Selenocystine inhibits colon cancer cell growth by promoting reactive oxygen species generation to trigger oxidative damage

doi:10.12122/j.issn.1673-4254.2026.03.07

Abstract

Abstract:

Objective To explore the molecular mechanism by which selenocystine (SeC) inhibits colon cancer cell growth in vitro. Methods Colon cancer cells (RKO, HCT-116, and LoVo) were cultured and treated with 5, 10, or 20 μmol/L SeC for 24 h and 48 h. MTT assay was used to detect the cell viability, and wound healing assay was used to examine changes in cell migration. Flow cytometry with PI staining was used to analyze cell cycle arrest and apoptosis. Fluorescence probes were employed to monitor reactive oxygen species (ROS) generation, mitochondrial morphology and membrane potential, and the changes in ferroptosis were evaluated by detecting malondialdehyde (MDA), glutathione (GSH) and ferrous ion (Fe²⁺) levels; Western blotting was used to detect the changes in protein expressions. Results SeC at all the 3 doses significantly inhibited proliferation and migration of colon cancer cells, down-regulated the expression of cell cycle-related proteins CDK2 and CDK4 and activated the apoptotic proteins PARP and caspase-9. Western blotting showed that SeC decreased the expression of ferroptosis proteins FTH1 and xCT and increased the expression of DMT1. The levels of MDA and Fe²⁺ were increased and GSH level was decreased in SeC-treated cells. Fluorescence staining results showed that SeC treatment induced mitochondrial structure damages and promoted cellular ROS production. SeC treatment also increased phosphorylation of oxidative damage proteins and lowered the expression levels of NRF2 and HO-1 proteins. ROS scavenger significantly reversed the up-regulation of DMT1, PARP and p-H₂A.X protein induced by SeC in colon cancer cells. Conclusion SeC induces apoptosis and ferroptosis of colon cancer cells by promoting ROS generation and initiating oxidative damage, suggesting the potential of SeC as a potential chemotherapeutic agent for colon cancer.

Key words: selenocystine, colon cancer, reactive oxygen species, oxidative stress, apoptosis, ferroptosis

Qile SONG, Yikai MIAO, Xiaotong FENG, Yifan WANG, Wei LIU, Qi WEI, Xinru YU, Wenwen CHEN, Xiaoyan FU. Selenocystine inhibits colon cancer cell growth by promoting reactive oxygen species generation to trigger oxidative damage[J]. Journal of Southern Medical University, 2026, 46(3): 532-540.

Figures/Tables 5

Fig.1 SeC inhibits growth of colon cancer cells in a dose-dependent manner. A: SeC inhibits viability of RKO, HCT-116 and LoVo cells detected by MTT assay. B: Changes in cell morphology (Original magnification: ×200). C: SeC inhibits the migration of RKO cells detected by wound healing assay. (×100). D: Quantitative analysis of cell migration. All experiments were repeated 3 times (n=3). All data were presented as Mean±SD. *P<0.05, **P<0.01 vs SeC 0 μmol/L.

Fig.2 SeC induces cell cycle arrest and apoptosis in colon cancer cells. A: SeC inhibits expressions of RKO cell cycle-related proteins detected by Western blotting. B: SeC-induced cell cycle arrest and apoptosis in RKO, HCT-116, and LoVo cells detected by flow cytometry with PI staining. C: Quantitative analysis of cell cycle proteins. D: Quantitative analysis of cell cycle arrest in RKO, HCT-116, and LoVo cells. All experiments were repeated three times (n=3). All data are presented as Mean±SD. *P<0.05 vs SeC 0 μmol/L.

Fig.3 SeC induces apoptosis and ferroptosis in colon cancer cells. A: PARP cleavage and caspase-9 activation. B: Effect of SeC on ferroptosis-related proteins in RKO cells detected by Western blotting. C: Quantitative analysis of apoptotic and ferroptosis-related proteins. D: Measurement of MDA, GSH and Fe2+ in RKO cells after SeC treatment. All experiments were repeated three times (n=3). All data are presented as Mean±SD. *P<0.05 vs SeC 0 μmol/L.

Fig.4 SeC induces mitochondrial dysfunction and oxidative stress damage in colon cancer cells. A: Effects of SeC on oxidative stress-related proteins in RKO cells detected by Western blotting. B: SeC induces ROS accumulation and mitochondrial dysfunction in RKO cells. DCFH-DA fluorescent probe was used to detect ROS production (×100). Mito-SOX fluorescent probe was used to detect mitochondrial superoxide production (×400). Mito-Tracker fluorescent probe was used to detect mitochondrial structural damage (×400). JC-1 fluorescent probe was used to detect mitochondrial membrane potential changes (×200). C: Quantitative analysis of oxidative stress-related protein and DNA damage-related protein results. D: Quantitative analysis of ROS, Mito-SOX and JC-1 fluorescence results. All experiments were repeated 3 times (n=3). All data are presented as Mean±SD. *P<0.05 vs SeC 0 μmol/L.

Fig.5 ROS elimination attenuates SeC-induced apoptosis and ferroptosis. A: NAC pretreatment inhibits SeC-induced DNA damage, apoptosis, and ferroptosis detected by Western blotting. B: NAC pretreatment reverses the effects of SeC on colon cancer cells. C: Changes in cell morphology (×200). D: Quantitative analysis of apoptosis-related proteins, ferroptosis-related proteins, and DNA damage-related proteins. All experiments were repeated 3 times (n=3). All data are presented as Mean±SD. *P<0.05 vs SeC 0 μmol/L.

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