Altered Capacity for H2S Production during the Spontaneous Differentiation of Caco-2 Cells to Colonocytes Due to Reciprocal Regulation of CBS and SELENBP1

Abstract

Hydrogen sulfide (H2S) has been proposed to promote tumor growth. Elevated H2S levels have been detected in human colorectal cancer (CRC) biopsies, resulting from the selective upregulation of cystathionine β-synthase (CBS). In contrast, the recently identified novel H2S-generating enzyme, selenium-binding protein 1 (SELENBP1), is largely suppressed in tumors. Here, we provide the first comparative analysis of the four human H2S-producing enzymes and the key H2S-catabolizing enzyme, sulfide:quinone oxidoreductase (SQOR), in Caco-2 human colorectal adenocarcinoma cells. The gene expression pattern of proliferating Caco-2 cells parallels that of CRC, while confluent cells undergo spontaneous differentiation to a colonocyte-like phenotype. SELENBP1 and SQOR were strongly upregulated during spontaneous differentiation, whereas CBS was downregulated. Cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase remained unaffected. Terminally differentiated cells showed an enhanced capacity to produce H2S from methanethiol and homocysteine. Differentiation induced by exposure to butyrate also resulted in the upregulation of SELENBP1, accompanied by increased SELENBP1 promoter activity. In contrast to spontaneous differentiation, however, butyrate did not cause downregulation of CBS. In summary, SELENBP1 and CBS are reciprocally regulated during the spontaneous differentiation of Caco-2 cells, thus paralleling their opposing regulation in CRC. Butyrate exposure, while imitating some aspects of spontaneous differentiation, does not elicit the same expression patterns of genes encoding H2S-modulating enzymes.

Methanethiol: A Scent Mark of Dysregulated Sulfur Metabolism in Cancer

“…In particular, the ingestion of dietary proteins that contain high amounts of methionine may result in increased methanethiol production by the gut microbiome, whereas activated charcoal and zinc salts have been demonstrated to neutralize sulfur-containing malodorous gases such as methanethiol and H 2 S [ 23 ]. Besides being excreted through flatus and feces, some of the bacteria-derived methanethiol diffuses into the epithelial cells lining the colonic lumen of the human host [ 9 ].…”

“…Cellular differentiation appears to be a major trigger of SELENBP1 expression: both the spontaneous and butyrate-induced differentiation of proliferating Caco-2 cells, a human intestinal adenocarcinoma cell line, to a colonocyte-like phenotype is associated with induction of SELENBP1 gene and protein expression and an increase in MTO activity in the terminally differentiated cells [ 9 , 38 , 39 ]. The downregulation of SELENBP1 via the treatment of Caco-2 cells with small interfering RNA (siRNA) resulted in lowered expression of a differentiation marker of colonic epithelial cells, carcinoembryonic antigen [ 38 ].…”

“…Several strains of gut bacteria may convert dietary methionine to H 2 S and/or methanethiol, thus representing the major source of exposure of humans to both VSCs. In addition, H 2 S and methanethiol are produced endogenously through enzymatic and non-enzymatic synthesis [ 9 , 10 ]. The cancer-associated dysregulation of sulfur metabolism results in excess levels of both VSCs in tumor tissue as well as in body fluids, breath, and/or excretions of cancer patients that are increasingly being exploited for the establishment of convenient non-invasive screening methods to detect early signs of cancer [ 11 , 12 , 13 , 14 ].…”

“…Excess H 2 S may promote cellular dedifferentiation and the growth of transformed cells as well as increase their potential to metastasize and develop resistance against chemotherapeutic agents [ 10 ]. The endogenous production of H 2 S in mammalian cells occurs largely through four enzymes: cystathionine β-synthase (CBS), cystathionine γ-lyase (CTH), 3-mercaptopyruvate sulfurtransferase (MPST), and selenium-binding protein 1 (SELENBP1) [ 9 , 17 , 18 ]. In particular, CBS has been shown to be upregulated in various types of cancer, including colorectal carcinoma (CRC), squamous cell carcinoma, and ovarian, breast, and thyroid cancers [ 10 ].…”

Selenium-binding protein 1 (SELENBP1) is a copper-dependent thiol oxidase

“…In humans, SELENBP1 is ubiquitously expressed, with highest levels in colon, lung and liver [ 3 ]. Numerous observations of its suppression in various tumors and its induction during terminal differentiation gave reason to the assumption that SELENBP1 may interfere with cellular proliferation, thus acting as a tumor suppressor [ [3] , [4] , [5] , [6] ]. In 2018, SELENBP1 was discovered to catalyze a novel enzymatic reaction in humans, acting as a methanethiol oxidase (MTO) that converts methanethiol to hydrogen sulfide (H 2 S), hydrogen peroxide (H 2 O 2 ) and formaldehyde [ 7 ].…”