Efficacy of Novel Aminooxyacetic Acid Prodrugs in Colon Cancer Models: Towards Clinical Translation of the Cystathionine ß-Synthase Inhibition Concept.

Upregulation of hydrogen sulfide (H2S) biosynthesis, at least in part related to the upregulation of cystathionine ß-synthetase (CBS) in cancer cells, serves as a tumor-promoting factor and has emerged as a possible molecular target for antitumor drug development. To facilitate future clinical translation, we have synthesized a variety of novel CBS-targeting, esterase-cleavable prodrugs based on the structure of the prototypical CBS inhibitor aminooxyacetic acid (AOAA). The pharmacological properties of these compounds were evaluated in cell-free assays with recombinant human CBS protein, the human colon cancer cell line HCT116, and in vivo using various tumor-bearing mice models. The prodrug YD0251 (the isopropyl ester derivative of AOAA) was selected for detailed characterization. YD0251 exhibits improved antiproliferative efficacy in cell culture models when compared to AOAA. It is up to 18 times more potent than AOAA at suppressing HCT116 tumor growth in vivo and is effective when administered to tumor-bearing mice either via subcutaneous injection or oral gavage. Patient-derived xenografts (PDTXs) with higher levels of CBS protein grew significantly larger than tumors with lower levels, and YD0251 treatment inhibited the growth of PDTXs with elevated CBS, whereas it had no significant effect on PDTXs with low CBS protein levels. The toxicity of YD0251 was assessed in mice subjected to subchronic administration of supratherapeutic doses the inhibitor; no significant alteration in circulating markers of organ injury or histopathological alterations were noted, up to 60 mg/kg/day × 5 days. In preparation to a future theranostic concept (to match CBS inhibitor therapy to high-CBS expressors), we identified a potential plasma marker of CBS-expressing tumors. Colon cancer cells produced significant levels of lanthionine, a rare metabolic intermediate of CBS-mediated H2S biosynthesis; forced expression of CBS into non-transformed epithelial cells increased lanthionine biogenesis in vitro and in vivo (measured in the urine of tumor-bearing mice). These current results may be useful to facilitate the translation of a CBS inhibition-based antitumor concept into the clinical space.

Drug resistance induces the upregulation of H2S-producing enzymes in HCT116 colon cancer cells.

Hydrogen sulfide (H2S) production in colon cancer cells supports cellular bioenergetics and proliferation. The aim of the present study was to investigate the alterations in H2S homeostasis during the development of resistance to 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent. A 5-FU-resistant HCT116 human colon cancer cell line was established by serial passage in the presence of increasing 5-FU concentrations. The 5-FU-resistant cells also demonstrated a partial resistance to an unrelated chemotherapeutic agent, oxaliplatin. Compared to parental cells, the 5-FU-resistant cells rely more on oxidative phosphorylation than glycolysis for bioenergetic function. There was a significant increase in the expression of the drug-metabolizing cytochrome P450 enzymes CYP1A2 and CYP2A6 in 5-FU-resistant cells. The CYP450 inhibitor phenylpyrrole enhanced 5-FU-induced cytotoxicity in 5-FU-resistant cells. Two major H2S-generating enzymes, cystathionine-ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST) were upregulated in the 5-FU-resistant cells. 5-FU-resistant cells exhibited decreased sensitivity to the CBS inhibitor aminooxyacetate (AOAA) in terms of suppression of cell viability, inhibition of cell proliferation and inhibition of oxidative phosphorylation. However, 5FU-resistant cells remained sensitive to the antiproliferative effect of benserazide (a recently identified, potentially repurposable CBS inhibitor). Taken together, the current data suggest that 5-FU resistance in HCT116 cells is associated with the upregulation of drug-metabolizing enzymes and an enhancement of endogenous H2S production. The anticancer effect of prototypical H2S biosynthesis inhibitor AOAA is impaired in 5-FU-resistant cells, but benserazide remains efficacious. Pharmacological approaches aimed at restoring the sensitivity of 5-FU-resistant cells to chemotherapeutic agents may be useful in the formulation of novel therapeutic strategies against colorectal cancer.

H2S-induced S-sulfhydration of lactate dehydrogenase a (LDHA) stimulates cellular bioenergetics in HCT116 colon cancer cells.

Cystathionine-ß-synthase (CBS) is upregulated and hydrogen sulfide (H2S) production is increased in colon cancer cells. The functional consequence of this response is stimulation of cellular bioenergetics and tumor growth and proliferation. Lactate dehydrogenase A (LDHA) is also upregulated in various colon cancer cells and has been previously implicated in tumor cell bioenergetics and proliferation. In the present study, we sought to determine the potential interaction between the H2S pathway and LDH activity in the control of bioenergetics and proliferation of colon cancer, using the colon cancer line HCT116. Low concentrations of GYY4137 (a slow-releasing H2S donor) enhanced mitochondrial function (oxygen consumption, ATP production, and spare respiratory capacity) and glycolysis in HCT116 cells. SiRNA-mediated transient silencing of LDHA attenuated the GYY4137-induced stimulation of mitochondrial respiration, but not of glycolysis. H2S induced the S-sulfhydration of Cys163 in recombinant LDHA, and stimulated LDHA activity. The H2S-induced stimulation of LDHA activity was absent in C163A LDHA. As shown in HCT116 cell whole extracts, in addition to LDHA activation, GYY4137 also stimulated LDHB activity, although to a smaller extent. Total cellular lactate and pyruvate measurements showed that in HCT116 cells LDHA catalyzes the conversion of pyruvate to lactate. Total cellular lactate levels were increased by GYY4137 in wild-type cells (but not in cells with LDHA silencing). LDHA silencing sensitized HCT116 cells to glucose oxidase (GOx)-induced oxidative stress; this was further exacerbated with GYY4137 treatment. Treatment with low concentrations of GYY4137 (0.3mM) or GOx (0.01U/ml) significantly increased the proliferation rate of HCT116 cells; the effect of GOx, but not the effect of GYY4137 was attenuated by LDHA silencing. The current report points to the involvement of LDHA in the stimulatory effect of H2S on mitochondrial respiration in colon cancer cells and characterizes some of the functional interactions between LDHA and H2S-stimulated bioenergetics under resting conditions, as well as during oxidative stress.

S-Sulfhydration of ATP synthase by hydrogen sulfide stimulates mitochondrial bioenergetics.

Mammalian cells can utilize hydrogen sulfide (H2S) to support mitochondrial respiration. The aim of our study was to explore the potential role of S-sulfhydration (a H2S-induced posttranslational modification, also known as S-persulfidation) of the mitochondrial inner membrane protein ATP synthase (F1F0 ATP synthase/Complex V) in the regulation of mitochondrial bioenergetics. Using a biotin switch assay, we have detected S-sulfhydration of the α subunit (ATP5A1) of ATP synthase in response to exposure to H2S in vitro. The H2S generator compound NaHS induced S-sulfhydration of ATP5A1 in HepG2 and HEK293 cell lysates in a concentration-dependent manner (50-300µM). The activity of immunocaptured mitochondrial ATP synthase enzyme isolated from HepG2 and HEK293 cells was stimulated by NaHS at low concentrations (10-100nM). Site-directed mutagenesis of ATP5A1 in HEK293 cells demonstrated that cysteine residues at positions 244 and 294 are subject to S-sulfhydration. The double mutant ATP synthase protein (C244S/C294S) showed a significantly reduced enzyme activity compared to control and the single-cysteine-mutated recombinant proteins (C244S or C294S). To determine whether endogenous H2S plays a role in the basal S-sulfhydration of ATP synthase in vivo, we compared liver tissues harvested from wild-type mice and mice deficient in cystathionine-gamma-lyase (CSE, one of the three principal mammalian H2S-producing enzymes). Significantly reduced S-sulfhydration of ATP5A1 was observed in liver homogenates of CSE-/- mice, compared to wild-type mice, suggesting a physiological role for CSE-derived endogenous H2S production in the S-sulfhydration of ATP synthase. Various forms of critical illness (including burn injury) upregulate H2S-producing enzymes and stimulate H2S biosynthesis. In liver tissues collected from mice subjected to burn injury, we detected an increased S-sulfhydration of ATP5A1 at the early time points post-burn. At later time points (when systemic H2S levels decrease) S-sulfhydration of ATP5A1 decreased as well. In conclusion, H2S induces S-sulfhydration of ATP5A1 at C244 and C294. This post-translational modification may be a physiological mechanism to maintain ATP synthase in a physiologically activated state, thereby supporting mitochondrial bioenergetics. The sulfhydration of ATP synthase may be a dynamic process, which may be regulated by endogenous H2S levels under various pathophysiological conditions.

Stimulatory effect of CSE-generated H2S on hepatic mitochondrial biogenesis and the underlying mechanisms.

We previously showed that hydrogen sulfide (H2S) upregulates peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α in primary hepatocytes. PGC-1α is a crucial regulator of mitochondrial biogenesis, a process required to maintain cellular energy homeostasis. We investigated the regulation of hepatic mitochondrial biogenesis by cystathionine γ-lyase (CSE)-generated H2S under physiological conditions. Primary hepatocytes isolated from CSE knockout (KO) and wild-type (WT) mice were used in all experiments. Mitochondrial DNA (mtDNA) and mRNA levels were measured via real-time PCR. Protein S-sulfhydration was determined via a modified biotin switch assay. MitoTracker Green was used to quantify mitochondrial content and distribution. CSE-KO hepatocytes produced less mtDNA compared to WT hepatocytes. Mitochondrial content was reduced in CSE-KO hepatocytes compared to WT hepatocytes, which was restored with NaHS (an H2S donor) treatment. CSE-KO hepatocytes exhibited lower levels of mitochondrial transcription factors and the mitochondrial transcription coactivator, peroxisome proliferator-activated receptor-γ coactivator-related protein (PPRC) compared to WT hepatocytes. NaHS administration upregulated PPRC, yet downregulated PGC-1ß protein level in mouse hepatocytes. Exogenous H2S induced the S-sulfhydration of PPRC, which was lower in untreated CSE-KO hepatocytes, but not that of PGC-1ß. Finally, knockdown of either PGC-1α or PPRC significantly decreased NaHS-stimulated mitochondrial biogenesis in hepatocytes, where knockdown of both genes were required to abolish NaHS-induced mitochondrial biogenesis. Endogenous H2S-induced liver mitochondrial biogenesis is dependent upon PGC-1α and PPRC signaling in primary hepatocytes. This study may offer clues to the regulation of energy homeostasis under physiological conditions as well as mitochondrial dysregulation.

Decreased Gluconeogenesis in the Absence of Cystathionine Gamma-Lyase and the Underlying Mechanisms.

AIMS: To investigate the regulation of hepatic glucose production by cystathionine γ-lyase (CSE)-generated hydrogen sulfide (H2S) in hepatic glucose production under physiological conditions. RESULTS: We found that CSE knockout (KO) mice had a reduced rate of gluconeogenesis, which was reversed by administration of NaHS (an H2S donor) (i.p.). Interestingly, isolated CSE KO hepatocytes exhibited a reduced glycemic response to chemical-induced activation of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and glucocorticoid pathways compared with wild-type (WT) hepatocytes. Treatment with the inhibitors for PKA (KT5720) or glucocorticoid receptor (GR) (RU-486) significantly reduced H2S-stimulated glucose production from both WT and CSE KO mouse hepatocytes. NaHS treatment upregulated the protein levels of key gluconeogenic transcription factors, such as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and CCAAT-enhancer-binding protein-ß (C/EBP-ß). Moreover, exogenous H2S augmented the S-sulfhydration of the rate-limiting gluconeogenic enzymes and PGC-1α and increased their activities, which were lower in untreated CSE KO hepatocytes. Finally, knockdown of PGC-1α, but not C/EBP-ß, significantly decreased NaHS-induced glucose production from the primary hepatocytes. INNOVATION: This study demonstrates the stimulatory effect of endogenous H2S on liver glucose production and reveals three underlying mechanisms; that is, H2S upregulates the expression levels of PGC-1α and phosphoenolpyruvate carboxykinase via the GR pathway; H2S upregulates the expression level of PGC-1α through the activation of the cAMP/PKA pathway as well as PGC-1α activity via S-sulfhydration; and H2S upregulates the expression and the activities (by S-sulfhydration) of glucose-6-phosphatase and fructose-1,6-bisphosphatase. CONCLUSION: This study may offer clues for the homeostatic regulation of glucose metabolism under physiological conditions and its dysregulation in metabolic syndrome.

Increased renal methylglyoxal formation with down-regulation of PGC-1α-FBPase pathway in cystathionine γ-lyase knockout mice.

We have previously reported that hydrogen sulfide (H(2)S), a gasotransmitter and vasodilator has cytoprotective properties against methylglyoxal (MG), a reactive glucose metabolite associated with diabetes and hypertension. Recently, H(2)S was shown to up-regulate peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, a key gluconeogenic regulator that enhances the gene expression of the rate-limiting gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase). Thus, we sought to determine whether MG levels and gluconeogenic enzymes are altered in kidneys of 6-22 week-old cystathionine γ-lyase knockout (CSE(-/-); H(2)S-producing enzyme) male mice. MG levels were determined by HPLC. Plasma glucose levels were measured by an assay kit. Q-PCR was used to measure mRNA levels of PGC-1α and FBPase-1 and -2. Coupled-enzymatic assays were used to determine FBPase activity, or triosephosphate levels. Experimental controls were either age-matched wild type mice or untreated rat A-10 cells. Interestingly, we observed a significant decrease in plasma glucose levels along with a significant increase in plasma MG levels in all three age groups (6-8, 14-16, and 20-22 week-old) of the CSE(-/-) mice. Indeed, renal MG and triosephosphates were increased, whereas renal FBPase activity, along with its mRNA levels, were decreased in the CSE(-/-) mice. The decreased FBPase activity was accompanied by lower levels of its product, fructose-6-phosphate, and higher levels of its substrate, fructose-1,6-bisphosphate in renal extracts from the CSE(-/-) mice. In agreement, PGC-1α mRNA levels were also significantly down-regulated in 6-22 week-old CSE(-/-) mice. Furthermore, FBPase-1 and -2 mRNA levels were reduced in aorta tissues from CSE(-/-) mice. Administration of NaHS, a H(2)S donor, increased the gene expression of PGC-1α and FBPase-1 and -2 in cultured rat A-10 cells. In conclusion, overproduction of MG in CSE(-/-) mice is due to a H(2)S-mediated down-regulation of the PGC-1α-FBPase pathway, further suggesting the important role of H(2)S in the regulation of glucose metabolism and MG generation.