CK2 and PI3K are direct molecular targets of quercetin in chronic lymphocytic leukaemia

Abstract

Despite the encouraging results of the innovative therapeutic treatments, complete remission is uncommon in patients affected by chronic lymphocytic leukaemia, which remains an essentially incurable disease. Recently, clinical trials based on BH3-mimetic drugs showed positive outcomes in subjects with poor prognostic features. However, resistance to treatments occurs in a significant number of patients. We previously reported that the multi-kinase inhibitor quercetin, a natural flavonol, restores sensitivity to ABT-737, a BH3-mimetic compound, in both leukemic cell lines and B-cells isolated from patients. To identify the molecular target of quercetin, we employed a new cell line, HG3, obtained by immortalization of B-cells from a chronic lymphocytic leukaemia patient at the later stage of disease. We confirmed that quercetin in association with ABT-737 synergistically enhances apoptosis in HG3 (combination index < 1 for all fractions affected). We also reported that the cellular uptake of quercetin is extremely rapid, with an intracellular concentration of about 38.5 ng/106 cells, after treatment with 25 μM for 5 min. We demonstrated that the activity of protein kinase CK2, which positively triggers PI3K/Akt pathway by inactivating PTEN phosphatase, is inhibited by quercetin immediately after its addition to HG3 cells (0-2 min). PI3K activity was also inhibited by quercetin within 60 min from the treatment. The combined inhibition of CK2 and PI3K kinase activities by quercetin restored ABT-737 sensitivity and increased lethality in human leukemia cells.

Keywords: Mcl-1; PI3K; chronic lymphocytic leukaemia; protein kinase CK2; quercetin.

Figure 1. Quercetin in association with ABT-737 induces apoptosis in HG3 cells

(A) Cells (0.5 × 10⁶/ml) were treated with different doses of ABT-737, quercetin or their combination as indicated for 24 h. Cell death, measured by neutral red assay, is reported as percentage of DMSO (0.1% v/v) treated cells, as described in Materials and Methods. Symbols (a, b, c, d, f, g) indicate significance with respect to DMSO (a) and treated cells (b = 0.25 μM ABT-737; c = 0.5 μM ABT-737; d = 1 μM ABT-737; e = 10 μM quercetin; f = 20 μM quercetin, g = 40 μM quercetin); p < 0.001 for all determinations except for a versus e, where p < 0.05 (one-way ANOVA test). (B) Combination Index (C.I.) isobologram. C.I. values, obtained from neutral red experiment (panel A) using a 1:40 concentration ratio of ABT-737 and quercetin, were plotted against the fraction affected (Fa). (C) Proteolytic activation of caspase-3 was measured after 6 h of incubation with the indicated concentrations of ABT-737 and quercetin and their combination. Immunoblot was performed using a specific antibody against caspase-3 (C3 = caspase-3; Cl-C3 = cleaved caspase-3). (D) Annexin V measurement in HG3 cells after 18 h incubation with quercetin (20 μM), ABT-737 (0.5 μM) and their combination, as described in Materials and Methods. Symbols (a, b, c) indicate significance; p < 0.001 with respect to DMSO (a) and treated cells (b = 0.5 μM ABT-737; c = 20 μM quercetin; d = ABT-737 + quercetin) (one-way ANOVA test).

Figure 2. Quercetin down-regulates Mcl-1 and inhibits Akt phosphorylation in HG3

Cells (0.5 × 10⁶/ml) were treated for the indicated time (min) with quercetin (25 μM) or DMSO (0.1% v/v). Immunoblots were incubated for 16 h at 4°C with anti-phospho-Akt (pAkt) antibody (upper panel), stripped and re-probed with anti-Mcl-1 antibody (lower panel). Densitometric analyses were obtained measuring optical density of bands normalized respect to the expression of α-tubulin (numbers below top and middle panels). Immunoblots are representative of at least four independent experiments.

Figure 3. Quercetin stability in HG3 cells

(A) Cells were incubated in the presence of 25 μM quercetin or vehicle control (DMSO) at different times (5, 60, 120, 240 min until 18 h); subsequently, quercetin was quantified in cells as described in Materials and Methods. (B) Cells (1 × 10⁶/ml) were treated with 25 μM quercetin or DMSO (0.1% v/v) before DPBA staining (5 min) performed as described in Materials and Methods. Cells were visualized using a fluorescent microscopy and photographed in phase contrast (a and c) and in FITC filter with 400× magnification (b and d).

Figure 4. Quercetin inhibits PI₃K activity in vitro and in HG3 cells

(A) In vitro kinase assay was performed using a commercially available kit (Abcam) by measuring the amount of radioactively labeled [γ-³²P] ATP incorporated into a lipid substrate, as described in the manufacturer's protocol in the presence of the range of quercetin concentrations indicated. The enzymatic activity was determined using as catalytic subunit the human recombinant PI₃K-γ (His tagged) produced in sf9 insect cells and included in the kit. Symbols (a, b, c, d) indicate significance with respect to DMSO (a) and treated cells (b = 0.1 μM quercetin; c = 1 μM quercetin; d = 50 μM quercetin); p < 0.01 for all determinations, except for a versus b where p < 0.05 (one-way ANOVA test). (B) Cells (5 × 10⁶/ml) were incubated in the presence of 25 μM quercetin, or DMSO (0.1% v/v) as control, for the indicated time (0 and 1 h). Immunoprecipitation was performed as reported in Materials and Methods section on 500 μg of total proteins using an antibody reacting against the p85-α and -β regulatory subunits of class I PI₃Ks. The immunoprecipitates were used as enzymatic source to detect PI₃K activity using the commercial kit described in panel A. The insert on top of the graph shows a representative autoradiogram of ³²P-phosphorylated lipid substrate, following separation using TLC, as reported in Materials and Methods. Symbols (a, b, c) indicate significance; p < 0.05 with respect to DMSO t = 0 (a); DMSO t = 1 h (c) and treated cells (b = 25 μM quercetin at t = 0; d = 25 μM quercetin at t = 1 h) (one-way ANOVA test). (C). After the kinase assay, the same immunoprecipitates used in panel B were washed to eliminate the reaction buffer remaining after the kinase reactions, added with loading buffer, boiled for 5 minutes and loaded on a 4–12% pre-cast gel before immunoblotting. The membrane was incubated with primary antibody against p85-α and -β regulatory subunits of class I PI₃Ks, or α-tubulin. Band intensities were quantified measuring optical density on Gel Doc 2000 and analysed by Multi-Analyst Software. Numbers between the panels indicate p85 expression normalized respect to α-tubulin. The immunoblot is representative of at least two independent experiments.

Figure 5. Quercetin inhibits CK2 activity restoring the control of PTEN on PI₃K-Akt pathway

(A) HG3 cells, after treatment with 0.1% DMSO, 25 μM quercetin, 12.5 μM TBBz for the indicated times, were lysed and used to assay CK2 activity as described in Materials and Methods. Symbols (a, d) indicate significance with respect to DMSO (a) and treated cells (d = 25 μM quercetin at t = 120 min; g = 12.5 μM TBBz at t = 120 min); p < 0.001 for all determinations, except for d versus g where p < 0.05 (one-way ANOVA test). The immunoblot on the bottom of the panel shows the expression of CK2α subunit detected using a not commercial, anti-CK2α antibody. The membrane was re-probed with the anti α-tubulin antibody. Band intensities were quantified measuring optical density on Gel Doc 2000 and analysed by Multi-Analyst Software. Numbers between the panels indicate CK2α expression normalized respect to α-tubulin. The image is representative of one out of two experiments performed. (B) and (C) HG3 cells were treated as in panel A for 0–2 min (B) or 60 min (C). After immunoblotting, the membranes were incubated for 16 h at 4°C with anti-phospho-Akt (p-Akt^Ser473), anti-phospho-PTEN (p-PTEN^Ser380), anti-Akt1/2/3, anti-PTEN, or α-tubulin antibodies. In panel C, the treatment with CAL-101 (5 μM) was also included. Band intensities were quantified measuring optical density on Gel Doc 2000 and analysed by Multi-Analyst Software. In panel B, numbers between panels indicate the expression of p-Akt^Ser473 and p-PTEN^Ser380, Akt1/2/3 and PTEN normalized respect to α-tubulin. In panel C, numbers between top and middle panels indicate the expression of p-Akt^Ser473 normalized respect to Akt1/2/3. Immunoblots are representative of at least three independent experiments performed. (D) HG3 cells were treated at the concentrations indicated for 24 h and the effect of TBBz, ABT-737 and their combination on cell viability (neutral red assay) was measured. Symbols (a, b, c, d) indicate significance with respect to DMSO (a) and treated cells (b = 12.5 μM TBBz; c = 0.25 μM ABT-737; d = TBBz+ABT-737); p < 0.001 (one-way ANOVA test).

Figure 6. Quercetin and BH3-mimetics enhance caspase-3 activity in HG3 cells

Activation of caspase-3 was measured after 6 h of incubation with the indicated reagents (0.25 μM ABT-263, 0.25 μM Whei, 25 μM quercetin) using the enzymatic assay described in Materials and Methods. Specific caspase-3 activity was expressed as nmol AFC/min/μg protein. Symbols (a, b, c, d, f, g) indicate significance with respect to DMSO (a) and treated cells (b = 0.25 μM ABT-263; c = 0.25 μM WHEI-539; d = 25 μM quercetin; e = 0.25 μM ABT-263 + 25 μM quercetin; f = 0.25 μM WHEI-539 + 25 μM quercetin); p < 0.001 for all determinations except for a versus b where p < 0.05 (one-way ANOVA test).

Figure 7. Scheme summarizing the key targets of quercetin in HG3 cells

The double inhibitory activity of quercetin on CK2 and PI₃K converges on Akt pathway, which is inactivated and unable to phosphorylate and stabilize anti-apoptotic Mcl-1. In parallel, ABT-737 (or other BH3-mimetics) can trigger and inhibit different anti-apoptotic members of Bcl-2 family. The combined effect of quercetin plus ABT-737 restores sensitivity to apoptosis in HG3 and CLL (see text for details).