Natural small-molecule compounds from Chinese herbal medicine are of interest as potent adjuvants to conventional cancer therapy, with lower toxicity and fewer side effects. Morusinol is a monomer compound from herbal medicine. This study aims to establish the therapeutic potential of Morusinol in the treatment of diffuse large B cell lymphoma (DLBCL), clarify its mechanism of action, and explore novel combination treatment options for this disease. 

1.     The half-maximal inhibitory concentration (IC50) of three DLBCL cell lines (SU-DHL-8, SU-DHL-2, and Farage) to Morusinol was detected by CCK8 to determine the inhibitory effect of Morusinol. The cell proliferation, apoptosis, cell cycle distribution, and Hoechst 33258 staining were detected after Morusinol treatment. High-throughput mRNA sequencing was used to screen out the differential protein-coding genes after Morusinol treatment. Differential genes enrichment analysis based on GO (Gene Ontology Resource), KEGG (Kyoto Encyclopedia of Genes and Genomes), and GSEA (Gene Set Enrichment Analysis) databases were used to clarify the signaling pathways and biological processes affected by Morusinol.  

2.     Drug affinity responsive target stability (DARTS) and Mass spectrometry (MS) were applied to screen out the potential target of Morusinol. The expression of the target protein was examined across multiple malignancies using data from the GEPIA database. The ability of Morusinol to bind to the target protein was detected by molecular docking and cellular thermal shift assay (CETSA). To explore the role of the target protein in DLBCL, it was subjected to loss-of-function analysis in DLBCL cells by transfecting siRNAs. Kaplan–Meier curve and Cox regression models were used to analyze overall survival (OS) (GSE10846). The concordance index (C-index) was used to assess the predicted validity of the National Comprehensive Cancer Network-International Prognostic Index (NCCN-IPI) model and the NCCN-IPI + the target model.

3.     The therapeutic effects of Morusinol in combination with established chemotherapeutics were detected by CCK8 assay. Combination index (CI) values were calculated based on the Chou–Talalay equation using CompuSyn software.  

4.     Xenograft mice were established to investigate the inhibitory effect of Morusinol on tumor growth. The toxic effect on important organs was observed by H&E staining.

1.     Morusinol exerted significant inhibitory effects on three DLBCL cells and affected a variety of biological processes such as apoptosis and cell cycle.   

2.     With the results of DARTS and MS, fucosyltransferase 8 (FUT8) was screened out as the target protein of Morusinol that mediates antitumor effects in DLBCL. The expression of FUT8 in DLBCL from the GEPIA database suggested that FUT8 acted as an oncogenic role in DLBCL. Molecular docking and cellular thermal shift assay (CETSA) suggested that Morusinol could bind to FUT8. Interference of FUT8 suppressed cell proliferation. Kaplan-Meier analysis indicated that high FUT8 expression was associated with poor OS of DLBCL patients (GSE10846). Likewise, the elevated expression of FUT8 in DLBCL is significantly correlated with poor OS in a univariate analysis and is statistically significant after adjusting for the NCCN-IPI in the multivariate analysis.     

3.     The quantitative analysis indicates that the combination of Morusinol and Chidamide exhibited potent synergistic cytotoxicity against three DLBCL cell lines, especially in the SU-DHL-2 cell line, as evidenced by predominantly <1.0 combination index (CI) values.  

4.     Morusinol synergizes Chidamide-mediated cytotoxicity in DLBCL in vivo. Treatment with Morusinol resulted in no toxicity to important organs, as evidenced by the absence of significant histological damage in the heart, liver, spleen, lung, and kidney tissues. 

The Natural small-molecule, Morusinol, exerts antitumor effects on DLBCL by inducing cell cycle arrest and apoptosis in vitro and reducing tumor burden in vivo. FUT8 is identified as the target protein that binds to Morusinol, thereby elucidating the mechanism of action of Morusinol. Furthermore, Morusinol synergizes chidamide-mediated cytotoxicity. In summary, Morusinol shows great potential as an anti-tumor agent for clinical applications in the management of DLBCL, with the potential to complement existing therapeutic strategies.