Shuangshu Decoction inhibits growth of gastric cancer cell xenografts by promoting cell ferroptosis via the P53/SLC7A11/GPX4 axis

doi:10.12122/j.issn.1673-4254.2025.07.02

Abstract

Abstract:

Objective To explore the mechanism of Shuangshu Decoction (SSD) for inhibiting growth of gastric cancer xenografts in nude mice. Methods Network pharmacology analysis was conducted to identify the common targets of SSD and gastric cancer cell ferroptosis, and bioinformatics analysis and molecular docking were used to validate the core targets. In the cell experiment, AGS cells were treated with SSD-medicated serum, Fer-1 (a ferroptosis inhibitor), or both, and the changes in cell viability, ferroptosis markers (ROS, Fe²⁺ and GSH), expressions of P53, SLC7A11 and GPX4, and mitochondrial morphology were examined. In a nude mouse model bearing gastric cancer xenografts, the effects of gavage with SSD, intraperitoneal injection of Fer-1, or their combination on tumor volume/weight, histopathology, and expressions of P53, SLC7A11 and GPX4 levels were evaluated. Results The active components in SSD (quercetin and wogonin) showed strong binding affinities to P53. In AGS cells, SSD treatment dose-dependently inhibited cell proliferation, increased ROS and Fe²⁺levels, upregulated P53 expression, and downregulated the expressions of SLC7A11 and GPX4, but these effects were effectively attenuated by Fer-1 treatment. SSD also induced mitochondrial shrinkage and increased the membrane density, which were alleviated by Fer-1. In the tumor-bearing mouse models, gavage with SSD significantly reduced tumor size and weight, caused tumor cell necrosis, upregulated P53 and downregulated SLC7A11 and GPX4 expression in the tumor tissue, and these effects were obviously mitigated by Fer-1 treatment. Conclusion SSD inhibits gastric cancer growth in nude mice by inducing cell ferroptosis via the P53/SLC7A11/GPX4 axis.

Key words: gastric cancer, network pharmacology, bioinformatics, molecular docking, Shuangshu Decoction, ferroptosis, P53/SLC7A11/GPX4 pathway

Xinyuan CHEN, Chengting WU, Ruidi LI, Xueqin PAN, Yaodan ZHANG, Junyu TAO, Caizhi LIN. Shuangshu Decoction inhibits growth of gastric cancer cell xenografts by promoting cell ferroptosis via the P53/SLC7A11/GPX4 axis[J]. Journal of Southern Medical University, 2025, 45(7): 1363-1371.

Figures/Tables 6

Tab.1 Primers sequences and the product length

Primer	The sequence (5'→3')
P53-F	TCAGATAGCGATGGTCTGGC
P53-R	CTCATAGGGACCCACCACAC
SLC7A11-F	GGCAGTTGCTGGGCTGATTT
SLC7A11-R	GGGCAACCATGAAGAGGCAT
GPX4-F	GCGGGCTACAACGTCAAATTC
GPX4-R	TCCACTTGATGGCATTTCCCAG
β-actin-F	CCTGGCACCCAGCACAAT
β-actin-R	GGGCCGGACTCGTCATAC

Tab.2 Top 10 active ingredients of Shuangshu Decoction

Molecule name	Degree	Molecular weight	Atom-based log P	Oral bioavailability (%)	Drug-likeness	Main source
Quercetin	199	302	1.5	46.43	0.28	Yanhusuo, Chuanlianzi
Wogonin	146	284.28	2.59	30.68	0.23	Cangshu
Naringenin	127	314.31	2.57	41.17	0.30	Chenpi
Cavidine	125	353.45	3.72	35.64	0.8	Banxia, Yanhusuo
Hyndarin	107	355.47	3.6	73.93	0.64	Yanhusuo
Mandenol	108	308.56	6.99	41.99	0.19	Yanhusuo, Chuanlianzi
Cryptopin	109	369.45	3.15	78.74	0.72	Yanhusuo
(r)-canadine	106	339.42	3.40	55.37	0.77	Yanhusuo
Bicuculline	105	367.38	2.83	69.67	0.88	Yanhusuo
Berberine	105	336.39	3.39	36.86	0.77	Yanhusuo

Fig.1 Network pharmacology analysis of gastric cancer-related genes and the mechanism of Shuangshu Decoction. A: Differential gene screening for gastric cancer. B: Wayne diagram of Shuangshu Decoction, gastric cancer (GC) and ferroptopsis. C: PPI diagram of the core targets. D: Differential analysis of P53. E: Representative P53 protein expression pattern in human gastric cancer and normal tissues (Original magnification: ×400). F: Survival analysis of gastric cancer patients with different P53 expression levels. G: Molecular docking analysis of the interaction between the compounds and the core proteins. ****P<0.0001.

Fig.2 Shuangshu Decoction-medicated serum promotes ferroptosis of AGS cells. A: Determination of the optimal concentration and time of the medicated serum for cell treatment. B: Effect of Shuangshu Decoction on ferroptosis of AGS cells. C: Effect of Shuangshu Decoction-medicated serum on AGS cell morphology (×100). D: Observation of ROS production in the cells under fluorescence microscope (×200). E: Influence of serum of Shuangshu Decoction on ROS production analyzed by flow cytometry and fluorescence intensity. F: Expression of GSH in AGS cells in each group. G: Expression of Fe2+ in AGS cells in each group. *P<0.05 vs control group; #P<0.05 vs SST group; ▲P<0.05 vs SST+Fer-1 group.

Fig.3 Shuangshu Decoction promotes ferroptosis in AGS cells via the P53/SLC7A11/GPX4 pathway. A: Protein expression levels of P53, SLC7A11 and GPX4 in the treated cells. B: P53, SLC7A11 and GPX4 mRNA expressions in the treated cells. C: Mitochondrial matrix density in each group. D: Mitochondrial damage in each group (TEM, ×20000). *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs SST group; ▲P<0.05 vs SST+Fer-1 group; M: Mitochondria.

Fig.4 Inhibitory effect of Shuangshu Decoction on xenograft tumor growth in nude mice and its regulation of the P53/SLC7A11/GPX4 pathway. A: Tumor volume in each group. B: Tumor weight in each group. C: Tumor growth curve in each group. D: Pathological examination of the tumors in each group (HE staining, ×200). E: Protein expression levels of P53, SLC7A11, and GPX4 in the tumor tissues from each group detected by Western blotting. F: SLC7A11, GPX4, and P53 mRNA expression levels in the tumor tissues. *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.05 vs SST group; ▲P<0.05 vs SST+Fer-1 group.

References 31

[1]	Morgan E, Arnold M, Camargo MC, et al. The current and future incidence and mortality of gastric cancer in 185 countries, 2020-40: a population-based modelling study[J]. EClinicalMedicine, 2022, 47: 101404. doi：10.1016/j.eclinm.2022.101404
[2]	Zheng RS, Chen R, Han BF, et al. Cancer incidence and mortality in China, 2022[J]. Zhonghua Zhong Liu Za Zhi Chin J Oncol, 2024, 46(3): 221-31.
[3]	Wang FH, Zhang XT, Tang L, et al. The Chinese society of clinical oncology (CSCO): clinical guidelines for the diagnosis and treatment of gastric cancer, 2023[J]. Cancer Commun (Lond), 2024, 44(1): 127-72. doi：10.1002/cac2.12516
[4]	Zhang X, Li M, Chen S, et al. Effectiveness of endoscopic screening for gastric cancer in China: A population-based study[J]. Gastrointest Endosc, 2023, 97(2): 231-240.
[5]	Qiu HB, Cao SM, Xu RH. Cancer incidence, mortality, and burden in China: a time-trend analysis and comparison with the United States and United Kingdom based on the global epidemiological data released in 2020[J]. Cancer Commun (Lond), 2021, 41(10): 1037-48. doi：10.1002/cac2.12197
[6]	Camargo MC, Figueiredo C, Machado JC. Review: gastric malignancies: basic aspects[J]. Helicobacter, 2019, 24(): e12642. doi：10.1111/hel.12642
[7]	Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death[J]. Cell, 2012, 149(5): 1060-72. doi：10.1016/j.cell.2012.03.042
[8]	Xie Y, Hou W, Song X, et al. Ferroptosis: process and function[J]. Cell Death Differ, 2016, 23(3): 369-79. doi：10.1038/cdd.2015.158
[9]	Yang WS, Stockwell BR. Ferroptosis: death by lipid peroxidation[J]. Trends Cell Biol, 2016, 26(3): 165-76. doi：10.1016/j.tcb.2015.10.014
[10]	Liang C, Zhang XL, Yang MS, et al. Recent progress in ferroptosis inducers for cancer therapy[J]. Adv Mater, 2019, 31(51): e1904197. doi：10.1002/adma.201904197
[11]	Liang YY, Qiu SJ, Zou YW, et al. Ferroptosis-modulating natural products for targeting inflammation-related diseases: challenges and opportunities in manipulating redox signaling[J]. Antioxid Redox Signal, 2024, 41(13/14/15): 976-91. doi：10.1089/ars.2024.0556
[12]	Li S, Zhang B. Traditional Chinese medicine network pharm-acology: theory, methodology and application[J]. Chin J Nat Med, 2013, 11(2): 110-20. doi：10.3724/sp.j.1009.2013.00110
[13]	Li X, Yang G, Li X, et al. Traditional Chinese medicine in cancer care: a review of controlled clinical studies published in Chinese[J]. PLoS One, 2013, 8(4): e60338. doi：10.1371/journal.pone.0060338
[14]	Zhang LL, Zhang DJ, Shi JX, et al. Immunogenic cell death inducers for cancer therapy: an emerging focus on natural products[J]. Phytomedicine, 2024, 132: 155828. doi：10.1016/j.phymed.2024.155828
[15]	Yuan JW, Khan SU, Yan JF, et al. Baicalin enhances the efficacy of 5-Fluorouracil in gastric cancer by promoting ROS-mediated ferroptosis[J]. Biomed Pharmacother, 2023, 164: 114986. doi：10.1016/j.biopha.2023.114986
[16]	Hu LY, Zhang ZY, Zhu F, et al. Schizandrin A enhances the sensitivity of gastric cancer cells to 5-FU by promoting ferroptosis[J]. Cytotechnology, 2024, 76(3): 329-40. doi：10.1007/s10616-024-00623-4
[17]	杨建宇, 林才志, 冯利. 抗癌秘验方[M]. 3版. 北京: 化学工业出版社, 2019: 376.
[18]	王春鹏, 程伟玲, 戴明明, 等. 林才志治疗慢性萎缩性胃炎验案举隅[J]. 中国民间疗法, 2021, 29(5): 107-8. doi：10.19621/j.cnki.11-3555/r.2021.0543
[19]	陈浩彬, 曹育启, 林才志. 林才志治疗大肠癌临床经验采撷[J]. 广西中医药, 2024, 47(1): 35-8.
[20]	Huang J, Chen J, Li JN. Quercetin promotes ATG5-mediating autophagy-dependent ferroptosis in gastric cancer[J]. J Mol Histol, 2024, 55(2): 211-25. doi：10.1007/s10735-024-10186-5
[21]	Ding LX, Dang SW, Sun MJ, et al. Quercetin induces ferroptosis in gastric cancer cells by targeting SLC1A5 and regulating the p-Camk2/p-DRP1 and NRF2/GPX4 Axes[J]. Free Radic Biol Med, 2024, 213: 150-63. doi：10.1016/j.freeradbiomed.2024.01.002
[22]	Liu X, Peng XH, Cen S, et al. Wogonin induces ferroptosis in pancreatic cancer cells by inhibiting the Nrf2/GPX4 axis[J]. Front Pharmacol, 2023, 14: 1129662. doi：10.3389/fphar.2023.1129662
[23]	Warias P, Plewa P, Poniewierska-Baran A. Resveratrol, piceatannol, curcumin, and quercetin as therapeutic targets in gastric cancer-mechanisms and clinical implications for natural products[J]. Molecules, 2024, 30(1): 3. doi：10.3390/molecules30010003
[24]	Qian JY, Lou CY, Chen YL, et al. A prospective therapeutic strategy: GPX4-targeted ferroptosis mediators[J]. Eur J Med Chem, 2025, 281: 117015. doi：10.1016/j.ejmech.2024.117015
[25]	Huang WJ, Wen F, Yang PP, et al. Yi-qi-Hua-yu-Jie-du decoction induces ferroptosis in cisplatin-resistant gastric cancer via the AKT/GSK3β/NRF2/GPX4 axis[J]. Phytomedicine, 2024, 123: 155220. doi：10.1016/j.phymed.2023.155220
[26]	Zheng GT, Liu XY, Abuduwufuer A, et al. Poria cocos inhibits the invasion, migration, and epithelial-mesenchymal transition of gastric cancer cells by inducing ferroptosis in cells[J]. Eur J Med Res, 2024, 29(1): 531. doi：10.1186/s40001-024-02110-0
[27]	Liu YQ, Gu W. p53 in ferroptosis regulation: the new weapon for the old guardian[J]. Cell Death Differ, 2022, 29(5): 895-910. doi：10.1038/s41418-022-00943-y
[28]	Jiang L, Kon N, Li TY, et al. Ferroptosis as a p53-mediated activity during tumour suppression[J]. Nature, 2015, 520(7545): 57-62. doi：10.1038/nature14344
[29]	Zhou BS, Wang HL, Zhang B, et al. Licochalcone B attenuates neuronal injury through anti-oxidant effect and enhancement of Nrf2 pathway in MCAO rat model of stroke[J]. Int Immunopharmacol, 2021, 100: 108073. doi：10.1016/j.intimp.2021.108073
[30]	Koppula P, Zhang YL, Zhuang L, et al. Amino acid transporter SLC7A11/xCT at the crossroads of regulating redox homeostasis and nutrient dependency of cancer[J]. Cancer Commun (Lond), 2018, 38(1): 12. doi：10.1186/s40880-018-0288-x
[31]	Hirschhorn T, Stockwell BR. The development of the concept of ferroptosis[J]. Free Radic Biol Med, 2019, 133: 130-43. doi：10.1016/j.freeradbiomed.2018.09.043