Co-administration of sodium hydrosulfide and tadalafil modulates hypoxia and oxidative stress on bladder dysfunction in a rat model of bladder outlet obstruction.

PURPOSE: This study aimed to assess the possible healing effect of combination treatment with a hydrogen sulfide (H2S) donor, sodium hydrosulfide (NaHS) plus tadalafil on partial bladder outlet obstruction (PBOO)-induced bladder dysfunction. MATERIALS AND METHODS: A total of 75 male Sprague-Dawley rats aged 10-wk and 300-350g were divided into five groups; control; PBOO; PBOO+NaHS (5.6mg/kg/day, i.p., 6-wk); PBOO+tadalafil (2mg/kg/day, oral, 6-wk) and PBOO+NaHS+tadalafil. PBOO was created by partial urethral ligation. 6 weeks after obstruction, the in vitro contractile responses of the detrusor muscle and Western blotting, H2S and malondialdehyde assay were performed in bladder tissues. RESULTS: There was an increase in bladder weight(p<0.001) and a decrease in contractile responses to KCL(p<0.001), carbachol(p<0.01), electrical field stimulation(p<0.05) and ATP (p<0.001) in the detrusor smooth muscle of obstructed rats which was normalized after the combination treatment. Cystathionine γ-lyase and cystathionine ß-synthase, and nuclear factor kappa B protein levels did not significantly differ among groups. The obstruction induced decrement in 3-mercaptopyruvate sulfur transferase protein expression(p<0.001) and H2S levels(p<0.01) as well as increment in protein expressions of neuronal nitric oxide synthase (NO, p<0.001), endothelial NOS (p<0.05), inducible NOS(p<0.001), hypoxia-inducible factor 1-alpha (p<0.01), and malondialdehyde levels (p<0.01), when combined treatment entirely normalized. CONCLUSIONS: Combination therapy has beneficial effects on bladder dysfunction via regulating both H2S and nitric oxide pathways as well as downregulation of oxidative stress and hypoxia. The synergistic effect of H2S and nitric oxide is likely to modulate bladder function, which supports the combined therapy for enhancing clinical outcomes in men with BPH/LUTS.

Enzyme-mediated depletion of serum l-Met abrogates prostate cancer growth via multiple mechanisms without evidence of systemic toxicity.

Extensive studies in prostate cancer and other malignancies have revealed that l-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum l-Met, either via partial dietary restriction or with bacterial l-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial l-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode l-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of l-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum l-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.

De novo engineering of a human cystathionine-γ-lyase for systemic (L)-Methionine depletion cancer therapy.

It has been known for nearly a half century that human tumors, including those derived from the nervous system such as glioblastomas, medulloblastoma, and neuroblastomas are much more sensitive than normal tissues to l-methionine (l-Met) starvation. More recently, systemic l-Met depletion by administration of Pseudomonas putida methionine-γ-lyase (MGL) could effectively inhibit human tumors xenografted in mice. However, bacterial-derived MGLs are unstable in serum (t(1/2) = 1.9 ± 0.2 h) and highly immunogenic in primates. Since the human genome does not encode a human MGL enzyme, we created de novo a methionine degrading enzyme by reengineering the structurally homologous pyridoxal phosphate-dependent human enzyme cystathionine-γ-lyase (hCGL). hCGL degrades l-cystathionine but displays no promiscuous activity toward l-Met. Rational design and scanning saturation mutagenesis led to the generation of a variant containing three amino acid substitutions (hCGL-NLV) that degraded l-Met with a k(cat)/K(M) of 5.6 × 10(2) M(-1) s(-1) and displayed a serum deactivation t(1/2) = 78 ± 5 h (non-PEGylated). In vitro, the cytotoxicity of hCGL-NLV toward 14 neuroblastoma cell lines was essentially indistinguishable from that of the P. putida MGL. Intravenous administration of PEGylated hCGL-NLV in mice reduced serum l-Met from 123 µM to <5 µM for over 30 h. Importantly, treatment of neuroblastoma mouse xenografts with PEGylated hCGL-NLV resulted in near complete cessation of tumor growth. Since the mode of action of hCGL-NLV does not require breaching the blood-brain barrier, this enzyme may have potential application for sensitive tumors that arise from or metastasize to the central nervous system.