Britannin Induces Autophagy-Dependent Apoptosis via the Reactive Oxygen Species/Adenosine 5′-monophosphate-Activated Protein Kinase-unc-51-Like Autophagy Activating Kinase 1 Axis in Cervical Cancer as Detected Using Nanomagnetic Beads

Abstract

Cervical cancer is a unique and common malignancy that occurs in women. Britannin has been proven to inhibit the progression of human liver and breast cancers. However, its efficacy in cervical cancer is still unclear. Human cervical squamous cell carcinoma SiHa cells were used to analyze the effect of britannin administration on cervical tumors by xenotransplantation of human tumor cells. The Cell Counting Kit-8 was used to determine the half maximal inhibitory concentration (IC50) of britannin on SiHa, and the cell invasion ability was measured using Transwell invasion assay. Cell migration was measured using the scratch-healing assay, while the clonogenic assay was performed to determine cell proliferation. Dichloro-dihydro-fluorescein diacetate was used to label reactive oxygen species (ROS) in cells or tissues. Intracellular autophages were stained with monodansylcadaverine. Western blotting assisted by nanomagnetic beads was performed to study the expression of p62, light chain 3 beta (LC3B), Beclin 1, and autophagy-related protein 5 (ATG5) in cells or tissues, as well as the phosphorylation of AMP-activated protein kinase (AMPK) and unc-51-like autophagy activating kinase 1 (ULK1). Hematoxylin and eosin staining was performed to analyze the pathological changes in the tumor cells. The expression of Ki-67, B-cell lymphoma 2 (Bcl-2), and Bax in the tumor cells was analyzed by immunohistochemistry. The IC50 obtained for britannin against SiHa was 10.01 μM. Britannin inhibited the proliferation, invasion, and migration of SiHa cells, and promoted the generation and autophagy of ROS in tumor cells and tissues. Furthermore, p-AMPK/AMPK and p-ULK1/ULK1, LC3B, Beclin 1, and ATG5 were upregulated, whereas p62 was downregulated in cells and tissues. Tumor development was inhibited; tissue inflammation was reduced; Ki-67 and Bcl-2 expression was downregulated; and Bax expression was upregulated. The expression of cleaved caspase-3, cleaved poly-ADP-ribose polymerases, and voltage-dependent anion-selective channel 1 was upregulated. These effects can be partially reversed by the AMPK inhibitor dorsomorphin dihydrochloride (BML-275). In vivo and in vitro studies showed that britannin upregulated the of ROS content of human cervical squamous cell carcinoma cells, leading to significant autophagy, thereby inhibiting the occurrence and development of cervical squamous cell carcinoma. The mechanism may be related to the ROS/AMPK/ULK1 signaling pathway.