Bu Heqi, Liu Dianlei, Zhang Guolin, Chen Li, Song Zhangfa
Department of Colorectal Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, People's Republic of China.
Department of Coloproctology Surgery, Tongde Hospital of Zhejiang Province, Hangzhou 310012, People's Republic of China.
Onco Targets Ther. 2020 Aug 25;13:8533-8545. doi: 10.2147/OTT.S262022. eCollection 2020.
Oridonin has been demonstrated to exert strong antitumor activities in various types of human cancers. Our previous study established that oridonin induced the apoptosis of and exerted an inhibitory effect on colon cancer cells in vitro and in vivo. However, the mechanisms behind the antitumor effects of oridonin on colorectal cancer are not clearly known. This study explored whether autophagy was involved in antitumorigenesis effects caused by the usage of oridonin in colon cancer and examined whether the AMPK/mTOR/ULK1 signaling pathway was involved in this process.
Cell viability was determined using CCK-8 assay. The distribution of cell apoptosis was evaluated using flow cytometry. RT-PCR and Western blotting analysis were conducted to identify the key target genes and proteins involved in the AMPK/mTOR cascade. AMPK siRNA was used to disturb AMPK expression. A DLD-1 cell orthotopic transplantation tumor model was established to explore the anti-cancer effects in vivo.
Oridonin exhibited a suppressive effect on DLD-1 cells in a concentration- and time-dependent manner. Additionally, in a dose-dependent manner, oridonin induced cell apoptosis via inducing the protein expression levels of cleaved caspase-3, cleaved PARP and stimulated autophagy by increasing protein expression levels of Becin1, LC3-II, decreasing protein expression levels of LC3-I, p62, which were respectively attenuated and elevated by autophagy inhibitor 3-MA. Furthermore, oridonin upregulated the expression level of p-AMPK and downregulated the expression levels of p-mTOR, p-ULK1 in the DLD-1 cells in a dose-dependent manner. Moreover, knockdown of AMPK by a specific siRNA reversed the expression levels of proteins involved in the AMPK/mTOR pathway, autophagy and apoptosis. In addition, outcomes from the in vivo experiments also showed that oridonin treatment significantly repressed tumorigenic growth of DLD-1 cells without any side effects, which was accompanied by the upregulation of p-AMPK, LC3-II, active caspase-3 protein expression levels and the downregulation of p-mTOR and p-ULK1 protein expression levels.
This study demonstrated that oridonin induced apoptosis and autophagy of colon cancer DLD-1 cells via regulating the AMPK/mTOR/ULK1 pathway, which indicated that oridonin may be used as a novel therapeutic intervention for patients with colorectal cancer.
冬凌草甲素已被证明在多种人类癌症中具有强大的抗肿瘤活性。我们之前的研究表明，冬凌草甲素在体外和体内均可诱导结肠癌细胞凋亡并发挥抑制作用。然而，冬凌草甲素对结直肠癌抗肿瘤作用的机制尚不清楚。本研究探讨自噬是否参与冬凌草甲素对结肠癌的抗肿瘤作用，并检测AMPK/mTOR/ULK1信号通路是否参与此过程。
采用CCK-8法检测细胞活力。使用流式细胞术评估细胞凋亡分布。进行RT-PCR和蛋白质印迹分析以鉴定参与AMPK/mTOR级联反应的关键靶基因和蛋白质。使用AMPK siRNA干扰AMPK表达。建立DLD-1细胞原位移植瘤模型以探讨体内抗癌效果。
冬凌草甲素对DLD-1细胞具有浓度和时间依赖性抑制作用。此外，冬凌草甲素以剂量依赖性方式诱导细胞凋亡，通过诱导裂解的caspase-3、裂解的PARP蛋白表达水平升高，并通过增加Becin1、LC3-II蛋白表达水平，降低LC3-I、p62蛋白表达水平来刺激自噬，而自噬抑制剂3-MA分别减弱和增强了这些作用。此外，冬凌草甲素以剂量依赖性方式上调DLD-1细胞中p-AMPK的表达水平，下调p-mTOR、p-ULK1的表达水平。此外，用特异性siRNA敲低AMPK可逆转AMPK/mTOR通路、自噬和凋亡相关蛋白的表达水平。此外，体内实验结果还表明，冬凌草甲素治疗可显著抑制DLD-1细胞的致瘤生长且无任何副作用，同时伴随着p-AMPK、LC3-II、活性caspase-3蛋白表达水平的上调以及p-mTOR和p-ULK1蛋白表达水平的下调。
本研究表明，冬凌草甲素通过调节AMPK/mTOR/ULK1通路诱导结肠癌DLD-1细胞凋亡和自噬，这表明冬凌草甲素可能作为结直肠癌患者的一种新型治疗干预手段。
