RESUMO
Colon cancer cells contain high levels of cystathionine-beta-synthase (CBS). Its product, hydrogen sulfide (H2S) promotes the growth and proliferation of colorectal tumor cells. In order to improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle, AOAA (1, 3 or 9 mg/kg/day) or YD0171 (0.1, 0.5 or 1 mg/kg/day) for 3 weeks. Tumor growth was significantly reduced by 9 mg/kg/day AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/day. Thus, the in vivo efficacy of YD0171 is 9-times higher than that of AOAA. YD0171 (1 mg/kg/day) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/day) also reduced tumor growth in patient-derived tumor xenograft (PDTX) bearing athymic mice. YD0171 (3 mg/kg/day) induced the regression of established HCT116 tumors in vivo. A 5-day safety study in mice demonstrated that YD0171 at 20 mg/kg/day (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities.
RESUMO
Cystathionine-ß-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the 'Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: â¼60µM), tannic acid (IC50: â¼40µM) and benserazide (IC50: â¼30µM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: â¼3µM) and NSC67078 (IC50: â¼1µM), while aurintricarboxylic acid (IC50: â¼3µM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: â¼1µM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of â¼6µM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: â¼6µM), tannic acid (IC50: â¼20µM), benserazide (IC50: â¼20µM), and NSC67078 (IC50: â¼0.3µM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: â¼300µM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300µM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300µM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100µM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50mg/kg/days.q.) prevented tumor growth. In silico docking simulations showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.
Assuntos
Benserazida/farmacologia , Neoplasias do Colo/tratamento farmacológico , Cistationina beta-Sintase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cumarínicos/farmacologia , Reposicionamento de Medicamentos/métodos , Metabolismo Energético/efeitos dos fármacos , Feminino , Células HCT116 , Células HT29 , Humanos , Hidrazinas/farmacologia , Sulfeto de Hidrogênio/metabolismo , Masculino , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Morfolinas/farmacologia , Compostos Organotiofosforados/farmacologia , Terapias em Estudo/métodosRESUMO
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.
Assuntos
Ácido Amino-Oxiacético/farmacologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Cistationina beta-Sintase/antagonistas & inibidores , Pró-Fármacos/farmacologia , Animais , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
BACKGROUND/AIM: Patient-derived xenografting (PDX) of human colorectal cancer (CRC) is the preferred experimental model to study tumor response to therapeutic agents. Gradually, human stromal cells are replaced by mouse stromal cells; however, the exact timing of the replacement of human with murine stromal cells in human CRC xenograft has not been fully elucidated. We hypothesize that orthologous murine transcripts functionally substitutes for the loss due to replacement of human stromal genes. MATERIALS AND METHODS: Human CRC were implanted in athymic nude mice in replicates and followed-up over time. Using next-generation sequencing, we determined the temporal kinetics of human stromal cell replacement with the orthologous murine transcripts. RESULTS: CRC cell-induced re-organization of the normal, quiescent murine stromal cells into a protumorigenic phenotype supporting human CRC growth occurs at initial implantation. CONCLUSION: Murine cell replacement occurs in a time- and size-dependent manner.
Assuntos
Neoplasias Colorretais/patologia , Células Estromais/patologia , Animais , Feminino , Xenoenxertos/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Transplante Heterólogo/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The trans-sulfuration enzyme cystathionine-ß-synthase (CBS) and its product hydrogen sulfide (H2S) are aberrantly upregulated in colorectal cancers, where they contribute to tumor growth and progression by both autocrine and paracrine mechanisms. However, it is unknown whether the CBS/H2S axis plays a role in colorectal carcinogenesis. Here, we report upregulation of CBS in human biopsies of precancerous adenomatous polyps and show that forced upregulation of CBS in an adenoma-like colonic epithelial cell line is sufficient to induce metabolic and gene expression profiles characteristic of colorectal cancer cells. Differentially expressed metabolites (65 increased and 20 decreased) clustered into the glycolytic pathway, nucleotide sugars, intermediates of the pentose phosphate pathway, and lipogenesis, including primarily phospholipids, sphingolipids, and bile acids. CBS upregulation induced broad changes in the NCM356 cell transcriptome with over 350 differentially expressed genes. These genes overlapped significantly with gene sets related to glycolysis, hypoxia, and a colon cancer cell phenotype, including genes regulated by NF-κB, KRAS, p53, and Wnt signaling, genes downregulated after E-cadherin knockdown, and genes related to increased extracellular matrix, cell adhesion, and epithelial-to-mesenchymal transition. The CBS-induced switch to an anabolic metabolism was associated with increased NCM356 cell bioenergetics, proliferation, invasion through Matrigel, resistance to anoikis, and CBS-dependent tumorigenesis in immunocompromised mice. Genetic ablation of CBS in CBS heterozygous mice (CBS+/- ) reduced the number of mutagen-induced aberrant colonic crypt foci. Taken together, these results establish that activation of the CBS/H2S axis promotes colon carcinogenesis. Cancer Res; 77(21); 5741-54. ©2017 AACR.
Assuntos
Pólipos Adenomatosos/genética , Colo/metabolismo , Cistationina beta-Sintase/genética , Mucosa Intestinal/metabolismo , Regulação para Cima , Pólipos Adenomatosos/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular , Movimento Celular/genética , Colo/patologia , Cistationina beta-Sintase/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Sulfeto de Hidrogênio/metabolismo , Mucosa Intestinal/patologia , Masculino , Metabolômica/métodos , Camundongos Knockout , Camundongos Nus , Transplante HeterólogoRESUMO
Therapeutic manipulation of the gasotransmitter hydrogen sulfide (H2S) has recently been proposed as a novel targeted anticancer approach. Here we show that human lung adenocarcinoma tissue expresses high levels of hydrogen sulfide (H2S) producing enzymes, namely, cystathionine beta-synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), in comparison to adjacent lung tissue. In cultured lung adenocarcinoma but not in normal lung epithelial cells elevated H2S stimulates mitochondrial DNA repair through sulfhydration of EXOG, which, in turn, promotes mitochondrial DNA repair complex assembly, thereby enhancing mitochondrial DNA repair capacity. In addition, inhibition of H2S-producing enzymes suppresses critical bioenergetics parameters in lung adenocarcinoma cells. Together, inhibition of H2S-producing enzymes sensitize lung adenocarcinoma cells to chemotherapeutic agents via induction of mitochondrial dysfunction as shown in in vitro and in vivo models, suggesting a novel mechanism to overcome tumor chemoresistance.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Antineoplásicos/uso terapêutico , Reparo do DNA , Metabolismo Energético , Sulfeto de Hidrogênio/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Mitocôndrias/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Antineoplásicos/farmacologia , Reparo do DNA/efeitos dos fármacos , DNA Mitocondrial/genética , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , Células Tumorais CultivadasRESUMO
BACKGROUND: Historically, strategies to reduce acute rejection and improve graft survival in kidney transplant recipients included blood transfusions (BTs) before transplantation. While advents in recipient immunosuppression strategies have replaced this practice, the impact of BTs in the organ donor on recipient graft outcomes has not been evaluated. We hypothesize that BTs in organ donors after neurologic determination of death (DNDDs) translate into improved recipient renal graft outcomes, as measured by a decrease in delayed graft function (DGF). METHODS: Donor demographics, critical care end points, the use of BTs, and graft outcome data were prospectively collected on DNDDs from March 2012 to October 2013 in the United Network for Organ Sharing Region 5 Donor Management Database. Propensity analysis determined each DNDD's probability of receiving packed red blood cells based on demographic and critical care data as well as provider bias. The primary outcome measure was the rate of DGF (dialysis in the first week after transplantation) in different donor BT groups as follows: no BT, any BT, 1 to 5, 6 to 10, or greater than 10 packed red blood cell units. Regression models determined the relationship between donor BTs and recipient DGF after accounting for known predictors of DGF as well as the propensity to receive a BT. RESULTS: Data were complete for 1,884 renal grafts from 1,006 DNDDs; 52% received any BT, 32% received 1 to 5 U, 11% received 6 to 10, and 9% received greater than 10 U of blood. Grafts from transfused donors had a lower rate of DGF compared with those of the nontransfused donors (26% vs. 34%, p < 0.001). After adjusting for known confounders, grafts from donors with any BT had a lower odds of DGF (odds ratio, 0.76; p = 0.030), and this effect was greatest in those with greater than 10 U transfused. CONCLUSION: Any BT in a DNDD was associated with a 23% decrease in the odds of recipients developing DGF, and this effect was more pronounced as the number of BTs increased. LEVEL OF EVIDENCE: Therapeutic study, level III; epidemiologic/prognostic study, level II.