Sanguinarine induces ferroptosis of colorectal cancer cells by upregulating STUB1 and downregulating GPX4

doi:10.12122/j.issn.1673-4254.2024.08.12

Abstract

Abstract:

Objective To investigate the effect of sanguinarine (SAN) on proliferation and ferroptosis of colorectal cancer cells. Methods SW620 and HCT-116 cells treated with different concentrations of SAN were examined for cell viability changes using CCK8 assay to determine the IC₅₀ of SAN in the two cells. The inhibitory effects of SAN on proliferation, invasion and migration of the cells were evaluated using colony-forming assay and Transwell assays. ROS production in the treated cells was analyzed with flow cytometry, and lipid peroxide production was assessed by detecting malondialdehyde (MDA) level. Glutathione (GSH) levels in the cells were detected, and Western blotting was used to detect the expressions of ferroptosis-related proteins STUB1 and GPX4. Results SAN significantly inhibited the proliferation, invasion and migration of SW620 and HCT-116 cells. SAN treatment significantly promoted ROS production, increased intracellular MDA level, and lowered GSH level in the two cells (P<0.05). Western blotting showed that SAN significantly upregulated the expression of STUB1 and down-regulated the expression of its downstream protein GPX4 (P<0.05). Conclusion SAN induces ferroptosis in colorectal cancer cells by regulating STUB1/GPX4, which may serve as a new therapeutic target for colorectal cancer.

Key words: sanguinarine, colorectal cancer, ferroptosis, STUB1, GPX4

Yinliang ZHANG, Zetan LUO, Rui ZHAO, Na ZHAO, Zhidong XU, Di AO, Guyi CONG, Xinyu LIU, Hailun ZHENG. Sanguinarine induces ferroptosis of colorectal cancer cells by upregulating STUB1 and downregulating GPX4[J]. Journal of Southern Medical University, 2024, 44(8): 1537-1544.

Figures/Tables 6

Fig.1 SAN represses colorectal cancer (CRC) cell viability and induces cell death. A, B: CCK-8 assay of SW620 and HCT-116 cells treated with different concentrations of SAN for 24 h. C, D: SW620 and HCT-116 cell viability following treatment with different concentrations of SAN for 24 and 72 h. E, F: Morphologies of CRC cells treated with SAN in the presence or absence of Fer-1 (1 μmol/L) (Original magnification: ×10). *P<0.05, **P<0.01, ****P<0.0001 vs control group; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.05 vs 24 h group.

Fig.2 SAN inhibits colony formation ability of SW620 and HCT-116 cells. A, B: Representative cell colonies after 14 days of SAN treatment in the presence or absence of Fer-1 (1 μmol/L). C, D: Colonies numbers in the 4 groups, demonstrating that SAN-induced SW620 and HCT-116 cell death was effectively blocked by Fer-1. ****P<0.0001 vs control group; #P<0.05, ##P<0.01 vs SAN group.

Fig.3 SAN inhibits migration and invasion ability of SW620 and HCT-116 cells A, B: Transwell migration assay of SAN-treated cells (×10). C, D: Number of migrated SW620 and HCT-116 cells. E, F: Transwell invasion assay of SAN-treated cells in the presence or absence of Fer-1 (×10). G, H: Number of migrated SW620 and HCT-116 cells. ****P<0.0001 vs control group; #P<0.05, ##P<0.01, ####P<0.0001 vs SAN group.

Fig.4 SAN induces ROS generation and contributes to ferroptosis in CRC cells. A, B: DCFH-DA probe was used to detected ROS levels in SW620 cells by flow cytometry and statistical analysis by Graphpad Prism. C, D: Flow cytometry was used to detected ROS levels in HCT116 cells and statistical analysis by Graphpad Prism. ****P<0.0001 vs control group; ##P<0.01, ###P<0.001 vs SAN group.

Fig.5 Levels of MDA and GSH in the CRC cells treated with SAN in the presence or absence of Fer-1. A, B: LIP levels in SW620 and HCT-116 cells at 24 h. C, D: MDA content in the cells. E, F: Intracellular GSH content of the cells. **P<0.01, ***P<0.001, ****P<0.0001 vs control group; #P<0.05, ##P<0.01 vs SAN group.

Fig.6 Effects of SAN on STUB1 and GPX4 expressions in SW620 and HCT-116 cells with or without Fer-1 treatment. A, B: Western blotting for STUB1 and GPX4 expressions in SW620 (A) and HCT-116 cells (B). C-F: Quantitative analysis of protein expressions. *P<0.05, **P<0.01 vs control group; #P<0.05 vs SAN group.

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