In vivo and in vitro studies of Alloimperatorin induced autophagy in cervical cancer cells via reactive oxygen species pathway

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.