激活磷酸腺苷活化蛋白激酶（AMPK）信号通路在伊曲康唑诱导的抗食管癌活性中的作用和机制研究

The esophageal carcinoma is one of the most malignant human tumors with extremely poor prognosis in China and around the world. Our preliminary results found that low concentrations of itraconazole (Itra, at μg/ml), the anti-fungi drug, induced significant growth inhibition and autophagic cell death in cultured esophageal carcinoma cells, which was associated with AMPK signaling activation, ceramide production, mTOR complex 1 (mTORC1) inhibition as well as EGFR/PDGFR degradation and Hedgehog(Hh)-Gli1 signaling in-activation. We propose that Itra-induced anti-esophageal carcinoma efficiency requires ceramide-dependent AMPK signaling activation. The aim of this proposal is to investigate the anti- esophageal carcinoma effect of Itra both in vivo and in vitro, and to study the underlying mechanisms by focusing on the AMP-activated protein kinase (AMPK) signaling. By using multiple methods, we are set to manipulate ceramide-AMPK signaling to increase Itra's efficiency. Finally, we will test the expression and activation of AMPKα1 in human esophageal carcinoma tissues, and analyze its relationship with tumor staging, patients' prognosis and other clinical outcomes. This study suggests that AMPK signaling might be the key determinant of Itra's response in esophageal carcinoma and probably other cancer cells. The results of this study will provide theoretical and experimental evidence for using Itra as a novel anti-esophageal carcinoma agent.

食管癌是常见恶性肿瘤之一，预实验结果显示抗真菌药物伊曲康唑（Itra）能显著抑制食管癌细胞存活、增殖，并诱导癌细胞自噬。机制方面Itra诱导神经酰胺合成而激活酸腺苷活化蛋白激酶（AMPK），并诱导生长因子受体EGFR/PDGFR降解，同时阻断mTOR和Hh-Gli1信号活化；而AMPK抑制剂及AMPK-shRNA逆转上述作用。我们提出神经酰胺介导的AMPK活化是Itra抗食管癌作用的核心机制。本课题中，申请者将利用分子、细胞生物学及裸鼠荷瘤等方法，体内外观察Itra的抗食管癌作用。重点研究Itra抗癌的分子机制，阐明AMPK信号通路在其中的作用。并调节癌细胞内神经酰胺含量和AMPK活化水平，达到增敏Itra抗癌的目的。最后检测AMPKα1在食管癌组织中的表达及活化水平，并分析其与肿瘤各项临床指标的相关性。旨在明确Itra抗食管癌作用和分子机制，为开发Itra治疗食管癌提供理论和实验依据。

目的:研究抗真菌药物伊曲康唑抗食管癌细胞的活性。我们发现μg/ mL浓度的伊曲康唑能够有效抑制人食管癌细胞（TE-1和Eca-109）的存活和增殖。伊曲康唑激活AMP激活的蛋白激酶（AMPK）信号，从而介导食管癌细胞死亡。药理学AMPK抑制，AMPKα1 shRNA敲低或显性失活突变（T172A）几乎完全消除了伊曲康唑诱导的对食道癌细胞的细胞毒性。相反，AMPK激活剂AICAR或持续活化AMPKα1（T172D）的表达增强伊曲康唑杀伤癌细胞活性。伊曲康唑在食管癌细胞中引起AMPK依赖性自噬细胞死亡，但不引起细胞凋亡。进一步的机制研究表明，伊曲康唑激活AMPK诱导多种受体酪氨酸激酶（RTK即EGFR，PDGFRα和PDGFRβ）溶酶体易位和降解，导致下游Akt抑制和食管癌细胞死亡。在体内，伊曲康唑管饲严重联合免疫缺陷（SCID）小鼠有效抑制Eca-109肿瘤生长，但它对AMPKα1耗尽的Eca-109细胞的肿瘤无效。同时，伊曲康唑给药也抑制了原代人食管癌细胞的异种移植肿瘤的生长。值得注意的是，在伊曲康唑治疗的肿瘤中，还观察到AMPK活化，RTK下调和Akt抑制。总之，我们得出结论，伊曲康唑可能通过激活AMPK信号传导在体外和体内抑制食管癌细胞。伊曲康唑可能作为一种有前景的抗食管癌药物进一步检测。