ETHE1 overexpression promotes SIRT1 and PGC1α mediated aerobic glycolysis, oxidative phosphorylation, mitochondrial biogenesis and colorectal cancer.

Ethylmalonic Encephalopathy Protein 1 (ETHE1) is a sulfur dioxygenase that regulates cellular H2S levels. We previously demonstrated a significant increase of ETHE1 expression in "single-hit" colon epithelial cells from crypts of patients with Familial Adenomatous Polyposis (FAP). Here, we report elevated levels of ETHE1 expression and increased mitochondrial density occurring in-situ in phenotypically normal FAP colorectal mucosa. We also found that constitutive expression of ETHE1 increased aerobic glycolysis ("Warburg effect"), oxidative phosphorylation, and mitochondrial biogenesis in colorectal cancer (CRC) cell lines, thereby depleting H2S which relieved the inhibition of phosphodiesterase (PDE), and increased adenosine monophosphate (AMP) levels. This led to activation of the energy sensing AMP-activated protein kinase (AMPKp), Sirtuin1 (SIRT1) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a master regulator of mitochondrial biogenesis. By contrast, shRNA silencing of ETHE1 reduced PDE activity, AMPKp/SIRT1/PGC1α levels and mitochondrial biogenesis. Constitutive expression of ETHE1 accelerated both CRC cell xenograft and orthotopic patient derived xenograft CRC cell growth in vivo. Overall, our data nominate elevated ETHE1 expression levels as a novel biomarker and potential therapeutic target for the prevention of CRC tumorigenesis.

High-fructose corn syrup enhances intestinal tumor growth in mice.

Excessive consumption of beverages sweetened with high-fructose corn syrup (HFCS) is associated with obesity and with an increased risk of colorectal cancer. Whether HFCS contributes directly to tumorigenesis is unclear. We investigated the effects of daily oral administration of HFCS in adenomatous polyposis coli (APC) mutant mice, which are predisposed to develop intestinal tumors. The HFCS-treated mice showed a substantial increase in tumor size and tumor grade in the absence of obesity and metabolic syndrome. HFCS increased the concentrations of fructose and glucose in the intestinal lumen and serum, respectively, and the tumors transported both sugars. Within the tumors, fructose was converted to fructose-1-phosphate, leading to activation of glycolysis and increased synthesis of fatty acids that support tumor growth. These mouse studies support the hypothesis that the combination of dietary glucose and fructose, even at a moderate dose, can enhance tumorigenesis.

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.

More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/Kras(G12D) mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations.

S-sulfhydration/desulfhydration and S-nitrosylation/denitrosylation: a common paradigm for gasotransmitter signaling by H2S and NO.

Sulfhydryl groups on protein Cys residues undergo an array of oxidative reactions and modifications, giving rise to a virtual redox zip code with physiological and pathophysiological relevance for modulation of protein structure and functions. While over two decades of studies have established NO-dependent S-nitrosylation as ubiquitous and fundamental for the regulation of diverse protein activities, proteomic methods for studying H2S-dependent S-sulfhydration have only recently been described and now suggest that this is also an abundant modification with potential for global physiological importance. Notably, protein S-sulfhydration and S-nitrosylation bear striking similarities in terms of their chemical and biological determinants, as well as reversal of these modifications via group-transfer to glutathione, followed by the removal from glutathione by enzymes that have apparently evolved to selectively catalyze denitrosylation and desulfhydration. Here we review determinants of protein and low-molecular-weight thiol S-sulfhydration/desulfhydration, similarities with S-nitrosylation/denitrosylation, and methods that are being employed to investigate and quantify these gasotransmitter-mediated cell signaling systems.

Inhibitory substrate binding site of human indoleamine 2,3-dioxygenase.

Human indoleamine 2,3-dioxygenase (hIDO) is an intracellular heme-containing enzyme, which catalyzes the initial and rate-determining step of L-tryptophan (L-Trp) metabolism via the kynurenine pathway. Due to its immunosuppressive function, hIDO has been recognized as an important drug target for cancer. Here we report evidence supporting the presence of an inhibitory substrate binding site (S(i)) in hIDO that is capable of binding molecules with a wide variety of structures, including substrates (L-Trp and 1-methyl-L-tryptophan), an effector (3-indole ethanol), and an uncompetitive inhibitor (Mitomycin C). The data offer useful guidelines for future development of more potent hIDO inhibitors; they also call for the re-evaluation of the action mechanism of Mitomycin C (MtoC), a widely used antitumor chemotherapeutic agent.

Redox cycling of iron complexes of N-(dithiocarboxy)sarcosine and N-methyl-D-glucamine dithiocarbamate.

Iron(II)-dithiocarbamate complexes are used to trap nitrogen monoxide in biological samples, and the resulting nitrosyliron(II)-dithiocarbamate is detected and quantified by ESR. As the chemical properties of these compounds have been little studied, we investigated whether iron dithiocarbamate complexes can redox cycle. The electrode potentials of iron complexes of N-(dithiocarboxy)sarcosine (dtcs) and N-methyl-d-glucamine dithiocarbamate (mgd) are 56 and -25 mV at pH 7.4, respectively, as measured by cyclic voltammetry. The autoxidation and Fenton reaction of iron(II)-dtcs and iron(II)-mgd were studied by stopped-flow spectrophotometry with both iron(II) complexes and dioxygen or hydrogen peroxide in excess. In the case of excess iron(II)-dtcs and -mgd complexes, the rate constants of the autoxidation and the Fenton reaction are (1.6-3.2) x 10(4) and (0.7-1.1) x 10(5) M(-1) s(-1), respectively. In the presence of nitrogen monoxide, the oxidation of iron(II)-dtcs and iron(II)-mgd by hydrogen peroxide is significantly slower (ca. 10-15 M(-1) s(-1)). The physiological reductants ascorbate, cysteine, and glutathione efficiently reduce iron(III)-dtcs and iron(III)-mgd. Therefore, iron bound to dtcs and mgd can redox cycle between iron(II) and iron(III). The ligands dtcs and mgd are slowly oxidized by hydrogen peroxide with rate constants of 5.0 and 3.8 M(-1) s(-1), respectively.

Radiosensitization mechanism of riboflavin in vitro.

Riboflavin, suggested to be a radiosensitizer, was studied in murine thymocytes and human hepatoma L02 cell line in vitro with MTT method and fluorescence microscopy. When the murine thymocytes treated with 5-400 mumol/L riboflavin were irradiated by 5 Gy (60)Co gamma ionizing radiation, the low concentration groups, i.e. treated with 5-50 mumol/L riboflavin, showed a different surviving fractions-time relating correlation compared with the high concentration groups, i.e. treated with 100-400 mumol/L riboflavin. The former had a high survival level at the end of irradiation, but which, after 4-h incubation, decreased rapidly to a low level. On the contrary, the high concentration groups showed a low survival level at the end of irradiation, and a poor correlation was found between the surviving fraction and the incubation time, after 4 h a little difference was observed. The results of fluorescence microscopy indicated that under low concentration conditions, the riboflavin localized mainly in nucleus (both perinuclear area and inside of nuclear membrane), while under high concentration conditions, intensive riboflavin also localized around cytoplasmic membranes. Thus we can conclude: the riboflavin had radiosensitivity effect on DNA under low concentration conditions, and enhanced the damage to cytoplasmic membrane under high concentration conditions. Also the most effective concentration of riboflavin can be evaluated to be approximate 100 mumol/L.