Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China.
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University, Guilin 541004, PR China.
Phytomedicine. 2018 Mar 1;41:33-44. doi: 10.1016/j.phymed.2018.01.024. Epub 2018 Jan 31.
Hepatocellular carcinoma (HCC) is the leading cause of cancer death, and novel chemotherapeutic drugs for treating HCC are urgently needed. 16-O-caffeoyl-16-hydroxylhexadecanoic acid (CHHA) is a new phenylpropanoid isolated by our group from Euphorbia nematocypha which is commonly used to treat solid tumors. However, the underlying mechanisms responsible for the CHHA-induced apoptosis in cancer cells, particularly in HCC, remain unknown.
In the present work, we evaluated the growth inhibitory effect of CHHA on HCC cells and explored the underlying molecular mechanisms.
METHODS/STUDY DESIGNS: The anti-proliferative activity of CHHA was evaluated by MTT assay. Cell cycle, apoptosis, reactive oxygen species and mitochondrial membrane potential were determined by flow cytometry. ER localization was performed by ER-tracker red staining. The effect of CHHA on the expression of mRNA in HCC cells was detected by RT-PCR. The potential mechanisms for proteins level in ER pathway and apoptosis were analyzed by Western blot.
Our results showed that CHHA exerted strong anti-proliferative activity against both HepG2 and Bel-7402 cells in a concentration- and time-dependent manner. Mechanistic studies demonstrated that CHHA induced apoptosis through mitochondrial apoptotic pathway, and arrested the cell cycle at G1 phase. CHHA was also found to induce endoplasmic reticulum (ER) stress, accompanied by ROS production, increase of intracellular calcium and up-regulation of GRP78, CHOP, caspase-12 and p-PERK. Inhibition of endoplasmic reticulum stress by salubrinal pretreatment could suppress both apoptosis and ER stress, indicating that ER stress induction contributes to apoptosis and is required for the latter. Besides, the ROS scavenger N-acetyl cysteine (NAC) significantly attenuated apoptosis induced by CHHA and reversed CHHA-stimulated the expression of ER markers.
In conclusion, CHHA inhibited HCC cell growth and induced apoptosis through mitochondria-mediated pathway and ROS-mediated endoplasmic reticulum stress. This provides molecular bases for developing CHHA into a drug candidate for the treatment of HCC.
肝细胞癌（HCC）是癌症死亡的主要原因，迫切需要新的化疗药物来治疗 HCC。16-O-咖啡酰-16-羟基十六烷酸（CHHA）是我们从Euphorbia nematocypha 中分离出的一种新型苯丙素，常用于治疗实体瘤。然而，CHHA 诱导癌细胞凋亡的潜在机制，特别是在 HCC 中，尚不清楚。
本研究评估了 CHHA 对 HCC 细胞生长的抑制作用，并探讨了其潜在的分子机制。
方法/研究设计：通过 MTT 测定评估 CHHA 的抗增殖活性。通过流式细胞术测定细胞周期、凋亡、活性氧（ROS）和线粒体膜电位。通过 ER-tracker red 染色进行 ER 定位。通过 RT-PCR 检测 CHHA 对 HCC 细胞中 mRNA 表达的影响。通过 Western blot 分析 ER 通路和凋亡相关蛋白水平的潜在机制。
我们的结果表明，CHHA 以浓度和时间依赖的方式对 HepG2 和 Bel-7402 细胞表现出强烈的抗增殖活性。机制研究表明，CHHA 通过线粒体凋亡途径诱导细胞凋亡，并将细胞周期阻滞在 G1 期。还发现 CHHA 诱导内质网（ER）应激，伴随着 ROS 产生、细胞内钙增加和 GRP78、CHOP、caspase-12 和 p-PERK 的上调。用 salubrinal 预处理抑制内质网应激可以抑制凋亡和 ER 应激，表明 ER 应激诱导有助于凋亡，是后者所必需的。此外，ROS 清除剂 N-乙酰半胱氨酸（NAC）显著减弱 CHHA 诱导的细胞凋亡，并逆转 CHHA 刺激的 ER 标志物表达。
总之，CHHA 通过线粒体介导的途径和 ROS 介导的内质网应激抑制 HCC 细胞生长并诱导凋亡。这为将 CHHA 开发成治疗 HCC 的药物提供了分子基础。
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