莱菔硫烷对膀胱癌细胞运动的影响及靶向能量代谢重排的调控作用

Invasion, metastasis and recurrence are the inherent characteristics of urinary bladder cancer. Accumulating evidence showed that sulforaphane (SFN), derived from cruciferous vegetables, inhibited the metastasis of bladder cancer, while its internal mechanisms remain to be elucidated. Tumor migration involves a series of complex processes initiated by the formation of membrane protrusions, accompanied by energy metabolic reprogramming. We previously confirmed that SFN could prevent the development of protrusions. We also demonstrated that SFN inhibited glycolysis, as well as aerobic respiration. Based on these findings, we proposed the hypothesis “ SFN prevented the metastasis of bladder cancer cell via targeting energy metabolic reprogramming.” In this project, we firstly explore the modes of energy metabolism in patients with bladder cancer and its associations with the potential ability of metastasis. Secondly, the effects of SFN on the key components of protrusions and its mechanisms were conducted. And further regulatory mechanisms by SFN including glycolysis, de novo fatty acid(FA) synthesis and mitochondrial respiratory chain were studied. Moreover, at the cellular, live animal, organ and molecular levels, the effects of SFN on the cell migration were revealed via targeting energy metabolic reprogramming. Finally, postoperative intervention trial in bladder cancer patients by oral consumption of SFN tablet will be carried out to verify its validity and regulatory mechanisms by energy metabolism. Our findings will provide the theory basis of SFN on the adjuvant therapy and novel idea against the metastasis of bladder cancer, with great theoretical significance and application value.

浸润、转移、复发是膀胱癌的固有特性。食源性植物化学物莱菔硫烷（SFN）可抑制膀胱癌细胞转移，内在机制有待阐明。肿瘤转移是由肿瘤细胞运动（即伪足形成）引发并伴有能量代谢重排的复杂过程。申请者前期研究证实SFN可抑制膀胱癌细胞伪足形成、糖酵解和有氧呼吸作用。据此提出“SFN靶向能量代谢重排抑制膀胱癌转移”的假设。本项目首先探讨膀胱癌患者能量代谢模式及与肿瘤转移潜力的关系；随后在细胞水平研究SFN对细胞伪足核心成分的作用及机制，并从糖酵解、脂肪酸合成、线粒体呼吸链深入探讨SFN对能量代谢重排的调控；在细胞、动物活体、器官及分子水平揭示靶向能量代谢重排后SFN对肿瘤细胞运动的影响；最后在人群干预试验中观察SFN预防膀胱癌患者转移复发的效果及能量代谢调控机制。本项目的完成将为食源性植物化学物SFN应用于辅助抗膀胱癌转移提供理论依据，为防治膀胱癌转移复发提供新思路，具有重要的理论意义和应用价值。

膀胱癌是最常见的泌尿系统恶性肿瘤，侵袭和转移是膀胱癌致死的首要原因。通过重新编排能量代谢模式来满足肿瘤细胞快速增殖和转移所需的能量，已被认为是癌症的新特征之一。而膀胱癌细胞的能量代谢模式及特征目前尚未明确。莱菔硫烷(Sulforaphane, SFN)存在于多种十字花科蔬菜，是迄今研究最广泛的一种具有抗癌活性的异硫氰酸酯类。SFN对肿瘤转移的研究仅限于胞外基质降解等传统信号调控。本研究在分析了膀胱癌患者能量代谢特征的基础上，系统阐明了SFN通过靶向“能量代谢重编程”抑制膀胱癌转移的防控策略。主要结果如下：收集不同病理分期膀胱癌患者的肿瘤及癌旁组织，通过GC-MS检测发现肿瘤组织中ATP及多种糖代谢产物均高表达，且具有GLUT非依赖性Warburg效应。采用qPCR检测发现，膀胱肿瘤组织中多种糖代谢酶PFK-1，LDHA及PDH的基因表达异常升高。血清酶活检测发现患者体内HK2，LDHA，PDH和PKM2的活性均异常升高；且PKM2与TNM分期呈正相关，PDH呈负相关。随后，从糖酵解和线粒体氧化磷酸化角度，发现SFN强烈下调胞内ATP水平。SFN显著抑制浸润性膀胱癌细胞内HK2，PDH和LDHA的蛋白表达。瞬时转染AKT1过表达质粒可强烈逆转SFN对T24细胞中HK2的抑制，提示SFN对膀胱癌细胞中HK2的调控是通过AKT1起作用的。在BBN诱导的小鼠膀胱癌模型中进一步验证了离体细胞实验结果。此外，SFN还可通过减少T24细胞线粒体数量，并抑制线粒体复合物I，II，III和V的活性来调控膀胱癌细胞线粒体有氧氧化。最后，通过透射和扫描电镜观察发现SFN破坏膀胱癌细胞形态；采用鬼笔环肽标记F-actin，激光共聚焦显微镜观察发现SFN抑制细胞伪足形成。当AKT1过表达后，SFN对伪足形成调控因子Arp2的抑制削弱，且对伪足破坏作用消失，表明SFN可通过AKT1调控细胞代谢供能，抑制Arp2表达，阻断膀胱癌细胞伪足形成。此外，SFN显著抑制p-cortactin的蛋白表达，揭示调控cortactin的活化和表达是SFN破坏膀胱癌细胞伪足形成的另一主要方式。最后，在裸鼠体内通过尾静脉注射造膀胱癌细胞肺转移模型，在体内验证了SFN的调控机制；延缓并抑制膀胱肿瘤在肺部的形成及转移。