青蒿素调节自噬增强IFN-γ抑制肝癌细胞增殖的分子机制研究

Artemisinin, the worldwide used anti-malarial drug, has recently being focused as a potential anti-cancer agent as well as a chemosensitizer when used in combination with anti-cancer drugs. It has been shown to inhibit cell growth in various types of cancer. Interferon-gamma (IFN-γ) is a pleiotropic cytokine with immunomodulatory, antiviral and anti-cancer effects. IFN-γ has been reported to induce apoptosis in mangy tumors including HCC. Our previous study showed that artemisinin inhibited the growth of SMMC-7721 and SK-HEP-1 human hepatoma cells with G1-phase cell cycle arrest. Artemisinin induced autophagosome formation and conversion of microtubule associated protein 1 light chain 3 (LC3) protein. A relatively low concentration of artemisinin (80μM) induced autophagosome formation and blocking autophagosome degradation in SK-HEP-1 cells. We also found that treatment with artemisinin at a relatively low concentration, although without a direct inhibitory effect on SK-HEP-1 cell proliferation, increased the sensitivity to IFN-γ in the cells. In this study, to investigate the mechanism behind combination of artemisinin to potentiate the inhibitory effect of IFN-γ on hepatoma cells, we will examine the proteins with dual roles in autophagosome formation and the fusion between autophagosomes and lysosomes in IFN-γ-treated SK-HEP-1 cells with or without artemisinin. We next will focus on the effects of artemisinin-induced autophagy induction or blockage in downstream on the IFN-γ-induced cell death in SK-HEP-1 cells. The protein kinase inhibitors or siRNA should be used to block the core signaling pathway to determine whether the combination of artemisinin enhances IFN-γ-induced growth inhibition through the pathway. The proliferative ability of SK-HEP-1 cells was analyzed by the MTT assay. Expression levels of the core proteins should be quantified by western blot analysis. LC3 turnover should be quantified by western blotting. Autophagy was quantified by GFP-LC3 and mRFP-GFP-LC3 puncta formation assays ,immunoelectron microscopy, immunofluorescence staining and confocal microscopy, Cathepsin B activity assay and western blot analysis. This study suggest that the combination therapy with artemisinin should be a novel therapeutic modality to improve efficacy of IFN-γ-based chemotherapy, and the autophagy modulated by artemisinin may be an effective and attractive strategy to enhance the IFN-γ-induced growth inhibition in hepatoma cells.

青蒿素是众所周知的抗疟药物，也具有良好的抗肿瘤作用。IFN-γ具有免疫调节、抑制肿瘤生长的作用，但由于其不稳定、活性低等缺点，治疗效果有待改善。我们预实验结果显示相对低剂量的青蒿素（80μM）不引起肝癌细胞周期抑制或凋亡，但诱导肝癌细胞自噬体增多、阻断自噬体-溶酶体融合，并具有增强肝癌细胞对IFN-γ的药物敏感性的作用。本研究将进行自噬诱导信号检测，溶酶体蛋白酶表达及活性检测，电子显微镜检测，GFP-LC3斑点检测，mRFP-GFP-LC3斑点计数等各项实验，以判断自噬激活或阻断自噬体-溶酶体融合在青蒿素增强肝癌细胞对IFN-γ药物敏感性中所起的作用，并筛选出关键的信号通路。随后，使用siRNA或抑制剂阻断筛选出的信号通路，进一步确定青蒿素通过自噬增强IFN-γ抑制肝癌细胞增殖的分子机制。本研究旨在揭示抗肿瘤药物与自噬的相互作用机制，将给肿瘤的治疗提供新的思路。

IFN-γ具有抗病毒、免疫调节及抗肿瘤特性。青蒿素是众所周知的抗疟药物，亦具有一定的抗肿瘤作用。本研究通过自噬诱导信号检测，MTT等各项实验明确相对低剂量的青蒿素（80μM）联合IFN-γ，可通过诱导自噬增强肝癌细胞SK-HEP-1对IFN-γ药物敏感性，使用3-MA阻断自噬可明显削弱肝癌细胞对联合用药的药物敏感性。为了进一步确定青蒿素通过自噬增强IFN-γ抑制肝癌细胞增殖的分子机制，本研究通过lncRNA芯片结合生物信息学分析筛选出差异性表达lncRNA LINC00173,经表达验证实验发现青蒿素可抑制lncRNA LINC0017的转录水平。在SK-HEP-1细胞中用siRNA干扰lncRNA LINC00173后，可明显上调自噬关键蛋白LC3B的mRNA表达水平。发现青蒿素可通过抑制lncRNA LINC00173上调LC3B的表达，诱导自噬增强肝癌细胞对IFN-γ的药物敏感性，进一步明确了青蒿素通过自噬增强IFN-γ抑制肝癌细胞增殖的分子机制。