普萘洛尔通过同时激活自体NK细胞与抑制肿瘤AKT/MAPK信号通路阻滞胃癌发生发展的机制研究

Gastric cancer is one kind of disease correlated closely with the activity of central nervous system. The long-term activation state resulting from irregular life style, strong work stress and overdrinking could lead to a variety of brain neurotransmitter metabolic abnormalities and then bring about chronic gastritis or gastric ulcer which is the key risk factor of gastric carcinogenesis. Researches have showed that the long-term over activation of β- sympathetic adrenaline system would significantly reduce the number and activity of natural killer (NK) cells in human innate immune response, resulting in the occurrence and development of tumor. However, β-adrenergic receptor blockade propranolol could reverse the reduction of NK cell number and inactivation of function. Meanwhile, our preliminary study has shown that propranolol could kill tumor cells through suppressing the phosphorylation of AKT/MAPK pathway. Therefore, we put forward the hypothesis that propranolol could suppress the occurrence and development of gastric cancer through the mechanism of both activating the innate immunity and directly killing tumor cells. This project has planned to systematically elaborate the dual-anticancer molecular mechanism of propranolol in the process of gastric cancer genesis and development from the level of cell, mice and clinical cases using the method of western blot, immunohistochemistry, transcriptome Sequencing, etc. The development of this project would provide new thought and theoretic support for the prevention and clinical treatment of gastric cancer.

胃癌是一种与中枢神经系统活动密切相关的疾病。由于生活不规律，工作压力大，过度饮酒等带来的长期应激状态会引起脑内多种神经递质代谢异常进而引发慢性胃炎，胃溃疡等胃癌发生的重要危险因素。研究表明β-交感肾上腺素系统的长期过度激活将显著降低人体固有免疫反应中自然杀伤细胞（NK）的数量和活性，导致肿瘤发生发展。而β-肾上腺素受体抑制剂普萘洛尔可以逆转由于应激引起的NK细胞数量的减少以及功能的失活。同时，我们的前期研究显示普萘洛尔可以通过抑制AKT/MAPK信号通路的磷酸化杀伤肿瘤细胞。因此我们提出假说，普萘洛尔将可能通过激活机体固有免疫以及直接杀伤肿瘤的双重机制达到阻滞胃癌发生发展的作用。本项目拟运用免疫印迹，免疫组化，转录组测序等方法，从细胞、小鼠和临床病例三个研究层面，系统阐述普萘洛尔在胃癌发生发展过程双重抗癌作用的分子机制。本项目的开展将为胃癌的预防与临床治疗提供新的思路与理论支持。

背景：普萘洛尔通过AKT/MAPK信号通路抑制了黑色素瘤及多种实体肿瘤的发展；同时普萘洛尔可以显著提升免疫细胞浸润肿瘤组织程度。.目的：明确普萘洛尔能否通过激活人体固有免疫反应（靶向CD8+ T细胞）以及抑制肿瘤细胞增殖（靶向肿瘤细胞），阻滞胃肠癌的发展，并探索其机制。.方法：运用MTS检测普萘洛尔处理后胃肠癌细胞活力；运用免疫印迹观察普萘洛尔处理后癌细胞增殖相关蛋白AKT/p-AKT/MEK/p-MEK/ERK/p-ERK等的表达改变。运用免疫组化方法观察普萘洛尔处理后小鼠瘤体组织中增殖相关蛋白的表达改变；使用流式细胞技术观察普萘洛尔处理后小鼠肿瘤组织中免疫细胞数量及功能的影响。通过Case-control设计展开临床研究，在未接受新辅助化疗的胃肠癌患者中随机入组普萘洛尔或安慰剂组，之后通过D2术式切除胃肠癌患者的原发病灶。运用免疫组化方法观察，使用普萘洛尔治疗的患者组织样本中免疫细胞浸润程度以及肿瘤相关增殖蛋白表达的改变。.结果：普萘洛尔可以抑制鼠源性胃/肠癌细胞系（CT26WT/MFC）的细胞活力，呈浓度和时间依赖性。体内研究显示普萘洛尔（200 µg/100µL腹腔注射给药）显著抑制小鼠瘤体增长（P < 0.0001）；普萘洛尔组小鼠肿瘤组织中CD3+CD8+ T细胞显著提高，同时发现肿瘤组织中CD8+ T细胞释放IFN-γ、T-bet表达增加。普萘洛尔可以显著抑制人源性胃、肠癌细胞系AGS、HGC、SW480、HCT116细胞活力，对人正常胃、肠上皮细胞系作用不大；同时普萘洛尔还下调了细胞p-MEK和p-ERK的蛋白表达。前瞻性的临床研究中发现服用普萘洛尔的胃肠癌患者其肿瘤组织中的CD8+ T细胞比例显著增加p-AKT，p-MEK和p-ERK的蛋白表达下调。.结论：普萘洛尔经AKT/MAPK信号通路诱导胃肠癌细胞的凋亡同时激活CD8+ T细胞抑制胃肠癌的发展。