Inhibition of angiogenesis by quercetin in tamoxifen-resistant breast cancer cells
Introduction
Breast cancer, the most common malignancy in women, grows under hormone-dependent control. Because estrogen is essential for breast carcinogenesis as well as for mammary gland development (Mueller et al., 2002, Petrangeli et al., 1994), anti-estrogens have important therapeutic potential in endocrine therapy for breast cancer. Tamoxifen (TAM), a non-steroidal anti-estrogen, is the most widely used anti-estrogen for the treatment or prevention of estrogen receptor-positive breast cancer (Rose et al., 1985). Nonetheless, innate or acquired resistance to TAM is a critical problem for anti-estrogen therapy (Clemons et al., 2002). Our group and others have established an MCF-7 derived TAM-resistant cell (TAMR-MCF-7 cell) line by long-term (>9 months) culture of MCF-7 cells with 4-hydroxytamoxifen (Choi et al., 2007, Knowlden et al., 2003). Microvessel counts in patients are significantly higher in TAM-resistant breast tumor tissues, as compared to TAM-responsive tumor tissues (Marson et al., 2001). Our recent study has also revealed that the angiogenic potential is up-regulated through the enhanced expression of vascular endothelial growth factor (VEGF) in TAMR-MCF-7 cells (Kim et al., 2009b).
Pin1, a peptidyl prolyl isomerase, specifically recognizes phosphorylated serine or threonine preceding proline (pSer/Thr-Pro) and catalyzes the isomerization of the peptide bond (Bayer et al., 2003, Lu, 2004). Pin1 overexpression has been observed frequently in most types of cancer tissues including breast and prostate tumors (Bao et al., 2004). Although the exact role of Pin1 in angiogenesis has not been fully elucidated, it has been shown that ectopic expression of Pin1 siRNA decreases VEGF levels in prostate tumor tissues (Ryo et al., 2005); in contrast, Pin1 overexpression increases transcription of the VEGF gene via the activation of hypoxia inducible factor-1α (HIF-1α) and activator protein-1 (AP-1) (Kim et al., 2008a). We have also demonstrated that Pin1 overexpression in TAMR-MCF-7 cells is essentially required for VEGF-mediated angiogenesis (Kim et al., 2009b). Based on the hypothesis that a Pin1 inhibitor could block the angiogenic potential of TAMR-MCF-7 cells, we first tried to identify effective compounds to inhibit Pin1-mediated VEGF gene transcription by examining six different flavonoids (morin, naringin, naringenin, apigenin, quercetin, silibinin). These flavonoids are present in various vegetables and fruits at relatively high concentrations, and sufficient amounts with high purity (>90%) can be isolated easily. Also these substances have attracted much attention as potential chemopreventive agents (Moon et al., 2006, Ramos, 2007). We found that quercetin most effectively suppressed VEGF gene transcription by Pin1 overexpression. Further, we have shown that the exaggerated Pin1 expression and VEGF production in TAMR-MCF-7 cells are suppressed by quercetin and that dual blocking of c-Jun/AP-1 and HIF-1α is involved in VEGF inhibition by quercetin.
Section snippets
Materials
Antibodies against c-Jun, Pin1, and HIF-1α were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies that recognize phosphorylated or total Akt, extracellular signal regulated kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK) were obtained from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-conjugated donkey anti-rabbit IgG, anti-goat IgG, and alkaline phosphatase-conjugated donkey anti-mouse IgG were purchased from Jackson Immunoresearch Laboratories
Inhibition of VEGF gene transcription by quercetin in Pin1-overexpressing MCF-7 cells
Because Pin1 is a critical factor for angiogenesis of TAM-resistant breast cancer, and VEGF expression is essential for angiogenesis in diverse tumor types (Kim et al., 2009b, Kerbel, 2000), we first compared VEGF gene transcription in MCF-7 cells stably overexpressing Pin1 (Pin1-MCF-7) incubated with six different flavonoids (morin, naringin, naringenin, apigenin, quercetin, silibinin) at a concentration 30 μM, respectively. Reporter gene assays using the VEGF-Luc plasmid showed that quercetin
Discussion
TAM resistance is an obstacle in the management of estrogen receptor-positive breast cancer patients (Ali and Coombes, 2002), and one of the clinical characteristics of TAM-resistant breast cancer is increased angiogenesis (Marson et al., 2001). Recently, we have demonstrated that Pin1 induction in TAMR-MCF-7 cells functions as a master regulator for both the VEGF-mediated angiogenesis and the epithelial mesenchymal transition (Kim et al., 2009a, Kim et al., 2009b). In the current study, we
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST) (Keon Wook Kang, No. 2009-0070587).
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