Table 7_β-caryophyllene sensitizes hepatocellular carcinoma cells to chemotherapeutics and inhibits cell malignancy through targeting MAPK signaling pathway.xlsx

Backgroundβ-caryophyllene (BCP) is a naturally occurring bicyclic sesquiterpene extracted from various plants, and widely used as a medicinal agent for various diseases. During hepatocellular carcinoma (HCC) development, cancer cells generally exhibit increased cell proliferation due to mutations or aberrant expression of key regulatory genes. The current study determines the cytotoxic effects of BCP alone or in combination with doxorubicin (DOX) and cisplatin (DDP) on HCC cells, and elucidates the underlying mechanism of BCP to exert its anticancer activities.
Materials and methodsHepG2, SMMC-7721 HCC cells, and HL-7702 normal liver cells were treated with BCP, DOX, and DDP individually or combinatorially. Cell proliferation assay, flow cytometric assay, and Western blot were employed to evaluate the cytotoxic effects of these treatments. Transwell assays were used to examine BCP’s effects on HCC cell migration and invasion. RNA-seq analysis was used to determine BCP’s primary target genes in HepG2 cells. Integrative analysis of differentially expressed genes (DEGs) of RNA-seq data with an HCC TCGA dataset identified BCP-targeted genes that were verified by RT-qPCR analysis. Ectopic gene expression, cell viability, and colony formation assay were performed to validate the primary targets of BCP.
ResultsBCP selectively inhibited HCC cell proliferation while exhibited relatively low toxicity in normal liver cells; however, DOX and DDP showed higher toxicity in normal cells than that in HCC cells. In combinatorial treatments, BCP synergistically enhanced cytotoxicity of DOX and DDP in HCC cells but this effect was markedly reduced in HL-7702 cells. BCP treatment reduced migration and invasion of HCC cells. Furthermore, RNA-seq analyses of BCP-treated HepG2 cells identified 433 protein-coding DEGs. 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 BCP-mediated inhibition.
DiscussionBCP inhibits malignant properties of HCC and synergistically sensitizes the anticancer activity of DOX and DDP. In HCC cells, BCP primarily targets the PGF gene and MAPK signaling pathway.

【背景】β-石竹烯（β-caryophyllene, BCP）是一种天然存在的双环倍半萜，可从多种植物中提取，广泛用作治疗多种疾病的药用制剂。在肝细胞癌（hepatocellular carcinoma, HCC）发生发展过程中，癌细胞通常因关键调控基因的突变或异常表达而出现细胞增殖异常增强。本研究旨在探究BCP单独或与阿霉素（doxorubicin, DOX）、顺铂（cisplatin, DDP）联合使用时，对HCC细胞的细胞毒性作用，并阐明BCP发挥抗癌活性的潜在分子机制。
【材料与方法】将HepG2、SMMC-7721肝癌细胞及HL-7702正常肝细胞分别或联合用BCP、DOX及DDP进行处理。采用细胞增殖检测、流式细胞术检测及蛋白质印迹法（Western blot）评估上述处理的细胞毒性效应；通过Transwell实验检测BCP对HCC细胞迁移与侵袭能力的影响；利用RNA测序（RNA-seq）分析确定BCP在HepG2细胞中的核心靶基因。将RNA测序数据中的差异表达基因（differentially expressed genes, DEGs）与癌症基因组图谱（The Cancer Genome Atlas, TCGA）肝癌数据集进行整合分析，筛选出BCP靶向的差异表达基因，并通过实时定量聚合酶链反应（RT-qPCR）进行验证。通过外源基因过表达、细胞活力检测及集落形成实验，进一步验证BCP的核心靶基因。
【结果】BCP可选择性抑制HCC细胞增殖，且对正常肝细胞的毒性相对较低；而DOX与DDP对正常细胞的毒性则高于其对肝癌细胞的毒性。联合用药时，BCP可协同增强DOX与DDP对HCC细胞的细胞毒性，但该协同效应在HL-7702正常肝细胞中显著减弱。BCP处理可降低HCC细胞的迁移与侵袭能力。此外，对BCP处理后的HepG2细胞进行RNA测序分析，共鉴定出433个编码蛋白的差异表达基因。整合分析显示，有5个BCP靶向的差异表达基因参与调控丝裂原活化蛋白激酶（MAPK）信号通路。在这5个基因中，3个基因的表达水平与HCC患者的总生存期呈显著正相关。作为核心靶基因，胎盘生长因子（PGF）经BCP处理后显著下调，且外源性过表达PGF可使HCC细胞对BCP介导的增殖抑制作用产生耐药性。
【讨论】BCP可抑制HCC的恶性表型，并协同增敏DOX与DDP的抗癌活性。在HCC细胞中，BCP主要靶向PGF基因及MAPK信号通路。