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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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Metabolic profile of eupatorin (10 μM) metabolism by CYP1 family enzymes and identification of cirsiliol as the primary metabolite. (a) Typical high-pressure liquid chromatography (HPLC) traces of 20-minute incubation of CYP1 enzymes with eupatorin. (b) Expansion of (a) showing metabolites E2 and E3. (c) Co-elution studies of eupatorin with cirsiliol. A 20-minute CYP1B1 incubate of eupatorin was spiked with cirsiliol (0.2 μM). Reaction mixtures contained eupatorin, NADPH (nicotinamide adenine dinucleotide phosphate), and recombinant microsomes purchased from Gentest Corporation (now part of BD Biosciences). Reactions were terminated by the addition of 1% acetic acid in methanol. (d) HPLC trace of metabolism of eupatorin in MDA-MB-468 cells. Samples were analysed by HPLC using a UV detector at 350 nm. Experiments were performed in triplicate. A350: absorption of light at wavelength 350 nm. AU: arbitrary units.
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Figure 4: Metabolic profile of eupatorin (10 μM) metabolism by CYP1 family enzymes and identification of cirsiliol as the primary metabolite. (a) Typical high-pressure liquid chromatography (HPLC) traces of 20-minute incubation of CYP1 enzymes with eupatorin. (b) Expansion of (a) showing metabolites E2 and E3. (c) Co-elution studies of eupatorin with cirsiliol. A 20-minute CYP1B1 incubate of eupatorin was spiked with cirsiliol (0.2 μM). Reaction mixtures contained eupatorin, NADPH (nicotinamide adenine dinucleotide phosphate), and recombinant microsomes purchased from Gentest Corporation (now part of BD Biosciences). Reactions were terminated by the addition of 1% acetic acid in methanol. (d) HPLC trace of metabolism of eupatorin in MDA-MB-468 cells. Samples were analysed by HPLC using a UV detector at 350 nm. Experiments were performed in triplicate. A350: absorption of light at wavelength 350 nm. AU: arbitrary units.

Mentions: Following the first line of evidence that eupatorin is a substrate for CYP1 family enzymes, we investigated the metabolism of this natural product in microsomes expressing human CYP1 family enzymes and in MDA-MB-468 and MCF-10A cells. Microsomes expressing CYP1A1, CYP1A2, or CYP1B1 and NADPH reductase were incubated with eupatorin for 20 minutes with NADPH as a co-factor. Eupatorin was metabolised to a large extent by CYP1A1 and CYP1A2 (Figure 3a). The concentration of the parent compound decreased considerably following incubation with these enzymes, over the 20-minute period, compared with control microsomes that did not contain CYPs. In contrast, CYP1B1 was a weak metaboliser of eupatorin compared with the other two CYP1s (Figure 3a). After incubation with eupatorin, CYP1B1 produced one metabolite, assigned E1, which eluted at approximately 9.6 minutes in the HPLC assay used in this study (Figure 4a). CYP1A1 and CYP1A2 produced three metabolites, assigned E1, E2, and E3, including the one seen in the case of CYP1B1. E2 and E3 were produced in negligible amounts compared with E1 (Figure 4b). All metabolites eluted at retention times shorter than that corresponding to the parent compound. This indicates that they resulted from hydroxylation/demethylation of eupatorin catalysed by the cytochrome P450-reductase system, thereby becoming more polar than eupatorin.


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)

Metabolic profile of eupatorin (10 μM) metabolism by CYP1 family enzymes and identification of cirsiliol as the primary metabolite. (a) Typical high-pressure liquid chromatography (HPLC) traces of 20-minute incubation of CYP1 enzymes with eupatorin. (b) Expansion of (a) showing metabolites E2 and E3. (c) Co-elution studies of eupatorin with cirsiliol. A 20-minute CYP1B1 incubate of eupatorin was spiked with cirsiliol (0.2 μM). Reaction mixtures contained eupatorin, NADPH (nicotinamide adenine dinucleotide phosphate), and recombinant microsomes purchased from Gentest Corporation (now part of BD Biosciences). Reactions were terminated by the addition of 1% acetic acid in methanol. (d) HPLC trace of metabolism of eupatorin in MDA-MB-468 cells. Samples were analysed by HPLC using a UV detector at 350 nm. Experiments were performed in triplicate. A350: absorption of light at wavelength 350 nm. AU: arbitrary units.
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Related In: Results  -  Collection

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Figure 4: Metabolic profile of eupatorin (10 μM) metabolism by CYP1 family enzymes and identification of cirsiliol as the primary metabolite. (a) Typical high-pressure liquid chromatography (HPLC) traces of 20-minute incubation of CYP1 enzymes with eupatorin. (b) Expansion of (a) showing metabolites E2 and E3. (c) Co-elution studies of eupatorin with cirsiliol. A 20-minute CYP1B1 incubate of eupatorin was spiked with cirsiliol (0.2 μM). Reaction mixtures contained eupatorin, NADPH (nicotinamide adenine dinucleotide phosphate), and recombinant microsomes purchased from Gentest Corporation (now part of BD Biosciences). Reactions were terminated by the addition of 1% acetic acid in methanol. (d) HPLC trace of metabolism of eupatorin in MDA-MB-468 cells. Samples were analysed by HPLC using a UV detector at 350 nm. Experiments were performed in triplicate. A350: absorption of light at wavelength 350 nm. AU: arbitrary units.
Mentions: Following the first line of evidence that eupatorin is a substrate for CYP1 family enzymes, we investigated the metabolism of this natural product in microsomes expressing human CYP1 family enzymes and in MDA-MB-468 and MCF-10A cells. Microsomes expressing CYP1A1, CYP1A2, or CYP1B1 and NADPH reductase were incubated with eupatorin for 20 minutes with NADPH as a co-factor. Eupatorin was metabolised to a large extent by CYP1A1 and CYP1A2 (Figure 3a). The concentration of the parent compound decreased considerably following incubation with these enzymes, over the 20-minute period, compared with control microsomes that did not contain CYPs. In contrast, CYP1B1 was a weak metaboliser of eupatorin compared with the other two CYP1s (Figure 3a). After incubation with eupatorin, CYP1B1 produced one metabolite, assigned E1, which eluted at approximately 9.6 minutes in the HPLC assay used in this study (Figure 4a). CYP1A1 and CYP1A2 produced three metabolites, assigned E1, E2, and E3, including the one seen in the case of CYP1B1. E2 and E3 were produced in negligible amounts compared with E1 (Figure 4b). All metabolites eluted at retention times shorter than that corresponding to the parent compound. This indicates that they resulted from hydroxylation/demethylation of eupatorin catalysed by the cytochrome P450-reductase system, thereby becoming more polar than eupatorin.

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