Cystathionine-β-Synthase Inhibition for Colon Cancer: Enhancement of the Efficacy of Aminooxyacetic Acid via the Prodrug Approach

Abstract

Colon cancer cells contain high levels of cystathionine-β-synthase (CBS). Its product, hydrogen sulfide (H 2 S), promotes the growth and proliferation of colorectal tumor cells. 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 or AOAA (0.1, 0.5 or 1 mg/kg/d) for 3 wks. Tumor growth was significantly reduced by 9 mg/kg/d AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/d. Thus, the in vivo efficacy of YD0171 is nine times higher than that of AOAA. YD0171 (1 mg/kg/d) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/d) also reduced tumor growth in patient-derived tumor xenograft bearing athymic mice. YD0171 (3 mg/kg/d) induced the regression of established HCT116 tumors in vivo. A 5-d safety study in mice demonstrated that YD0171 at 20 mg/kg/d (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.

Upregulation of Cystathionine-β-Synthase in Colonic Epithelia Reprograms Metabolism and Promotes Carcinogenesis

“…H 2 S levels were determined using a 7-azido-4-methylcoumarin (AzMC) fluorescent probe (Sigma-Aldrich, # L511455) (12). Cells were lysed using non-denaturing lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 1% Triton X-100).…”

H 2 S-induced S -sulfhydration of lactate dehydrogenase a (LDHA) stimulates cellular bioenergetics in HCT116 colon cancer cells

“…The human colorectal carcinoma cell line, HCT116 (ATCC, Manassas, VA; Cat.# CCL-247) was cultured in McCoy’s 5A medium (ATCC) supplemented with 10% FBS, 100 IU/ml penicillin and 100 mg/ml streptomycin as described [1–3]. Cells were grown in a 37°C, 5% CO 2 atmosphere.…”

“…The XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent Technologies, North Billerica, MA) was used to measure bioenergetic function as described [1–3]. Cells were treated with GYY4137 (0.1–1 mM; a slow-releasing H 2 S donor) for 24 h, followed by analysis.…”

“…Cystathionine-β-synthase (CBS) is upregulated and hydrogen sulfide (H 2 S) production is increased in various types of cancer including colon cancer [1–3], ovarian cancer [4], breast cancer [5] and lung cancer [6]. The functional consequence of this response is stimulation of cellular bioenergetics, tumor growth and proliferation (overviewed in [7]).…”

“…Our current studies were inspired by our recent metabolomic analysis in HCT116 colon cancer cells, showing that pharmacological inhibitor of CBS decreases lactate/pyruvate ratio [3], as well as by the direct in vitro cell-free follow-up studies demonstrating that H 2 S donation increases the catalytic activity of LDH [3]. The results of the present studies confirm and extend these findings and unveil complex functional interactions between H 2 S and LDHA in colon cancer cells - both in resting conditions as well as in the presence of oxidative stress.…”