Objective:

2

To explore the specific pharmacological molecular mechanisms of Laoke Formula (LK) on treating advanced non-small cell lung cancer (NSCLC) based on clinical application

network pharmacology and experimental validation.

Methods:

2

Kaplan-Meier method and Cox regression analysis were used to evaluate the survival benefit of Chinese medicine (CM) treatment in 296 patients with NSCLC in Tianjin Medical University Cancer Institute and Hospital from January 2011 to December 2015. The compounds of LK were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform

and the corresponding targets were performed from Swiss Target Prediction. NSCLC-related targets were obtained from Therapeutic Target Database and Comparative Toxicogenomics Database. Key compounds and targets were identified from the compound-target-disease network and protein-protein interaction (PPI) network analysis

respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis were used to predict the potential signaling pathways involved in the treatment of advanced NSCLC with LK. The binding affinities between key ingredients and targets were further verified using molecular docking. Finally

A549 cell proliferation and migration assay were used to evaluate the antitumor activity of LK. Western blot was used to further verify the expression of key target proteins related to the predicted pathways.

Results:

2

Kaplan-Meier survival analysis showed that the overall survival of the CM group was longer than that of the non-CM group (36 months

vs

. 26 months)

and COX regression analysis showed that LK treatment was an independent favorable prognostic factor (

P

=0.027). Next

97 components and 86 potential targets were included in the network pharmacology

KEGG and GO analyses

and the results indicated that LK was associated with proliferation and apoptosis. Moreover

molecular docking revealed a good binding affinity between the key ingredients and targets.

In vitro

A549 cell proliferation and migration assay showed that the biological inhibition effect was more obvious with the increase of LK concentration (

P

<

0.05). And decreased expressions of nuclear factor κB1 (NF-κB1)

epidermal growth factor receptor (EGFR) and AKT serine/threonine kinase 1 (AKT1) and increased expression of p53 (

P

<

0.05) indicated the inhibitory effect of LK on NSCLC by Western blot.

Conclusion:

2

LK inhibits NSCLC by inhibiting EGFR/phosphoinositide 3-kinase (PI3K)/AKT signaling pathway

NFκB signaling pathway and inducing apoptosis

which provides evidence for the therapeutic mechanism of LK to increase overall survival in NSCLC patients.