Role and mechanism of hepatic stellate cells in regulating the apoptosis of hepatocellular carcinoma cells through cystathionine γ-lyase/hydrogen sulfide

Hongwei SHANG; Yanan MA; Xin LU; Lingna LYU; Huiguo DING

doi:10.12449/JCH241117

Volume 40 Issue 11

Nov. 2024

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Article Navigation > Journal of Clinical Hepatology > 2024 > 40(11): 2238-2245

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Role and mechanism of hepatic stellate cells in regulating the apoptosis of hepatocellular carcinoma cells through cystathionine γ-lyase/hydrogen sulfide

DOI: 10.12449/JCH241117

Hongwei SHANG¹,
Yanan MA^{2, 3},
Xin LU¹,
Lingna LYU²,
Huiguo DING^{2
,
,
,}

1.
School of Basic Medical Sciences，Capital Medical University，Beijing 100069，China
2.
Department of Hepatology and Gastroenterology，Beijing YouAn Hospital，Capital Medical University，Beijing 100069，China
3.
Oncology Center，Shanxi Bethune Hospital，Taiyuan 030000，China

Research funding:

Sino-German Cooperation Group (GZ1517)

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Corresponding author: DING Huiguo， dinghuiugo@ccmu.edu.cn （ORCID： 0000-0002-8716-4926）
Received Date: 2024-08-07
Accepted Date: 2024-08-26
Published Date: 2024-11-25

Abstract

Abstract

Objective As important components in the microenvironment of hepatocellular carcinoma （HCC）， hepatic stellate cells （HSCs） and hydrogen sulfide （H₂S） participate in various biological processes that regulate the development and progression of HCC. Through the co-culture of HSCs and HCC cells， this article aims to investigate the role and mechanism of HSCs in regulating the apoptosis of HCC cells by secreting H₂S. Methods The HSC cell line （LX-2） and HCC cell lines （HepG2 and PLC/PRF/5） were used for experiment. RT-qPCR and Western Blot （WB） were used to measure the mRNA and protein expression levels of cystathionine γ-lyase （CSE）， a key synthase for H₂S； ELISA was used to measure the concentration of H₂S in supernatant； next-generation sequencing， cell immunofluorescence assay， chromatin immunoprecipitation （ChIP）， and WB were used to measure the JNK/JunB-TNFSF14 signaling pathway genes， binding sites， and related proteins after HepG2 cells were treated by H₂S. LX-2 cells were co-cultured with HepG2 or PLC/PRF/5 cells in a Transwell chamber； CCK-8 assay and flow cytometry were used to measure the viability and apoptosis of HCC cells， and WB was used to measure the H₂S-TNFSF14 signaling pathway-related proteins. All cell experiments were repeated three times. The independent-samples t test was used for comparison of continuous data between two groups； a one-way analysis of variance or the analysis of variance with repeated measures was used for comparison between multiple groups， and the Dunnett-t test was used for further comparison between two groups. Results LX-2 cells synthesized H₂S mainly through CSE， and the concentration of H₂S in supernatant of LX-2 cells gradually increased over time （22.89±0.08 pg/mL vs 28.29±0.15 pg/mL vs 36.19±1.90 pg/mL， F=79.63， P<0.05）. In LX-2 cells， the mRNA expression level of CSE was significantly higher than that of CBS and MPST （1.008±0.13 vs 0.320±0.014 vs 0.05±0.02， F=80.84， P<0.05）. When CSE was inhibited by PPG， the concentration of H₂S decreased with the increase in the concentration of PPG （P<0.05）. LX-2 cells were co-cultured with HepG2 or PLC/PRF/5 cells， and over the time of culture， there were significant reductions in the viability of HepG2 cells （87.48%±0.82% vs 70.48%±0.641% vs 52.89%±0.57% vs 45.20%±0.69%， F=1 517.13， P<0.001） and PLC/PRF/5 cells （92.41%±0.48% vs 74.10%±0.73% vs 53.70%±0.60% vs 44.00%±0.27%， F=2626.21， P<0.001） and significant increases in the apoptosis of HepG2 cells （12.88%±0.64% vs 15.5%±0.16% vs 18.43%±0.37% vs 13.01%±0.58%， F=142.15， P<0.001） and PLC/PRF/5 cells （8.51±0.05 vs 12.80±0.33 vs 15.59±0.21 vs 10.72±0.30， F=676.40， P<0.001）， with the most significant changes on day 3. Next-generation sequencing showed that endogenous H₂S and NaHS （endogenous H₂S donor） were involved in regulating the expression of various genes in HepG2 cells. By releasing H₂S， NaHS and LX2 activated the JNK/JunB signaling pathway and upregulated the expression of the apoptosis gene TNFSF14 in HCC cells， with increased binding between p-JunB and the transcriptional regulatory regions of the TNFSF14 gene. Conclusion In the microenvironment of HCC， HSCs activate the JNK/JunB signaling pathway in HCC cells through the signal molecules CSE/H₂S， and there is an increase in the expression of TNFSF14， thereby promoting the apoptosis of HCC cells.
- Carcinoma， Hepatocellular,
- Hepatic Stellate Cells,
- Hydrogen Sulfide

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References

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Role and mechanism of hepatic stellate cells in regulating the apoptosis of hepatocellular carcinoma cells through cystathionine γ-lyase/hydrogen sulfide

DOI: 10.12449/JCH241117

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Role and mechanism of hepatic stellate cells in regulating the apoptosis of hepatocellular carcinoma cells through cystathionine γ-lyase/hydrogen sulfide

DOI: 10.12449/JCH241117

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