In this study, the expression, prognostic value and correlation with TILs of CTH in HCC were analyzed for the first time. Compared with normal tissues, the expression of CTH in HCC decreased significantly. In addition, CTH expression was related to T stage (T1&T2), pathologic stage (stage 1&stage 2), gender (male), histologic stage (grade 1), race (black or African American), AFP ( < = 400 ng/ml) and albumin (< 3.5g/dl). These results suggested that CTH played an important role in the progression and metastasis of HCC, which were consistent with previous studies (8–12; 22–24). The decrease of CTH activity will lead to the consumption of glutathione (GSH) in cancer patients (22), and GSH is extremely significant for the removal and detoxification of carcinogens (23). In addition, CTH was down-regulated in HCC patients, and individual cancer stage and T grade were negatively correlated with the expression of CTH (24).
As immunohistochemical images from HPA database verified, the CTH was highly expressed in normal liver tissues and low expressed in HCC tissues. Then, the top 50 up and down-regulated genes with the highest correlation with CTH were selected, and the five genes with the highest correlation between them were selected for verification in HCC, respectively. In addition, GO and KEGG analysis showed that CTH was mainly related to small molecule catabolism, mitochondrial matrix and coenzyme binding, enrichment and crosstalk of these genes in Phenylalanine metabolism, Fc gamma R − mediated phagocytosis and PPAR signaling pathway. As the GSEA results of reactome signaling by interleukins (NES = − 1.446, p = 0.009, FDR = 0.005) and the reactome neutrophil degranulation (NES = − 1.585, p = 0.009, FDR = 0.005) indicated, CTH was related to many pathways related to malignant tumors in HCC, especially immune related pathways, which was the same as previous studies (25–26). CTH silencing induced the expression of the natural-killer group 2, member D (NKG2D) ligands, major histcompatibility complex class I related chain A (MICA) and UL16 binding protein 2 (ULBP2), in the human breast cancer cell line (MDA-MB-231) triple-negative breast cancers (TNBC) cell line, which was associated with improved cytotoxicity of NK cell against H2S-depleted tumor targets, and CTH siRNAs caused the decrease of the production of tumor necrosis factor –α (TNF–α), which is a cytokine that may promote immune cell apoptosis in tumor microenvironment (25). H2S plays a role in regulating several pro-inflammatory cytokines levels in diabetic rat models including TNF - α tumor (26).
Survival analysis showed that low expression of CTH was correlated with poor OS (HR = 0.55, p = 0.00068), DSS (HR = 0.49 p = 0.0046) and PFS (HR = 0.64, p = 0.0045) in HCC. In addition, T stage (T3&T4), pathologic stage (stage 3&stage 4), tumor status (with tumor), alcohol consumption and hepatitis factors will make the prognosis of low expression group of CTH worse, which were consistent with previous reports (27–28). The low expression of a polymorphism of the alanine-glyoxylate aminotransferase 2 (AGXT2), D-amino acid oxidase (DAO) and CTH and the high expression of bisphosphoglycerate mutase (BPGM), cystathionine-beta-synthase (CBS), phosphoserine phosphatase (PSPH) and Acyl-CoA thioesterase 7 (ACOT7) are associated with the poor prognosis of HCC (24). In addition, knockout of CTH in mice will develop acute hepatitis in the case of intaking excessive methionine (27).
CTH expression was reported to be related to several TILs in HCC, especially CD56 (bright) NK cells, TFH, Th17 cells and Tcm. These results suggested that CTH was involved in the regulation of TILs. Han et al (28) revealed that pretreatment with D,L-propargylglycine, an inhibitor of CTH, attenuated plasma levels of TNF-α, IL-6, as well as urea and reduced H2S concentration in the kidney following hemorrhagic shock. According to Cui et al (29), the constitutive sulfurization of liver kinase B1 by H2S derived from CTH activates its target kinase adenosine monophosphate (AMP) activated protein kinase and promotes Treg differentiation and proliferation, so as to reduce the immune inflammation of blood vessels and kidneys and prevent hypertension. The results of studies above have the effect of mutual authentication.
The immune markers in HCC were further analyzed. After cell purity correction, CTH was negatively correlated with many immune cell markers in HCC, including that CTH was related to the immune infiltration of HCC. There is a significant correlation between CTH levels and several T lymphocyte markers (CD8 + T cell, general T cell, Th1, Th2, Th17 and Treg), monocyte and dendritic cell. These connections may indicate a potential mechanism by which CTH regulates T cell function in HCC. Kaplan-Meier and Cox proportional risk analysis showed that the high expression level of CTH enriched in various immune cell cohorts of HCC had a good prognosis. As Wu et al (30) reported, the effect of CTH/H2S on IL-1ese connections might indicate a potential mechanism by which CTH regulates T cell function in HCC. Kaplan-Meier and Cox proportional risk analysis showed that the high expression monocytes and dendritic cells, which is down-regulated in transplantation tolerance and may be involved in maintaining tolerance (31). Moreover, CTH / H2S pathway over inhibits JNK/NF-κB signaling plays an anti-inflammatory role in ox-LDL-stimulated macrophage (12). These results may explain that the high expression of CTH partially affects the prognosis of HCC patients via immune infiltration. Moreover, CTH trans-sulfur pathway converts cystathionine to cysteine and is responsible for the increase of total homocysteine (tHcy), which plays an important role in oxidative stress, resulting in the decrease of s-adenosyl-l-methionine (SAM) level and DNA demethylation (32–34). Therefore, we believe that CNV, somatic mutation and DNA methylation may result in the increase of CTH level, which may lead to HCC.
Five candidate drugs were found through TISIBD database (Pyridoxal phosphate, L-cysteinehosphate, 2-[(3-Hydroxy-2-Methyl-5-Phosphonooxymethyl-Pyridin-4- Ylmethyl)-Imino]-5-phosphono-pent-3-enoic acid, Carboxymethylthio-3- (3-Chlorophenyl)-1,2,4-Oxadiazol and L-2-amino-3-butynoic acid), which have targeted therapeutic effects on CTH. Pyridoxal phosphate is co-enzyme of cystathionine yssynthase (CBS) and CTH, and CTH shows greater loss of activity than CBS during pyridoxal phosphate deficiency (35). According to Yadav et al (36), L-cysteine inhibited the thcystathionine and H2S synthesis from cysteine by human CTH, which are consistent with our conjecture.