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

Van Hung Hoang , Thi Thanh Huong Le , Phu Hung Nguyen , Viet Hoang , Van Khang Pham , Thi Kieu Oanh Nguyen , Dinh Quang Hung Can
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Abstract

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.
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石菖蒲提取物在 HepG2 肝癌细胞中降低癌症干细胞特性并刺激 ROS 信号通路
背景石菖蒲在中国被称为 "石长普",用于传统中药已有两千多年的历史。根据《神农本草经》的记载,石菖蒲属的许多物种都可用于治疗人类的各种疾病,如癫痫、心血管问题、腹痛、记忆力减退等。石菖蒲属的多个品种已被证实对多种癌细胞株具有抗增殖作用。本研究旨在评估石菖蒲提取物(AGE)对 HepG2 肝癌细胞的抑制作用。迁移采用伤口愈合技术进行分析。采用三维培养技术评估肿瘤球的形成和癌细胞的侵袭潜力。实时 PCR 用于 mRNA 表达分析。采用荧光染色技术分析 CD44 蛋白和 ROS 表达水平的变化。结果表明,AGE 对细胞增殖有很强的抑制作用,其 IC50 值为 34.1 mg/mL。AGE 能明显抑制三维培养物中肿瘤球的形成和生长,减少肿瘤球的数量和大小。此外,AGE 还能抑制癌细胞的侵袭。AGE 可抑制干细胞相关标记物的下调,包括 NANOG、SOX2、OCT4 和 ALDH。研究还表明,AGE通过激活参与ROS信号通路的基因,增加了细胞内活性氧(ROS)的产生,这可能会导致细胞增殖、迁移和侵袭的减少,以及癌症干细胞特性的降低。最后,通过UPLC-QtoF-MS分析鉴定了AGE中的58种化合物,并评估了Mulberrofuran K、Akebonoic acid和Shancilin与肝癌干细胞相关蛋白质标记物的亲和性。
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