Recent advances in the role of endogenous hydrogen sulphide in cancer cells

Abstract Hydrogen sulphide (H2S) is a gaseous neurotransmitter that can be self‐synthesized by living organisms. With the deepening of research, the pathophysiological mechanisms of endogenous H2S in cancer have been increasingly elucidated: (1) promote angiogenesis, (2) stimulate cell bioenergetics, (3) promote migration and proliferation thereby invasion, (4) inhibit apoptosis and (5) activate abnormal cell cycle. However, the increasing H2S levels via exogenous sources show the opposite trend. This phenomenon can be explained by the bell‐shaped pharmacological model of H2S, that is, the production of endogenous (low concentration) H2S promotes tumour growth while the exogenous (high concentration) H2S inhibits tumour growth. Here, we review the impact of endogenous H2S synthesis and metabolism on tumour progression, summarize the mechanism of action of H2S in tumour growth, and discuss the possibility of H2S as a potential target for tumour treatment.


| INTRODUCTION
Hydrogen sulphide (H 2 S) is one of the three known gaseous signalling molecules in biological systems. Together with carbon monoxide (CO) and nitric oxide (NO), it forms a family of endogenous gases.
These gases are involved in regulating a variety of physiological and pathological processes [1][2][3] and show pleiotropy and dose dependence on a variety of diseases, including cancer. [4][5][6][7] At present, some compounds that can inhibit or induce the synthesis of these gases have been tested in preclinical research, including NO-releasing drugs for cancer prevention and treatment and CO-releasing drugs for immune inflammation or autoimmune diseases. [8][9][10][11][12][13] H 2 S mainly comes from different substrates catalysed by cystathionine (CTH) β-synthase (CBS), CTH γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). [14][15][16] In cancer cells, H 2 S shows cytoprotective or cytotoxic effects, depending on the concentration: that is, a low concentration (endogenous) of H 2 S can induce tumorigenesis, while a high level (exogenous) of H 2 S can inhibit tumorigenesis. [17][18][19] This provides two different ideas for the treatment of cancer, namely inhibiting the production of endogenous H 2 S or adding exogenous H 2 S. This review summarizes the effect of endogenous H 2 S on cancer, focuses on the impact of the change of endogenous H 2 S concentration on cancer cells, and expounds its implication on cancer treatments, hoping to provide insight for follow-up research and drug development.
2 | ENZYMES THAT SYNTHESIZE H 2 S

| The distribution of CBS and its catalysed reaction
In mammals, CBS is mainly found in the liver, brain, kidney, and pancreas. 20,21 In the liver, the content of CBS is most abundant in hepatocytes and least in the hepatic stellate cells (HSCs) and Kupffer cells. 22,23 CBS is expressed in all brain regions except the hippocampus, with the highest content present in the cerebellum and cerebral cortex. 24 CBS is also expressed in neural stem cells and regulates their differentiation. 25 In the kidney, CBS is mainly distributed in the glomeruli, the epithelium of the proximal tubules, collecting ducts, and the inter-lobular arteries of the kidney. 26,27 Moreover, CBS is abundantly expressed in acinar cells of the pancreas, and can also be detected in pancreatic islet cells and exocrine cells. 28,29 CBS content in other tissues is relatively low. In the digestive system, CBS exists in the gastric mucosa, colonic epithelium, small intestine, jejunum, and ileum. [30][31][32][33] CBS is also significantly expressed in the spleen. 34 CBS has been suggested to play an important role in the female reproductive system since it is well expressed in the ovary and uterus but is relatively low in the prostate and testis. 35 It is also expressed in the prostate epithelium, bladder, and urethra. [36][37][38] In the heart, CBS is expressed in cardiomyocytes, coronary arteries, and perivascular adipose tissue. 39,40 Meanwhile, in the lung, it is expressed in the epithelial cells of the alveoli, bronchiole, and trachea, as well as the endothelial cells (ECs) and smooth muscle cells of the pulmonary artery. [41][42][43][44] In addition, the content of CBS in the thyroid is low and it is significantly increased in thyroid cancer. 45 Likewise, CBS is not contained in breast tissue but is overexpressed in breast cancer (BC). 46 CBS can generate H 2 S through several condensation reactions including those of two molecules of L-cysteine into L-lanthionine, two L-homocysteine molecules into L-homolanthionine, and Lcysteine and L-homocysteine into L-cystathionine. 3,47 Although a large amount of cystathionine (CTH) can theoretically inhibit or even reverse the overall response of CBS, the level of CTH in most tissues is very low, so it is difficult to achieve this reverse reaction in vivo. 48 2.2 | The distribution of CSE and its catalysed reaction As the main H 2 S synthase, CSE is mainly expressed in the cardiovascular and respiratory systems, 49,50 including in the liver, kidney, pancreas, uterus, and prostate. [50][51][52] In addition, a small amount of CSE mRNA has also been detected in the brain, but because the inhibitor of CSE could not impede the production of H 2 S in the brain, it is thought that CSE is not the main H 2 S producing enzyme in the brain. 24 CSE can decompose cysteine into pyruvate, ammonia, and thiocysteine, and further catalyse thiocysteine to produce H 2 S. CSE can also use homocysteine as a substrate to generate H 2 S. CSE deficiency can lead to cystathioniuria and hyperhomocysteinemia. 53

| The distribution of 3-MST and its catalytic reaction
3-MST is found in almost all tissues of mammals; however, its expression is tissue-specific. In the central nervous system, 3-MST is mainly located in hippocampal vertebral neurons, cerebellar Purkinje cells, and olfactory bulb mitral valve cells. 54 In addition, 3-MST is also relatively high in the kidney, liver, testis, large intestine, and endocrine organs. 55 3-MST catalyses the production of H 2 S and requires the assistance of cysteine aminotransferase (CAT). 56 CAT converts cysteine to 3-mercaptopyruvate, and 3-MST transfers sulphur from 3-mercaptopyruvate to sulphite, sulphur acceptor, or sulphur. However, this method can only generate sulphane sulphur or combined sulphur, but for producing H 2 S, the action of reducing agents (e.g., thioredoxin, dihydrolipoic acid) or various enzymes in the cell is required. 57,58 3 | THE TUMOUR-PROMOTING MECHANISM OF H 2 S

| H 2 S promotes angiogenesis
Angiogenesis is a multi-step process involving ECs that is characterized by endothelial extracellular matrix remodelling, including initiation, migration, catheter formation, and differentiation. 59 When gene mutations accumulate and cause cancer, the solid tumour will form a highly vascularized state. These vessels provide oxygen and nutrition for the development or local spread of the tumour. 60 H 2 S promotes EC angiogenesis by regulating cyclic nucleotides, kinases, and ion channels. 61,62 H 2 S donors increase the phosphorylation levels of Akt, p38, and ERK1/2, while the pharmacological inhibition of PI3K/Akt and MAPK inhibits the proliferation and migration of EC. H 2 S also promotes angiogenesis through the K ATP channel. In addition, in human EC, the K ATP channel plays a role upstream of p38. [63][64][65][66][67] The inhibition of endothelial nitric oxide synthase, soluble guanylyl cyclase, or cyclic guanosine monophosphate (cGMP) dependent protein kinase weakens H 2 S-stimulated angiogenesis, indicating H 2 S can interact with multiple molecules of the NO/cGMP pathway to promote angiogenesis. 68,69 Vascular endothelial growth factor (VEGF) can promote vascular permeability, extracellular matrix degeneration, vascular EC migration, proliferation, and angioplasty. 70,71 Many studies have shown that there is extensive interaction between H 2 S and VEGF.
In particular, the incubation of human EC with VEGF increases the concentration of H 2 S, and the silencing or pharmacological inhibition of CSE weakens the angiogenesis of VEGF-stimulated EC. 66 Although the mechanism of this phenomenon has not been clarified, some experiments show that it may be caused by CSEmediated Ca 2+ /calmodulin-dependent activation. 69 In addition, the inhibition of CSE can markedly block the activation of p38 and ERK1/2 stimulated by VEGF. 66 CBS silencing also reduced the expression of VEGFR2 and neuropilin-1, thereby reducing the signal intensity of VEGF. The S-sulfhydration of specificity protein 1 (Sp1) at Cys68 and Cys755 by H 2 S enhances the stability of Sp1, and subsequently, promotes the transcription of VEGFR2. 72 H 2 S can also enhance binding of VEGF to VEGFR2 (thereby increasing activity of the latter). 73

| H 2 S inhibits apoptosis
Apoptosis refers to the autonomous and orderly death of cells controlled by genes to maintain the stability of the internal environment.
It involves the activation, expression, and regulation of a series of genes. 74 Evasion of apoptosis is an important mechanism in the development of cancer, allowing cancer cells to survive under physiological stress. 75 H 2 S has been found to play an anti-apoptotic effect in the cardiovascular system, ischaemia-reperfusion injury, and various cancers. [76][77][78][79][80] One of the potential anti-apoptotic mechanisms of H 2 S is its anti-oxidant effect achieved by scavenging reactive oxygen species (ROS) and reactive nitrogen species (RNS). Although H 2 S is usually at a low concentration under baseline conditions, its small molecular structure and ability to penetrate freely on the cell membrane make it a more effective antioxidant than glutathione (GSH).
However, it is reasonable to believe that H 2 S-mediated antioxidant protection is caused by a wide range of intermediate signals it regulates rather than direct ROS/RNS clearance. 81 Another potential mechanism is the activation of anti-apoptotic pathways via Ssulfhydrating NF-κB, Kelch-like ECH-associated protein 1, and mitogen-activated protein kinase kinase 1 (MEK1). 82-84

| H 2 S boosts cellular bioenergetics
Cellular bioenergetics plays an important role in the occurrence and development of different types of cancer. 85,86 Initially, H 2 S was reported to exhibit cytotoxic effects on mitochondria by inhibiting the cytochrome c oxidase system, but recent studies demonstrate a more complex, concentration-dependent regulation of mitochondrial and cellular bioenergetics by H 2 S. In normal intestinal epithelial cells, H 2 S acts as a substrate for bioenergy production. 87,88 Further research shows that in colon and ovarian cancer, H 2 S can serve both as a regulator and a substrate of bioenergetics. 89,90 CBS silencing reduces oxygen consumption and adenosine triphosphate (ATP) production. Silencing of 3-MST in hepatoma cells also shows similar effects. Likewise, the pharmacological inhibition of CBS and 3-MST blocks electron transport and mitochondrial energy production in various cancer cells, whereas replenishment of substrates for these enzymes reversed this process. [91][92][93][94][95] It is worth adding that, H 2 S by itself cannot initiate or maintain the mitochondrial electron transport system, but can affect glycolysis-derived electron donors.
The H 2 S-mediated mitochondrial electron transport requires the participation of sulphide quinone oxidoreductase (SQR), 87,96,97 and the expression of SQR in tumour cells is up-regulated under hypoxic conditions that may be a potential mechanism for tumour cells to use H 2 S to generate energy. 98 On the other hand, electrons from SQR can also be transported in reverse when cells are exposed to higher concentrations of H 2 S. 87 In cancer cells, this mechanism does not aid in electron transport, proton pump, or ATP generation, but instead it stimulates mitochondrial ROS production. 99 In addition, H 2 S can directly S-sulfhydrate glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to enhance its activity in ATP generation. 52

| H 2 S promotes DNA repair and tumour growth
Recent studies have shown that cell cycle checkpoints, DNA damage and repair, and the expression of proteins involved in maintaining gene stability are regulated by both exogenous and endogenous H 2 S. [100][101][102] The effect is suggested to be due to be associated with activities of MEK1 and poly [ADP-ribose] polymerase 1 (PARP-1).
Specifically, PARP1 can sense DNA single-strand or double-strand breaks and initiate DNA damage repair pathways. PARP inhibitors have been developed to block DNA repair in BRCA-mutated cancers, thereby initiating signalling pathways that trigger apoptosis and ultimately inhibit tumour growth. 103 H 2 S is abnormally elevated in a variety of cancers, and inhibition of CBS or CSE activity suppresses tumour growth in colon, lung, prostate, and BCs. 89,93,104,105 MEK1 belongs to the classical MAPK kinase pathway, and the activation of MEK1 is closely related to cell proliferation and tumorigenesis. 106 S-sulfhydration of MEK1 by H 2 S at Cys341 promotes phosphorylation and nuclear translocation of MEK1, thereby activating PARP-1-mediated DNA damage repair, which is most likely a key driver of tumour growth due to CBS or CSE overexpression. 84 In addition, H 2 S can S-sulfhydrate Exo/endonuclease G at Cys76 in mitochondria to mediate DNA damage repair in mitochondria. 107 4 | ENDOGENOUS H 2 S, CELL SIGNAL TRANSDUCTION, AND CANCER

| Endogenous H 2 S and BC
BC is the most common malignancy in women and is divided into different subtypes with widely varying prognoses and treatment modalities. 108,109 Endocrine therapy is suitable for hormone receptor (HR)positive patients, and targeted therapy is suitable for human epidermal growth factor receptor 2 (HER2)-positive patients. 110 Triplenegative breast cancer (TNBC) is a highly aggressive subtype lacking oestrogen receptor, progesterone receptor, and HER2, and it is easy to metastasize to the nervous system and lungs. 111 which improves the targeting of NK cells to BC cells. At the same time, the expression of co-stimulatory ligands CD86 and 41BBL on BC cells increased, and these ligands bind to homologous receptors CD28 and 41BB on T cells to activate T cells and enhance their function. Tumour necrosis factor α (TNF-α) promotes immune cell apoptosis in the tumour microenvironment, and TNF-α expression is also reduced after CBS and CSE knockdown. 114 In addition, the level of reactive aldehyde (such as 4-hydroxynonenal and malondialdehyde) adducts in BC cells co-cultured with macrophages increases after CBS silencing, resulting in cytotoxicity. 119 The recently discovered novel CSE inhibitor I157172 up-regulates sirtuin 1 (SIRT1) and inhibits the phosphorylation and deacetylation of STAT3, the expression of MMP2/9, p-Akt, and Bcl-2, which in turn inhibits the migration and invasion of BC cell line MCF7. 120 In addition, CTH, an intermediate metabolite of the CBScatalysed synthesis of H 2 S, has recently been found to exert antiapoptotic effects in cells. 121

| Endogenous H 2 S and hepatoma
The role of endogenous H 2 S in hepatoma was shown to be twofold, which appears to be related to the cell type and the reaction mechanism of H 2 S synthase. In addition, a variety of factors also affect hepatoma by regulating endogenous H 2 S. CSE is highly expressed in hepatoma cell lines HepG2 and PLC/PRF/5, but low in Hep3B, and its silencing shows an inhibitory effect in HepG2 and PLC/PRF/5, while the effect on Hep3B was not obvious. Subsequent experiments showed that the tumour suppressor effect caused by the knockdown of CSE was achieved by regulating apoptotic proteins (p53, Bax, Bcl-2, p21, and caspase-3), key proteins of EGFR and MAPK signalling pathways, and increasing the production of ROS to induce RNA damage. 122 High expression and over-activation of indoleamine 2,3-dioxygenase 1 (IDO1) are important reasons for the immune evasion of cancer cells. 123,124 In hepatocellular carcinoma (HCC) patients, the expression of IDO1 is negatively correlated with the expression of CSE. The deletion of H 2 S in CSE À/À mice leads to the increased expression and activity of IDO1. Exogenously added H 2 S downregulates the expression of IDO1 through the NF-κB and STAT3 pathways and inhibits the activity of IDO1 through the nitric oxide synthase/NO pathway. In addition, exogenously added H 2 S also inhibited tumour growth in H22 hepatoma mice by inducing effector T cells and suppressing myeloid-derived suppressor cells. 125 The effect of other factors on cancer may also play a role through the CSE/H 2 S axis. Irradiation increased the long-term migration and invasion ability of HepG2 cells. This effect was due to the increased expression of CBS and CSE caused by radiation, and the activation of epithelialmesenchymal transition (EMT) and P38/MAPK signalling pathways.
After knocking down CBS and CSE, the p38/EMT signalling pathway was inhibited, and the effect was more obvious after knocking down CSE. 126 The PI3K/Akt signalling pathway plays a role in promoting invasive phenotype, malignancy, angiogenesis, and so forth in a variety of cancers. 127,128 The activated PI3K /Akt pathway promotes the occurrence and development of HCC through Sp1-mediated regulation of CSE promoter and protein expression, indicating a positive feedback loop between CSE and PI3K/Akt. 129  acts as a substrate for 3-MST with higher efficiency than cysteine, but its role in cancer is bidirectional. 141,142 In SW480 cells, exogenous supplementation of NAC increased the expression and activity of F I G U R E 2 In addition to the hydrogen sulphide (H 2 S) synthesized by the hepatoma cells themselves, the H 2 S secreted by the hepatic stellate cells also has an effect on the hepatoma cells. CDK4, recombinant cyclin dependent Kinase 4; E2F1, E2F transcription factor 1; FOX3a, forkhead box O3; FOXP3, forkhead box P3; HSC, hepatic stellate cell; IDO1, indoleamine 2, 3 -dioxygenase 1; IL-6, interleukin-6; iNOS, nitric oxide synthase; JNK, c-Jun N-terminal kinase; JunB, JunB Proto-Oncogene; MMP, matrix metallopeptidase; NF-κB, nuclear factor kappa-B; NO, nitric oxide; P27, CDK inhibitor P27; Rb, retinoblastoma; STAT3, signal transducer and activator of transcription 3; TNFSF14, pro-apoptotic factor; VEGF, vascular endothelial growth factor 3-MST and SQR. Since 3-MST exists as a cancer promoter in colorectal cancer, it seems that NAC can lead to the development of colorectal cancer, but whether it can lead to drug resistance in colorectal cancer cells by regulating H 2 S and oxidative stress needs further evaluation. 143 When converted to cysteine, xCT (also known as SLC7A11) acts as a precursor for GSH biosynthesis, and increased xCT expression is associated with chemoresistance and nutrient dependence in a variety of cancers. 144,145 In human colon cancer cells HCT116 and HT29, xCT is highly expressed, and CES-derived H 2 S S-sulfhydrates OTU domain-containing ubiquitin aldehydebinding protein 1 (OTUB1) at Cys91 to regulate its binding to xCT.
Inhibition of CSE attenuates the S-sulfhydration of OTUB1 and reduces xCT production. In addition, the production of GSH and H 2 S was reduced after the inhibition of xCT and CSE, which may lead to cell oxidative stress-induced apoptosis. In vivo experiments also showed that after the knockdown of CSE and xCT, the expression of PCNA was decreased, and the expression of prostaglandinendoperoxide synthase was increased, which significantly inhibited tumour growth. 146 Microorganisms in the gut environment can degrade cysteine to generate H 2 S, resulting in high levels of H 2 S in the gut environment. 147 There may be some crosstalk between these H 2 S and intracellular H 2 S so that the three H 2 S-producing enzymes exhibit such a complex and biphasic role in colorectal cancer (Figure 3).

| Endogenous H 2 S and ovarian cancer
The synthesis of H 2 S in ovarian cancer is mainly regulated by CBS and CBS is highly expressed in both primary epithelial ovarian cancer and ovarian cancer cell lines, and down-regulation of CBS in vitro induces oxidative stress to trigger apoptosis cascade by regulating GSH, ROS, p53, and NF-κB in ovarian cancer cells, and reduces NAD/NADH ratio and ATP production by inhibiting mitochondrial respiration. In vivo CBS silencing inhibits tumour angiogenesis by reducing Ki67 and CD31. Meanwhile, down-regulation of CBS enhances ovarian cancer sensitivity to cisplatin both in vivo and in vitro. 90 Mitomycin 2 (MFN2) plays an important role in cell proliferation and death. 154 High expression of CBS and MFN2 has a poor prognosis for ovarian cancer. Metabolites GSH and H 2 S of CBS can increase the expression of MFN2. 155 Also, Nrf2 enhances the expression of CBS through antioxidant response element (ARE) and high expression of CBS mitigates ferroptosis induced by erastin (xCT-specific inhibitors) in ovarian cancer by regulating S-adenosyl homocysteine, homocysteine, and CTH. 156 In addition, inhibition of CBS-induced and CSE-induced cell death in ES2 cell line. 157 Selenium-containing chrysin can inhibit cancer by inhibiting CBS 158 ( Figure 5).

| Endogenous H 2 S and prostate cancer
The presence of 3-MST was not detected in prostate cancer tissues. 159 However, the expression of the other two H 2 S synthases is also influenced by a variety of factors. Studies have shown that CBS is not detected in benign prostatic epithelium, while low and high levels of CBS are detected in benign hyperplasia prostate cell lines and androgen-dependent prostate cancer cell lines (LNCaP and DU145), respectively, which seems to indicate that high expression of CBS promotes the progression of prostate cancer, while low expression of CBS is found in bone metastatic cell lines of prostate cancer PC-3.
This may also be due to the fact that the expression of CBS is regu-  161 However, it has been reported that H 2 S can increase LNCap mitosis by activating T-type calcium channels, 162 and dihydrotestosterone can down-regulate CBS and CSE expression. 36 In general, H 2 S plays a different role in prostate cancer by regulating different molecules.

| Endogenous H 2 S and other cancers
Endogenous H 2 S also has different effects on other types of cancer.
In non-small cell lung cancer (NSCLC) cell lines A549 and 95D, the suppressing effect. HIF-1α is critical for H 2 S-mediated EMT and angiogenesis, and up-regulation of HIF-1α resulted in up-regulation of CBS and CSE expression followed by increased production of VEGF, PI3K, and p-PI3K, which was reversed by AOAA and PAG. 163 165 In addition, in various types of thyroid cancer, the expression of CBS is up-regulated to varying degrees, and the highly expressed CSE can also activate the hedgehog signalling pathway to promote the occurrence and development of thyroid papillary carcinoma. 45,166 In the adoptive cell transfer mouse model, T cells that overexpressed CSE showed better tumour inhibitory effect than normal T cells, due to the fact that in T cells, the overexpression of CSE does not promote its proliferation and change its phenotype, but rather enhances the inhibition of tumour growth by regulating the concentration of serine, proline, and glycine in the metabolic environment.
Inhibiting tumour growth by changing its microenvironment represents a novel approach for tumour immunotherapy. 167

| Pharmacological inhibitor
The commonly used inhibitors of endogenous H 2 S are usually inhibitors of H 2 S-generating enzymes, mainly PAG, BCA, AOAA, HA, and HMPSNE. AOAA was first thought to be a specific inhibitor of CBS.
Recent studies have found that AOAA can act as a bidirectional inhibitor of CBS and CSE, possibly due to its inhibitory effect on pyridoxal- 5 0 -phosphate, which is a catalytically active cofactor for various enzymes, including CBS and CSE. 168 PAG is an irreversible and specific inhibitor of CSE, but it also inhibits several transamination reactions in muscle and exhibits some nephrotoxicity, and it cannot cross the blood-brain barrier. 168 BCA is a reversible CSE inhibitor with broad bioavailability, but it has inevitable neurotoxicity. 169 HA is a cellular metabolite that can release NO and has antioxidant properties. It can be used as an inhibitor of heme-containing enzymes including CBS, 170 but it has an inhibitory effect on CSE at low concentrations. 171 I157172 is a novel CSE inhibitor that exerts a tumour suppressor effect in BC. 120 HMPSNE is a newly discovered specific inhibitor of 3-MST, which acts on the activated cysteine residues in the active site of 3-MST. 172 In addition, trifluoroalanine can also inhibit CBS and CSE, and aminoethoxyvinylglycine can inhibit CSE, but these two inhibitors have not been used in cancer research. 171  Furthermore, in addition to the endogenous H 2 S in tumour cells themselves, H 2 S in T cells and HSCs, for example, can also exert tumoursuppressive effects by regulating the microenvironment.
In conclusion, although there are various difficulties and challenges, the inhibition of endogenous H 2 S production is a potential cancer treatment.

CONFLICT OF INTEREST STATEMENT
The authors declare that they have no competing interests.

DATA AVAILABILITY STATEMENT
Not applicable.