自噬是一种基本的循环途径，可增强细胞的适应性，促进细胞存活。然而，这种生存机制可能阻碍抗癌治疗策略，因为这些策略旨在诱导细胞死亡。在本研究中，我们从中药青藤中分离出一种新型的自噬抑制剂-方肌碱（Fan）。我们推测，当Fan阻断自噬时，癌细胞在治疗过程中失去了大量的自我保护能力。首先，我们详细研究了Fan抑制自噬的机制。具体而言，Fan通过GFP-LC3标记导致自噬体显著增加，LC3-II表达升高进一步证实了这一点。自噬受体蛋白p62也上调，提示可能存在自噬流的抑制。我们进一步排除了溶酶体和自噬体之间的融合障碍的可能性，通过双重荧光染色法证实它们的共定位存在。然而，溶酶体的酸性环境可能受损，这由溶酶体内酸敏染料荧光减弱和成熟的溶酶体卡他普汀数量减少所提示。为了测试Fan的抗癌潜力，我们将其与顺铂（Cis）或紫杉醇（PTX）联合应用于肺癌细胞治疗中。这种联合治疗表现出协同增强的杀伤效应。这些有希望的抗肿瘤结果也在移植瘤模型中得到了复制。本研究的意义在于确定Fan作为一种有效的自噬抑制剂，以及它增强现有抗癌药物疗效的潜力。通过揭示Fan对自噬的作用机制，并在联合治疗中展现出协同效应，我们的研究为克服癌症治疗中的自噬介导耐药提供了宝贵的见解。版权所有 © 2023。由Elsevier Inc.出版。

Autophagy is a fundamental recycling pathway that enhances cellular resilience, promoting survival. However, this survival mechanism can impede anti-cancer treatment strategies designed to induce cell death. In this study, we identified a novel autophagy inhibitor, Fangchinoline (Fan) isolated from the traditional Chinese medicine Stephania tetrandra. We speculated that when Fan blocks autophagy, cancer cells lose substantial self-preservation abilities during treatment. Firstly, we examined in detail the mechanism through which Fan inhibits autophagy. Specifically, Fan induced a significant increase in autophagosomes, as indicated by GFP-LC3 labeling, confirmed by the up-regulation of LC3-II. The autophagy receptor protein p62 was also up-regulated, suggesting a potential inhibition of autophagy flux. We further ruled out the possibility of fusion barriers between lysosomes and autophagosomes, as confirmed by their co-localization in double fluorescence staining. However, the lysosomal acid environment might be compromised, as suggested by the diminished fluorescence of acidity-sensitive dyes in the lysosomes and the corresponding decrease in mature forms of lysosomal cathepsin. To test the anti-cancer potential of Fan, we combined it with Cisplatin (Cis) or Paclitaxel (PTX) for lung cancer cell treatment. This combined treatment demonstrated a synergistically enhanced killing effect. These promising anti-tumor results were also replicated in a xenografted tumor model. The significance of this research lies in the identification of Fan as a potent autophagy inhibitor and its potential to enhance the efficacy of existing anti-cancer drugs. By unraveling the mechanisms of Fan's action on autophagy and demonstrating its synergistic effect in combination therapies, our study provides valuable insights for developing novel strategies to overcome autophagy-mediated resistance in cancer treatment.Copyright © 2023. Published by Elsevier Inc.