Integrated Network Pharmacology, Transcriptomics, and Experimental Validation Identify PI3K-AKT and STAT3 as Key...

Pien Tze Huang (PZH), a classic Traditional Chinese Medicine (TCM), has long been used for treating inflammation and cancer, yet its pharmacological mechanisms against liver cancer and Colorectal Cancer (CRC) remain incompletely understood. This study aims to elucidate the potential antitumor targets, signaling pathways, and functional effects of PZH against liver cancer and CRC.

Network pharmacology was applied to identify the active components of PZH from the TCMSP database and predict their potential targets, while disease-associated targets were retrieved from GeneCards. Core targets and pathways were screened via Protein-Protein Interaction (PPI) network analysis and functional enrichment (GO/KEGG) using Metascape. Molecular simulation was performed to assess the binding of key components to core targets. Experimental validation in Huh7, HCT116, and RKO cells included assessments of cell viability, cell cycle, protein expression, and transcriptomic profiling using CCK-8, flow cytometry, Western blot, and RNA sequencing, respectively.

A total of 15 active components of PZH were identified, corresponding to 353 potential targets, among which 138 and 102 overlap with liver cancer and CRC, respectively. Network analysis identified AKT1, STAT3, EGFR, and ESR1 as core targets, which were enriched in PI3K-AKT, STAT3, ErbB, and PD-L1/PD-1 checkpoint pathways. Molecular simulation suggested favorable binding affinities of muscone and quercetin with AKT1, and of ginsenoside F2 and ginsenoside Rh2 with STAT3. In vitro, PZH inhibited cell proliferation, induced G1/S phase arrest, and downregulated Cyclin D1, PCNA, c-Myc, and PD-L1. Mechanistically, PZH selectively suppressed PI3K-AKT and STAT3 signaling, and combined treatment with four active components produced consistent inhibition of these pathways and downstream targets.

These findings suggest that PZH may exert anti-tumor effects through coordinated modulation of PI3K-AKT and STAT3 signaling pathways, which are key regulators of tumor cell proliferation and immune evasion. The suppression of PD-L1 expression further implies a potential role of PZH in modulating tumor immune escape in addition to inhibiting tumor growth. Moreover, the integration of network pharmacology, molecular simulation, and experimental validation provides a framework for understanding the multi-target nature of traditional Chinese medicine and may facilitate the discovery of synergistic therapeutic mechanisms in complex diseases such as cancer.

PZH exerts multi-component and multi-target anti-tumor effects against liver cancer and CRC, primarily by inhibiting PI3K-AKT and STAT3 signaling, thereby suppressing tumor cell proliferation and PD-L1 expression. These findings provide a mechanistic rationale for the traditional use of PZH and suggest its potential as a candidate for further preclinical investigation in liver cancer and CRC.