HDAC Inhibitors Potentiate Doxorubicin Cytotoxicity in HepG2 Cells and Alter DNA-Repair- and p53-Associated Transcriptional Programs

Histone deacetylase inhibitors (HDACis) can enhance responses to DNA-damaging chemotherapy, but how they alter doxorubicin (DOX)-induced transcriptional programs remains incompletely understood. We investigated the effects of valproic acid (VPA) and vorinostat (SAHA) on DOX cytotoxicity and associated molecular responses in HepG2 cells. HDAC inhibition, cell viability, apoptosis, and DOX IC50 values were assessed using histone H3 acetylation, cell counting, Alamar Blue assays, and cleaved caspase-3 immunofluorescence. Transcriptomic responses were analyzed by RNA sequencing, differential expression analysis, and Hallmark gene set enrichment analysis, with selected findings evaluated by RT-qPCR, Western blotting, and comparison with published CRISPR/Cas9 DOX-response data. Both HDAC inhibitors increased histone H3 acetylation and enhanced DOX cytotoxicity, markedly reducing DOX IC50 values. VPA co-treatment also increased DOX-induced cleaved caspase-3 signaling. RNA-seq showed broad transcriptional remodeling, including depletion of the DNA-repair gene set following VPA treatment and attenuation of canonical p53-responsive genes during HDACi–DOX co-treatment. Several HDACi-repressed DNA-repair genes overlapped with genes whose loss sensitized cells to DOX in a published CRISPR screen. HDAC inhibition therefore enhances DOX cytotoxicity and is accompanied by alterations in DNA-repair-associated and p53-responsive transcriptional programs.