Background

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.

Methods

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).

Results

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.

Conclusions

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.

中文翻译：

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Cs Severin抑制肝癌PLC细胞中Ca ^{2 +}稳态异常触发的线粒体介导途径抑制细胞凋亡

背景

许多实验和流行病学研究已经证明之间的链路华支睾吸虫（Ç。冬虫夏草以及肝细胞癌（HCC））感染和胆管癌（CCA）。涉及CCA和HCC恶性肿瘤的潜在分子机制尚未得到解决。Cs severin是C. sinensis（Cs ESPs）的排泄/分泌产物的成分，已证实在人HCC细胞株PLC中引起明显的凋亡抑制。但是，抗凋亡的机制尚不清楚。在本研究中，我们调查了Cs Severin基因转染后抗凋亡机制的细胞特征。

方法

在本研究中，我们使用Annexin PE / 7-AAD分析评估了Cs severin基因过表达对PLC细胞凋亡的影响。Western印迹用于定量cs severin在PLC细胞中过表达时caspase-3和caspase-9的激活，Bax的线粒体易位和Cyt c的释放。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变孔（MPTP）的变化。

结果

诱导凋亡后，PLC细胞中Cs Severin的过表达表现出凋亡抗性。此外，在Cs severin过表达的PLC细胞中，caspase-3和caspase-9的激活被特别削弱。Cs severin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，凋亡的Cs severin过表达PLC细胞中Bax线粒体易位和随后的Cyt c释放被下调。

结论

目前的发现表明，Cs Severin是Cs ESP的一个组成部分，它通过膜Ca ²⁺通道的失活赋予了对人类HCC细胞凋亡的保护作用。Cs severin可能通过C参与了HCC的过程。患病的中华感染。