Red ginseng polysaccharide exhibits anticancer activity through GPX4 downregulation-induced ferroptosis

Abstract

Context: Red ginseng polysaccharide (RGP) is an active component of the widely used medicinal plant Panax ginseng C. A. Meyer (Araliaceae), which has displayed promising activities against cancer cells. However, the detailed molecular mechanism of RGP in ferroptosis is still unknown.

Objective: This study evaluates the effects of RGP in cancer cells.

Materials and methods: A549 and MDA-MB-231 cells were used. Cell proliferation was measured by CCK-8 assay after being treated with RGP at concentrations of 0, 50, 100, 200, 400, 800 and 1600 μg/mL at 0, 12, 24 and 48 h. Lipid reactive oxygen species (ROS) levels were assessed by C11-BODIPY assay. The control group was treated with PBS.

Results: RGP inhibited human A549 (IC₅₀: 376.2 μg/mL) or MDA-MB-231(IC₅₀: 311.3 μg/mL) proliferation and induced lactate dehydrogenase (LDH) release, promoted ferroptosis and suppressed the expression of GPX4. Moreover, the effects of RGP were enhanced by the ferroptosis inducer erastin, while abolished by ferroptosis inhibitor ferrostatin-1.

Discussion and conclusions: Our study is the first to demonstrate (1) the anticancer activity of RGP in human lung cancer and breast cancer. (2) RGP presented the anti-ferroptosis effects in lung and breast cancer cells via targeting GPX4.

Keywords: Lung cancer; breast cancer; traditional Chinese medicine.

Figure 1.

RGP inhibited cancer cell proliferation in A549 and MDA-MB-231 cells. Lung cancer cell line A549 (A) and breast cancer cell line MDA-MB-231 (B) were incubated with increasing concentrations of RGP for 24 and 48 h. Cell viability was measured using CCK-8 assay and normalized to 0 h using the following equation: OD₄₅₀ nm at 0, 12, 24 and 48 h/average OD₄₅₀ nm at 0 h in 0 µg/mL group. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 2.

RGP treatment dose-dependently resulted in LDH release, ROS accumulation and GPX4 downregulation in A549 and MDA-MB-231 cells. A549 and MDA-MB-231 cells were treated with 0, 100, 200 and 400 μg/mL of RGP and incubated for 48 h. (A, B) Levels of LDH release were measured. (C, D) Lipid ROS accumulation was determined by BODIPYTM 581/591 C11 using a flow cytometer. (E, F) GPX4 expression was examined by Western blotting. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 3.

Ferroptosis induction mediated the underlying effects of RGP treatment. A549 and MDA-MB-231 cells were pre-treated with 200 μg/mL of RGP, and then ferrostatin-1 (2 μM) or erastin (35 μM) was added to cells. (A, B) Cell viability assay, (C, D) LDH release assay and (E, F) C11-BODIPY assay were performed. (G, H) GPX4 expression level was determined. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 4.

GPX4 overexpression abrogated the effects of RGP. GPX4 or empty vector was overexpressed in A549 and MDA-MB-231 cells, and cells were incubated for 48 h, with or without 200 μg/mL of RGP treatment. (A, B) Western blotting, (C, D) cell viability assay and (E, F) LDH release assay were performed. (G, H) Lipid ROS accumulation was determined according to C11-BODIPY signal shift. ***p < 0.001.