Proteomic Analysis of Quercetin-Treated K562 Cells

Abstract

Among natural products under investigation for their additive potential in cancer prevention and treatment, the flavonoid quercetin has received attention for its effects on the cell cycle arrest and apoptosis. In the past, we addressed this issue in K562 cells, a cellular model of the human chronic myeloid leukemia. Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) proteomics with the aim to increase knowledge on the regulative and metabolic pathways modulated by quercetin in these cells. After 24 h of quercetin treatment, we observed that apoptosis was not completely established, thus we selected this time range to capture quantitative data. As a result, we were able to achieve a robust identification of 1703 proteins, and to measure fold changes between quercetin-treated and untreated cells for 1206 proteins. Through a bioinformatics functional analysis on a subset of 112 proteins, we propose that the apoptotic phenotype of K562 cells entails a significant modulation of the translational machinery, RNA metabolism, antioxidant defense systems, and enzymes involved in lipid metabolism. Finally, we selected eight differentially expressed proteins, validated their modulated expression in quercetin-treated K562 cells, and discussed their possible role in flavonoid cytotoxicity. This quantitative profiling, performed for the first time on this type of tumor cells upon treatment with a flavonoid, will contribute to revealing the molecular basis of the multiplicity of the effects selectively exerted by quercetin on K562 cells.

Keywords: K562; SILAC; apoptosis; chronic myeloid leukemia; flavonoids; lipid metabolism; oxidative stress; quantitative proteomics; quercetin.

Figure 1

Effect of 25 µM quercetin on K562 cell growth (A), apoptosis (B), and caspase-3 activity (C). (A) Cells were counted and the number of trypan blue-negative cells was determined at the indicated times. (B) The percentage of condensed and fragmented nuclei was estimated by fluorescence microscope analysis of acridine orange and ethidium bromide double-stained cells at the indicated times. At least 400 cells were counted for each determination. (C) The caspase-3 activity was measured spectrofluorimetrically using DEVD-aminomethylcoumarin as substrate. Results represent the mean ± SD of three independent experiments. Statistical evaluation was achieved by Student’s t-test. *, data are significantly different from untreated cells (p < 0.05).

Figure 2

Schematics of the workflow developed in this work. The entire strategy employed in profiling protein fold changes in quercetin-treated K562 cells consists of four main steps (horizontal levels). (A) Cell growing. Orange culture dishes codes for K562 cells grown in a “heavy” medium supplemented with L-[4,4,5,5-D4]-lysine and L-[¹³C6¹⁵N4]-arginine (marked as K4, R10); grey culture dishes codes for K562 cells grown in a “light” medium supplemented with L-lysine and L-arginine (marked as K0, R0). (B) Treatment scheme in “forward” and “reverse” SILAC experiments. In “forward” experiments, heavy isotope labelled cells (in orange code) were treated for 24 h with 25 μM quercetin (red lightning bolt), whereas control cells (grey coded) where treated with a vehicle solution (DMSO; cyan lightning bolt). In “reverse” SILAC experiments, media were swapped. The three replicates for each status are also shown. (C) Scheme of the pairwise merging into the SILAC samples. For sake of clarity, merging of only a treated and control replicate over three for each SILAC experiment is shown. Arrows with a cyan tail refers to the aliquot from a control cultured cell, whereas the aliquot from a quercetin treated sample is coded by an arrow with a red tail; colors of the arrow heads code for the stable isotope labelling of the cell culture, as described in line A. (D) Summary of the proteomics strategy. Figures of the SDS-PAGE of the three replicates for each “forward” and “reverse” SILAC experiments are shown. Dashed lines refer to slice excision selection. At this final step, two further replicates (“technical”) were obtained by splitting each slice, and each submitted to LC-MSMS analysis.

Figure 3

Summary of SILAC data. (A) Volcano plot of quantitative data. One sample t-test analysis was performed on the protein relative abundances measured among data from 12 replicates between quercetin treated and control cells (Q/C ratios; columns headed as “H/L normalized ratio” in Table S2, according to the original MaxQuant output). Log₂ Q/C values were plotted against the −log₁₀ of the FDR adjusted p-values (according to the Benjamini–Hochberg method; y axis, –log₁₀ p-value). Vertical and horizontal lines mark Q/C and p-values used as arbitrary thresholds in DEPs selection. Blue dots show proteins upregulated in quercetin-treated cells, red dots those downregulated. (B) Western blotting for SLC3A2 and HMGCS1 proteins, employed in validating SILAC data. Immunoblots are representative of three independent experiments with similar results. C: Control, untreated cells; Q: Quercetin-treated cells. (C) Quantification of relative abundance. In the densitometric analysis, after normalization with tubulin or actin protein levels, values have been obtained by the ratio between the intensities of SLC3A2 and HMGCS1 bands in quercetin-treated and -untreated cells, assigning the value 1 to the control. Results represent the mean ± SD of three independent experiments. Statistical evaluation was achieved by Student’s t-test. *, data significantly different from untreated cells (p < 0.05).

Figure 4

Enrichment analysis of the 112 differentially expressed proteins (DEPs) in K562 cells upon 24 h treatment with quercetin. GO terms found enriched among biological processes (ORA analysis by WebGestalt). X-axis—enrichment ratios. Process categories are listed on the left bar side. p-values and FDR values are also showed right next to each bar. The 2.22 × 10⁻¹⁶ value is the smallest positive floating-point number in the R platform. Blue bars refer to downregulated, and red bars to upregulated proteins, as listed in Table 1 and Table 2. Statistics are shown in Table S3.

Figure 5

Western blotting analyses in K562 cells treated for 24 h with 25 µM quercetin. (A) Representative immunoblots of three independent experiments with similar results; C: control, untreated cells, Q: quercetin-treated cells. (B) In the densitometric analysis, after normalization with tubulin or actin protein levels, relative protein expression values have been determined as ratios between the intensities of protein bands in treated and untreated cells, assigning the value 1 to the control. Results represent the mean ± SD of three independent experiments. Statistical evaluation was achieved by Student’s t-test. *, p < 0.05 vs. control; **, p < 0.01 vs. control.

Figure 6

Analysis of lipid content in quercetin-treated K562 cells by Nile Red staining. (A) Intracellular lipid content was quantified after Nile Red staining by spectrofluorimetric analysis. Nile Red displays different emission maxima, depending on the hydrophobicity of the bound lipids; Ex/Em 485/570: Nile-Red-stained neutral lipids; Ex/Em 485/620: Nile-Red-stained polar lipids. Values have been obtained by the ratios between the fluorescence of treated and untreated cells, assigning the value 1 to the control. Results represent the mean ± SD of three independent experiments. Statistical evaluation was achieved by Student’s t-test. *, Data are significantly different from untreated cells (p < 0.05). (B–D) Neutral-Red-stained neutral lipids of untreated, quercetin-treated for 24 h and 48 h K562 cells, respectively, were visualized by fluorescence microscopy. Scale bars, 25 μm.