Search for content and authors

Mercapturic acids as a naturally occurring isothiocyanates prodrug form with site-directed delivery system built-in based on an oxidative environment of the tumor

Mateusz Psurski 1,2Jarosław Ciekot 2Joanna Wietrzyk 2Józef Oleksyszyn 1

1. Wroclaw University of Technology, Division of Medicinal Chemistry and Microbiology (PWR), Wybrzeże Wyspiańskiego 27, Wrocław 50-370, Poland
2. Polish Academy of Sciences, Institute of Immunology and Experimental Therapy (IITD), Rudolfa Weigla 12, Wrocław 53-114, Poland


Background. Naturally occurring isothiocyanates are a well-known constituents present in cruciferous vegetables exhibiting high anticancer activity in vivo which is associated with relatively low toxicity.  Their mode of action involves multiple mechanisms of which glutathione depletion appears to be the most important. These process proceeds though mercapturic acids pathway that start from isothiocyanate conjugation with glutathione and ends once its final product – dithiocarbamate (mercapturic acids) is excreted. Outside the cell it can undergo slow hydrolysis resulting in isothiocyanate’s moiety release which can re-enter the cell via passive diffusion. These metabolic cycle appears to play a pivotal role in isothiocyanates biological activity, thus, enhancement of these process in the vicinity of cancer cells (tumor) might be an important mechanism of their selective killing.

Among many metabolic, phenotypic and genetic changes involved in malignant transformation one can find increased reactive oxygen species production. These feature is responsible for higher mutation rate and is generally recognized as beneficial for cancer growth. However, such attribute might be useful as a mechanism of prodrugs activation specificity in the vicinity of a tumor tissue. We believe that the stability of dithiocarbamate moiety is correlated with environment redox status, thus, mercapturic acids might be recognized as a redox sensitive prodrugs for isothiocyanates.

Aim of the study. To investigate a potential role of reactive oxygen species (ROS) and oxidative stress in mercapturic acids decomposition to isothiocyanates.

Methods. A series of mercapturic acids were synthesized from parental isothiocyanates using well established methods. The stability of representative compounds in water with or without hydrogen peroxide added was assessed using HPLC system with UV or MS/MS detection methods. Antiproliferative activity in vitro was analyzed with SRB (sulforhodamine B) assay. Caspase-3/7 activity was used as an indicator of apoptosis and was detected by the analysis of the sample proteolytic activity using Ac-DEVD-AMC as a fluorogenic substrate.

Results. A series of mercapturic acids was analyzed for the antiproliferative activity after 72 hours of drug treatment using a series of concentrations. Activity observed for almost all tested compounds (expressed as IC50 value) was similar to the activity exhibited by corresponding parental isothiocyanates. The most active compounds were mercapturic acids, derivatives of previously not tested 3,4-dimetoxybenzyl isothiocyanate (1), 6-benzoyloxyhexyl isothiocyanate (2) and 6-hydroxyhexyl isothiocyanate (3) as well as previously tested benzyl (4) and phenethyl isothiocyanate (5). The stability of three representative compounds (2, 4 and 5) was significantly reduced (2-3 times, expressed as t1/2) when hydrogen peroxide was present (5 molar equivalents). For example for 5 t½ dropped down from 306 minutes to 126 minutes (k = 0.3252 and 0.1320, respectively). For all analyzed compounds, parental isothiocyanate and bis-N-acetyl-cystine were recognized as a final breakdown products both in sample with or without H2O added. Interestingly, when hydrogen peroxide was present, additional intermediates were observed indicating a striking differences in the mechanism of decomposition. These intermediates were identified as likely oxidized form of dithiocarbamate (M + 16) and desulfurized form of oxidized dithiocarbamate (M + 16 – 32). Addition of hydrogen peroxide (non-toxic concentrations) to the culture medium enhance mercapturic acids decomposition leading to faster induction of apoptosis – significant caspase-3/7 activity was observed after 6 hour and 15 hour of treatment with or without H2O2 added, respectively (for parental isothiocyanates such activity occurred after 4-5 hour).

Conclusions. The results of our studies clearly indicates that mercapturic acids stability is influenced by the presence of hydrogen peroxide. In vitro studies provide evidence that the feature observed using HPLC method is probably associated with a more rapid mercapturic acids activation (caspase-3/7 assay). Moreover, mercapturic acids showed high antiproliferative activity comparable to parental isothiocyanate’s activity, thus, the addition of the selectivity mechanism does not interfere with biological activity. As the result of the ROS overproduction by the cancer cells, the accumulation of isothiocyanates should occur preferentially inside the cancer cells. Therefore, mercapturic acids could be consider as form of the prodrug for natural isothiocyanates responsible for the site-directed delivery of isothiocyanate directly to the cancer cells.

This work was supported by a grant of National Science Centre, grant no. 2011/01/N/NZ4/03361.


Legal notice
  • Legal notice:

Related papers

Presentation: Poster at IX Multidyscyplinarna Konferencja Nauki o Leku, by Mateusz Psurski
See On-line Journal of IX Multidyscyplinarna Konferencja Nauki o Leku

Submitted: 2014-03-14 16:18
Revised:   2014-05-02 17:34