Lung cancer is a common malignant tumor that seriously threatens human life and health [1]. Its morbidity and mortality are at the top of the list in most countries [2]. As the early clinical symptoms of lung cancer are not obvious, most of the patients are diagnosed with a serious condition. According to the pathological classification, lung cancer can be divided into small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). SCLC is the most malignant type of lung cancer, with an incidence of about 15–20%. NSCLC is mainly divided into squamous cell carcinoma, adenocarcinoma and large cell carcinoma, with an incidence about 80–85% [3,4,5]. At present, the main strategies for the treatment of lung cancer are surgery combined with radiotherapy and chemotherapy; however, the side effects are obvious. Therefore, it is urgent to develop novel methods for the treatment of lung cancer.

H₂S is a new type of gas signal molecule after nitric oxide (NO) and carbon monoxide (CO) [6], and it is a colorless, flammable, and water-soluble gas with a rotten egg smell [7,8,9]. H₂S is mainly produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) [10,11,12]. The third enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST), can also generate endogenous H₂S in the presence of a reductant using 3-mercaptopyruvate (3-MP) as substrate [13,14]. In addition, tobacco cigarette smoke, industrial gases, NaHS, GYY4137, AP39, etc., may produce exogenous H₂S. H₂S is reported to be involved in the development of many diseases [15] and it has both pro-apoptotic and anti-apoptotic effects in cultured cells [16,17]. H₂S can participate in the occurrence and development of tumors through the mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK1/2) pathway, endoplasmic reticulum stress, and ion channels [18,19]. H₂S has been shown to be involved in the occurrence and development of lung cancer [20]. 

H₂S is mainly produced by CBS and CSE [10,11,12]. The third enzyme, 3-MST, can also promote the production of endogenous H₂S from 3-MP in the presence of reducing agents [13,14]. 3-MST exists in both mitochondria and cytoplasm, while CBS and CSE mainly exist in the cytoplasm [21]. Analysis of NSCLC biopsies and adjacent non-tumor tissues showed selectively high levels of endogenous H₂S-producing enzymes, namely CBS, CSE, and 3-MST [22,23].

CSE is a homo-tetramer composed of pyridoxal 5′-phosphate (PLP)-bound 45 kDa subunits, and is the second enzyme to form H₂S in the transsulfuration pathway. CSE mainly decomposes cysteine, a byproduct of CBS, into cysteine, α-ketobutyrate, and ammonia. Like CBS, CSE can also decompose cysteine and produce H₂S (Figure 1). The PLP-CSE interaction is required for enzymic activity [23]. Hypoxia is a typical feature of solid tumors, including NSCLC [24]. CSE activity is a key driver of the transsulfuration pathway, cysteine catabolism, and H₂S production. Tumor angiogenesis is induced by H₂S mediated via hypoxia. H₂S can increase the ability of endothelial cell invasion and duct formation [22]. H₂S also plays a protective role in tumor-induced oxidative stress [25].

The typical role of CSE in the transsulfuration pathway is to cleave cysteine to form cysteine, ammonia, and α-ketobutyrate. Different from CBS, only homocysteine is used as the substrate of CSE in the formation of H₂S [14]. Due to the low specificity of the CSE substrate, cystathionine, cysteine, and homocysteine can be regulated in the same binding capsule, where they compete with PLP to form Schiff base, while CBS has no binding site for PLP; therefore, H₂S produced by CSE is more sensitive to homocysteine [12].

The protein and mRNA levels of CSE in tumor tissues are higher than in adjacent tissues. Compared with the corresponding levels in normal lung epithelial cell line BEAS-2B, NSCLC cell lines (A549 and 95D) showed the selective up-regulation of protein and mRNA expression of all three H₂S-producing enzymes. It can be concluded that NSCLC cells selectively over-express CSE, thus inducing H₂S production and promoting cell proliferation, migration, and invasion [22]. However, some studies have shown that compared with CBS, the clinical correlation between the expression of CSE in tumors and the prognosis of patients is not significant. There is no difference in clinical outcomes between high and low CSE expression in most cancers [26].

CBS is a homologous tetrameric enzyme with about 63 kDa subunits, which binds to two cofactors, PLP and heme [27]. The first and committed step in the transsulfuration pathway of the catalytic conversion of H₂S by CBS is to use homocysteine instead of serine to form cystathionine and water. When the substrate is cysteine instead of serine, the products of the reaction are cysteine and H₂S. CBS also catalyzes other reactions of cysteine to H₂S [14]. Through the gene knockout experiment, it can be concluded that CSE is necessary for the synthesis of cysteine through sulfur transfer, and its gene destruction deprives CBS of one of the substrates needed to produce H₂S. Thus, the disruption of CSE genes affects CSE- and CBS-dependent H₂S synthesis and reduces H₂S production. In contrast, disruption of the CBS gene results in the accumulation of homocysteine, a substrate for H₂S production by CSE [10,14].

CBS exists mainly in the cytoplasm under normal physiological conditions [21]; however, CBS can be transferred to mitochondria in response to hypoxia or ischemia, a process that is partly the result of Lon protease regulating mitochondrial CBS stability [28]. Several studies have shown that hypoxia can be a condition that induces CBS translocation into the mitochondria. In fact, CBS can be detected in both the cytosolic and mitochondrial parts of HCT116 cells and A2780 cells [29,30]. The translocation of CBS to mitochondria may have important implications for the regulation of cancer cell bioenergy and survival [31]. It may be the result of threonine acylation that CBS enters the nucleus [32].

NSCLC cells selectively over-express CBS, which induces H₂S production and promotes cell proliferation, migration, and invasion [22]. For example, in human non-small-cell lung adenocarcinoma, Western blot analysis of tumor tissue compared with normal adjacent lung tissue (n = 20) showed that CBS protein is significantly increased about five-fold in tumor homogenate; furthermore, these tissues also produce approximately two times as much H₂S as their surrounding normal tissues [33]. Increased levels of CBS protein or mRNA have also been reported in two different collections of lung cancer clinical specimens [8]. Hypoxia, glucose deprivation, and hydrogen peroxide treatment have been applied to various HCC and breast cancer cells to generate repaired cells, which are partially resistant to subsequent injury and exhibit the up-regulation of CBS; therefore, in this experimental model, oxidative stress seems to be the most important factor leading to the up-regulation of CBS [34,35]. CBS mRNA and protein levels are slightly up-regulated in chemically hypoxic A549 lung cancer cells treated with cobalt chloride [22]. Lung adenocarcinoma cell lines A549, H522, H1944, Calu-6 cells, A549, and 95D cells are used to examine the functional role of CBS. The results show that the expression of CBS in cancer cell lines is significantly higher than that in the untransformed lung epithelial cell line Beas2B control [8,22,33]. Studies have shown that CBS mRNA is induced in response to the transcription factor Nrf2 [36], and CBS protein levels are regulated by both transcriptional and post-transcriptional processes, including ubiquitination [31].

3-MST is a 33 kDa zinc-dependent enzyme and H₂S-producing enzyme. As early as 1959, Hylin and Wood confirmed that 3-MST could produce polysulfides [37]. Kamoun thought that 3-MST had the ability to produce H₂S as early as 2004. 3-MST is a PLP-independent enzyme that catalyzes L-cysteine to form H₂S. In addition, 3-MST can bind to cysteine aminotransferase in the presence of α-ketoglutarate [38].

3-MST is structurally expressed in all kinds of somatic cells, as well as various cancer cells. Wrobel’s team has performed related earlier studies on the expression of 3-MST in cancer cells: in human tumor cell lines (U373 astrocytoma cell line; SH-SY5Y neuroblastoma cell line; and two melanoma cells lines, A375 and WM35), they found a large amount of 3-MST expression and enzymatic activity. The expression and activity of 3-MST in these cell lines are significantly higher than those of CSE, so it can be concluded that 3-MST is a more important source of H₂S [39,40]. Western blotting indicates [33] that 3-MST expression is either considerably greater or marginally higher in human lung cancer tumors compared with adjacent non-cancer tissues. However, the expression of 3-MST in human papillary thyroid carcinoma tumors is not different from that in surrounding non-cancer tissues [41], and the expression level of 3-MST in renal cell carcinoma tumors is highly variable [42]. At present, a variety of lung adenocarcinoma cell lines (A549 H522~H1944) have been proven to have 3-MST expression/catalytic activity [33,43].

One of the characteristics of lung cancer is angiogenesis, and this process can supply the oxygen and nutrients required for the growth of lung cancer cells; furthermore, it also creates the potential for tumor spread. It is reported that H₂S has the ability to promote new blood vessels [44,45]. Related studies have shown that 3-MST-derived H₂S produced by endothelial cells plays a certain role in vasodilation, endothelial cell proliferation, migration, and angiogenesis, especially under hypoxic conditions [13,46,47,48]. Some studies have shown that silent 3-MST can reduce the proliferation rate of A549 cells (human lung adenocarcinoma cell line) and decrease the repair rate of mitochondrial DNA [37]. To summarize, 3-MST participates in the generation and development of lung cancer and plays an important role. Furthermore, researchers compare CSE, CBS, and 3-MST in Table 1.

CSE and CBS are primarily expressed in human airway smooth muscle cells (SMCs), pulmonary blood vessels and endothelial cells, and the airway SMCs of mouse lung. Some studies have shown a higher expression of CSE in the airway and peripheral lung tissue of rat pulmonary blood vessels [53]. The primary steps in H₂S catabolism include exhalation, lung ventilation, methylation modification, and oxidation. Large quantities of H₂S cause the mitochondria to generate thiosulfate, which is converted to sulfate by rhodanese. Thiol S-methyltransferase, on the other hand, catalyzes the methylation of H₂S in the cytoplasm, where it is transformed into methanethiol and dimethyl sulfide [23]. Methanethiol is converted into dimethyl sulfide by S-methyltransferase. In conclusion, H₂S may be quickly expelled from the body as a gas in the respiratory system and can be eliminated in the urine as sulfate and thiosulfate.