Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets

Abstract

Flavonoids constitute the largest class of dietary phytochemicals, adding essential health value to our diet, and are emerging as key nutraceuticals. Cellular targets for dietary phytochemicals remain largely unknown, posing significant challenges for the regulation of dietary supplements and the understanding of how nutraceuticals provide health value. Here, we describe the identification of human cellular targets of apigenin, a flavonoid abundantly present in fruits and vegetables, using an innovative high-throughput approach that combines phage display with second generation sequencing. The 160 identified high-confidence candidate apigenin targets are significantly enriched in three main functional categories: GTPase activation, membrane transport, and mRNA metabolism/alternative splicing. This last category includes the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), a factor involved in splicing regulation, mRNA stability, and mRNA transport. Apigenin binds to the C-terminal glycine-rich domain of hnRNPA2, preventing hnRNPA2 from forming homodimers, and therefore, it perturbs the alternative splicing of several human hnRNPA2 targets. Our results provide a framework to understand how dietary phytochemicals exert their actions by binding to many functionally diverse cellular targets. In turn, some of them may modulate the activity of a large number of downstream genes, which is exemplified here by the effects of apigenin on the alternative splicing activity of hnRNPA2. Hence, in contrast to small-molecule pharmaceuticals designed for defined target specificity, dietary phytochemicals affect a large number of cellular targets with varied affinities that, combined, result in their recognized health benefits.

Keywords: FRET; cancer; inflammation; nanosensor.

Fig. 1.

Synthesis of apigenin beads and PD-Seq strategy outline. (A) Scheme for the chemical synthesis of A- (orange) and C-beads (blue). The coupling of apigenin to the beads occurred at the end of a polyethylene glycol linker (PEGA beads). Depending on the apigenin –OH group participating in the coupling to the phenyl bromoacetate group, A-beads consist of a combination of three products. (B) Schematic representation of the biopanning steps in the screening of a phage display cDNA library generated from human breast tumor cells mRNA. Three rounds of biopanning (3×), each including binding to the beads, washing, elution, and amplification, were performed in parallel using A- or C-beads. (C) Schematic representation of the fractions used to make the libraries for Illumina GAII sequencing. The preclearing and washing steps were skipped here for simplicity. The original library was an aliquot of a single amplified library purchased from Novagen. Original library (Ori-lib) and input and elution fractions (referred as E) obtained from the first and second rounds of biopanning using A- and C-beads (A-E1 and A-E2 and C-E1 and C-E2, respectively) were used to generate libraries for sequencing. (D) Schematic representation of Illumina GAII libraries preparation. PCR primers (indicated by arrows) at the cDNA insert and vector boundaries were used to amplify the cDNA-containing region, and they were subsequently ligated to Illumina adapters (gray areas) and indexed sequences (red area). (E) Heat map of nICPGs for each biopanning step was generated based on hierarchical clustering (Fig. S2). Cluster I, shown here, consisting of 160 genes is significantly enriched in the A-E2 fraction.

Fig. 2.

hnRNPA2 directly binds apigenin through the GRD. (A) Lysates from HeLa cells expressing full-length hnRNPA2-GFP or GFP alone were pulled down with A- or C-beads. Pull-down assays were resolved by SDS/PAGE and analyzed by Western blot using anti-GFP antibodies. (B) Schematic representation of GST-hnRNPA2 clones used in the pull-down assays with C- or A-beads. RRM, RNA recognition motifs of hnRNPA2. hnRNPA2^C corresponds to the C-terminal 78-aa fragment present in ϕ-hnRNPA2^C identified by conventional phage display screening. hnRNPA2^∆C corresponds to a clone in which this C-terminal 78-aa fragment was deleted. hnRNPA2^∆GRD corresponds to a clone in which the GRD domain was deleted. GST-hnRNPA2^∆C corresponds to hnRNPA2 lacking the C-terminal region. All forms of hnRNPA2 were GST-tagged, E. coli-expressed, and affinity-purified forms. (C) Different versions of recombinant affinity-purified GST-hnRNPA2 proteins were pulled down with A- or C-beads (indicated as A or C, respectively). Pull-down assays (bound) and supernatants fractions were resolved by SDS/PAGE and analyzed by Western blot using anti-GST antibodies. Arrows indicate the correctly sized products; smaller bands present in some of the lanes correspond to degradation products.

Fig. 3.

Binding affinity of the interaction of hnRNPA2 with apigenin. (A) Apigenin (10 μM) was titrated with increasing concentrations of purified GST-hnRNPA2 (0, 0.2, 0.5, 1, 2, 5, 10, and 20 μM) or GST (Inset). Changes in absorption across the UV-visible spectrum were determined over the 250- to 450-nm range. (B) Changes in absorbance of apigenin at 370 nm as determined in A. (C) Dissociation constant (K_D) of the apigenin–hnRNPA2 complex calculated using the Benesi–Hilderbrand method as described in Materials and Methods. Data represent the mean ± SEM (n = 3). (D) hnRNPA2^C (Fig. 2B) was cloned into the pFLIP2 vector in frame between the regions coding for the N-terminal CFP and C-terminal YFP. The affinity-purified FLIP2-3-hnRNPA2^C protein was incubated with increasing concentrations of apigenin (0, 1, 5, 10, 25, 50, and 100 μM) for 3 h at 37 °C. Relative fluorescence units (RFUs) were determined by spectrofluorometry (λ_ext = 405 nm; λ_emi = 460–600 nm) and represented as emission spectra. The absence of an isosbestic point in the nanosensor spectra is likely a consequence of flavonoids, particularly flavones, absorbing light at a wavelength that partially overlaps with the CFP excitation spectrum (Fig. S6). YFP fluorescence is not affected by the various flavonoids over the broad concentration range tested (Fig. S6). (E) The calculated YFP/CFP fluorescent ratios (530/480 nm) are represented over the 0- to 100-μM concentration range for each flavonoid. (F) Flavonoid-dependent changes in YFP/CFP ratios were transformed into saturation curves as described in Materials and Methods. K_D value was determined by using nonlinear regression. Data represent the mean ± SEM (n = 3).

Fig. 4.

Apigenin affects hnRNPA2 dimerization. (A) Purified 6xHis-hnRNPA2 (125 nM) protein was incubated with 125 nM native GST-hnRNPA2, 125 nM boiled GST-hnRNPA2, or 125 nM GST for 1 h at room temperature followed by the addition of GSH-acceptor and anti–His-donor beads for 6 h. (B) GST-hnRNPA2 (125 nM) was incubated with 125 nM 6xHis-hnRNPA2 for 1 h at room temperature followed by the addition of GSH-acceptor and anti–His-donor beads for 6 h at room temperature. Apigenin (Api; 100 μM), naringenin (Nar; 100 μM), or diluent control (DMSO) was added for 15 min at room temperature. Data represent the mean ± SEM (n = 4). *P < 0.05.

Fig. 5.

Apigenin regulates alternative splicing of hnRNPA2 substrates in breast cancer cells. (A–C) MDA-MB-231 breast cancer cells were treated with 50 μM apigenin (Api), luteolin (Lut), or naringenin (Nar) or diluent DMSO for 48 h. Total RNA was isolated, and alternative splicing was analyzed by RT-PCR using specific primers for (A) c-FLIP, (B) caspase-9, and (C) BIRC5. GAPDH expression was used as the loading control. Reactions were resolved in 2% (wt/vol) agarose gels. Positions of the primers (arrows) and splicing variants (boxes) are represented schematically on the right. (D–F) Graphs represent the percent of the indicated splice isoform. Data represent the mean ± SEM (n = 3). *P < 0.05 compared with DMSO control samples.