Quercetin potentiates doxorubicin mediated antitumor effects against liver cancer through p53/Bcl-xl

Abstract

Background: The dose-dependent toxicities of doxorubicin (DOX) limit its clinical applications, particularly in drug-resistant cancers, such as liver cancer. In this study, we investigated the role of quercetin on the antitumor effects of DOX on liver cancer cells and its ability to provide protection against DOX-mediated liver damage in mice.

Methodology and results: The MTT and Annexin V/PI staining assay demonstrated that quercetin selectively sensitized DOX-induced cytotoxicity against liver cancer cells while protecting normal liver cells. The increase in DOX-mediated apoptosis in hepatoma cells by quercetin was p53-dependent and occurred by downregulating Bcl-xl expression. Z-VAD-fmk (caspase inhibitor), pifithrin-α (p53 inhibitor), or overexpressed Bcl-xl decreased the effects of quercetin on DOX-mediated apoptosis. The combined treatment of quercetin and DOX significantly reduced the growth of liver cancer xenografts in mice. Moreover, quercetin decreased the serum levels of alanine aminotransferase and aspartate aminotransferase that were increased in DOX-treated mice. Quercetin also reversed the DOX-induced pathological changes in mice livers.

Conclusion and significance: These results indicate that quercetin potentiated the antitumor effects of DOX on liver cancer cells while protecting normal liver cells. Therefore, the development of quercetin may be beneficial in a combined treatment with DOX for increased therapeutic efficacy against liver cancer.

Figure 1. Quercetin potentiates the effect of DOX on proliferation and apoptosis in liver cancer cells, but not in normal liver cells.

(A) Effect of quercetin on the proliferation of SMMC7721 and QGY7701 liver cancer cells, and L02 normal liver cells. The cells were incubated with quercetin (0 µM to 150 µM) for 48 h and subjected to an MTT assay to determine the proliferation rate. (B) Quercetin sensitized SMMC7721 cells to DOX. (C) Quercetin sensitized QGY7701 cells to DOX. (D) Quercetin partially reduced the DOX-induced growth inhibition in L02 cells. The cells were incubated with DOX (1 µM) and/or quercetin (20 µM) (B, C, and D) for 24 h, and then subjected to an MTT assay. Data are presented as mean ± S.D. of three independent experiments. (E) SMMC7721 and L02 cells were incubated with DOX (1 µM) and/or quercetin (20 µM) for 24 h, and then analyzed by flow cytometry using Annexin V/PI staining to discriminate the live cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−), necrosis or late apoptotic cells (Annexin V+/PI+), and dead cells (Annexin V−/PI+). *P<0.05 vs. cells co-treated with quercetin.

Figure 2. Quercetin enhances DOX-induced caspase activation.

(A) Cleaved caspases-3, -8, and -9 and PARP expression were assessed by western blotting in SMMC7721 cells treated with DOX (1 μM) and/or quercetin (20 μM) for 24 hours. β-actin was used as an internal control. (B) Caspase-3 activity assay of DOX- (1 µM) and/or quercetin-(20 µM) treated SMMC7721 cells for 24 h. Cell lysates were incubated with fluorogenic caspase-3 substrate for 1 h at 37°C. Caspase-3 activity was normalized to cell lysate protein and expressed as fold activation compared with the control. *P<0.05 vs. DOX-treated cells. (C) Caspase inhibitor Z-VAD-fmk reduced the effect of quercetin on DOX-induced apoptosis in QGY7701 cells examined by PI staining. Control: 0.5% dimethyl sulfoxide; quercetin: 20 µM; DOX: 1 µM; DOX + quercetin: DOX 1 µM plus quercetin 20 µM; Z-VAD-fmk + DOX + quercetin: cells were pretreated with 25 µM of z-VAD-fmk for 1.5 h and further treated with DOX plus quercetin for 24 h. Data are presented as mean ± S.D. of three independent experiments. *P<0.05 vs. DOX-treated cells. **P<0.01 vs. DOX + quercetin-treated cells.

Figure 3. Quercetin potentiates DOX-induced apoptosis in liver cancer cells through Bcl-xl/Bax-mediated mitochondrial pathway.

(A, B) Effect of DOX and/or quercetin on the mitochondrial membrane potential breakdown in SMMC7721 cells treated with DOX (1 µM) and/or quercetin (20 µM) for 24 h. JC-1 is observed as green fluorescing monomers in the cytosol or as red fluorescing aggregates in intact mitochondria. The reduction of red fluorescence intensity indicates mitochondrial breakdown with intact membrane potential. *P<0.01 vs. DOX-treated cells. (C) Western blot analysis of the total expression of Bid, Bcl-2, and Bcl-xl, as well as the mitochondrial distribution of Bax and the cytosol distribution of cytochrome c in SMMC7721 cells treated with DOX (1 µM) and/or quercetin (20 µM) for 24 h. β-actin was used as an internal control for the total protein and cytosol protein. Hsp60 was used as an internal control for the mitochondrial protein. (D) Western blot analysis of the mitochondrial distribution of Bax and the total expression of Bcl-xl in SMMC7721 cells transfected with Bcl-xl expression vector and treated with DOX (1 µM) and/or quercetin (20 µM) for 24 h. (E) Effect of DOX and/or quercetin on the apoptosis of SMMC7721/Bcl-xl and SMMC7721/neo cells assayed by PI staining after DOX (1 µM) and/or quercetin (20 µM) was administered for 24 h. *P<0.01, SMMC7721/neo vs. SMMC7721/Bcl-xl cells. Data are presented as mean ± S.D. of three independent experiments.

Figure 4. Quercetin potentiates DOX-induced apoptosis of SMMC7721 cells in a p53-dependent manner.

(A) p53 and PUMA expressions were assessed by western blot in SMMC7721 cells treated with DOX (1 µM) and/or quercetin (20 µM) for 24 h. β-actin was used as an internal control. (B) The luciferase assay of DOX- and quercetin-induced p53 activation. SMMC7721 cells were pretreated for 1 h with 2 µM of pifithrin-α, and then treated with DOX (1 µM) or quercetin (20 µM) for 12 h. p53 activity was determined by luciferase activity assay. Data are presented as mean ± S.D. of three independent experiments. *P<0.01 vs. DOX-treated cells. **P<0.001 vs. DOX + quercetin-treated cells. (C) SMMC7721 cells were pretreated with 2 µM of pfithrin-α for 1 h, and then treated with DOX (1 µM) and/or quercetin (20 µM) for 24 h. Apoptosis rates were assayed by Annexin V/PI staining. Data are presented as mean ± S.D. of three independent experiments. *P<0.001 vs. DOX-treated cells. **P<0.01 vs. DOX-treated cells. ***P<0.001 vs. DOX + quercetin-treated cells.

Figure 5. Quercetin potentiates SMMC7721 tumor growth inhibition by DOX in vivo.

SMMC7721 cell-derived tumors were developed in nude mice and treated with saline, quercetin, DOX, and DOX + quercetin. (A) Tumor growth was monitored by measuring the tumor volume for three weeks. (n = 5 mice per group). *P<0.01 vs. the control and quercetin-treated groups. **P<0.01 vs. DOX-treated group. (B) At the end of three weeks, the tumors were collected and weighed. DOX reduced the tumor size compared with the control and quercetin-treated groups (*P<0.05). Co-treatment significantly reduced the tumor size compared with other treatment groups (**P<0.001). (C) Tumor samples were subjected to hematoxylin and eosin staining and immunohistochemical analysis using Ki67, Bcl-xl, and p53 antibodies. Co-treated tumors showed a significant reduction in Ki67 and Bcl-xl expression as well as an increase in p53 expression compared with other treated tumors (P<0.05).

Figure 6. Histological changes in mice livers that received DOX and/or quercetin.

(A) C57BL/6 mice were treated with quercetin (100 mg/kg/day, p.o.) four days before the i.p. administration of DOX (20 mg/kg). Livers were removed five days after DOX treatment, and the sections were stained with hematoxylin and eosin (H&E; magnification 200×). (B) SMMC7721 cell-derived tumors were developed in nude mice and treated with saline, quercetin, DOX, and DOX + quercetin. At the end of three weeks, livers were collected and stained with H&E. (C) A model of the effect of quercetion on DOX in liver cells. In this model, quercetin increases DOX-induced p53 expression in liver cancer cells and reduces Bcl-xl expression, thereby increasing caspase −3/−9 activity and potentiating DOX-induced cell death in liver cancer cells. Quercetin decreases DOX-induced oxidative stress and increases cell survival in normal liver cells.