Abstract：
BACKGROUND: Autophagy could sense metabolic conditions and safeguard cells against nutrient deprivation, ultimately supporting the survival of cancer cells. Nobiletin (NOB) is a kind of bioactive component of the traditional Chinese medicine Citri Reticulatae Pericarpium and has been proven to induce GC cell death by reducing de novo fatty acid synthesis in our previous study. Nevertheless, the precise mechanisms by which NOB induces cell death in GC cells still need further elucidation.
OBJECTIVE: To examine the mechanism by which NOB inhibits gastric cancer progression through the regulation of autophagy under the condition of lipid metabolism inhibition.
UNASSIGNED: Proliferation was detected by the CCK-8 assay. RNA sequencing (RNA-seq) was used to examine signaling pathway changes. Electron microscopy and mRFP-GFP-LC3 lentiviral transfection were performed to observe autophagy in vitro. Western blot, plasmid transfection, immunofluorescence staining, and CUT & Tag-qPCR techniques were utilized to explore the mechanisms by which NOB affects GC cells. Molecular docking and molecular dynamics simulations were conducted to predict the binding mode of NOB and SREBP1. CETSA was adopted to verify the predicted of binding model. A patient-derived xenograft (PDX) model was employed to verify the therapeutic efficacy of NOB in vivo.
RESULTS: We conducted functional studies and discovered that NOB inhibited the protective effect of autophagy via the PI3K/Akt/mTOR axis in GC cells. Based on previous research, we found that the overexpression of ACLY abrogated the NOB-induced autophagy-dependent cell death. In silico analysis predicted the formation of a stable complex between NOB and SREBP1. In vitro assays confirmed that NOB treatment increased the thermal stability of SREBP1 at the same temperature conditions. Moreover, CUT&TAG-qPCR analysis revealed that NOB could inhibit SREBP1 binding to the ACLY promoter. In the PDX model, NOB suppressed tumor growth, causing SREBP1 nuclear translocation inhibition, PI3K/Akt/mTOR inactivation, and autophagy-dependent cell death.
CONCLUSIONS: NOB demonstrated the ability to directly bind to SREBP1, inhibiting its nuclear translocation and binding to the ACLY promoter, thereby inducing autophagy-dependent cell death via PI3K/Akt/mTOR pathway.
OBJECTIVE: To examine the mechanism by which NOB inhibits gastric cancer progression through the regulation of autophagy under the condition of lipid metabolism inhibition.
UNASSIGNED: Proliferation was detected by the CCK-8 assay. RNA sequencing (RNA-seq) was used to examine signaling pathway changes. Electron microscopy and mRFP-GFP-LC3 lentiviral transfection were performed to observe autophagy in vitro. Western blot, plasmid transfection, immunofluorescence staining, and CUT & Tag-qPCR techniques were utilized to explore the mechanisms by which NOB affects GC cells. Molecular docking and molecular dynamics simulations were conducted to predict the binding mode of NOB and SREBP1. CETSA was adopted to verify the predicted of binding model. A patient-derived xenograft (PDX) model was employed to verify the therapeutic efficacy of NOB in vivo.
RESULTS: We conducted functional studies and discovered that NOB inhibited the protective effect of autophagy via the PI3K/Akt/mTOR axis in GC cells. Based on previous research, we found that the overexpression of ACLY abrogated the NOB-induced autophagy-dependent cell death. In silico analysis predicted the formation of a stable complex between NOB and SREBP1. In vitro assays confirmed that NOB treatment increased the thermal stability of SREBP1 at the same temperature conditions. Moreover, CUT&TAG-qPCR analysis revealed that NOB could inhibit SREBP1 binding to the ACLY promoter. In the PDX model, NOB suppressed tumor growth, causing SREBP1 nuclear translocation inhibition, PI3K/Akt/mTOR inactivation, and autophagy-dependent cell death.
CONCLUSIONS: NOB demonstrated the ability to directly bind to SREBP1, inhibiting its nuclear translocation and binding to the ACLY promoter, thereby inducing autophagy-dependent cell death via PI3K/Akt/mTOR pathway.
摘要：
背景：自噬可以感知代谢状况，保护细胞免受营养剥夺，最终支持癌细胞的生存。Nobiletin（NOB）是中药陈皮的一种生物活性成分，在我们之前的研究中已被证明可以通过减少从头脂肪酸的合成来诱导GC细胞死亡。然而，NOB诱导GC细胞死亡的确切机制仍需进一步阐明.
目的：探讨在脂质代谢抑制条件下，NOB通过调节自噬抑制胃癌进展的机制。
■通过CCK-8测定检测增殖。RNA测序(RNA-seq)用于检查信号传导途径的变化。电镜观察和mRFP-GFP-LC3慢病毒转染在体外观察自噬。蛋白质印迹,质粒转染，免疫荧光染色，利用CUT和Tag-qPCR技术探索NOB影响GC细胞的机制。通过分子对接和分子动力学模拟来预测NOB与SREBP1的结合模式。采用CETSA对预测的结合模型进行验证。采用患者来源的异种移植物(PDX)模型来验证NOB在体内的治疗功效。
结果：我们进行了功能研究，发现NOB通过PI3K/Akt/mTOR轴抑制GC细胞自噬的保护作用。在前人研究的基础上,我们发现ACLY的过表达消除了NOB诱导的自噬依赖性细胞死亡。计算机模拟分析预测了NOB和SREBP1之间稳定复合物的形成。体外测定证实NOB处理在相同温度条件下增加SREBP1的热稳定性。此外，CUT&TAG-qPCR分析揭示NOB可以抑制SREBP1与ACLY启动子的结合。在PDX模型中，NOB抑制肿瘤生长，引起SREBP1核易位抑制，PI3K/Akt/mTOR失活,和自噬依赖性细胞死亡。
结论：NOB表现出直接结合SREBP1的能力，抑制其核易位并结合ACLY启动子，从而通过PI3K/Akt/mTOR途径诱导自噬依赖性细胞死亡。
目的：探讨在脂质代谢抑制条件下，NOB通过调节自噬抑制胃癌进展的机制。
■通过CCK-8测定检测增殖。RNA测序(RNA-seq)用于检查信号传导途径的变化。电镜观察和mRFP-GFP-LC3慢病毒转染在体外观察自噬。蛋白质印迹,质粒转染，免疫荧光染色，利用CUT和Tag-qPCR技术探索NOB影响GC细胞的机制。通过分子对接和分子动力学模拟来预测NOB与SREBP1的结合模式。采用CETSA对预测的结合模型进行验证。采用患者来源的异种移植物(PDX)模型来验证NOB在体内的治疗功效。
结果：我们进行了功能研究，发现NOB通过PI3K/Akt/mTOR轴抑制GC细胞自噬的保护作用。在前人研究的基础上,我们发现ACLY的过表达消除了NOB诱导的自噬依赖性细胞死亡。计算机模拟分析预测了NOB和SREBP1之间稳定复合物的形成。体外测定证实NOB处理在相同温度条件下增加SREBP1的热稳定性。此外，CUT&TAG-qPCR分析揭示NOB可以抑制SREBP1与ACLY启动子的结合。在PDX模型中，NOB抑制肿瘤生长，引起SREBP1核易位抑制，PI3K/Akt/mTOR失活,和自噬依赖性细胞死亡。
结论：NOB表现出直接结合SREBP1的能力，抑制其核易位并结合ACLY启动子，从而通过PI3K/Akt/mTOR途径诱导自噬依赖性细胞死亡。
