Degradation of 2-(3-aminopropylamino)-ethanethiol (WR-1065) by Cu-dependent amine oxidases and influence on glutathione status of chinese hamster ovary cells

doi:10.1016/0006-2952(95)00164-U

Biochemical Pharmacology

Volume 50, Issue 4, 8 August 1995, Pages 489-496

https://doi.org/10.1016/0006-2952(95)00164-U Get rights and content

Abstract

The radioprotective drug 2-(3-aminopropylamino)ethanethiol (WR-1065) can be degraded when incubated in cell culture medium in vitro. The degradation reaction consumes oxygen and results from the action of Cu-dependent amine oxidases present in the serum content of the medium. Analysis of the degradation products of WR-1065 demonstrates the formation of cysteamine, acrolein and H₂O₂. WR-2721, the inactive prodrug of WR-1065, is not a substrate for these enzymes. Extracellular degradation of WR-1065 by Cu-dependent amine oxidases leads to an intracellular depletion of glutathione (GSH) in Chinese hamster ovary (CHO) cells and to reduction of clonogenic cell survival. Addition of aminoguanidine, an inhibitor of Cu-dependent amine oxidases, protects CHO cells from the toxic effects of WR-1065 and under these conditions an increase of intracellular GSH levels occurs. These data demonstrate that WR-1065 can be degraded to toxic compounds by the presence of Cu-dependent amine oxidases which might have further implications for the clinical use of WR-2721.

References (52)

D Glover et al.
Phase I trials of WR-2721 and cis-platinum
Int J Radiat Oncol Biol Phys
(1984)
JH Glick et al.
Phase I controlled trials of WR-2721 and cyclophosphamide
Int J Radiat Oncol Biol Phys
(1984)
EP McGovern et al.
HPLC assay for 2-(3-aminopropylamino)ethanethiol (WR-1065) in plasma
Int J Radiat Oncol Biol Phys
(1984)
JF Utley et al.
Pharmacokinetics of WR-1065 in mouse tissue following treatment with WR-2721
Int J Radiat Oncol Biol Phys
(1984)
N Seiler et al.
Determination of amino acids by separation of their ion pairs with dodecyl sulphate
J Chromatogr
(1985)
DJ Reed et al.
High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides
Anal Biochem
(1980)
RD Issels et al.
Influence of thiols on thermosensitivity of mammalian cells in vitro
Meth Enzymol
(1990)
OH Lowry et al.
Protein measurement with the Folin phenol reagent
J Biol Chem
(1951)
G Witz
Biological interactions of alpha,beta-unsaturated aldehydes
Free Radic Biol Med
(1989)
RD Issels et al.
Promotion of cystine uptake and its utilization for glutathione biosynthesis induced by cysteamine and N-acetylcysteine
Biochem Pharmacol
(1988)

LM Shaw et al.

Human pharmacokinetics of WR-2721

Int J Radiat Oncol Biol Phys

(1986)

KW Anderson et al.

Analysis of S-35 labeled WR-2721 and its metabolites in biological fluids

Int J Radiat Oncol Biol Phys

(1984)

JD Butler et al.

Cystine depletion by WR-1065 in cystinotic cells. Mechanism of action

Biochem Pharmacol

(1985)

W Gahl et al.

Reversal by aminoguanidine of the inhibition of proliferation of human fibroblasts by spermidine and spermine

Chem Biol Interact

(1978)

N Seiler

Inhibition of Enzymes Oxidizing Polyamines

B Mondovi et al.

Purification of Bovine Plasma Amine Oxidase

Meth Enzymol

(1983)

RA Alarcon

Acrolein. IV. Evidence for the formation of the cytotoxic aldehyde acrolein from enzymatically oxidized spermine or spermidine

Arch Biochem Biophys

(1970)

H Esterbauer et al.

The reaction of cysteine with α,β-unsaturated aldehydes

Tetrahedron

(1976)

N Brock et al.

The development of mesna for regional detoxification

Cancer Treat Rev

(1983)

JM Patel et al.

Acrolein-induced injury of cultured pulmonary artery endothelial cells

Toxicol Appl Pharmacol

(1993)

G Multhoff et al.

Differential effects of ifosfamide on the capacity of cytotoxic T lymphocytes and natural killer cells to lyse their target cells correlate with intracellular glutathione levels

Blood

(1995)

NF Schor

Depletion of glutathione by the radio-protective agent S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR2721)

Biochem Pharmacol

(1988)

PM Harari et al.

Heat shock stimulates polyamine oxidation by two distinct mechanisms in mammalian cell cultures

Int J Radiat Oncol Biol Phys

(1989)

JM Yuhas

Active versus passive absorption kinetics as the basis for selective protection of normal tissues by S-2-(3-aminopropylamino)-ethylphosphorothioic acid

Cancer Res

(1980)

MM Kligerman et al.

Phase I trials of WR2721 in combination with radiation therapy and with the alkylating agents cyclophosphamide and cis-platinum

Cancer Clin Trials

(1981)

D Glover et al.

WR-2721 and high-dose cisplatin: an active combination in the treatment of metastatic melanoma

J Clin Oncol

(1987)

Cited by (41)

In vitro study of doxorubicin-induced oxidative stress in spermatogonia and immature Sertoli cells
2018, Toxicology and Applied Pharmacology
Citation Excerpt :
DXO treatment in antioxidant experiments lasted 24 h for Ser-W3 and 48 h for GC-6Spg. To activate amifostine to its active metabolite WR-1065 and prevent its degradation, culture media was also supplemented with 1 U/ml alkaline phosphatase and 1 mM aminoguanidine bicarbonate (Meier and Issels, 1995; Muller et al., 2004). Pre-treatment with BSO or NAC was done the day after seeding for the indicated time before DXO exposure, then DXO was added for 12 h.
Pediatric chemotherapy treatments can impair long-term male fertility. Unfortunately, no fertility preservation solution is available for pre-pubertal boys. Studies suggest that doxorubicin, used against pediatric cancers, induces oxidative stress in the testis. However, the targeted testicular cell types remain unknown. The goal of this study was to determine whether doxorubicin can induce oxidative stress in rat spermatogonia (GC-6Spg) and immature Sertoli (Ser-W3) cell lines, and to assess their protection by antioxidants. Using the MTT assay, we have shown that doxorubicin induces a time- and dose-dependent cytotoxicity in these two cell lines, Ser-W3 being more sensitive than GC-6Spg. After 3 h of treatment, reactive oxygen species and nuclear 8-oxo-deoxyguanosine increase in Ser-W3, but not in GC-6Spg. Moreover, after 6 h of treatment, intracellular reduced glutathione levels decrease significantly in Ser-W3 cells. These results show that doxorubicin induces oxidative stress in the Ser-W3 cell line. However, a depletion in glutathione does not affect their survival, and supplementation only offers a weak protection after exposure to doxorubicin, suggesting that the glutathione system is not essential for Ser-W3 cell line's defense against doxorubicin. On the other hand, among four antioxidants selected from the literature, none reduces the cytotoxicity of doxorubicin in Ser-W3 cells. Together, our data suggest that oxidative stress may not be a major pathway for doxorubicin's cytotoxicity in GC-6Spg and Ser-W3 lines. This study provides new insights in the mechanisms by which chemotherapies affect the pre-pubertal testis, with the long-term goal to help improve the quality of life of pediatric cancer survivors.
Amine oxidases of the quinoproteins family: Their implication in the metabolic oxidation of xenobiotics
2011, Annales Pharmaceutiques Francaises
Citation Excerpt :
Nevertheless, the loss of the amino group upon incubation of amlodipine, an antihypertensive agent, in dog and human plasma, is compatible with the implication of SSAOs in amlodipine metabolism (Fig. 2) [15,16]. Similarly, the cytoprotective drug WR 1065 (2-[3-aminopropylamino] ethanethiol), administered as the thiophosphate derivative amifostine and utilized to protect tissues against the damaging effects of radiotherapy and chemotherapy, is converted by CuAOs to an aldehyde which spontaneously decomposes into cysteamine and acrolein (Fig. 3) [14,17]. The term SSAOs obviously is a confusing name because DAOs and LOs are also inhibited by semicarbazide, and it has often been used to refer specifically to AOs that are active toward primary amines.
Copper amine oxidases (CuAOs) are ubiquitous enzymes, which play a vital role in the physiology and pathology of mammals in controlling the metabolism of various primary monoamines, diamines and polyamines of endogenous or xenobiotic origin. CuAOs, which belong to the quinoproteins family, possess two cofactors: tightly bound Cu^II and a quinone residue, which catalyzes the oxidative deamination of primary amines with concomitant production of aldehyde, ammonia and hydrogen peroxide through a “ping-pong” mechanism. Interest in human enzymes of the CuAOs class has increased in recent years driven by the discovery that the human vascular adhesion protein-1 (VAP-1), which regulates leucocyte trafficking and glucose transport, is a CuAO enzyme. The activities of CuAOs are increased in various human disorders such as diabetes, Alzheimer's disease and many inflammation-associated diseases leading to the overproduction of toxic metabolites, especially hydrogen peroxide and aldehyde compounds. As most consequences are pathological, effective and selective inhibitors of CuAOs should be of great interest as therapeutic agents. Nevertheless, the utilization of CuAOs to generate enzymatic toxic products into cancer cells for selective in situ killing, deserves to be considered in cancer therapy. This paper briefly highlights recent progress in the study of physiological, pathological and molecular aspects of CuAOs in mammals. Furthermore, a small molecule, that mimics the metabolic activity of CuAOs toward endogenous and exogenous amines, is described because it could be used as a surrogate of enzymes for a preliminary screening of potential inhibitors of CuAO enzymes.
Les enzymes amine-oxydase à cuivre (CuAOs) sont des enzymes ubiquitaires qui jouent un rôle essentiel chez les mammifères, tant au plan physiologique que pathologique, puisqu’elles contrôlent le métabolisme de diverses monoamines primaires, diamines et polyamines, d’origine endogène ou exogène. Les CuAOs, qui appartiennent à la famille des quinoprotéins, possèdent deux cofacteurs : un ion cuivrique et un résidu quinonique qui catalyse la désamination oxydante des amines primaires, avec production concomitante d’aldéhydes, ammoniac et peroxyde d’hydrogène au travers d’un mécanisme « ping-pong ». Récemment, un intérêt particulier a été porté à la famille des CuAOs humaines depuis la découverte de leur identité avec la protéine d’adhésion vasculaire humaine (VAP-1), qui joue un rôle régulateur dans la circulation des leucocytes et l’assimilation du glucose. Dans de nombreuses pathologies telles que le diabète, la maladie d’Alzheimer et les maladies inflammatoires, l’activité des CuAOs est accrue et conduit à une surproduction de métabolites toxiques, en particulier de peroxyde d’hydrogène et d’aldéhydes. Comme la plupart des conséquences sont pathologiques, la recherche d’inhibiteurs efficaces et sélectifs des CuAOs présente un intérêt en thérapeutique. Néanmoins, l’utilisation des CuAOs pour générer des produits toxiques, peroxyde d’hydrogène et aldéhydes, à l’intérieur des cellules cancéreuses, ouvre de nouvelles perspectives en thérapie anticancéreuse. Cet article décrit brièvement les progrès récemment réalisés dans la connaissance de la physiologie, de la pathologie et des mécanismes moléculaires des CuAOs chez les mammifères. De plus, une petite molécule qui mime l’activité métabolique des CuAOs envers les amines primaires, endogènes et exogènes, est présentée. Elle pourrait en effet être utilisée pour sélectionner de nouveaux inhibiteurs d’enzymes CuAO.
Amifostine does not preferentially stimulate the growth of residual polyclonal progenitor cells in myelodysplastic syndromes
2003, Leukemia Research
Amifostine is a phosphorylated aminothiol that has besides anti-oxidative and cytoprotective properties, also survival- and growth-promoting effects on hematopoietic progenitor cells. Clinical studies have demonstrated that infusions with amifostine are able to increase erythro-, myelo-, and thrombopoiesis in some patients with myelodysplastic syndromes (MDS). Since clonal and non-clonal progenitors can coexist in early phase MDS, we have studied if amifostine exerts a selective growth-promoting effect on clonal or non-clonal cells. For this purpose, purified CD34⁺ marrow progenitors from nine female MDS patients were grown in short- and long-term cultures. Clonality was studied on individual colonies using polymorphisms in the human androgen receptor assay (HUMARA) locus. Three patients had growth of residual non-clonal progenitors at baseline. Continuous exposure to 100 nM amifostine exerted a growth-promoting effect on progenitors in 50% of the patients. HUMARA patterns of individual colony-forming unit granulocyte macrophage (CFU-GM; 5/9) and 5 week long-term culture-initiating cells (LTC-IC; 2/9) were compared without and with amifostine exposure. We did not observe preferential stimulation of clonal or non-clonal progenitors. Based on these results, the stimulation of committed and immature progenitor growth in MDS by amifostine, is non-selective and does not favor nor suppress the growth of residual non-clonal cells.
The cytoprotective aminothiol WR1065 activates p53 through a non-genotoxic signaling pathway involving c-Jun N-terminal kinase
2003, Journal of Biological Chemistry
Citation Excerpt :
Fig. 1shows that WR1065 induced a rapid (detectable after 15 min, Fig.1A) and long lasting (over 60 h, Fig. 1C) accumulation of wild-type p53 in MCF-7 cells, which correlated with enhanced DNA binding activity. As WR1065 is rapidly degraded by copper-dependent amine oxidases in culture medium to form toxic metabolites, all experiments were performed in the presence of aminoguanidine (AG) at 4 mm, a concentration shown previously to inhibit copper-dependent amine oxidases (22,63). Fig. 1 shows that AG alone did not affect p53 levels (Western blot, A and C) and activity (DNA binding activity, B and C).
WR1065 is an aminothiol with selective cytoprotective effects in normal cells compared with cancer cells. In a previous study (North, S., El-Ghissassi, F., Pluquet, O., Verhaegh, G., and Hainaut, P. (2000) Oncogene 19, 1206–1214), we have shown that WR1065 activates wild-type p53 in cultured cells. Here we show that WR1065 induces p53 to accumulate through escape from proteasome-dependent degradation. This accumulation is not prevented by inhibitors of phosphatidylinositol 3-kinases and is not accompanied by phosphorylation of Ser-15, -20, or -37, which are common targets of the kinases activated in response to DNA damage. Furthermore, WR1065 activates the JNK (c-Jun N-terminal kinase), decreases complex formation between p53 and inactive JNK, and phosphorylates p53 at Thr-81, a known site of phosphorylation by JNK. A dominant negative form of JNK (JNK-APF) reduces by 507 the activation of p53 by WR1065. Thus, WR1065 activates p53 through a JNK-dependent signaling pathway. This pathway may prove useful for pharmacological modulation of p53 activity through non-genotoxic mechanisms.
Activation of p53 by the cytoprotective aminothiol WR1065: DNA-damage-independent pathway and redox-dependent modulation of p53 DNA-binding activity
2003, Biochemical Pharmacology
Citation Excerpt :
All experiments were performed in presence of aminoguanidine (AG) at a concentrations of 4 mM as determined previously [21]. Indeed, it has been reported by Meier and Issels [33] that in the culture medium, WR1065 may undergo degradation into cytotoxic metabolites (H2O2, acrolein) by copper-dependent amine oxidases. Time-course experiments with MCF-7 cells indicated that WR1065 (1 mM+AG) did not generate detectable DNA fragmentation after up to 8 hr of treatment (Fig. 1A).
WR1065 is an aminothiol with selective cytoprotective effects in normal compared to cancer cells, which is used to protect tissues against the damaging effect of radiation and chemotherapeutic drugs. WR1065 has been shown to induce wild-type p53 accumulation and activation in cultured cells, suggesting a role of p53 in cytoprotection. However, the molecular mechanisms by which WR1065 activates p53 remain unclear. Here, we demonstrated that p53 accumulation by WR1065 in MCF-7 cells did not result from the formation of DNA-damage as measured by DNA fragmentation and Comet assay, nor from oxidative stress as detected by measurement of glutathione levels, lipid peroxidation and reactive oxygen species production. p53 activation by WR1065 was not prevented by inhibition of PI-3 kinases, and was still detectable in MCF-7 cells stably transfected with the oncoprotein E6, which repressed p53 induction by DNA damage. These data provided evidence that WR1065 induces p53 by a pathway different than the one elicited by DNA-damage. Direct reduction by WR1065 of key cysteines in p53 may play an important role in this alternative pathway, as shown by the fact that WR1065 activated the redox-dependent, DNA-binding activity of p53 in vitro.
Amifostine: A tonic or toxin to myeloid progenitors
2000, Leukemia Research

View all citing articles on Scopus

^☆: This work was supported by Grant Is 31/3-2 of the Deutsche Forschungsgemeinschaft.

View full text

Research paperDegradation of 2-(3-aminopropylamino)-ethanethiol (WR-1065) by Cu-dependent amine oxidases and influence on glutathione status of chinese hamster ovary cells☆

Abstract

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

J Chromatogr

Anal Biochem

Meth Enzymol

J Biol Chem

Free Radic Biol Med

Biochem Pharmacol

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Biochem Pharmacol

Chem Biol Interact

Meth Enzymol

Arch Biochem Biophys

Tetrahedron

Cancer Treat Rev

Toxicol Appl Pharmacol

Blood

Biochem Pharmacol

Int J Radiat Oncol Biol Phys

Active versus passive absorption kinetics as the basis for selective protection of normal tissues by S-2-(3-aminopropylamino)-ethylphosphorothioic acid

Cancer Res

Phase I trials of WR2721 in combination with radiation therapy and with the alkylating agents cyclophosphamide and cis-platinum

Cancer Clin Trials

WR-2721 and high-dose cisplatin: an active combination in the treatment of metastatic melanoma

J Clin Oncol

Research paper
Degradation of 2-(3-aminopropylamino)-ethanethiol (WR-1065) by Cu-dependent amine oxidases and influence on glutathione status of chinese hamster ovary cells☆