硝唑尼特（NTZ）通过抑制自噬-溶酶体通路增强膀胱癌对吉西他滨敏感性的分子机制研究

Gemcitabine is widely used in perfusion chemotherapy for bladder cancer, while development of tumor resistance has already critically limited its efficacy. Multiple evidence suggests that autophagy may be the key regulator in gemcitabine resistance. mTORC1-TFEB pathway is one of the important signaling pathways regulating autophagy and lysosomal function. Therefore, autophagy inhibition through regulating this pathway will be helpful for alleviating gemcitabine resistance. In the preliminary study of this project, we found that Nitazoxanide (NTZ), an FDA-approved antiprotozoal drug, might enhance the cytotoxic effect of gemcitabine by suppressing gemcitabine-induced autophagy. Further studies indicated that NTZ impaired the degradation activity of lysosomes and mTORC1-TFEB pathway, indicating that this pathway may associate with NTZ-mediated autophagy inhibition in bladder cancer. In this project, we will: 1) Further confirm the inhibitory effect of NTZ on gemcitabine-induced autophagy at the molecular and cellular levels; 2) Clarify the key role and regulatory mechanism of mTORC1-TFEB pathway in NTZ-mediated autophagy impairment through inhibiting lysosome activity. 3) Evaluate whether NTZ could enhance the efficacy of gemcitabine against bladder cancer in vivo. Our research will provide an experimental and theoretical basis for combination of NTZ with gemcitabine in clinics for bladder cancer treatment.

吉西他滨广泛用于膀胱癌灌注化疗，但继发性耐药严重影响其治疗效果。研究表明，吉西他滨诱导的自噬能引发耐药。mTORC1-TFEB通路是调控自噬和溶酶体功能的重要信号通路之一，通过调控该通路抑制自噬将有助于缓解吉西他滨耐药。前期研究发现，一种抗原虫药硝唑尼特可能抑制了吉西他滨诱导的自噬过程，从而增强吉西他滨对膀胱癌细胞的杀伤作用；初步机理研究表明NTZ抑制了溶酶体的降解能力并参与调控mTORC1-TFEB通路，提示该通路可能与NTZ抑制膀胱癌细胞自噬有关。基于上述研究本项目拟：1）在分子和细胞水平进一步明确NTZ对吉西他滨诱导的自噬的抑制作用；2）阐明mTORC1-TFEB通路在NTZ抑制溶酶体活性进而导致自噬损伤过程中的关键作用及调控机制；3）在动物水平评价NTZ能否增强吉西他滨对膀胱癌的治疗效果。本研究为临床上将NTZ联合吉西他滨用于膀胱癌灌注化疗以增强其疗效提供了实验与理论依据。

膀胱癌是泌尿系统最常见恶性肿瘤之一，易复发和转移导致膀胱癌患者预后较差，迫切需要寻求有效治疗方法。硝唑尼特（Nitazoxanide，NTZ）是美国FDA批准的抗原生虫药，因其良好的安全性和抗肿瘤潜能受到关注，但其在膀胱癌中的作用和潜在机制尚不清楚。RNA-seq 结合细胞学实验发现NTZ引起细胞线粒体损伤及线粒体自噬启动，该过程不依赖经典的自噬调控蛋白Atg5/Beclin1，而由PINK1/pS65-Ub/receptor通路介导。同时NTZ抑制溶酶体降解活性，导致线粒体自噬流受损并加重受损线粒体累积。进一步研究发现，受损线粒体产生的大量ROS可同时调节上述两个过程，因为NAC清除ROS可有效抑制PINK1信号通路介导的自噬启动，并减轻溶酶体功能损伤。更重要的是NTZ联合自噬晚期抑制剂氯喹（Chloroquine,CQ）加重细胞自噬流损伤增强了NTZ诱导的细胞凋亡，而联合ROS清除剂减轻细胞自噬流损伤及细胞毒性，说明NTZ诱导的线粒体和线粒体自噬流损害增强了细胞毒性。同样NTZ在三维膀胱肿瘤球中也显示出类似的线粒体自噬应答信号。最后，体内研究表明口服NTZ能够显著抑制小鼠原位膀胱肿瘤的生长，并且没有明显的全身毒性。我们的研究揭示了NTZ在不同阶段参与ROS介导的线粒体自噬调节，并证明了它是一种潜在的抗膀胱肿瘤活性的候选药物。