Glaucarubulone glucoside from Castela macrophylla suppresses MCF-7 breast cancer cell growth and attenuates benzo[a]pyrene-mediated CYP1A gene induction

Abstract

Quassinoids often exhibit antioxidant and antiproliferative activity. Emerging evidence suggests that these natural metabolites also display chemopreventive actions. In this study, we investigated the potential for the quassinoid glaucarubulone glucoside (Gg), isolated from the endemic Jamaican plant Castela macrophylla (Simaroubaceae), to display potent cytotoxicity and inhibit human cytochrome P450s (CYPs), particularly CYP1A enzymes, known to convert polyaromatic hydrocarbons into carcinogenic metabolites. Gg reduced the viability of MCF-7 breast adenocarcinoma cells (IC₅₀ = 121 nm) to a greater extent than standard of care anticancer agents 5-fluorouracil, tamoxifen (IC₅₀ >10 μm) and the tamoxifen metabolite 4-hydroxytamoxifen (IC₅₀ = 2.6 μm), yet was not cytotoxic to non-tumorigenic MCF-10A breast epithelial cells. Additionally, Gg induced MCF-7 breast cancer cell death. Gg blocked increases in reactive oxygen species in MCF-10A cells mediated by the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) metabolite B[a]P 1,6-quinone, yet downregulated the expression of genes that promote antioxidant activity in MCF-7 cells. This implies that Gg exhibits antioxidant and cytoprotective actions in non-tumorigenic breast epithelial cells and pro-oxidant, cytotoxic actions in breast cancer cells. Furthermore, Gg inhibited the activities of human CYP1A according to non-competitive kinetics and attenuated the ability of B[a]P to induce CYP1A gene expression in MCF-7 cells. These data indicate that Gg selectively suppresses MCF-7 breast cancer cell growth without impacting non-tumorigenic breast epithelial cells and blocks B[a]P-mediated CYP1A induction. Taken together, our data provide a rationale for further investigations of Gg and similar plant isolates as potential agents to treat and prevent breast cancer. Copyright © 2017 John Wiley & Sons, Ltd.

Keywords: breast cancer; chemoprevention; cytochrome P450; cytotoxicity; natural product.

Figure 1

Structures of isolated constituents from Castela macrophylla.

Figure 2

Gg demonstrates greater anticancer activity than 5-Fluorouracil, tamoxifen or its metabolite 4OH-tamoxifen in MCF-7 breast cancer cells. In A-C, cells were analysed for cell survival using the Alamar Blue™ assay following treatment, as outlined in Materials and Methods. Statistical significance as indicated by * P < 0.05, **P < 0.01 or ***P < 0.01 versus vehicle control.

Figure 3

Gg displays no appreciable cytotoxicity in MCF-10A breast epithelial cells and apoptosis in MCF-7 breast cancer cells.(A) MCF-10A were exposed to Gg (1 nM-10 μM) 4 OH-Tam (1 nM-10 μM) or vehicle for 72 h before Alamar Blue™ assay analysis was employed, as outlined in Materials and Methods. Statistical significance as indicated by *** P < 0.001 versus vehicle control.(B) MCF-7 cells were exposed to media containing Gg (0.01-1.0 μM) or 0.025% DMSO for 24 h before being analysed for apoptosis using the AnnexinV-7AAD assay, as described in Materials and Methods. Data represent the mean percentage ± SEM of three independent experiments performed in triplicate. Statistical significance as indicated by * P < 0.05 versus vehicle control.

Figure 4

Gg inhibits CYP1 enzyme activities and suppresses B[a]P-induced CYP1A gene expression in non-invasive MCF-7 breast cancer cells. (A) Human recombinant CYP1B1-catalysed 7-ethoxyresorufin activity (0.37μM), CYPs 1A1 catalysed 7-ethoxy-3-cyanocoumarin deethylase activity (0.5μM), were determined in the presence of varying concentrations of Gg (0-20μM, as described in Materials and Methods for IC₅₀ determinations. Control enzyme activity (mean ± SEM) for CYPs 1B1 and 1A1 were 0.34 ± 0.08, 0.86 ± 0.01 μM/min/pmol of CYP respectively. (B) Human recombinant CYP activity (as indicated by relative fluorescence) for isoforms CYP1A1, CYP1B1, CYP2C19, CYP2D6 and CYP3A4 following treatment with Gg reported as IC₅₀ values in accordance with Materials and Methods. Results are represented as the mean of at least three independent experiments ±SEM. (C) MCF-7 cells were exposed to B[a]P alone or in combination with Gg at indicated concentrations for 24 h. Cells were harvested, RNA extracted and quantitative real-time PCR analysis performed in accordance with Materials and methods to evaluate CYP1A1 and CYP1A2 mRNA expression. Data represent the mean ± SEM of three independent experiments. Statistical significance as indicated by ** P< 0.01 or ***P < 0.001 versus treatment with B[a]P only.

Figure 4

Gg inhibits CYP1 enzyme activities and suppresses B[a]P-induced CYP1A gene expression in non-invasive MCF-7 breast cancer cells. (A) Human recombinant CYP1B1-catalysed 7-ethoxyresorufin activity (0.37μM), CYPs 1A1 catalysed 7-ethoxy-3-cyanocoumarin deethylase activity (0.5μM), were determined in the presence of varying concentrations of Gg (0-20μM, as described in Materials and Methods for IC₅₀ determinations. Control enzyme activity (mean ± SEM) for CYPs 1B1 and 1A1 were 0.34 ± 0.08, 0.86 ± 0.01 μM/min/pmol of CYP respectively. (B) Human recombinant CYP activity (as indicated by relative fluorescence) for isoforms CYP1A1, CYP1B1, CYP2C19, CYP2D6 and CYP3A4 following treatment with Gg reported as IC₅₀ values in accordance with Materials and Methods. Results are represented as the mean of at least three independent experiments ±SEM. (C) MCF-7 cells were exposed to B[a]P alone or in combination with Gg at indicated concentrations for 24 h. Cells were harvested, RNA extracted and quantitative real-time PCR analysis performed in accordance with Materials and methods to evaluate CYP1A1 and CYP1A2 mRNA expression. Data represent the mean ± SEM of three independent experiments. Statistical significance as indicated by ** P< 0.01 or ***P < 0.001 versus treatment with B[a]P only.

Figure 5

Gg suppresses 1,6-BPQ-mediated increases in ROS in non-malignant MCF-10A cells. MCF-10A cells were exposed to Gg (1 μM) alone or in combination with 1,6-BPQ (2 μM) for 2 hours before being analysed for ROS production using flow cytometry as described in Materials and methods. Data represent the mean ± SEM of three independent experiments. Statistical significance as indicated by * P < 0.05 or ** P < 0.01 when comparing indicated data points.