Na+K+ATP酶α亚型表达分布与蟾皮非极性组份抑制肝癌细胞增殖侵袭敏感性的关联分析与功能论证

Effective treatment medicine is still lacking for liver cancer. Traditional Chinese medicine Toad skin and Huachansu have been widely used for liver cancer treatment. However the molecular mechamism remains unclear. It has been accepted that the non-polar fraction (especially the cardiac glycosides such as bufalin )from toad skin is the mainly anti-tumor fractions and it functions via Na+K+ATPase in tumor cells. However, study from our group have previouly found that the response rates varied when bufalin was treated on different liver cancer cell lines, which is similar to that have been observed in clinical liver cancer treatment. To identify the mechanism involved, we could found that the effects of bufalin on liver cancer cells correlated positively with the expression pattern of Na+K+ATPase.Therefore, in this study we will further evalute the correlation between Na+K+ATPase α subunits expression pattern and sensitivity of liver cancer cells to non-polar fraction from toad skin. We will also evaluated the anti-tumor effects in liver cancer cells after the a3 is ectopically expressed or RNAi-mediated silenced.Collectively we hope to confirm whether Na+K+ATPase α subunits mediate the effects of the non-polar fraction of toad skin on liver cancer and to unveil the mechanism involved.We also try to identify a biomarker that could predict treatment response, which could finally lead to improvement in liver cancer treatment.

肝癌目前缺乏有效治疗药物。中药蟾皮及其制剂华蟾素已广泛用于肝癌治疗，并取得疗效，但作用机制不清。研究证实蟾皮非极性组份（主要是强心苷类物质，如bufalin）是其主要抗瘤活性成份，该抗瘤作用可能与肿瘤细胞Na+K+ATP酶有关。课题组在前期研究中发现，不同人肝癌细胞应用bufalin后虽均出现生长抑制，但敏感性却有很大差异，这与临床上不同患者治疗后出现不同的疗效反应相一致。为寻找导致该差异的原因，我们发现，肝癌细胞Na+K+ATP酶α亚型在不同肝癌细胞中表达分布差异与bufalin抗肝癌作用有相关性。因此，本研究中我们将系统评价肝癌细胞Na+K+ATP酶α亚型表达分布是否与蟾皮非极性组份治疗敏感性相关，并通过功能学研究证实α亚型表达改变是否可引起治疗敏感性变化，以期明确α亚型是否介导了肝癌治疗敏感性差异，最终为明确蟾皮制剂药物作用机制、寻找疗效预测标志物、进一步提高临床疗效提供依据。

本研究发现Na+/K+-ATPase α亚基在人肝癌细胞、肝脏组织中的表达以α1亚型为主，肿瘤细胞对蟾毒灵的敏感性与其Na+/K+-ATPase α1（ATP1A1）的表达量成负相关。下调肝癌细胞ATP1A1的表达使其对多种强心苷类物质，包括蟾毒灵、华蟾蜍精、华蟾蜍它灵、华蟾素注射液和乌本苷的敏感性增强。体外实验通过观察细胞形态、细胞周期检测、TUNEL实验均显示与对照组相比，蟾毒灵作用于转染了ATP1A1-siRNA的细胞会引起更显著的细胞凋亡。体内实验也证明了抑制ATP1A1能够增强蟾毒灵抑制肿瘤生长的作用，蟾毒灵在转染对照siRNA组里的抑瘤率为33.6%，而在转染ATP1A1-siRNA组里的抑瘤率为55.1%。进一步的机制研究发现，抑制ATP1A1可通过增强p53的活性增强蟾毒灵的作用。这部分研究为明确蟾皮制剂药物作用机制、寻找疗效预测标志物、进一步提高临床疗效提供了重要的理论依据。鉴于ATP1A1在肝癌组织中高表达，我们进一步研究了ATP1A1对肝癌细胞生物学行为的影响，评估其作为肝癌治疗靶点的价值。研究发现，在肝癌细胞中抑制ATP1A1的表达会抑制细胞增殖、迁移、体内成瘤能力，诱导细胞凋亡，其机制与抑制ATP1A1的表达使胞内ROS升高，细胞处于氧化应激状态有关。全基因组表达谱芯片显示，下调肝癌细胞ATP1A1的表达影响的基因主要参与调控细胞周期、细胞应激、DNA修复、DNA复制及细胞代谢。尽管近几年蟾毒灵抗肿瘤作用的分子机制报道已经很多，但仍存在很多未解之谜，在研究中课题组揭示了蟾毒灵抑制肝癌细胞增殖的新机制。通过全基因组表达谱芯片及进一步验证，我们发现蟾毒灵能够抑制肝癌细胞神经再生相关蛋白（NREP）的表达，在肝癌细胞中过表达NREP会减弱蟾毒灵对细胞的增殖抑制作用。我们也首次发现NREP的mRNA水平在肝癌组织中高表达，抑制NREP的表达会抑制肝癌细胞的增殖。