腺苷抗胰腺癌效应中线粒体自噬的作用及分子机制

Discovery of effective anti-pancreatic cancer drug is of great clinical value. Adenosine is a pivotal intra- and extra-cellular signal molecule, whose anti-tumor effect was highly concerned. We first reported that adenosine could induce multiple modes of death like apoptosis, senescence and autophagy, remarkably inhibiting pancreatic cancer cells proliferation, indicating that adenosine harbored an anti-pancreatic cancer potential. Our subsequent studies showed that senescence happened in advance of apoptosis, and autophagy was proved to be mitophagy by electronmicroscopy. Interestingly, adenosine could induce mitochondrial ROS formation, disrupting mitochondrial membrane potential and thus triggering mitophagy. Moreover, autophagy inhibitors could enhance adenosine’s cytotoxicity. Additionally, our previous work found that adenosine could trigger mitochondrial apoptosis in other gastrointestinal tumors, suggesting that mitochondrion was the important target of adenosine’s anti-tumor effect. Therefore, we speculated that adenosine could cause cell senescence by mitochondrial ROS formation, and meanwhile, induce protective mitophagy, thus finally leading to mitochondrial apoptosis via function decompensation. To confirm this ratiocination, we will investigate the role and mechanism of adenosine in mitophagy as well as the function of mitophagy in adenosine-induced cell senescence and apoptosis by various experimental methods, and verify the anti-cancer effect of adenosine alone or combined with autophagy inhibitors in orthotopic pancreatic cancer model. Our expected results will provides promising evidence for adenosine clinical translation as a novel anti-pancreatic drug.

寻找有效抗胰腺癌的新药具有重要临床价值。腺苷作为细胞内外的重要信号分子，其抗肿瘤效应备受关注。申请人首次报道腺苷可诱导凋亡、衰老、自噬等多种死亡方式显著抑制胰腺癌细胞增殖，提示腺苷具有抗胰腺癌潜能。后续研究显示衰老早于凋亡发生，自噬被电镜证实为线粒体自噬。进一步发现腺苷诱导线粒体ROS产生导致线粒体膜电位破坏从而诱发线粒体自噬，且自噬抑制剂能增强腺苷的细胞毒性。结合申请人既往发现腺苷在其它消化道肿瘤中可诱导线粒体凋亡，提示线粒体是腺苷抗肿瘤效应的重要靶标。故推测腺苷通过线粒体ROS产生致细胞衰老的同时诱导保护性线粒体自噬，最终功能失代偿导致线粒体凋亡。为验证该推论，拟应用多种实验手段，明确腺苷诱导线粒体自噬的作用及分子机制，探讨线粒体自噬在腺苷诱导细胞衰老与凋亡中的调控作用，最后在原位胰腺癌模型中明确腺苷单独及与自噬抑制剂联合的抗癌效应。预期成果将为腺苷作为抗胰腺癌药物的转化提供坚实证据。

腺苷作为一种在人体内广泛分布的核苷，是嘌呤能信号途径中的重要分子。本项目主要围绕阐明外源腺苷在体内外的抗癌效应及其分子机制，通过PDX模型和原位荧光模型等两种胰腺癌动物模型多角度多方位验证了腺苷的抗癌效应，同时对其肝脏和肾脏等脏器的毒副作用也进行了系统性评估，结果发现腺苷能显著地抑制体内肿瘤的生长速率并同时有效地提高小鼠的生存率；进一步在两株胰腺癌细胞系里也发现随着腺苷浓度的增加，细胞凋亡信号的激活和细胞衰老关键蛋白p21的逐渐累积，显著抑制了细胞增殖率。通过RNAi技术下调p21，发现胰腺癌细胞衰老表型减少而细胞凋亡比例明显升高，并导致细胞总体增殖速度进一步下降。通过激酶小分子抑制剂文库的筛选，发现p21调控蛋白Akt被抑制后，腺苷诱导细胞凋亡的作用被放大增强。通过药物联合作用解析发现Akt抑制剂GSK690693和腺苷联用存在很强相关性，并在胰腺癌原位模型中证实Akt抑制剂通过增强细胞凋亡进一步提高腺苷对胰腺癌的治疗效果。另一方面，阐明腺苷诱导细胞自噬与细胞凋亡之间的关系，发现自噬抑制剂CQ在腺苷诱导的细胞毒性中的作用，进一步阐明了腺苷诱导的细胞自噬是线粒体自噬，验证了FUNDC1是腺苷诱导线粒体自噬的关键分子，并探究了FUNDC1在线粒体自噬中的作用。本项目首次报道了外源腺苷在体外及体内对胰腺癌的治疗作用，并初步发现Akt-p21信号轴促进胰腺癌的腺苷耐受功能。因此，我们的研究为腺苷及Akt抑制剂GSK690693用于胰腺癌治疗提供了可靠的临床前研究支撑，并为今后改善腺苷抗癌效应找到了潜在分子靶点。