ABSTRACT

Alloimperatorin (Alloi) has been shown to have anti-proliferative effects in our previous studies. we aimed to investigate whether Alloimperatorin induces autophagy through the reactive oxygen species (ROS) pathway and anticancer activity in vivo. The anti-proliferative effect of Alloimperatorin was evaluated using a cell counting kit (CCK-8 kit). Apoptosis was detected using flow cytometry. Confocal microscopy, immunofluorescence, and mRFP-GFP-LC3 lentivirus transfection were used to verify autophagy. Electron microscopy detection of autophagosomes was induced by Alloimperatorin. Western blotting was used to detect autophagy proteins in HeLa and SiHa cells. A xenograft model was used to monitor the inhibitory effect of Alloimperatorin on tumor growth in nude mice. The results showed that Alloimperatorin induced ROS production and inhibited the proliferation of HeLa and SiHa cells. Furthermore, Alloimperatorin increased the apoptosis rate in HeLa and SiHa cells. Confocal microscopy fluorescence indicated that Alloimperatorin increased autophagy fluorescence of HeLa and SiHa cells. mRFP-GFP-LC3 lentivirus transfection and electron microscopy demonstrated that Alloimperatorin increased autophagy in HeLa and SiHa cells. Western blotting showed that Alloimperatorin induced the expression of autophagy proteins in HeLa and SiHa cells. However, N-acetylcysteine reversed the autophagy. These results demonstrate that Alloimperatorin can induce autophagy in HeLa and SiHa cells through the ROS pathway. In vivo xenograft experiments showed that Alloimperatorin could inhibit tumor growth in nude mice. Alloimperatorin is expected to be an effective new drug for cervical cancer treatment.

Abbreviations: ROS, reactive oxygen species; Alloi, Alloimperatorin; CCK-8, Cell Counting Kit-8; NAC, N-acetyl-L-cysteine; DCFH-DA, 2,7-dichlorodihydrofluorescein diacetate; OD, optical density; PBS, phosphate buffer solution; BCA, bicinchoninic acid; DAPI, 4,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide.

摘要

在我们之前的研究中，别欧前胡素 (Alloi) 已被证明具有抗增殖作用。我们的目的是研究别欧前胡素是否通过活性氧（ROS）途径诱导自噬以及体内抗癌活性。使用细胞计数试剂盒（CCK-8试剂盒）评估别欧前胡素的抗增殖作用。使用流式细胞术检测细胞凋亡。使用共聚焦显微镜、免疫荧光和mRFP-GFP-LC3慢病毒转染来验证自噬。电子显微镜检测自噬体是由别欧前胡素诱导的。Western blotting用于检测HeLa和SiHa细胞中的自噬蛋白。采用异种移植模型监测别欧前胡素对裸鼠肿瘤生长的抑制作用。结果表明，别欧前胡素诱导ROS产生并抑制HeLa和SiHa细胞的增殖。此外，Alloimperatorin 增加了 HeLa 和 SiHa 细胞的凋亡率。共聚焦显微镜荧光表明 Alloimperatorin 增加了 HeLa 和 SiHa 细胞的自噬荧光。mRFP-GFP-LC3 慢病毒转染和电镜证明 Alloimperatorin 增加 HeLa 和 SiHa 细胞的自噬。蛋白质印迹显示别欧前胡素诱导 HeLa 和 SiHa 细胞中自噬蛋白的表达。然而，N-乙酰半胱氨酸逆转了自噬。这些结果表明 Alloimperatorin 可以通过 ROS 途径诱导 HeLa 和 SiHa 细胞自噬。体内异种移植实验表明Alloimperatorin可以抑制裸鼠肿瘤的生长。别欧前胡素有望成为治疗宫颈癌的有效新药。

缩写： ROS，活性氧；Alloi，Allo欧前胡素；CCK-8，细胞计数试剂盒-8；NAC，N-乙酰基-L-半胱氨酸；DCFH-DA，2,7-二氯二氢荧光素二乙酸酯；OD，光密度；PBS，磷酸盐缓冲溶液；BCA、二辛可宁酸；DAPI，4,6-二脒基-2-苯基吲哚；DMSO，二甲基亚砜。