Molecular and Cellular Pharmacology
Differential effects of esculetin and daphnetin on in vitro cell proliferation and in vivo estrogenicity

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Abstract

Esculetin (6,7-dihydroxycoumarin) and daphnetin (7,8-dihydroxycoumarin) are secondary metabolites of plants used in folk medicine. These compounds have showed great antiproliferative activity in several tumor cell lines and have been proposed as potential anticancer agents. However, the estrogenic potential of these two compounds has to date not been reported. The present study compared esculetin and daphnetin on the inhibition of cell proliferation and cell cycle progression of the MCF-7 estrogen-responsive human carcinoma cell line. In vivo and in vitro estrogenic activity for both compounds was also evaluated. Esculetin inhibited cell proliferation after 72 h exposure (IC50 = 193 ± 6.6 μM), while daphnetin evidenced inhibiting effects starting at 24-h exposure (72 h, IC50 = 73 ± 4.1 μM). Both effects showed changes in cyclin D1 gene expression. In non-estrogenic conditions (E-screening assay), esculetin produced biphasic response on proliferation of the MCF-7 cells; at 10 8–10 6 M, concentrations induced proliferative effects as EC50 = 4.07 × 10 9 M (E2 = 2.91 × 10 12 M); at higher concentrations (10 5–10 4 M), cell proliferation was inhibited. Relative proliferative effect at E2 was 52% (E2 = 100), relative proliferative potency was 0.072 (E2 = 100). Additionally, esculetin tested in vivo showed estrogenic effects at 50–100 mg/kg doses; relative uterotrophic effect at E2 was 37%, with relative uterotrophic potency registered at 0.003. In contrast, daphnetin did not induce estrogenic effects in vitro or with in vivo models. The low estrogenic activity of esculetin could prove useful in postmenopausal therapy but not as a safe antitumor agent in estrogen-dependent tumors. Daphnetin-based antiproliferative selectivity with MCF-7 cells showed that daphnetin is a promising antitumoral agent also acting on estrogen dependent tumors.

Introduction

Clinical studies have reported antitumor activity of coumarin in several cancer types (Marshall et al., 1991, Mohler et al., 1992, Thornes et al., 1994). Coumarin acts as a prodrug creating active metabolites which could be responsible for observed effects (Egan et al., 1990). Experimental assays showed that coumarin producing antiproliferative effect in tumor cell lines with mM concentrations. However, 7-hydroxycoumarin (its main biotransformation product in humans) has greater antiproliferative activity (Jiménez-Orozco et al., 1999, Jiménez-Orozco et al., 2001, Lopez-Gonzalez et al., 2004). The addition of two hydroxyl groups in the meta- or para-positions of the coumarin nucleus increases antiproliferative activity to ranges of μM concentration (Kolodziej et al., 1997).

Dihydroxy-coumarin derivatives, esculetin (6,7-dihydroxycoumarin) and daphnetin (7,8-dihydroxycoumarin), are secondary metabolites of plants used in folk medicine to counter inflammatory and allergic diseases (Fylaktakidou et al., 2004, Riveiro et al., 2010). Esculetin possesses diverse pleiotropic actions (Chu et al., 2001): it inhibits smooth vascular muscle cell proliferation through the inhibition of kinases and mitogen signal pathways mediated by Ras protein (Pan et al., 2003). The antiproliferative effect of esculetin has been reported to be greater in tumor cells than in non-malignant cells (Finn et al., 2002, Kawaii et al., 2001, Kawase et al., 2003). Esculetin has been considered a promising anticancer agent due to its multiple properties (Lacy and O'Kennedy 2004).

Daphnetin has been clinically used in the treatment of coagulation disorders, rheumatoid arthritis and has been shown to possess anti-malarian and anti-pyretic properties (Liang et al., 2010). The daphnetin compound also induces cell differentiation (Finn et al., 2004) and inhibits kinase activity (Yang et al., 1999). The antiproliferative effects of daphnetin have been studied to a lesser extent than those induced by esculetin. However, the daphnetin is also capable of producing notable antiproliferative activity among different malignant cell lines (Finn et al., 2001, Finn et al., 2002).

Therapeutic strategies for breast cancer-dependent hormone tumors typically involve the inhibition of the estrogen receptor by selective estrogen receptor modulators (SERMs), such as tamoxifen. A new 3, 4 substituted coumarin-based SERM was designed based on the structural similarity of coumarin molecules to the AB ring of natural estradiol hormones (McKie et al., 2004). A second complementary strategy in breast cancer therapy consists in the inhibition of enzymes found within the steroid biosynthetic cascade. In this respect, some 7-hydroxycoumarin analogs have been considered to be promissory anti-breast cancer agents for their sulfatase and aromatase inhibitory activity (Musa et al., 2008).

While the estrogenic effect of certain coumarin derivatives such as coumestrol has been firmly documented, the estrogenic properties of esculetin and daphnetin have yet to be reported. Evaluation of esculetin and daphnetin estrogenic activity should be obligatory, given that high estrogen levels or exposition to estrogenic substances are recognized factors in breast cancer (Fernández and Russo, 2010). The aim of the present study was to compare the effects of esculetin and daphnetin on MCF-7 cell proliferation under estrogenic and non-estrogenic conditions and their effects on the cell cycle. Estrogenicity for both compounds was also tested by means of uterotrophic assays.

Section snippets

Reagents

17β-Estradiol (E2; 1,3,5(10)-estratrien-3,17-β-diol), esculetin, (6,7-dihydroxycoumarin) and daphnetin (7,8-dihydroxycoumarin) from Sigma (St. Louis, MO, USA) were dissolved in an ethanol absolute (Merck, Darmstadt, Germany). Anti-cyclin D1 mouse monoclonal, anti-UBF, anti-cyclin E, and anti-p27 rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All cell culture reagents, media, as well as the TRIzol reagent, were purchased from Gibco (Invitrogen

Antiproliferative effects

In MCF-7 cells, esculetin exerted low antiproliferative activity; only exposures to larger concentrations (200 μM during 72-h) inhibited proliferation by more than 50% (IC50 = 193 ± 6.6 μM). At concentrations of 50 μM or less, esculetin elicited consistently small, not significant increases in proliferation (Fig. 1). In contrast, daphnetin inhibited proliferation at exposures as short as 24-h, while at 72-h it had an IC50 of 73 ± 4.1 μM; smaller concentrations did not stimulate proliferation.

Effects on cell cycle proteins

Levels of

Discussion

Our results show that esculetin and daphnetin have differential effects on MCF-7 cell proliferation in estrogenic and non estrogenic conditions. In estrogenic conditions, concentrations of esculetin of 50 μM and lower were able to produced small increases in cell proliferation rate, which correlate with increases in cyclins D1 and E. Cyclin D1 in particular is a key protein for the initiation of the cell cycle, working as a sensor for mitogenic signals (Witzel et al., 2010). It has been widely

Conclusion

Despite the fact that the antiproliferative activity of esculetin has been widely reported, in this work we observed that the molecule produced biphasic effects, similar to those observed with other pythoestrogens in breast carcinoma MCF-7 cells. The inhibitory activity of esculetin occurred only at μM concentrations. Meanwhile, lower concentrations produced estrogenic effects in vitro and in vivo that would limit its use as an anticancer agent in estrogen-dependent tumors.

Our data suggest that

Acknowledgments

We thank Dr. Ingrid Grummt and Dr. Renata Voit from the German Cancer Research Center (DKFZ) for their assistance in performing the immunoblot and the kinase assays as well as for the DAAD scholarship A/01/05883. The authors are grateful to ME Avila Aguirre, M Medina-Jiménez and A Espinoza-Sánchez for their technical assistance. This research was supported by the PAPITT-UNAM IN209010-2, the CONACYT 52175 and the DAAD.

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