Shi Mengchen, Zhou Lina, Zhao Lu, Shang Mei, He Tongtong, Tang Zeli, Sun Hengchang, Ren Pengli, Lin Zhipeng, Chen Tingjin, Yu Jinyun, Xu Jin, Yu Xinbing, Huang Yan
Department of Parasitology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
Key Laboratory for Tropical Disease Control, Ministry of Education, Sun Yat-Sen University, Guangzhou, China.
PLoS Negl Trop Dis. 2017 Nov 10;11(11):e0006074. doi: 10.1371/journal.pntd.0006074. eCollection 2017 Nov.
Numerous experimental and epidemiological studies have demonstrated a link between Clonorchis sinensis (C. sinensis) infestation and cholangiocarcinoma (CCA) as well as hepatocellular carcinoma (HCC). The underlying molecular mechanism involved in the malignancy of CCA and HCC has not yet been addressed. Csseverin, a component of the excretory/secretory products of C. sinensis (CsESPs), was confirmed to cause obvious apoptotic inhibition in the human HCC cell line PLC. However, the antiapoptotic mechanism is unclear. In the present study, we investigated the cellular features of the antiapoptotic mechanism upon transfection of the Csseverin gene.
In the present study, we evaluated the effects of Csseverin gene overexpression on the apoptosis of PLC cells using an Annexin PE/7-AAD assay. Western blotting was applied to quantify the activation of caspase-3 and caspase-9, the mitochondrial translocation of Bax and the release of Cyt c upon Csseverin overexpression in PLC cells. Laser scanning confocal microscopy was used to analyze the changes of intracellular calcium. Fluorescence assay and immunofluorescence assays were performed to observe the changes of the mitochondrial permeability transition pore (MPTP).
The overexpression of Csseverin in PLC cells showed apoptosis resistance after the induction of apoptosis. Additionally, the activation of caspase-3 and caspase-9 was specifically weakened in Csseverin overexpression PLC cells. The overexpression of Csseverin reduced the increase in intracellular free Ca2+, thereby inhibiting MPTP opening in PLC cells. Moreover, Bax mitochondrial translocation and the subsequent release of Cyt c were downregulated in apoptotic Csseverin overexpression PLC cells.
The present findings suggest that Csseverin, a component of CsESPs, confers protection from human HCC cell apoptosis via the inactivation of membranous Ca2+ channels. Csseverin might be involved in the process of HCC through C. sinensis infestation in affected patients.
大量实验和流行病学研究表明，华支睾吸虫（华支睾吸虫）感染与胆管癌（CCA）以及肝细胞癌（HCC）之间存在联系。CCA和HCC恶性肿瘤所涉及的潜在分子机制尚未得到解决。华支睾吸虫排泄/分泌产物（CsESPs）的一种成分Csseverin被证实可导致人肝癌细胞系PLC中明显的凋亡抑制。然而，抗凋亡机制尚不清楚。在本研究中，我们研究了转染Csseverin基因后抗凋亡机制的细胞特征。
在本研究中，我们使用Annexin PE/7 - AAD检测法评估了Csseverin基因过表达对PLC细胞凋亡的影响。应用蛋白质印迹法来定量PLC细胞中Csseverin过表达时caspase - 3和caspase - 9的激活、Bax的线粒体转位以及细胞色素c的释放。使用激光扫描共聚焦显微镜分析细胞内钙的变化。进行荧光测定和免疫荧光测定以观察线粒体通透性转换孔（MPTP）的变化。
PLC细胞中Csseverin的过表达在诱导凋亡后显示出抗凋亡能力。此外，Csseverin过表达的PLC细胞中caspase - 3和caspase - 9的激活被特异性减弱。Csseverin的过表达减少了细胞内游离Ca2 +的增加，从而抑制了PLC细胞中MPTP的开放。此外，在凋亡的Csseverin过表达PLC细胞中，Bax的线粒体转位以及随后细胞色素c的释放被下调。
本研究结果表明，CsESPs的一种成分Csseverin通过膜性Ca2 +通道的失活赋予对人肝癌细胞凋亡的保护作用。Csseverin可能通过受影响患者的华支睾吸虫感染参与HCC的发生过程。
