BIOACTIVATION OF NATURAL DIETARY PRODRUGS BY SPECIFIC CYTOCHROME P450 ENZYMES

Tumour specific cytochrome P450 enzymes (CYPs) have recently been established as a novel target in anticancer drug discovery. The enzymes are used to activate nontoxic prodrugs with latent activity. Thus, the combination of tumour specific CYPs as ‘rescue enzymes’ with a suitable substrate provides a platform to develop selective anticancer prodrugs. This thesis describes the bioactivation of natural prodrugs by tumour specific CYPl enzymes. An initial exploratory investigation was conducted on a variety of natural products including all-/raz7.y-retinoic acid (/-RA), vitamin D3, and the isoflavonoids genistein and daidzein. These compounds were found to be poor substrates for CYPl enzymes. In contrast, synthetic chaicone analogues of a natural chaicone butein, DMU2123 and DMU2127, were found to be substrates of the CYPl Al enzyme. In a cell-based cytotoxicity assay these chaicones were activated by over 50-fold in CYPl expressing cell lines compared with non-expressing cells. This effect was reversed by co-application of the CYPl inhibitor acacetin. The most promising natural CYPl substrates in the initial screening assay were found to be the citrus flavonoids tangeretin and nobiletin. Nobiletin was the most potently activated compound. It was found to have very low toxicity toward the normal breast cell line MCFIOA (IC50 = 50ju.M), but was highly growth inhibitory toward the CYPl expressing breast cancer cell line MDA-MB-468 (IC50 = 0.1 pM). Similar, but slightly less, bioactivation was also seen for tangeretin. The cytotoxicity of these compounds in MDA-MB-468 cells was abrogated by the co-application of CYPl inhibitors, acacetin and a-naphthoflavone (a-NF). This result implies that the bioactivation by CYPl enzymes in this cell line is involved in the mechanism of anticancer activity ofthese citrus flavonoids, and these compounds are acting as natural anticancer prodrugs. A biphasic dose-response curve was observed for these flavonoids, indicating there is probably more than one bioactivation step involved, and the putative active cytotoxic species is a secondary metabolite. Western blotting analysis showed that CYPl Al may have been responsible for the bioactivation of the latent anticancer activity of the flavones. This was confirmed using recombinant CYPlAl microsomes which showed that CYPlAl is the major enzyme catalysing the bioconversion of these citrus flavonoids. CYPl Bl could also metabolise these compounds but to a lesser extent. LC-MS studies showed that tangeretin undergoes demethylation and hydroxylation to at least four (one primary and three secondary) metabolites by the CYPlAl enzyme, whereas nobiletin mainly undergoes demethylation to at least four (one primary and three secondary) metabolites. Two of the secondary metabolites of these citrus flavonoids were exclusively generated by CYPlAl, supporting the hypothesis of two-stage metabolism to generate the active growth inhibitory metabolites. Tangeretin and nobiletin induced cell cycle accumulation at G1 phase in the breast cancer cell line MDA-MB-468. Furthermore, tangeretin and nobiletin were found to induce expression of CYPlAl, and were thus able to induce their own metabolism in cancer cells, leading to inhibition of cell proliferation. Hence both tangeretin and nobiletin hold promise as lead compounds for the development of non-toxic and tumour-selective drugs that can be used in both cancer prevention and therapy.

Name dc:type.qualificationname

PhD

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

De Montfort University

Year dc:date.issued

2008

Author dc:creator

• SURICHAN, SOMCHAIYA

Licence

• https://dora.dmu.ac.uk/bitstreams/381d85d3-b36e-4fd7-a6dd-dbb3479f6812/download