven core targets (TP53, AKT1, STAT3, JUN, TNF, HSP90AA1, IL-6, MAPK3, CASP3, EGFR, MYC) were found by PPI network analysis constructed of 142 common targets. The results of KEGG enrichment displayed PI3K/AKT signaling pathway as core pathway. After the treatment of WTX, the inhibitory of viability, metastases and cell cycle arrest at G2/M phase were observed in HCT116 cells. Moreover, WTX induced an increase in the expression of apoptosis proteins (Bak, cytochrome c, cleaved caspase-9/caspase-9 and cleaved caspase-3/caspase-3) and the levels of ROS and MDA, a decrease in the expression of PI3K/AKT signaling related proteins (PI3K, p-PI3K, p-AKT/AKT and p-mTOR/mTOR) and the level of SOD. WTX treatment significantly reduced the tumor weight, increased cleaved caspase-3 positive area and decreased that of ki67 in xenograft mouse model. Through a network pharmacology approach and in vitro experiments, we predicted and verified the effect of WTX on colorectal cancer cells mainly depended on the regulation of intrinsic apoptosis via PI3K/AKT signaling pathway, and further animal experiments proved that WTX has a good anti-colon cancer effect in vivo.

Lin F ，Zhang G ，Yang X ，Wang M ，Wang R ，Wan M ，Wang J ，Wu B ，Yan T ，Jia Y ... -  《-》