β-caryophyllene sensitizes hepatocellular carcinoma cells to chemotherapeutics and inhibits cell malignancy through targeting MAPK signaling pathway

In hepatocellular carcinoma (HCC) development, cancer cells generally exhibit increased cell proliferation due to mutations and aberrant expressions of key regulatory genes. The current study determines the cytotoxic effects of β-caryophyllene (BCP) alone or in combination with doxorubicin (DOX) and cisplatin (DDP) on HCC cells and also elucidates the underlying mechanism of BCP to exert its anticancer activities. HepG2, SMMC-7721 HCC cells, and HL-7702 normal liver cells were treated with BCP, DOX, and DDP individually or in their combinations. Cell proliferation assay, flow cytometric assay, and Western blot analysis were used to evaluate the cytotoxic effects of these treatments. Transwell assays were used to examine the effects of BCP on migration and invasion of HCC cells. RNA-seq studies were used to determine the primary target genes of BCP-treated HepG2 cells. Integrative analysis of differentially expressed genes (DEGs) of RNA-seq data with a TCGA dataset of HCC patients identified BCP-targeted genes that were verified by RT-qPCR. Ectopic gene expression, cell viability, and colony formation assay were performed to validate the primary targets of BCP. The results found that, BCP selectively inhibited HCC cell proliferation while exhibited relatively low toxicity to normal liver cells; however, DOX and DDP showed higher toxicity in normal cells than that in HCC cells. Importantly, in combinatorial treatments, BCP synergistically enhanced cytotoxicity of DOX and DDP in HCC cells but this effect is markedly reduced in HL-7702 cells. BCP-treated HCC cells exhibited decreased migration and invasion ability versus the control. Furthermore, RNA-seq analyses of BCP-treated HepG2 cells identified 433 protein-coding DEGs with over 1.5-fold change. Integrative analyses revealed five BCP-targeted DEGs regulating the MAPK signaling pathway. Among these five genes, three displayed a significantly positive correlation of their expression with the overall survival of HCC patients. As a primary target, PGF was significantly downregulated by BCP treatment, and its exogenous expression desensitized HCC cells to the inhibition of BCP treatment. In summary, BCP inhibits malignant properties of HCC and synergistically sensitizes the anticancer activity of DOX and DDP. In HCC cells, BCP primarily targets PGF and MAPK signaling pathway.