ESCC is an aggressive disease with an unpromising outlook (Harada et al., 2020). Traditional treatments, such as surgery or radiotherapy, have not been effective, and a new treatment is urgently needed. With the wide application of TCM in different diseases, clinical efficacy has been proven. This study offers an attempt to combine network pharmacology and bioinformatics to offer an innovative approach to treating ESCC. The intersection of 226 ZQFZ targets and the ESCC datasets GSE38129 and GSE20347 revealed a total of 35 core targets for this study.
According to GO analysis, a majority of the ZQFZ and ESCC differential genes were linked to biological functions such as protein phosphorylation, signal transduction, and cell proliferation control. Aberrant ANO1 expression may influence the ESCC process via the signal transduction mechanism(Liu et al., 2021). PTEN and PD-L1 have been demonstrated to affect the course of ESCC through the control of cell proliferation in both in vitro and in vivo investigations(Qiu et al., 2022). As a result of KEGG signaling pathway enrichment studies, Hub genes were mainly enriched in Pathways in cancer, AGE-RAGE signaling pathway in diabetic complications, Cellular senescence, and other signaling pathways. Notably, each research added to the confirmation of our findings(Wang et al., 2021a, Wang et al., 2021b, Zheng et al., 2020). GSEA analysis revealed that the majority of the ESCC's genes were enriched in REACTOME_BUDDING_AND_MATURATION_OF_HIV_VIRION, REACTOME_LATE_ENDOSOMAL_MICROAUTOPHAGY, KEGG_HISTIDINE_ METABOLISM, and other signaling pathways, which are directly linked to the emergence, growth, and metastasis of cancer. This further strengthens our belief in the use of ZQFZ for ESCC.
The immune cells that make up the tumor microenvironment are mostly found in tumor cells, and they are linked to the growth of tumors. The results of the immunoassay showed that T cells CD4 memory resting, M0 Macrophage, M1 Macrophage, and Mast resting cells have a high correlation with ESCC relative to other immune cells. Tumor-associated M1 macrophages in immune cells can inhibit and kill cancer cells (Xiao et al., 2020). CMSP may inhibit ESCC in vivo and in vitro by inducing M1-type macrophage polarization (Wang et al., 2023b). Additionally, a good prognosis for esophageal squamous carcinoma was significantly and positively linked with an increase in M1 macrophage density(Jiang et al., 2021). Meanwhile, PRDM5 low expression can produce a poor prognosis for esophageal squamous carcinoma through T cells CD4 memory resting (Guo et al., 2022). However, the link between ESCC and other immune-invading cells has only sometimes been described and needs to be thoroughly investigated. Interestingly, in exploring the relationship between core genes and common immunosuppressive checkpoints HVEM, TIGIT, PD-1, TIM-3, CTLA4, CD47, and CD200R1 (Di et al., 2022), we found significant correlations between SERPINE1, CHEK1, CXCL8 and CD47, IGFBP3, MMP9, and TIGIT, and most importantly, CXCL10 and HVEM, TIGIT, PD-1, TIM-3, CTLA4, CD47, and CD200R1 were almost all positively correlated. The immunological response to ESCC is suppressed by these inhibitory immune checkpoints, indicating a feasible use of CXCL10 in immunotherapy. The TIMER database was further validated and CXCL10 was found to correlate with B Cell, CD4 + T Cell, Macrophage, Neutrophil, and Dendritic Cell. This has shown that CXCL10 influences immune cells in a way that affects ESCC development, offering CXCL10 a theoretical basis for being a potential therapy for ESCC.
CXCL10 is a member of the CXC chemokines, which are associated with various diseases, including autoimmune diseases and tumors (Lee et al., 2017). CXCL10 acts by attaching to its receptor CXCR3, and it is crucial for inducing effector CD8 + T cells, Th1 cells, and NK cells to infiltrate inflammatory areas(Antonelli et al., 2014). Interestingly, CXCL10 and its receptor CXCR3 may also have some antitumor effects; CXCL10 binds to CXCR3 and induces CD4+/CD8 + T cells to infiltrate into tumor sites, inducing apoptosis and thus inhibiting tumor growth (Sahin et al., 2013). Additionally, it has been noticed that ESCC patients' prognoses are strongly correlated with CXCL10 expression, which promotes ESCC growth(Sato et al., 2016). In summary, our findings are supported by the available literature and experiments, which point to the possible application of ZQFZ in the treatment of ESCC.
Next, we performed univariate and multivariate Cox analyses of the 11 screened genes and plotted prognostic survival KM curves for the differential gene CHEK1, and the results showed P < 0.05, demonstrating that the prognosis for ESCC was significantly influenced by CHEK1. The anticipated ROC was plotted using the survROC program, and the AUC values and calibration curves were produced (Fig. 11-F) to further confirm our proposed diagnostic model. The results showed some accuracy in the 3-year survival rate (AUC = 0.984). The limitation is that the TCGA included in this study had fewer survival data on ESCC, making the model lack some convincing power.
Finally, we docked quercetin, kaempferol, luteolin, 7-O-methylisomucronulstol, and beta-sitosterol, the five most closely linked compounds in ZQFZ, with 11 differential genes molecule by molecule, showing good results. Quercetin is a natural flavonoid that shows anticancer effects by inhibiting the PI3K pathway (Maurya and Vinayak, 2015). The natural polyphenol kaempferol, which is primarily present in fruits and vegetables, has been demonstrated in several studies to destroy cancer cells by altering cell signaling pathways, triggering apoptosis, and causing cell cycle arrest(Almatroudi et al., 2023). Most of the luteolin is present in various plants in the form of glycosides. Through the inhibitor lidocaine, VRK1 reduces the biological malignant propensity of ESCC cells (Liu et al., 2017). Beta-sitosterol is the most prevalent phytosterol present in a wide range of plant-based foods. Some malignancies, including liver, cervical, colon, stomach, lung, and pancreatic tumors, are resistant to it in preclinical studies during the past few decades(Wang et al., 2023). This reinforces our vision of ZQFZ for ESCC.
Of course, the lack of further validation with experiments is a limitation of this study. Moreover, due to the absence of experimental circumstances, molecular dynamics simulations were not performed for molecular docking, which is a pity for us. We hope to verify our speculations in subsequent animal experiments.
In conclusion, the network pharmacology method offers an innovative viewpoint on how ZQFZ works to treat ESCC. This work advances our understanding of how ZQFZ particles modulate ESCC through many pathways and multiple targets. It offers a theoretical foundation for using ZQFZ particles in ESCC clinical settings.