Study on the Mechanism of Jianpi Yangzheng Formula Regulating Tumor-Associated Macrophage Exosomes to Induce Anoikis in Gastric Cancer Cells

OBJECTIVE To investigate the effect of Jianpi Yangzheng (JPYZ) formula regulating tumor-associated macrophage (TAM) exosomes on anoikis in gastric cancer and its mechanism.

METHODS TAM model was established by inducing human mononuclear THP-1 cells in vitro; M0, TAM and TAM+JPYZ formula exosomes were extracted by ultracentrifugation and co-incubated with gastric cancer cells. Flow cytometry was used to detect the effect of exosomes in each group on anoikis in gastric cancer cells. A BALB/c transplanted tumor mouse model was constructed, and the expression level of apoptotic proteins in transplanted tumors was detected by Western blot. Label-free mass spectrometry proteomics and bioinformatics were used to analyze the differential proteins in gastric cancer cells before and after intervention; Western blot and qPCR experiments were used to detect the expression level of differential protein isocitrate dehydrogenase 1 (IDH1), and ubiquitination experiments were used to detect the ubiquitination level of IDH1; kits were used to detect the levels of α-ketoglutarate (α-KG), NADPH/NADP⁺, glutathione (GSH/GSSG) and reactive oxygen species (ROS) content.

RESULTS The anoikis rate of gastric cancer cells was reduced after TAM exosomes intervention (P < 0.05, P < 0.01), while it was significantly increased after TAM+JPYZ exosomes intervention (P < 0.05, P < 0.01). In the in vivo mouse experiment, the ratio of Cleaved Caspase-3/Caspase-3 in the tumor of the TAM exosome group was reduced (P < 0.05), while the ratio was significantly increased after TAM+JPYZ exosome intervention (P < 0.05). Proteomic analysis showed that IDH1 was significantly different after intervention, and was related to tricarboxylic acid cycle metabolism. Compared with the M0 group, the IDH1 ubiquitination level of gastric cancer cells in the TAM group with exosome intervention was increased, the levels of α-KG, NADPH/NADP⁺ and GSH/GSSG were significantly increased, and the ROS content was reduced (P < 0.05), while TAM+JPYZ exosomes could reverse the above phenomenon.

CONCLUSION JPYZ Formula can regulate TAM exosomes to cause ubiquitin degradation of IDH1 in gastric cancer cells, reduce the level of tricarboxylic acid cycle metabolism in gastric cancer cells, promote ROS accumulation, induce anoikis in gastric cancer, and thus inhibit the development of gastric cancer.