Trichodermin, an endophytic fungal sesquiterpene, suppresses colorectal cancer cell migration and invasion by targeting the PKC-ERK-Sp1-CTSV axis

Background: Trichodermin (TCD), a sesquiterpene initially isolated from the fungus Trichoderma viride, has previously shown potent activity against pancreatic tumors and chondrosarcoma. However, its therapeutic potential in colorectal cancer (CRC) remains poorly understood. Methods: Cell proliferation, cytotoxicity, and cell cycle distribution were assessed using the MTT assay, LDH assay, and Annexin V/PI staining via flow cytometry, respectively. The migratory and invasive capabilities of the cells were evaluated using in vitro migration and invasion assays. The human protease array is engineered to assess the expression levels of multiple proteases. Gene expression and regulation were analyzed using quantitative RT-PCR and siRNA techniques, while protein expression was evaluated via immunoblot analysis. Finally, TCGA was utilized to evaluate the clinical relevance of our molecular findings by correlating gene expression patterns with patient outcomes and other clinical parameters. Results: We found that TCD markedly inhibited the cell migration and invasion of human CRC DLD1 and HT-29 cells, without affecting cell viability, cell cycle progression, or apoptotic response of either normal or CRC cells. Proteomic analysis indicated that downregulation of cathepsin V (CTSV) was the major mechanism of TCD anti-metastatic activity. Further investigation revealed that TCD reduced Sp1 transcriptional activity, thereby suppressing CTSV expression. Additionally, TCD inhibited the phosphorylation levels of PKCα, PKCδ, and ERK, which in turn disrupted Sp1 and CTSV expression. Knockdown of ERK (siRNA-ERK) or CTSV (siRNA-CTSV) increased the anti-migration and anti-invasion effects of TCD treatment. Investigations using the PKC activator TPA shown that TCD counteracted TPA-induced activation of the PKC-ERK-Sp1-CTSV pathway, effectively limiting CRC cell migration and invasion. Conclusion: Our findings robustly provide compelling evidence that TCD exerts potent anti-migration and anti-invasion effects on CRC cells by targeting PKCα/PKCδ/ERK/Sp1/CTSV axis. These results highlight TCD as a promising therapeutic candidate for advanced or metastatic CRC.