Quercetin induces tumor-selective apoptosis through downregulation of Mcl-1 and activation of Bax

Abstract

Purpose: To investigate the in vivo antitumor efficacy of quercetin in U937 xenografts and the functional roles of Mcl-1 and Bax in quercetin-induced apoptosis in human leukemia.

Experimental design: Leukemia cells were treated with quercetin, after which apoptosis, Mcl-1 expression, and Bax activation and translocation were evaluated. The efficacy of quercetin as well as Mcl-1 expression and Bax activation were investigated in xenografts of U937 cells.

Results: Administration of quercetin caused pronounced apoptosis in both transformed and primary leukemia cells but not in normal blood peripheral mononuclear cells. Quercetin-induced apoptosis was accompanied by Mcl-1 downregulation and Bax conformational change and mitochondrial translocation that triggered cytochrome c release. Knockdown of Bax by siRNA reversed quercetin-induced apoptosis and abrogated the activation of caspase and apoptosis. Ectopic expression of Mcl-1 attenuated quercetin-mediated Bax activation, translocation, and cell death. Conversely, interruption of Mcl-1 by siRNA enhanced Bax activation and translocation, as well as lethality induced by quercetin. However, the absence of Bax had no effect on quercetin-mediated Mcl-1 downregulation. Furthermore, in vivo administration of quercetin attenuated tumor growth in U937 xenografts. The TUNEL-positive apoptotic cells in tumor sections increased in quercetin-treated mice as compared with controls. Mcl-1 downregulation and Bax activation were also observed in xenografts.

Conclusions: These data suggest that quercetin may be useful for the treatment of leukemia by preferentially inducing apoptosis in leukemia versus normal hematopoietic cells through a process involving Mcl-1 downregulation, which, in turn, potentiates Bax activation and mitochondrial translocation, culminating in apoptosis.

Figure 1

Quercetin markedly induces apoptosis in U937 human leukemia cells in dose-and time-dependent manners, which is associated with down-regulation of Mcl-1. (A) U937 cells were treated with 0, 10, 20, 30, and 40 µM of quercetin for 9 h. (B) The cells were treated with 40 µM of quercetin for 0, 1, 2, 4, 6, 9, 12, and 24 h. For A and B, after treatment the cells were stained with annexin V/propidium iodide (PI), and the percentage of apoptotic cells was determined using flow cytometry. (C) The cells were treated with 0, 10, 20, 30, and 40 µM of quercetin for 6 h and 12 h. (D) The cells were treated with 40 µM quercetin for 0, 1, 2, 4, 6, 9, 12, and 24 h. After treatment in C and D, immunoblot analysis was done to monitor expression of Mcl-1, Bcl-2, and Bcl-xL. Blots were subsequently stripped and reprobed with antibody against β-actin to ensure equivalent loading.

Figure 2

Quercetin induces Bax conformational change and mitochondrial translocation in U937 cells. (A) U937 cells were treated with 0, 10, 20, 30, and 40 µM of quercetin for 6 h and 12 h, after which immunoblot analysis was done to monitor the total levels of Bax. (B) U937 cells were treated with 40 µM of quercetin for 0, 3, 6, and 12 h, after which cells were lysed in CHAPS buffer and subjected to immunoprecipitation (IP) using anti-Bax (6A7) and then immunoblotted with anti-Bax antibody (N-20). For comparison, the lower panel (designated as Lysate) was loaded with whole-cell lysate. Alternatively, cells were treated with 40 µM of quercetin for 9 h, after which cells were stained with FITC conjugated anti-confirmationally changed Bax (6A7) and subjected to flow cytometry. This is the representative histogram (solid, control; dotted, quercetin treatment) of flow data. Untreated control was set up as 100%. (C) U937 cells were treated with 0, 10, 20, 30, and 40 µM of quercetin for 9 h, after which cytosolic and mitochondrial fractions were isolated and subjected to immunoblot analysis using an anti-Bax antibody. For Western blot analysis blots were subsequently stripped and reprobed with an antibody against β-actin (cytosolic fraction) or Cox IV (mitochondrial fraction) to ensure equivalent loading. (D) U937 cells were untreated (panels a–c) or treated (panels d–f) with 40 µM of quercetin for 9 hours. Bax was stained green with FITC conjugated anti-Bax antibody (panel a and d). Mitochondria were stained red with MitoTracker (panel b and e). Panel (c) is the overlay of panels (a) and (b). Panel (f) is the overlay of panels (d) and (e). Co-localization of Bax and mitochondria is shown as yellow.

Figure 3

Quercetin down-regulates Mcl-1 and promotes Bax activation in multiple human leukemia cell lines and primary leukemia blasts, but not in normal human peripheral blood mononuclear cells. (A) U937, Jurkat, and HL-60 cells were exposed to 0, 20, 40, and 60 µM of quercetin for 9 h, after which the percentage of apoptotic cells (Annexin V/PI staining) was determined by flow cytometry. Untreated U937, HL-60, and Jurkat cells were lysed and subjected to immunoblot analysis to detect basic protein levels of Mcl-1 (inset). (B) Jurkat and HL-60 cells were treated with 40 µM of quercetin for 0, 2, 4, 8, and 12 h, after which immunoblot analysis was done to monitor Mcl-1 expression. For Western blot analysis blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading. Alternatively, cells were treated with 40 µM of quercetin for 9 h, and flow cytometry analysis was performed to detect the percentage of cells with active Bax (6A7 positive). Untreated control was set up as 100%. (C) Mononuclear cells were isolated from the BM (bone marrow) or PM (peripheral blood) of five leukemia patients (designated as # 1–5), including two AML (acute myeloid leukemia), one MM (multiple myeloma), and two CLL (chronic lymphocytic leukemia) patients. Cells then incubated with 0, 20, 40, and 60 µM of quercetin for 9 h. At the end of this period, the percentage of apoptotic cells (Annexin V/PI staining) was determined by flow cytometry. The blasts were incubated with 40 µM of quercetin and then lysed for immunoblot using Mcl-1 primary antibody (Patient #2 and Patient #5) (inset, left) or analyzed by flow cytometry using FITC-Bax 6A7 antibody (Patient #5) (inset, right). For Western blot analysis blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading. For flow cytometry analysis, untreated control was set up as 100%. (D) Mononuclear cells were isolated from the PM of two healthy donors. Cells then incubated with 0, 20, 40, 60 and 80 µM of quercetin for 9 h. At the end of this period, the percentage of apoptotic cells (Annexin V/PI staining) was determined by flow cytometry. The blasts were incubated with 40 and 80 µM of quercetin and then lysed for immunoblot using Mcl-1 primary antibody (Donor #1) (inset, left) or analyzed by flow cytometry using FITC-Bax 6A7 antibody (Donor #1) (inset, right). For Western blot analysis blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading. For flow cytometry analysis, untreated control was set up as 100%.

Figure 4

Ectopic expression of Mcl-1 markedly protects cells from quercetin-induced apoptosis, while Mcl-1 siRNA transfection renders cells more susceptible to quercetin-induced apoptosis. U937 cells stably transfected with an empty vector (pCEP) and Mcl-1 construct were performed. (A) Cells were treated with 0, 20, 40, and 60 µM of quercetin for 9 h, after which apoptosis was analysed using Annexin V/PI assay. *Values for Mcl-1 cells were significantly decreased compared to those for pCEP cells after quercetin treatment at concentrations of 40 and 60 µM by ANOVA; p < 0.05. (B) Cells were treated with 0, 10, 20, 30, and 40 µM of quercetin for 9 h, after which total cellular extracts were prepared and subjected to Western blot analysis using antibodies against Mcl-1, Bcl-2, and Bcl-xL. Blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading. (C) and (D) U937 cells were transfected with Mcl-1 siRNA or Control siRNA and incubated for 24 h at 37°C, after which cells were treated with 20 µM of quercetin for additional 9 h, after which (C) the percentage of apoptotic cells was determined using the Annexin V/PI assay by flow cytometry. *Values for quercetin-treated cells were significantly increased compared to those for untreated cells after transfected with Mcl-1 siRNA by ANOVA; p < 0.05. (D) cells were lysed and subjected to immunoblot analysis using antibodies against Mcl-1, Bcl-2, and Bcl-xL. For Western blot analysis blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading.

Figure 5

(A) and (B) Down-regualation of Bax successfully protects cells from cell death induced by quercetin. U937 cells were transfected with either Bax siRNA or Control siRNA for 24 h, after which cells were treated with 40 µM of quercetin for additional 9 h., after which (A) apoptosis was determined using the Annexin V/PI assay by flow cytometry. *Values for Bax siRNA-treated cells were significantly decreased compared to those for Control siRNA-treated cells after treatment with quercetin by ANOVA; p < 0.05, (B) total cellular extracts were prepared and subjected to immunoblot analysis using antibodies against Bax, Mcl-1, and PARP. Blots were subsequently stripped and reprobed with an antibody against β-actin to ensure equivalent loading. (C), (D) and (E) Mcl-1 inhibits Bax transformational change and translocation. (C) WT U937, U937 stably-over-expressing Mcl-1, and U937 transfected with Mcl-1 siRNA cells were treated with 40 µM quercetin for 9 h, after which the percentage of 6A7 Bax positive cells (U937 WT untreated cells were set up as 100%) was determined by flow cytometry. *Values for U937/Mcl-1 cells were significantly decreased compared to those for U937 WT cells after treatment with quercetin by ANOVA; p < 0.05. *Values for Mcl-1 siRNA-treated cells were significantly increased compared to those for U937 WT cells after treatment with quercetin by ANOVA; p < 0.05. U937/Mcl-1 and its empty-vector control (U937/pCEP) cells (D) and U937 transfected with Mcl-1 siRNA or Control siRNA cells (E) were treated with 0 and 40 µM of quercetin, after which cytosolic (designated as C) and mitochondrial (designated as M) fractions were prepared and subjected to immunoblot analysis using an anti-Bax antibody. For western blot analysis, blots were subsequently stripped and reprobed with an antibody against β-actin (cytosolic fraction) or Cox IV (mitochondrial fraction) to ensure equivalent loading.

Figure 6

Quercetin inhibits tumor growth and induces apoptosis in the xenograft animal model. 6- to 8-week-old nude mice received subcutaneous transplants of 6 × 10⁶ U937 cells. From the second day, mice were randomized into a control group (6 mice/group) and two treated groups (6 mice/group, quercetin 20 mg/kg and 40 mg/kg). Quercetin i.p. administration and tumor volume assessment were conducted as described in “Methods”. Representive animals with solid tumor volume were shown in (A), tumor volume measured in day 11 and day 16 was shown in (B), body weight was shown in (C), and representative IHC images for TUNEL staining and percentage of apoptotic cells (TNUNEL positive cells) in tumor tissue were shown in (D), representative IF images for Mcl-1 expression were shown in (E) and Bax activation using IP (anti-Bax 6A7) following immunoblotting (anti-Bax N-20) was shown in (F) in tumor samples. *Values of tumor volumn for quercetin treatment groups were significantly decreased compared to those for non-treatment group by Student’s t-test; p < 0.05. *Values of TUNEL positive cells for quercetin treatment groups were significantly increased compared to those for non-treatment group by Student’s t-test; p < 0.05.