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

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.

GRAPHICAL ABSTRACT

KEYWORDS:

• Alloimperatorin
• cervical cancer cells
• autophagy
• reactive oxygen species

Ethical statement

All animal tumor model protocols were approved by the Animal Care and Use Committee of the First Hospital of Lanzhou University (approval).

Availability of data and materials

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Authors’ contributions

Conceptualization: Yongxiu Yang and Yingying Bai

Data curation: Lijuan Yang and Yuemei Cheng

Methodology: Yuemei Chen and Wenhua Wang

Writing-original draft: Yingying Bai

Writing-review editing: Yongxiu yang and Yingying Bai

Project: Yongxiu yangAll authors reviewed the manuscript

Acknowledgements

We thank Professor Yong Xiu Yang for editoing the manuscript, Taylor & Francis Group for grammar and sentence modification and polishing.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by grants from the Construction of the Gansu Provincial Administration of Traditional Chinese Medicine [GZKG-2020-46].