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.

References 35

1	Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-49.
2	Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death[J]. Cell, 2012, 149(5): 1060-72.
3	Ren XP, Zhang L, Xia SJ, et al. A new visual transient elastography technique for grading liver fibrosis in patients with chronic hepatitis B[J]. Ultrasound Q, 2020, 37(2): 105-10.
4	Huang LL, Yu XP, Li JL, et al. Effect of liver inflammation on accuracy of FibroScan device in assessing liver fibrosis stage in patients with chronic hepatitis B virus infection[J]. World J Gastroenterol, 2021, 27(7): 641-53.
5	张云飞, 张特, 马文静, 等. 神农架中草药博落回提取物血根碱的抗肿瘤作用研究进展[J]. 湖北医药学院学报, 2018, 4(5): 476-83.
6	朱丽飞, 王小莉, 曲书昊. 血根碱的药理作用研究进展[J]. 广东化工, 2020, 47(22): 69-70.
7	Gong XL, Chen ZH, Han QR, et al. Sanguinarine triggers intrinsic apoptosis to suppress colorectal cancer growth through disassociation between STRAP and MELK[J]. BMC Cancer, 2018, 18(1): 578.
8	Olofinsan K, Abrahamse H, George BP. Therapeutic role of alkaloids and alkaloid derivatives in cancer management[J]. Molecules, 2023, 28(14): 5578.
9	Wei GX, Xu YH, Peng T, et al. Sanguinarine exhibits antitumor activity via up-regulation of Fas-associated factor 1 in non-small cell lung cancer[J]. J Biochem Mol Toxicol, 2017, 31(8):1002.
10	Hałas-Wiśniewska M, Zielińska W, Izdebska M, et al. The synergistic effect of piperlongumine and sanguinarine on the non-small lung cancer[J]. Molecules, 2020, 25(13): 3045.
11	Su Q, Wang JJ, Wu Q, et al. Sanguinarine combats hypoxia-induced activation of EphB4 and HIF-1α pathways in breast cancer[J]. Phytomedicine, 2021, 84: 153503.
12	Zhu M, Gong ZY, Wu Q, et al. Sanguinarine suppresses migration and metastasis in colorectal carcinoma associated with the inversion of EMT through the Wnt/β-catenin signaling[J]. Clin Transl Med, 2020, 10(1): 1-12.
13	Su Q, Wang JJ, Liu F, et al. Blocking Parkin/PINK1-mediated mitophagy sensitizes hepatocellular carcinoma cells to sanguinarine-induced mitochondrial apoptosis[J]. Toxicol In Vitro, 2020, 66: 104840.
14	Xu RZ, Wu JC, Luo YB, et al. Sanguinarine represses the growth and metastasis of non-small cell lung cancer by facilitating ferroptosis[J]. Curr Pharm Des, 2022, 28(9): 760-8.
15	Alakkal A, Thayyullathil F, Pallichankandy S, et al. Sanguinarine induces H₂O₂-dependent apoptosis and ferroptosis in human cervical cancer[J]. Biomedicines, 2022, 10(8): 1795.
16	Zou YL, Palte MJ, Deik AA, et al. A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis[J]. Nat Commun, 2019, 10(1): 1617.
17	何丹, 李琳霈, 谭小宁, 等. 基于p53/SLC7A11/GPX4信号轴探讨七叶内酯诱导小鼠乳腺癌4T1细胞铁死亡的作用机制[J]. 中国病理生理杂志, 2024, 40(2): 282-90.
18	朱权, 黄柏胜, 位磊艳, 等. 过表达LncRNA MEG3通过促进铁死亡增强肝癌细胞对顺铂的化疗敏感性[J]. 南方医科大学学报, 2024, 44(1): 17-24.
19	Torii S, Shintoku R, Kubota C, et al. An essential role for functional lysosomes in ferroptosis of cancer cells[J]. Biochem J, 2016, 473(6): 769-77.
20	Yu HT, Guo PY, Xie XZ, et al. Ferroptosis, a new form of cell death, and its relationships with tumourous diseases[J]. J Cell Mol Med, 2017, 21(4): 648-57.
21	Wu W, Geng ZX, Bai HR, et al. Ammonium Ferric Citrate induced Ferroptosis in Non-Small-Cell Lung Carcinoma through the inhibition of GPX4-GSS/GSR-GGT axis activity[J]. Int J Med Sci, 2021, 18(8): 1899-909.
22	Jiang XJ, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease[J]. Nat Rev Mol Cell Biol, 2021, 22(4): 266-82.
23	Khan AQ, Mohamed EAN, Hakeem I, et al. Sanguinarine induces apoptosis in papillary thyroid cancer cells via generation of reactive oxygen species[J]. Molecules, 2020, 25(5): 1229.
24	Zhang Y, Huang WR. Sanguinarine induces apoptosis of human lens epithelial cells by increasing reactive oxygen species via the MAPK signaling pathway[J]. Mol Med Rep, 2019, 19(5): 4449-56.
25	Kim S, Lee TJ, Leem J, et al. Sanguinarine-induced apoptosis: generation of ROS, down-regulation of Bcl-2, c-FLIP, and synergy with TRAIL[J]. J Cell Biochem, 2008, 104(3): 895-907.
26	Su LJ, Zhang JH, Gomez H, et al. Reactive oxygen species-induced lipid peroxidation in apoptosis, autophagy, and ferroptosis[J]. Oxid Med Cell Longev, 2019, 2019: 5080843.
27	Wang WM, Green M, Choi JE, et al. CD8⁺ T cells regulate tumour ferroptosis during cancer immunotherapy[J]. Nature, 2019, 569(7755): 270-4.
28	Hirschhorn T, Stockwell BR. The development of the concept of ferroptosis[J]. Free Radic Biol Med, 2019, 133: 130-43.
29	Xia HB, Wu Y, Zhao J, et al. N6-Methyladenosine-modified circSAV1 triggers ferroptosis in COPD through recruiting YTHDF1 to facilitate the translation of IREB2[J]. Cell Death Differ, 2023, 30(5): 1293-304.
30	Kong PZ, Yang MM, Wang Y, et al. Ferroptosis triggered by STAT1- IRF1-ACSL4 pathway was involved in radiation-induced intestinal injury[J]. Redox Biol, 2023, 66: 102857.
31	Song XX, Zhu S, Chen P, et al. AMPK-mediated BECN1 phosphorylation promotes ferroptosis by directly blocking system x_c ^- activity[J]. Curr Biol, 2018, 28(15): 2388-99.e5.
32	Ursini F, Maiorino M. Lipid peroxidation and ferroptosis: the role of GSH and GPx4[J]. Free Radic Biol Med, 2020, 152: 175-85.
33	Ding YH, Chen XP, Liu C, et al. Identification of a small molecule as inducer of ferroptosis and apoptosis through ubiquitination of GPX4 in triple negative breast cancer cells[J]. J Hematol Oncol, 2021, 14(1): 19.
34	Sun XF, Zhang Q, Lin XH, et al. Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination[J]. Cell Death Dis, 2023, 14(12): 839.
35	Wang J, Zhang HY, Chen LM, et al. CircDCBLD2 alleviates liver fibrosis by regulating ferroptosis via facilitating STUB1-mediated PARK7 ubiquitination degradation[J]. J Gastroenterol, 2024, 59(3): 229-49.

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