Responses to Nei-Xiao-Luo-Li-San in Toledo Diffuse Large B-Cell Lymphoma Cells: Potential Involvement of ATF5- and HDAC-Associated Epigenetic Changes

Abstract

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma in adults and is characterized by aggressive progression and substantial molecular and clinical heterogeneity. Despite advances in immunochemotherapy, a considerable proportion of patients develop relapsed or refractory disease, highlighting the need to better understand the molecular mechanisms underlying DLBCL. Nei-Xiao-Luo-Li-San (NXLLS), a traditional herbal formulation historically used for nodules and tumor-like masses, provides a pharmacological model for investigating molecular responses and candidate regulatory mechanisms in DLBCL. Methods: NXLLS was chemically characterized to assess its compositional profile and compositional similarity among batches. Its biological effects were evaluated by assessing cell viability, migration, invasion, and cell-cycle progression in Toledo DLBCL cells. Molecular responses to NXLLS were investigated through an integrated approach combining transcriptomic analysis, network pharmacology, and molecular assays. The functional role of ATF5 was further evaluated using lentiviral overexpression and knockdown. Results: Chromatographic profiling provided an exploratory assessment of compositional similarity among NXLLS samples and supported the selection of eight representative compounds using liquid chromatography–mass spectrometry (LC–MS) annotation and high-performance liquid chromatography (HPLC) authentic-standard confirmation. NXLLS reduced cell viability, migration, and invasion in Toledo DLBCL cells within the tested concentration range. At the molecular level, NXLLS was associated with altered cell-cycle distribution and potential G1-phase accumulation at later time points, reduced vascular endothelial growth factor (VEGF)-associated enzyme-linked immunosorbent assay (ELISA) signal, and modulated stress-response genes. RNA sequencing (RNA-seq) revealed transcriptional changes involving stress-response and histone deacetylase (HDAC)-associated epigenetic pathways. Correspondingly, HDAC expression was reduced, whereas acetylated histone H3 lysine 9 (Ac-H3K9) and acetylated histone H3 lysine 27 (Ac-H3K27) levels were increased. ATF5 knockdown recapitulated several cellular and epigenetic changes observed following NXLLS treatment, supporting ATF5 as a potential downstream mediator of the NXLLS response. Conclusions: This study identified potential molecular targets associated with the response to NXLLS in Toledo DLBCL cells, with ATF5 further investigated as a potential downstream mediator. The findings also suggest an association between the NXLLS response and HDAC-related epigenetic changes, providing a basis for further investigation of their mechanistic relationships.