Chemical proteomics identifies heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as the molecular target of quercetin in its anti-cancer effects in PC-3 cells

Abstract

Quercetin, a flavonoid abundantly present in plants, is widely used as a phytotherapy in prostatitis and prostate cancer. Although quercetin has been reported to have a number of therapeutic effects, the cellular target(s) responsible for its anti-cancer action has not yet been clearly elucidated. Here, employing affinity chromatography and mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as a direct target of quercetin. A specific interaction between quercetin and hnRNPA1 was validated by immunoblotting and in vitro binding experiments. We found that quercetin bound the C-terminal region of hnRNPA1, impairing the ability of hnRNPA1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. In addition, hnRNPA1 was recruited to stress granules after treatment of cells with quercetin for up to 48 h, and the levels of cIAP1 (cellular inhibitor of apoptosis), an internal ribosome entry site translation-dependent protein, were reduced by hnRNPA1 regulation. This is the first report that anti-cancer effects of quercetin are mediated, in part, by impairing functions of hnRNPA1, insights that were obtained using a chemical proteomics strategy.

Keywords: Chemical Proteomics; Molecular Target; Natural Product; Prostate Cancer; Protein Targeting; Proteomics; Quercetin; RNA-binding Protein; hnRNPA1.

FIGURE 1.

Schematic depiction of the workflow used to identify and characterize quercetin-binding proteins. Quercetin-specific binding proteins were captured by quercetin-Sepharose beads, and eluted fractions were resolved by SDS-PAGE. Distinct proteins in gel-eluted bands were identified using MS and validated by immunoblotting analyses and surface plasmon resonance binding assays. Specific targets were further characterized using a series of approaches, including confocal microscopy, IP, RIP, RT-qPCR, and immunoblotting analysis. Q, quercetin; T, total cell lysates; W, proteins that did not bind quercetin; E, bound proteins eluted.

FIGURE 2.

Quercetin uptake, cell viability, and apoptosis assay in quercetin-treated PC-3 cells. A, quercetin uptake was determined by quantifying the intracellular radioactivity of ³H-labeled quercetin at designated time points. B, cells were treated with the indicated concentrations of quercetin for 24, 48, and 72 h, and cell viability was analyzed by quantifying intracellular ATP level using a CellTiter-Glo kit. C, cells were stained with annexin V and caspase-3/7 together with 7-aminoactinomycin D for staining apoptotic cells and dead cells, respectively, after treatment with control or 100 μm quercetin for 24 and 48 h. *, p < 0.05; **, p < 0.01; ***, p < 0.001 with respect to control; Student's t test. QCT, quercetin; Ctrl, vehicle control. Error bars, S.E.

FIGURE 3.

Identification and validation of quercetin binding targets using immunoblotting and surface plasmon resonance binding assays. A, silver-stained gel showing proteins bound to the affinity column in the presence or absence of quercetin. T, total cell lysates; Un, unbound fraction; W, proteins that did not bind quercetin; E, bound proteins eluted. The indicated protein bands were excised from the gel and digested with trypsin, and the peptides were analyzed by MS. The identified quercetin-binding proteins are listed in Table 1. B, both unbound and bound proteins were separated by SDS-PAGE on 10% gels and immunoblotted with antibodies against vinculin, nucleolin, EF-1α, and hnRNPA1. C and E, recombinant full-length hnRNPA1 (aa 1–320) protein and the C-terminal region of hnRNPA1 (aa 268–320) were individually covalently coupled to a Biacore CM5 chip following the manufacturer's instructions. Serial dilutions of quercetin (final concentrations, 10 to 0.15 μm) were perfused over the immobilized proteins to allow association to occur, and dissociation was then monitored. Solvent correction was performed by subtracting vehicle-alone signals and those of quercetin perfused over uncoupled dextran matrix. D and F, saturation curve fitting of quercetin-full-length hnRNPA1 and quercetin-C-terminal region hnRNPA1 (aa 268–320) was plotted using GraphPad Prism software. The K_d values were calculated to be about 8.9 μm for full-length hnRNPA1 and about 1.7 μm for the C-terminal region of hnRNPA1. QCT, quercetin. RU, response units. Error bars, S.E.

FIGURE 4.

Cytoplasmic accumulation of hnRNPA1 and reduced Tnpo1 binding after quercetin treatment. A, PC-3 cells were treated with 100 μm quercetin for 18 and 24 h. Immunofluorescence analyses showed the localization of hnRNPA1 (red), and DAPI was used to stain nuclei. Merge, merged images. Scale bar, 20 μm. B, left, PC-3 cells were treated with 100 μm quercetin for 24 and 48 h. After treatment, cell lysates were immunoprecipitated with anti-hnRNPA1 antibodies, and proteins in immunoprecipitates were resolved by SDS-PAGE on 10% gels and probed with anti-Tnpo1 and anti-hnRNPA1 antibodies. In the bottom panel, one-third of the total protein lysate was loaded. Right, quantification of three independent immunoblotting analyses. The intensity of Tnpo1 was quantified by densitometry and normalized to actin. ***, p < 0.001 with respect to control. IB, immunoblot. Error bars, S.E.

FIGURE 5.

Localization of WT hnRNPA1 and F2mt-hnRNPA1 in transiently transfected PC-3 cells following quercetin treatment. A, mass spectra of F-peptides of cytoplasmic hnRNPA1 treated with vehicle (data not shown) or 100 μm quercetin for 24 h. B, schematic representation of the FLAG WT hnRNPA1 and FLAG F2mt-hnRNPA1 used in this work. The mutations of serines (positions 309–312) to alanines in the F-peptide are shown. C, PC-3 cells were transfected with FLAG WT hnRNPA1 or FLAG F2mt-hnRNPA1, and pull-down fractions were separated by SDS-PAGE, immunoblotted, and probed with anti-FLAG antibody. D, PC-3 cells co-transfected with FLAG WT hnRNPA1 or GFP-F2mt-hnRNPA1 were treated with quercetin for 24 h and then immunostained with antibodies against FLAG (red) and imaged. Nuclei were visualized by counterstaining with DAPI (blue). Merge, merged images. Scale bar, 20 μm. QCT, quercetin; OSM, osmotic stress; T, total cell lysates; W, proteins that did not bind quercetin; E, bound proteins eluted. Error bars, S.E.

FIGURE 6.

Quercetin induces hnRNPA1 localization to SGs and reduces cIAP1 protein expression level. A, the mRNA level of cIAP1 in cytosolic fractions after treatment with 100 μm quercetin was analyzed. by RT-qPCR. B, cytoplasmic mRNA obtained from cytosolic extracts of cell lysates was immunoprecipitated with hnRNPA1 antibodies, purified, and quantified by RT-qPCR. C, PC-3 cells were treated with 100 μm quercetin for 24 and 48 h. Subcellular fractionation and lysates were separated by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies. Respective cytoplasmic and nuclear fractions were loaded and analyzed by immunoblotting as indicated. α-Tubulin and lamin were used as cytosol and nucleus markers, respectively. Quantification of three independent analyses of cIAP1 protein is shown below the blot. The intensity was quantified by densitometry and normalized to that of actin. D, PC-3 cells transfected with shRNAs targeting hnRNPA1 and a non-targeting shRNA (shCtrl) were incubated with quercetin for 48 h. The cells were lysed and analyzed by immunoblotting accordingly with the indicated antibodies. E, PC-3 cells transfected with indicated shRNAs were analyzed. Cells were incubated with vehicle or quercetin (PC-3 control cells and shCtrl) for 48 h. The cells were stained with annexin V and caspase-3/7 together with 7-aminoactinomycin D for staining apoptotic cells and dead cells, respectively. F, PC-3 cells transfected with shRNAs targeting hnRNPA1, cIAP1, and shCtrl were incubated with quercetin for 48 h. Cell viability was analyzed by quantifying intracellular ATP level using a CellTiter-Glo kit. G, cells were fixed and double immunostained with antibodies against hnRNPA1 (green) or PABP (red). Nuclei were visualized by counterstaining with DAPI (blue). Merge, merged images. Scale bar, 20 μm. Arrows, formation of SGs. *, p < 0.05; **, p < 0.01 with respect to control. Error bars, S.E.

FIGURE 7.

Model of quercetin binds to hnRNPA1 and induces apoptotic mechanism in PC-3 cells. A, in normal conditions, hnRNPA1 is released from mRNAs in the cytoplasm through binding Tnpo1 and returns back to the nucleus. B, upon quercetin treatment, quercetin binds to hnRNPA1 and hampers its returning back to the nucleus, resulting in accumulation of hnRNPA1 in the cytoplasm.