Abstract：
OBJECTIVE: Nitidine chloride (NC), a natural phytochemical alkaloid derived from Zanthoxylum nitidum (Roxb.) DC, exhibits multiple bioactivities, including antitumor, anti-inflammatory, and other therapeutic effects. However, the primary targets of NC and the mechanism of action (MOA) have not been explicitly defined.
METHODS: We explored the effects of NC on mTORC1 signaling by immunoblotting and fluorescence microscopy in wild-type and gene knockout cell lines generated by the CRISPR/Cas9 gene editing technique. We identified IGF2R as a direct target of NC via the drug affinity-responsive target stability (DARTS) method. We investigated the antitumor effects of NC using a mouse melanoma B16 tumor xenograft model.
RESULTS: NC inhibits mTORC1 activity by targeting amino acid-sensing signaling through activating transcription factor 4 (ATF4)-mediated Sestrin2 induction. NC directly binds to IGF2R and promotes its lysosomal degradation. Moreover, NC displayed potent cytotoxicity against various cancer cells and inhibited B16 tumor xenografts.
CONCLUSIONS: NC inhibits mTORC1 signaling through nutrient sensing and directly targets IGF2R for lysosomal degradation, providing mechanistic insights into the MOA of NC.
METHODS: We explored the effects of NC on mTORC1 signaling by immunoblotting and fluorescence microscopy in wild-type and gene knockout cell lines generated by the CRISPR/Cas9 gene editing technique. We identified IGF2R as a direct target of NC via the drug affinity-responsive target stability (DARTS) method. We investigated the antitumor effects of NC using a mouse melanoma B16 tumor xenograft model.
RESULTS: NC inhibits mTORC1 activity by targeting amino acid-sensing signaling through activating transcription factor 4 (ATF4)-mediated Sestrin2 induction. NC directly binds to IGF2R and promotes its lysosomal degradation. Moreover, NC displayed potent cytotoxicity against various cancer cells and inhibited B16 tumor xenografts.
CONCLUSIONS: NC inhibits mTORC1 signaling through nutrient sensing and directly targets IGF2R for lysosomal degradation, providing mechanistic insights into the MOA of NC.
摘要：
目标：氯化尼替丁（NC），一种天然的植物化学生物碱，来自两面针（Roxb。)DC,表现出多种生物活性，包括抗肿瘤,抗炎,和其他治疗效果。然而,尚未明确定义NC的主要目标和作用机制（MOA）。
方法：我们通过免疫印迹和荧光显微镜在CRISPR/Cas9基因编辑技术产生的野生型和基因敲除细胞系中探索了NC对mTORC1信号传导的影响。我们通过药物亲和响应靶标稳定性（DARTS）方法将IGF2R鉴定为NC的直接靶标。我们使用小鼠黑色素瘤B16肿瘤异种移植模型研究了NC的抗肿瘤作用。
结果：NC通过激活转录因子4（ATF4）介导的Sestrin2诱导靶向氨基酸感应信号来抑制mTORC1活性。NC直接结合IGF2R并促进其溶酶体降解。此外，NC对各种癌细胞表现出有效的细胞毒性并抑制B16肿瘤异种移植物。
结论：NC通过营养感知抑制mTORC1信号传导，并直接靶向IGF2R进行溶酶体降解，提供对NCMOA的机械学见解。
方法：我们通过免疫印迹和荧光显微镜在CRISPR/Cas9基因编辑技术产生的野生型和基因敲除细胞系中探索了NC对mTORC1信号传导的影响。我们通过药物亲和响应靶标稳定性（DARTS）方法将IGF2R鉴定为NC的直接靶标。我们使用小鼠黑色素瘤B16肿瘤异种移植模型研究了NC的抗肿瘤作用。
结果：NC通过激活转录因子4（ATF4）介导的Sestrin2诱导靶向氨基酸感应信号来抑制mTORC1活性。NC直接结合IGF2R并促进其溶酶体降解。此外，NC对各种癌细胞表现出有效的细胞毒性并抑制B16肿瘤异种移植物。
结论：NC通过营养感知抑制mTORC1信号传导，并直接靶向IGF2R进行溶酶体降解，提供对NCMOA的机械学见解。
