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Antiproliferative and cytostatic effects of the natural product eupatorin on MDA-MB-468 human breast cancer cells due to CYP1-mediated metabolism.

Androutsopoulos V, Arroo RR, Hall JF, Surichan S, Potter GA - Breast Cancer Res. (2008)

Bottom Line: The antiproliferative effect, as measured by EROD (ethoxyresorufin-O-deethylase) assay and Western immunoblotting, was attributed mainly to CYP1A1 expression in MDA-MB-468 cells but not in MCF-10A cells.Eupatorin was further shown to arrest the cell cycle of the CYP1-expressing cell line MDA-MB-468 in G2/M phase, whereas no effect was observed in MCF-10A cells, which do not express CYP1 enzymes.The effect of eupatorin on the MDA-MB-468 cell cycle could be reversed by co-application of the CYP1 inhibitor acacetin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester, UK.

ABSTRACT

Introduction: The natural product eupatorin has been reported to have antiproliferative activity in tumour cell lines, but the exact mechanism is unclear. The cytochromes P450 CYP1B1, CYP1A1, and CYP1A2 have been shown to participate in the activation of various xenobiotics, compounds derived from the diet as well as chemotherapeutic drugs. CYP1B1 and CYP1A1 have also been proposed as targets for cancer chemotherapy for their differential and selective overexpression in tumour cells. In this study, we aimed to identify a possible mechanism of action for the antiproliferative effect of eupatorin, which can be attributed to CYP1 family-mediated metabolism.

Methods: The study focuses on the antiproliferative action of eupatorin on the human breast carcinoma cell line MDA-MB-468 and on a cell line derived from normal mammary tissue, MCF-10A. The cytotoxicity of the flavone, its effect on the cell cycle of the abovementioned cell lines, and its metabolism by CYP1 family enzymes were examined.

Results: Eupatorin showed a dose-dependent inhibitory effect of cell growth on MDA-MB-468 cells with a submicromolar median inhibition concentration (IC50) whereas the IC50 of this compound in MCF-10A cells was considerably higher. The antiproliferative effect, as measured by EROD (ethoxyresorufin-O-deethylase) assay and Western immunoblotting, was attributed mainly to CYP1A1 expression in MDA-MB-468 cells but not in MCF-10A cells. Moreover, CYP1 family enzymes were shown to metabolise eupatorin in vitro to the flavone cirsiliol and two other unidentified metabolites. Metabolism of eupatorin was also detected in MDA-MB-468 cell cultures, whereas metabolism by MCF-10A cells was negligible. Eupatorin was further shown to arrest the cell cycle of the CYP1-expressing cell line MDA-MB-468 in G2/M phase, whereas no effect was observed in MCF-10A cells, which do not express CYP1 enzymes. The effect of eupatorin on the MDA-MB-468 cell cycle could be reversed by co-application of the CYP1 inhibitor acacetin.

Conclusion: The flavone eupatorin is selectively activated in breast cancer cells, but not in normal breast cells, due to CYP1 family metabolism. This provides a basis for selectivity which is desired against breast tumour cells. In this sense, eupatorin is shown by this study to be a very promising chemopreventative candidate that should be examined further in an in vivo study.

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Chemical structures of eupatorin and cirsiliol and possible structures of the metabolites E2 and E3.
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Figure 7: Chemical structures of eupatorin and cirsiliol and possible structures of the metabolites E2 and E3.

Mentions: Cirsiliol, or 3',4'-dihydroxy-5,6,7-trimethoxy flavone (Figure 7), results from 4'-demethylation of eupatorin. The IC50 values for cirsiliol in MDA-MB-468 and MCF-10A cells show a 7-fold difference (Figure 1d), which indicates that CYP1-mediated metabolism further activates this compound. Nevertheless, this initial eupatorin metabolite is at least partially responsible for its antiproliferative activity as it showed lower IC50 values in MCF-10A cells than eupatorin did. In addition, the metabolites E2 and E3 contribute to the ultimate submicromolar toxicity of eupatorin in MDA-MB-468 cells, which was not observed for cirsiliol. The unidentified metabolites E2 and E3 may be produced by hydroxylation reactions at positions 5' of the B ring and 8' of the A ring or by demethylation reactions at positions 6' and 7' of the A ring (Figure 7).


Antiproliferative and cytostatic effects of the natural product eupatorin on MDA-MB-468 human breast cancer cells due to CYP1-mediated metabolism.

Androutsopoulos V, Arroo RR, Hall JF, Surichan S, Potter GA - Breast Cancer Res. (2008)

Chemical structures of eupatorin and cirsiliol and possible structures of the metabolites E2 and E3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2481486&req=5

Figure 7: Chemical structures of eupatorin and cirsiliol and possible structures of the metabolites E2 and E3.
Mentions: Cirsiliol, or 3',4'-dihydroxy-5,6,7-trimethoxy flavone (Figure 7), results from 4'-demethylation of eupatorin. The IC50 values for cirsiliol in MDA-MB-468 and MCF-10A cells show a 7-fold difference (Figure 1d), which indicates that CYP1-mediated metabolism further activates this compound. Nevertheless, this initial eupatorin metabolite is at least partially responsible for its antiproliferative activity as it showed lower IC50 values in MCF-10A cells than eupatorin did. In addition, the metabolites E2 and E3 contribute to the ultimate submicromolar toxicity of eupatorin in MDA-MB-468 cells, which was not observed for cirsiliol. The unidentified metabolites E2 and E3 may be produced by hydroxylation reactions at positions 5' of the B ring and 8' of the A ring or by demethylation reactions at positions 6' and 7' of the A ring (Figure 7).

Bottom Line: The antiproliferative effect, as measured by EROD (ethoxyresorufin-O-deethylase) assay and Western immunoblotting, was attributed mainly to CYP1A1 expression in MDA-MB-468 cells but not in MCF-10A cells.Eupatorin was further shown to arrest the cell cycle of the CYP1-expressing cell line MDA-MB-468 in G2/M phase, whereas no effect was observed in MCF-10A cells, which do not express CYP1 enzymes.The effect of eupatorin on the MDA-MB-468 cell cycle could be reversed by co-application of the CYP1 inhibitor acacetin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester, UK.

ABSTRACT

Introduction: The natural product eupatorin has been reported to have antiproliferative activity in tumour cell lines, but the exact mechanism is unclear. The cytochromes P450 CYP1B1, CYP1A1, and CYP1A2 have been shown to participate in the activation of various xenobiotics, compounds derived from the diet as well as chemotherapeutic drugs. CYP1B1 and CYP1A1 have also been proposed as targets for cancer chemotherapy for their differential and selective overexpression in tumour cells. In this study, we aimed to identify a possible mechanism of action for the antiproliferative effect of eupatorin, which can be attributed to CYP1 family-mediated metabolism.

Methods: The study focuses on the antiproliferative action of eupatorin on the human breast carcinoma cell line MDA-MB-468 and on a cell line derived from normal mammary tissue, MCF-10A. The cytotoxicity of the flavone, its effect on the cell cycle of the abovementioned cell lines, and its metabolism by CYP1 family enzymes were examined.

Results: Eupatorin showed a dose-dependent inhibitory effect of cell growth on MDA-MB-468 cells with a submicromolar median inhibition concentration (IC50) whereas the IC50 of this compound in MCF-10A cells was considerably higher. The antiproliferative effect, as measured by EROD (ethoxyresorufin-O-deethylase) assay and Western immunoblotting, was attributed mainly to CYP1A1 expression in MDA-MB-468 cells but not in MCF-10A cells. Moreover, CYP1 family enzymes were shown to metabolise eupatorin in vitro to the flavone cirsiliol and two other unidentified metabolites. Metabolism of eupatorin was also detected in MDA-MB-468 cell cultures, whereas metabolism by MCF-10A cells was negligible. Eupatorin was further shown to arrest the cell cycle of the CYP1-expressing cell line MDA-MB-468 in G2/M phase, whereas no effect was observed in MCF-10A cells, which do not express CYP1 enzymes. The effect of eupatorin on the MDA-MB-468 cell cycle could be reversed by co-application of the CYP1 inhibitor acacetin.

Conclusion: The flavone eupatorin is selectively activated in breast cancer cells, but not in normal breast cells, due to CYP1 family metabolism. This provides a basis for selectivity which is desired against breast tumour cells. In this sense, eupatorin is shown by this study to be a very promising chemopreventative candidate that should be examined further in an in vivo study.

Show MeSH
Related in: MedlinePlus