Acorus gramineus extract decreases cancer stem cell properties and stimulates ROS signaling pathway in HepG2 hepatocellular carcinoma cells

Abstract

ABTRACT: Background: Acorus gramineus is called “Shi Chang Pu'” in Chinese and has been used in traditional Chinese medicine (TCM) for over 2,000 years. The records in the 'Shennong Bencao Jing' show that many species in the genus Acorus are used to treat various diseases in humans such as epilepsy, cardiovascular issues, abdominal pain, memory loss, etc. Several species of the genus Acorus have been demonstrated to exhibit antiproliferative effects against various cancer cell lines. This study aims to evaluate the inhitory effects of Acorus gramineus extract (AGE) on HepG2 liver cancer cells. Methods: The inhibitory effect of AGE on HepG2 cell proliferation was assessed using the MTT assay. Migration was analyzed using the wound healing technique. The 3D culture technique was employed to evaluate tumorsphere formation and the invasive potential of cancer cells. Real-time PCR was used for mRNA expression analysis. Changes in the expression levels of CD44 protein and ROS were analyzed using fluorescence staining techniques. Chemical compounds of AGE were identified using UPLC-QToF-MS. Results: The results indicate that AGE exhibits potent inhibitory activity against cell proliferation, with an IC50 value of 34.1 mg/mL. AGE significantly inhibited both the formation and growth of tumorspheres in 3D cultures, reducing both their number and size. Additionally, AGE was found to suppress the invassion of cancer cells. AGE regulated the downregulation of stem cell-related markers including NANOG, SOX2, OCT4, and ALDH. AGE was also shown to increase the production of reactive oxygen species (ROS) within cells by activating genes involved in ROS signaling pathways, which may lead to a decrease in cell proliferation, migration, and invasion, as well as a reduction in cancer stem cell properties. Finally, 58 compounds present in AGE were identified by UPLC-QtoF-MS analysis, and Mulberrofuran K, Akebonoic acid, and Shancilin were evaluated for their affinity with protein markers related to liver cancer stem cells. Conclusion: This study is the first report on the inhibitory activity of AGE against cancer stem cell characteristics in HepG2 cells, highlighting the potential of this plant species as an anti-liver cancer agent.

Keywords

• Liver cancer cells
• ROS signaling
• Cancer stem cells
• CD44 marker
• tumorsphere