Jian Pi Hua Tan Fang Reverses Trastuzumab Resistance of HER2-Positive Gastric Cancer Through PI3K/AKT/mTOR Pathway: Integrating Network Pharmacology, Molecular Docking and Experimental Validation

Abstract

Background: Currently, trastuzumab resistance significantly impacts the treatment outcome for individuals with HER2-positive gastric cancer. In clinical practice, Jian Pi Hua Tan Fang (JPHTF) has been shown to be effective in preventing recurrences and metastases caused by gastric cancer. Yet, the treatment process remains unknown. We aim to evaluate the potential pharmacological mechanism of JPHTF in interfering with resistance to trastuzumab in HER2-positive gastric cancer (GC).

Methods: In this study, network pharmacology and molecular docking techniques were used to forecast the potential active ingredients, pathways, and targets of JPHTF in overcoming trastuzumab resistance in HER2-positive GC. Then, in vitro models of NCI-N87/TR was developed, and JPHTF-containing serum was utilized for intervention to confirm these crucial targets.

Results: Network pharmacology showed that 92 potential active compounds and 420 therapeutic targets of JPHTF. SRC, EGFR, TP53, and AKT1 were identified as the main targets associated with the PI3K/Akt, MAPK, and Ras pathways, playing crucial roles in angiogenesis, cell apoptosis, cell proliferation, and resistance to chemotherapy in the GC microenvironment. Molecular docking analysis showed that quercetin, formononetin, and luteolin, which are the main active ingredients, exhibit high binding affinity to the central targets PI3K, AKT, and mTOR. In vitro experiment, the JPHTF-containing serum has a significant alleviating effect on reversing trastuzumab resistance and cell apoptotic and proliferation of NCI-N87/TR. Further molecular biological experiments showed that JPHTF could regulate the expression of PI3K/AKT/mTOR pathway.

Conclusion: JPHTF has the ability to overcome trastuzumab resistance in NCI-N87 cells through the regulation of the PI3K/AKT/mTOR pathway.

Keywords: HER2‐positive gastric cancer; Jian Pi Hua Tan Fang; PI3K/AKT/mTOR pathway; Trastuzumab resistance; molecular docking; network pharmacology.

Figure 1

The workflow of the study.

Figure 2

(A) The “herb–ingredient–target‐pathway” network of JPHTF. The tangerine square, circular, green arrow, and red rectangle corresponded to the herbs, active components, predicted targets, and top 20 pathways respectively. (B) Venn diagram showing the intersection of JPHTF‐related genes and GC‐related genes. The GC‐related targets are shown in the green circle, and the JPHTF‐related targets are shown in the blue circle. The intersection of the two circles indicates potential targets of JPHTF in GC treatment. (C) Protein–protein interaction (PPI) network of common targets were performed by STRING database, each edge represented the connection between targets.

Figure 3

(A) The PPI network of the 420 cross‐targets were constructed using Cytoscape. Nodes represent targets, and lines represent interactions between two targets. PPI network: the darker the color, the larger the node, the larger its degree value and the greater its importance within the network. (B) PPI network hub gene screening process: The red nodes represent the targets that meet the screening conditions.

Figure 4

(A) Barplot performed the top 20 enriched terms of BP, CC, and MF. The horizontal axis represented the name of each term, and the vertical axis represented the counts of genes in the enriched term. (B) The bubble plots of the 20 most significant signaling pathways based on KEGG enrichment analysis. The bubble size represents the number of genes enriched in this pathway, the bubble color difference represents the level of gene enrichment in this pathway, and the circle size represents the number of targets contained in the pathway. The redder the color, the smaller the p value.

Figure 5

Molecular docking of hub targets and active components of JPHTF. (A) PI3K‐formononetin (binding site: ARG‐18, ILE‐53, LYS‐3); (B) PI3K‐luteolin (binding site: ARG‐18, ARG‐4, ILE‐53, LYS‐3, TRP‐55, ASP‐68); (C) PI3K‐quercetin (binding site: TYR‐76, TYR‐12, ALA‐48, GLU‐51); (D) PI3K‐alpelisib (PI3K inhibitor) (binding site: TYR‐76, ALA‐48); (E) AKT‐formononetin (binding site: GLU‐198); (F) AKT‐luteolin (binding site: LYS‐10, ASP‐13); (G) AKT‐quercetin (binding site: GLN‐153, GLU‐36, ALA‐51); (H) AKT‐ipatasertib (AKT inhibitor) (binding site: HIS‐73); (I) mTOR‐formononetin (binding site: MET‐219, PHE‐217, GLU‐162, LYS‐160); (J) mTOR‐luteolin (binding site: SER‐203, GLN‐216, HIS‐201, HIS‐206); (K) mTOR‐quercetin (binding site: GLN‐39, LYS‐119, PHE‐221, ARG‐138); (L) mTOR‐rapamycin (mTOR inhibitor) (binding site: GLN‐137, ARG‐138, ARG‐41).

Figure 6

NCI‐N87/TR construction, preliminary investigation of trastuzumab resistance. (A) Examining HER2 using Western blot in MGC803, MKN145, NCI‐N87, and MKN28 cell lines; (B) HER2 expression levels were measured relative to GAPDH through normalization; (C) Trastuzumab induces morphological changes in NCI‐N87 cells; (D) MTS assay was used to detect the viability of NCI‐N87 cells treated with trastuzumab.; (E) and (F) NCI‐N87 and NCI‐N87/TR cells were subjected to Western blot analysis for PI3K, p‐AKT, AKT, mTOR, PTEN, P27, P38‐MAPK, and p‐P38‐MAPK; (G) and (H) NCI‐N87 and NCI‐N87/TR cells, both treated and untreated with trastuzumab, were subjected to Western blot analysis for HER2 and p‐HER2 (Tyr1221/1222). Results are shown as the average ± standard deviation (n = 3), with statistical significance indicated by *p < 0.01, **p < 0.001 when compared to the control group.

Figure 7

(A) The cell viability of NCI‐N87 and NCI‐N87/TR cells at diferent concentrations of JPHTF containing serum. (B) The cell viability of NCI‐N87/TR cells containing 5% JPHTF serum at different concentrations of trastuzumab. (C) NCI‐N87/TR celll western blot analysis of PI3K, p‐AKT, AKT, mTOR, PTEN, P27, P38‐MAPK and p‐P38‐MAPK at 48 h of culture with 5% blank serum and different JPHTF containing serum. (D) The relative protein levels of (C). (E) NCI‐N87/TR celll western blot analysis above proteins with 190 μg/mL trastuzumab, 5% JPHTF‐containing serum or combination of both, respectively. (F) The relative protein levels of (E). T: trastuzumab, J: JPHTF, T + J: trastuzumab + JPHTF. Data are presented as the mean ± SD (n = 6), *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group.

Figure 8

Schematic representation of Jian Pi Hua Tan Fang reverses trastuzumab resistance of HER2‐positive gastric cancer through PI3K/AKT/mTOR pathway. In the in vitro experiments, JPHTF serum noticeably enhances trastuzumab sensitivity, upregulates PTEN and P27 expression, effectively inhibits the activation of the PI3K/Akt/mTOR pathway, and subsequently reverses NCI‐N87 cell resistance to trastuzumab.