Center of Research and Development on Life Sciences and Environmental Sciences, Harbin University of Commerce, Harbin, Heilongjiang 150076, P.R. China.
Institute of Materia Medica and Postdoctoral Programme of Harbin University of Commerce, Harbin, Heilongjiang 150076, P.R. China.
Oncol Rep. 2018 Mar;39(3):1475-1484. doi: 10.3892/or.2018.6200. Epub 2018 Jan 8.
Cytisine, a quinolizidine alkaloid, is one of the major bioactive components found in the small tree Sophora Alopecuraides L., and is a traditional Chinese medicine that is used for treating hepatitis and liver cancer. In the 1960s, quinolizidine alkaloids were reported to exhibit inhibitory effects on tumour cell proliferation in several types of cancer cells. However, few studies have investigated the effect of cytisine on liver cancer. Our team confirmed that cytisine induced apoptosis in HepG2 cells via a mitochondrial pathway. The primary aim of the present study was to evaluate the endoplasmic reticulum (ER) stress caused by calcium overload in cytisine‑induced apoptosis in HepG2 cells and the molecular mechanisms of this phenomenon. In addition, the present study was undertaken to evaluate the expression of α7‑nAChR when apoptosis was induced by cytisine in HepG2 cells. In the present study, transmission electron microscopy was used to observe the morphological appearance of HepG2 cells. The apoptosis of the cells with cytoplasmic vacuolization was significant under electron microscopy. Apoptotic bodies, the expansion of the ER, and swelling of mitochondria were observed in the HepG2 cells after cytisine treatment. Flow cytometric analysis demonstrated that the apoptosis rate of HepG2 cells was upregulated. In addition, the intracellular calcium concentration was detected by laser confocal fluorescence microscopy. The laser confocal fluorescence microscopy showed that the calcium concentration was increased in a dose‑dependent manner. The activity of caspase‑4 was evaluated by an enzyme‑linked analyser, and the expression levels of CHOP, JNK, p‑JNK and α7‑nAChR were assessed via western blot analysis. In the present study, we observed that cytisine induced ER stress‑inducing factors and CHOP and p‑JNK1/2 protein expression, and it increased the JNK protein expression in the HepG2 cells. Furthermore, α7‑nAChR protein expression was promoted in a dose‑dependent manner after cytisine treatment. These findings suggest that cytisine induced the ER stress‑mediated apoptotic pathway via activation of CHOP, JNK and caspase‑4 in HepG2 cells, and cytisine is a potential new target compound for nAchRs (nicotinic acetylcholine receptors) to treat liver cancer.
野靛碱，一种喹诺里西啶生物碱，是小乔木槐属苦参中的主要生物活性成分之一，是一种用于治疗肝炎和肝癌的传统中药。20 世纪 60 年代，有报道称喹诺里西啶生物碱对几种癌细胞的肿瘤细胞增殖具有抑制作用。然而，很少有研究调查野靛碱对肝癌的影响。我们的团队证实，野靛碱通过线粒体途径诱导 HepG2 细胞凋亡。本研究的主要目的是评估野靛碱诱导的 HepG2 细胞内质网（ER）应激引起的钙超载在细胞凋亡中的作用及其分子机制。此外，本研究还评估了野靛碱诱导 HepG2 细胞凋亡时α7-nAChR 的表达。在本研究中，使用透射电子显微镜观察 HepG2 细胞的形态外观。电镜下细胞质空泡化的细胞凋亡明显。野靛碱处理后，HepG2 细胞中观察到凋亡小体、内质网扩张和线粒体肿胀。流式细胞术分析表明 HepG2 细胞的凋亡率上调。此外，通过激光共聚焦荧光显微镜检测细胞内钙离子浓度。激光共聚焦荧光显微镜显示，钙离子浓度呈剂量依赖性增加。通过酶联分析评估 caspase-4 的活性，并通过 Western blot 分析评估 CHOP、JNK、p-JNK 和α7-nAChR 的表达水平。在本研究中，我们观察到野靛碱诱导内质网应激诱导因子和 CHOP 和 p-JNK1/2 蛋白表达，并增加了 HepG2 细胞中 JNK 蛋白的表达。此外，野靛碱处理后，α7-nAChR 蛋白表达呈剂量依赖性增加。这些发现表明，野靛碱通过激活 HepG2 细胞中的 CHOP、JNK 和 caspase-4 诱导内质网应激介导的凋亡途径，并且野靛碱是治疗肝癌的 nAchRs（烟碱型乙酰胆碱受体）的潜在新靶化合物。
