Oxyprenylated Phenylpropanoids Bind to MT1 Melatonin Receptors

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Oxyprenylated Phenylpropanoids Bind to MT1 Melatonin Receptors and Inhibit Breast Cancer Cell Proliferation and Migration Mahmud Hasan,† Salvatore Genovese,‡ Serena Fiorito,‡ Francesco Epifano,‡ and Paula A. Witt-Enderby*,† †

Division of Pharmaceutical, Administrative and Social Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282, United States Department of Pharmacy, University “G. d’Annunzio” of Chieti e Pescara, Chieti 66100, Italy

‡

ABSTRACT: Oxyprenylated compounds (i.e., ferulic acid and coumarin derivatives) demonstrate neuroprotection and anticancer properties as reported in previous studies. We have tested the affinity of oxyprenylated ferulic acid (1−4) and umbelliferone derivatives (5−11) to melatonin receptors as well as their antiproliferation and antimigratory properties against breast cancer (BC) cell lines. All the compounds except for ferulic acid, boropinic acid, and umbelliferone had binding affinities to melatonin receptors in the nM to μM range, and both auraptene and umbellinprenin reduced BC cell proliferation and migration in phenotypically diverse BC including triple negative.

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oxyprenylated coumarin derivatives were also tested. Myriad coumarin derivatives (∼1300) have been found in 150 different plant species from 30 different plant families including Rutaceae, Umbelliferae, and Apiaceae.14 In the United States, the most common source of coumarin comes from Cinnamomum cassia, otherwise known as cassia cinnamon.15 Coumarin derivatives have gained in popularity because of their reported health benefits, especially against cancer.16,17 For example, coumarin and its derivatives were reported as having anticancer activity by inhibiting the synthesis of estrogen, and, as such, a coumarin-based selective estrogen receptor modulator was developed and demonstrated to have a similar potency to tamoxifen.18 Melatonin, like coumarin, also has been shown to possess antiestrogen properties19−21 and produce anticancer effects in the body. Moreover, coumarins were found to be potent inhibitors of melatonin metabolism, suggesting that structural similarities between melatonin and coumarins may exist.22 Therefore, coumarin-based melatonin analogues were developed.23 In this study, oxyprenylated ferulic acid and coumarin derivatives (compounds 1−11) were chosen for study because these compounds demonstrate similar neuroprotective and anticancer

lants have been used therapeutically for treating a variety of diseases ranging from neurodegenerative diseases1 to cancer.2,3 The biochemical mechanisms within plants produce secondary metabolites, which are used as host defense mechanisms against herbivores, bacteria, and other invading pathogens.4 Unlike primary metabolites, secondary metabolites are not involved in a plant’s growth, development, or reproduction;5 however, these secondary metabolites (e.g., alkaloids, terpenoids, glycosides, polyketides, phenylpropanoids, and others) have been used for medicinal purposes for centuries to treat diseases such as cancer, leukemia, diabetes, anemia, and inflammation.6,7 Oxyprenylated compounds, a class of secondary metabolites produced during the prenylation of an alkaloid or a phenylpropanoid,8 have demonstrated efficacy against cancer, inflammation, microbes, and fungi possibly through their antioxidant, receptor, or transporter-mediated properties.9−11 As of 2007, about 300 oxyprenylated derivatives have been isolated from plants; some of these derivatives as well as their semisynthetic analogues show promise as anticancer, anti-inflammatory, antimicrobial, and antifungal therapies.9 Ferulic acid, a cinnamic acid derivative, was first isolated in 1866 from Ferula fetida.12 This compound has been demonstrated to prevent mammary tumor formation in Sprague− Dawley rats, perhaps through its antigenotoxic or antioxidant action.13 In addition to the four ferulic acid derivatives 1−4, seven © 2017 American Chemical Society and American Society of Pharmacognosy

Received: October 7, 2017 Published: November 16, 2017 3324

DOI: 10.1021/acs.jnatprod.7b00853 J. Nat. Prod. 2017, 80, 3324−3329

Journal of Natural Products

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Figure 1. Competition of melatonin or 1−11 for 2-[125I]-iodomelatonin binding to human MT1Rs expressed in CHO cells (1A−1L). Each point represents the mean ± SEM of 2-[125I]-iodomelatonin binding normalized against vehicle (total) binding from three independent experiments.

actions to melatonin14,24−28 and they display similar structural similarities to melatonin (specifically in their ring structures; graphical abstract). For most of these compounds, their mechanisms of action are unknown; however, some of the oxyprenylated derivatives, (e.g., 2 and 6) have been shown to activate the aryl hydrocarbon receptor,10 promoting mammary gland differentiation while inhibiting breast cancer (BC) cell proliferation and migration.29−32 In addition, 6 demonstrated growth inhibitory effects in human cancer cell lines (HeLa and HT-29) and neuroprotective effects in astrocytes and neurons perhaps through the activation of the aryl hydrocarbon receptor.10 In this study, oxyprenylated ferulic acid (1−4) and umbelliferone (5−8) derivatives and their semisynthetic analogues (9−11) were tested for their binding affinities to MT1 melatonin receptors and for their effects on various breast cancer cells (Figure 1; Table 1). Using competition binding assays, we assessed the affinity of compounds 1−11 for human MT1Rs expressed in CHO cells. Out of the 11 natural product extracts screened, compounds 1, 2, and 5 did not demonstrate any

concentration-dependent inhibition of 2-[125I]-iodomelatonin binding. Compounds 3 (Ki = 59.54 μM), 4 (Ki = 785.8 nM), 6 (Ki = 5.176 μM), 7 (Ki = 1.894 μM), 9 (Ki = 888.3 nM), 10 (Ki = 445 nM), and 11 (Ki = 369.9 nM) all produced monophonic curves with nM to μM affinity, slightly less than the affinity of melatonin for MT1Rs described in this study (Ki = 303.7 pM). Out of all the compounds screened, 8 bound to MT1Rs in a biphasic manner and with the highest affinity (KiLow = 3.3 nM, KiHigh = 1.6 nM), similar to what was reported previously33 (Figure 1; Table 1). Next, all compounds were screened for their effects on BC cell proliferation to ascertain if binding to MT1Rs correlated with their efficacy against breast cancer cell proliferation. Four breast cancer cell lines were screened: one was an ER+/PR+ line (MCF-7); another was a HER2+ line (MMC); and two were triple-negative (ER−/PR−/HER2−) breast cancer cell lines (MDA-MB-231 and BT549). Out of all compounds screened and when compared to control cells, only 7 and 8 decreased MMC, MDA-MB-231, and BT-549 but not MCF-7 BC cell 3325



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Table 1. Natural Product Composite Binding Affinity to MT1Rs and Potency and Efficacy to Inhibit Cell Proliferationa

a Data represent the mean ± SEM of three independent experiments. Data were analyzed by one-way ANOVA followed by Newman−Keuls post hoc t-test where significance was defined as p < 0.05. * denotes significance vs vehicle. N.C. = not converged.

Figure 2. Cell proliferation in response to melatonin or compounds 1−11. MTT assays were performed on (A) MCF-7, (B) MMC, (C) MDA-MB-231, or (D) BT-549 breast cancer cells. Each point represents the mean ± SEM of three independent experiments. 3326



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Figure 3. Breast cancer cell migration in response to vehicle, 100 μM melatonin or 100 μM 7 or 8 on (A) MCF-7, (B) MMC, (C) MDA-MB-231, or (D) BT-549 breast cancer cells. Each bar represents the mean ± SEM of the border width change that occurred over a 24 h period normalized against baseline (0 h). Change in border width after 24 h exposure to compound 7: MCF = 3% ± 3, MMC = 5% ± 2, MDA-MB-231 = −36% ± 14, BT-549 = 7% ± 3 and compound 8: MCF = 9% ± 4, MMC = 7% ± 3, MDA-MB-231= −52% ± 15, BT-549 = 23% ± 7. Data were analyzed by one-way ANOVA followed by Newman−Keuls post hoc t test, where significance was defined as p < 0.05. “a” denotes p < 0.05 vs vehicle; “b” denotes p < 0.05 vs Mel.

cell proliferation and migration. These novel findings that coumarin derivatives bind to MT1 melatonin receptors and inhibit diverse breast cancer cell lines, including triple negative, suggest that melatonin/umbelliferone/coumarin derivatives may represent novel breast cancer drug candidates to be developed and tested in the future.

proliferation (Table 1; Figure 2). Because 7 and 8 bound to MT1Rs and inhibited breast cancer cell proliferation, they were further tested for their ability to inhibit cell migration using scratch “wound” assays. As shown in Figure 3, both 7 and 8 inhibited MCF-7, MMC, and BT-549 cell migration compared to vehicle and melatonin, and only 7 reduced cell migration in the triple-negative cell line, MDA-MB-231. In this study, we found that the binding affinity at MT1Rs was greatest for compounds 8−11, with 8 displaying the highest affinity (1.6 nM) compared to all the natural products that displayed high nM or low μM affinity. Among compounds 8− 11, only 7 and 8 showed significant reduction of breast cancer cell proliferation (10−20% inhibition of MCF-7 cell proliferation; 40−45% inhibition of MMC cell proliferation; 30−40% inhibition of MDA-MB-231 cell proliferation; and ∼15% inhibition of BT-549 cell proliferation) compared to vehicle-treated cells. Further analysis of 7 and 8 to inhibit breast cancer cell migration demonstrated that they were most efficacious for MCF-7, MMC, and BT-549 cells, suggesting that these compounds may be working through myriad mechanisms that include antioxidative, antiestrogenic, and, perhaps, melatonin receptors; the role of melatonin receptors would have to be further validated by use of selective melatonin receptor antagonists in future studies. Perhaps, the addition of the farnesyl group on the seventh position of umbelliferone/coumarin (8) facilitated high binding affinity to the MT1Rs compared to the other derivatives, whereas geranylation (7) of the auraptene molecule or farnesylation (8) on the seventh position of umbelliferone/ coumarin molecule increased their efficacy to inhibit breast cancer

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EXPERIMENTAL SECTION

Chemical Synthesis. The chemical syntheses of all compounds as well as sources of naturally occurring coumarins 1−8 have been recently reported.34 Briefly, commercially available umbelliferone was alkylated at the phenolic function with the suitable alkyl bromide using acetone as the solvent and in the presence of dry K2CO3 as the base at 80 °C for 1 h, affording the desired adduct. It is noteworthy that 1−11 have been purified by a simple acid−base workup followed by crystallization (n-hexane) from every reaction in very good yields (>92%) and purity (>97.6% assessed by HPLC) without the need of any SiO2 gel based chromatographic step. 1H and 13C NMR data for all samples exactly matched those reported in the literature for the same compounds.10 Melatonin Receptor Binding. 2-[125I]-Iodomelatonin was used to measure the binding affinities of melatonin and compounds 1−11 as described previously with modification.34 Competition binding assays were performed on MT1 CHO whole cell lysates prepared by washing MT1 CHO cells with 5 mL of phosphate-buffered saline grown on a 10 cm cell culture plate (Corning Inc., Corning, NY, USA) followed by lifting them into buffer (10 mM KPO4, 1 mM EDTA, pH 7.4). Cell pellets were then prepared by centrifugation (500g for 5 min) and then resuspended in 50 mM Tris-HCl buffer (pH 7.4). Next, 200 μL of the cell suspension was added to each tube containing 115 pM of 2-[125I]-iodomelatonin in the absence (total binding) or 3327


Journal of Natural Products presence of melatonin (1 pM to 100 μM) or 1−11. Cells were incubated for an hour at room temperature and then filtered rapidly over glass-fiber filters (0.22 μm; Schleicher & Schuell, Keene, NH, USA) presoaked in 0.5% polyethylenimine solution (v/v) (Sigma-Aldrich, St. Louis, MO, USA). Each filter paper was washed twice with 5 mL of ice-cold buffer [50 mM Tris-HCl buffer (pH 7.4)] and then counted in a gamma counter. Cell Culture. MCF-7, MDA-MB-231, and BT-549 cell lines were obtained from American Type Culture Collection (ATCC). MCF-7, MMC (mouse mammary carcinoma), and MDA-MB-231 cells were cultured in DMEM:F12 (Thermo Scientific, Pittsburgh, PA, USA) supplemented with 10% fetal bovine serum (Thermo Scientific) and 1% penicillin/streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere with 5% CO2. BT-549 cells were cultured under the same conditions except that the medium was RPMI-1640 (Thermo Scientific). Cell Proliferation Assay. Cell proliferation assays were conducted using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) per the manufacturer’s instruction. Cells were plated in 48-well plates (Corning Inc.) at a density of 2 × 105 cells/cm2. The next day, cells were exposed to vehicle, melatonin (1 nM, 1 μM, 10 μM, and 100 μM), or 1−11 (1 nM, 1 μM, 10 μM, and 100 μM) in medium for an additional 24 h. The next day, 25 μL of MTT solution (5 mg dissolved in 5 mL of sterile water) was added to each well (0.5 mg/mL final concentration) and then incubated for 3 h (5% CO2 and 37 °C). Next, the medium was aspirated and 250 μL of DMSO (Fisher Scientific) was added to each well to terminate the reaction. The plate was then wrapped in foil, placed on a shaker, and incubated at room temperature for 15 min to dissolve the MTT-formazan crystals. Absorbance readings at 570 nm were made using a spectrophotometer (VICTOR3 1420 multilabel counter, PerkinElmer). Cell Migration Assay. Scratch assays were performed on breast cancer cells plated on 24-well cell plates (Corning Inc.) to assess the effect of 1−11 on migration activity. Twenty-four hours prior to the assay, cells were plated to allow for them to attach and grow. The next day, a uniform scratch was made on the bottom of each well using a sterile 200 μL pipet tip. The media was then removed and replaced with fresh medium containing vehicle (0.01% ethanol) or 100 μM 1−11. An image of the scratch was taken by an EVOS digital inverted fluorescence microscope and designated at baseline (0 h). After 24 h, another image of the same scratch was taken. The border widths at 0 h and 24 h were measured in mm, and the change of border width was expressed as percentages of baseline using the formula (24 h − 0 h/0 h) × 100. A negative number at 24 h indicates cell migration or a narrowing of the border, while a more positive number at 24 h indicates an inhibition of cell migration. Statistical Analysis. All the statistical analyses were performed in Prism software (version 6, GraphPad, La Jolla, CA, USA). Data were represented as the mean ± standard error of mean unless mentioned otherwise. One-way ANOVA with Newman−Keuls multiple comparisons post hoc test was conducted for statistical comparisons among treatment groups. The Ki values were obtained using the nonlinear regression model with one-site fit except for 8, where a two-site fit was used. Mean difference between treatment groups was considered significant at p < 0.05.

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ACKNOWLEDGMENTS

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REFERENCES

Note

This work was supported by the Marie-Clement Rodier, C.S.Sp. Endowed Chair fund awarded to P.A.W.-E.

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AUTHOR INFORMATION

Corresponding Author

*Phone (P. A. Witt-Enderby): +1-412-396-4346. E-mail: [email protected]. ORCID

Francesco Epifano: 0000-0002-0381-7812 Paula A. Witt-Enderby: 0000-0002-1844-4457 Notes

The authors declare no competing financial interest. 3328



Note

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Oxyprenylated Phenylpropanoids Bind to MT1 Melatonin Receptors

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