背景:非小细胞肺癌(NSCLC)占肺癌的85%,成为全球所有癌症中死亡率最高的癌症。在NSCLC中阻断自噬代表了抑制血管生成和克服耐药性的有希望的治疗策略。抗肿瘤佐剂中的天然成分越来越多地被报道以更少的副作用和增加化疗药物敏感性的潜力促进细胞死亡。黄芩苷,黄芩提取物黄酮苷,据报道可以诱导NSCLC细胞死亡,然而,其对NSCLC细胞自噬的影响尚不清楚.
目的:本研究旨在探讨黄芩苷对NSCLC细胞自噬通量的影响,揭示其潜在的作用机制,包括潜在的靶点及其在NSCLC细胞死亡中的作用。
方法:黄芩苷的体外抗癌作用通过增殖评价来验证,克隆形成,细胞周期,和三种NSCLC细胞系(A549、H1299和PC-9)中的细胞迁移。在裸鼠皮下移植瘤模型中评价黄芩苷的体内抗肿瘤效果。NSCLC细胞的自噬特征包括通过蛋白质印迹和免疫荧光染色检测自噬标志物,通过TEM观察亚细胞结构,通过RNA-seq和荧光染色的溶酶体功能(LysoTracker®,LysoSensor®,和吖啶橙)。基于RNA-seq和使用凋亡的分子生物学验证,自噬,和溶酶体抑制剂,通过Ca2+通量测定验证了黄芩苷的潜在靶分子,MCLN3被shRNA敲除,和虚拟分子对接。
结果:黄芩苷抑制NSCLC细胞增殖和迁移,抑制体内肿瘤生长。黄芩苷通过激活溶酶体膜阳离子通道MCLN3阻断自噬通量,破坏其Ca2+平衡并诱导溶酶体功能障碍,导致自溶体降解失败。细胞质Ca2+失衡进一步导致NSCLC细胞线粒体膜电位去极化和ROS积累,介导自噬相关凋亡。
结论:这项研究表明,黄芩苷通过激活MCLN3抑制自溶酶体降解,导致溶酶体pH升高功能障碍,从而抑制NSCLC中的自噬,导致NSCLC细胞凋亡。这些发现丰富了现有的基于自噬抑制的癌症治疗理论和黄酮类化合物作为抗肿瘤药物的潜在机制。为其今后的临床应用铺平了道路。
BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 85 % of lung cancer, becoming the most mortality of all cancers globally. Blockage of autophagy in NSCLC represents a promising therapeutic strategy that inhibits angiogenesis and overcomes drug resistance. Natural ingredients in anti-tumor adjuvants are increasingly reported to promote cell death with less side effects and the potential to increase chemotherapeutic drugs sensitivity. Baicalin, a Scutellaria baicalensis-extracted flavonoid glycoside, is reported to induce death of NSCLC cells, however, its effects on autophagy in NSCLC cells remain unclear.
OBJECTIVE: This study aimed to investigate the effect of baicalin on autophagic flux in NSCLC cells, unraveling the underlying mechanism including potential target and its role in cell death of NSCLC cells.
METHODS: In vitro anti-cancer effects of baicalin were verified by evaluating proliferation, clone formation, cell cycle, and cell migration in three NSCLC cell lines (A549, H1299, and PC-9). In vivo anti-tumor efficacies of baicalin were evaluated in subcutaneous xenograft tumor model in nude mice. Autophagy characterization in NSCLC cells included autophagic marker detection by western blot and immunofluorescence staining, subcellular structure observation by TEM, lysosomal function by RNA-seq and fluorescence staining (LysoTracker®, LysoSensor®, and acridine orange). Based on RNA-seq and molecular biological verification using apoptotic, autophagic, and lysosomal inhibitors, potential target molecule of baicalin was verified via Ca2+ flux assay, MCOLN3 knockdown by shRNA, and virtual molecular docking.
RESULTS: Baicalin inhibited NSCLC cell proliferation and migration, and suppressed tumor growth in vivo. Baicalin blocked the autophagic flux via activating the membranal cation channel MCOLN3 of lysosome, which disrupted its Ca2+ balance and induced lysosome dysfunction, leading to failure of autolysosome degradation. The cytoplasmic Ca2+ imbalance further resulted in depolarization of mitochondrial membrane potentials and ROS accumulation in NSCLC cells, mediating autophagy-related apoptosis.
CONCLUSIONS: This study demonstrated that baicalin inhibited autolysosome degradation by activating MCOLN3, leading to dysfunction in lysosomal pH elevation, thereby inhibiting autophagy in NSCLC, leading to apoptotic death of NSCLC cells. These findings enriched the existing theories of cancer therapy based on autophagy inhibition and underlying mechanisms of flavonoids as antitumor agents, paving the way for their clinical application in future.