Daphnetin sensitizes hepatocellular carcinoma cells to chemotherapeutics and suppresses malignant phenotypes through modulating DDIT3-associated stress-response

Many natural products can both suppress tumor growth and promote human health. Daphnetin (DAP) is a natural coumarin and possesses suppressive activities against various cancers, but the molecular mechanisms underlying its anticancer action remain poorly understood. In the treatments of hepatocellular carcinoma (HCC), many generic chemotherapeutic agents, such as doxorubicin (DOX) and cisplatin (DDP), manifest significant side effects on patients. In the present study, we first investigated the cytotoxic effects of DAP alone or in combination with DOX or DDP in HCC HepG2 and SMMC-7721 cells, and in normal liver HL-7702 cells. Based on isobologram analysis, DAP synergistically enhanced cytotoxic activities of DDP and DOX in HCC cells but not in HL-7702 cells. RNA-seq analysis of DAP-treated HepG2 cells identified 484 protein-coding DEGs involved in multiple pathways, such as PI3K-AKT signaling, amino acid deprivation, and unfolded protein response. Especially, DAP treatment upregulated a stress-responsive pro-apoptotic transcription factor DDIT3. Consistently, DDIT3 knockdown desensitized HCC cells to DAP, while constitutively active AKT did not rescue DAP-induced defective phenotypes. Collectively, we demonstrate DAP as a multi-pathway stress-modulating agent that can both attenuate malignant phenotypes of HCC cells and sensitize the cells to clinical chemotherapeutics.