Quercetin-induced ubiquitination and down-regulation of Her-2/neu

Abstract

Her-2/neu (ErbB2) is a transmembrane tyrosine kinase and acts as a co-receptor for the other EGFR family members. It is well known that high expression of Her-2/neu is associated with a poor prognosis in breast cancer. Quercetin, a flavonoid present in many vegetables and fruits, has been studied extensively as a chemoprevention agent in several cancer models. In this study, we observed that quercetin decreased the level of Her-2/neu protein in time- and dose-dependent manners and also inhibited the downstream survival PI3K-Akt signaling pathway in Her-2/neu-overexpressing breast cancer SK-Br3 cells. We also observed that quercetin induced polyubiquitination of Her-2/neu. When the proteasome pathway was blocked by MG-132 during quercetin treatment, accumulation of the NP-40 insoluble form of Her-2/neu occurred. Interestingly, data from immunocomplex studies revealed that quercetin promoted interaction between Her-2/neu and Hsp90 which is a molecular chaperone involved in stabilization of Her-2/neu. In this condition, inhibition of Hsp90 activity by a specific inhibitor, geldanamycin (GA), or intracellular ATP depletion caused dissociation of Hsp90 from Her-2/neu and promoted ubiquitination and down-regulation of Her-2/neu protein. In addition, the carboxyl terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, played a crucial role in the quercetin-induced ubiquitination of Her-2/neu. Inhibition of tyrosine kinase activity of Her-2/neu by quercetin could indicate an lateration in the Her-2/neu structure which promotes CHIP recruitments and down-regulation of Her-2/neu. We believe that by using quercetin, new therapeutic strategies can be developed to treat Her-2/neu overexpressing cancers.

Fig. 1

Quercetin down-regulates Her-2/neu in SK-Br3. A: SK-Br3 cells were treated with 100 μM or 200 μM of quercetin for the indicated days. B: SK-Br3 cells were treated with 100 μM or 200 μM of quercetin for the indicated h. C: SK-Br3 cells were treated with indicated amount of quercetin for 24 h. Cellular levels of Her-2/neu protein and phosphorylation of the downstream kinases, phosphoinositide-3 kinase (PI3K) and Akt, were compared from whole cell lysate. D: Cell surface expression of Her-2/neu protein in SK-Br3 cells was determined by flow cytometry using a mouse anti–Her-2/neu antibody directed against the extracellular domain of Her-2/neu. SK-Br3 cells were incubated in the absence or presence of 200 μM of quercetin for 24 h. E: SK-Ov3 cells were treated with 100 μM or 200 μM of quercetin for the indicated days. F: MCF-7 cells were treated with indicated amount of quercetin for 24 h. Cellular levels of Her-2/neu protein was compared from whole cell lysate.

Fig. 1

Quercetin down-regulates Her-2/neu in SK-Br3. A: SK-Br3 cells were treated with 100 μM or 200 μM of quercetin for the indicated days. B: SK-Br3 cells were treated with 100 μM or 200 μM of quercetin for the indicated h. C: SK-Br3 cells were treated with indicated amount of quercetin for 24 h. Cellular levels of Her-2/neu protein and phosphorylation of the downstream kinases, phosphoinositide-3 kinase (PI3K) and Akt, were compared from whole cell lysate. D: Cell surface expression of Her-2/neu protein in SK-Br3 cells was determined by flow cytometry using a mouse anti–Her-2/neu antibody directed against the extracellular domain of Her-2/neu. SK-Br3 cells were incubated in the absence or presence of 200 μM of quercetin for 24 h. E: SK-Ov3 cells were treated with 100 μM or 200 μM of quercetin for the indicated days. F: MCF-7 cells were treated with indicated amount of quercetin for 24 h. Cellular levels of Her-2/neu protein was compared from whole cell lysate.

Fig. 2

Her-2/neu down-regulation by quercetin is not regulated at transcription level. A: SK-Br3 cells were treated with 100 μM of quercetin for the indicated times and then total RNA was extracted. Her-2/neu mRNA levels were compared by Northern blot analysis. Ethidium bromide stained rRNA bands are shown as a loading control. B: Her-2/neu mRNA levels were compared by real-time RT-PCR analysis using 18S rRNA as an internal control. Error bars represent standard error from the mean (SEM) for three independent experiments.

Fig. 2

Her-2/neu down-regulation by quercetin is not regulated at transcription level. A: SK-Br3 cells were treated with 100 μM of quercetin for the indicated times and then total RNA was extracted. Her-2/neu mRNA levels were compared by Northern blot analysis. Ethidium bromide stained rRNA bands are shown as a loading control. B: Her-2/neu mRNA levels were compared by real-time RT-PCR analysis using 18S rRNA as an internal control. Error bars represent standard error from the mean (SEM) for three independent experiments.

Fig. 3

Quercetin induces ubiquitination of Her-2/neu and concomitantly enhances the recruitment of Hsp90 to Her-2/neu. A: SK-Br3 cells were cotreated with 200 μM of quercetin and MG-132 (5 μM) or NH₄Cl (10 mM) for 24 hr. Cell extract was prepared by resuspending in lysis buffer (NP-40 soluble) and the insoluble proteins were extracted with SDS sample buffer and then boiled (NP-40 insoluble). Her-2/neu protein level was compared by Western blot analysis. B: SK-Br3 cells were treated with 200 μM of quercetin for the indicated times After Her-2/neu proteins were immunoprecipitated, ubiquitination level and interacting amount of Hsp90 were compared by Western blot analysis. C: SK-Br3 cells were treated with increased amounts of quercetin for 4 h and then analysed as above. D: SK-Br3 cells were either left untreated or treated with 200 μM of quercetin for 4 h and then whole cell lysate was prepared. After immunoprecipitation with normal mouse serum, anti-EGFR, or anti-Her-2/neu antibody, the ubiquitination level and interacting amount of Hsp90 were compared by Western blot analysis.

Fig. 3

Quercetin induces ubiquitination of Her-2/neu and concomitantly enhances the recruitment of Hsp90 to Her-2/neu. A: SK-Br3 cells were cotreated with 200 μM of quercetin and MG-132 (5 μM) or NH₄Cl (10 mM) for 24 hr. Cell extract was prepared by resuspending in lysis buffer (NP-40 soluble) and the insoluble proteins were extracted with SDS sample buffer and then boiled (NP-40 insoluble). Her-2/neu protein level was compared by Western blot analysis. B: SK-Br3 cells were treated with 200 μM of quercetin for the indicated times After Her-2/neu proteins were immunoprecipitated, ubiquitination level and interacting amount of Hsp90 were compared by Western blot analysis. C: SK-Br3 cells were treated with increased amounts of quercetin for 4 h and then analysed as above. D: SK-Br3 cells were either left untreated or treated with 200 μM of quercetin for 4 h and then whole cell lysate was prepared. After immunoprecipitation with normal mouse serum, anti-EGFR, or anti-Her-2/neu antibody, the ubiquitination level and interacting amount of Hsp90 were compared by Western blot analysis.

Fig. 3

Quercetin induces ubiquitination of Her-2/neu and concomitantly enhances the recruitment of Hsp90 to Her-2/neu. A: SK-Br3 cells were cotreated with 200 μM of quercetin and MG-132 (5 μM) or NH₄Cl (10 mM) for 24 hr. Cell extract was prepared by resuspending in lysis buffer (NP-40 soluble) and the insoluble proteins were extracted with SDS sample buffer and then boiled (NP-40 insoluble). Her-2/neu protein level was compared by Western blot analysis. B: SK-Br3 cells were treated with 200 μM of quercetin for the indicated times After Her-2/neu proteins were immunoprecipitated, ubiquitination level and interacting amount of Hsp90 were compared by Western blot analysis. C: SK-Br3 cells were treated with increased amounts of quercetin for 4 h and then analysed as above. D: SK-Br3 cells were either left untreated or treated with 200 μM of quercetin for 4 h and then whole cell lysate was prepared. After immunoprecipitation with normal mouse serum, anti-EGFR, or anti-Her-2/neu antibody, the ubiquitination level and interacting amount of Hsp90 were compared by Western blot analysis.

Fig. 4

Iron chelation effect of quercetin is not involved in Her-2/neu down-regulation. A: SK-Br3 cells were pretreated with various amount of FeSO₄ for 30 min and then 200 μM of quercetin was applied for additional 24 h. Cellular Her-2/neu protein level was compared by Western blot analysis. B: SK-Br3 cells were treated with 200 μM of desferrioxamine (DFX) for the indicated time. Cellular Her-2/neu protein level was compared by Western blot analysis. Accumulation of HIF-1α is shown as confirmation of the biological effect of DFX. C: SK-Br3 cells were treated with 200 μM of quercetin or 200 μM of FeSO₄ for 4 h. For the samples with combined treatment, FeSO₄ was applied 30 min before (Fe + Q) or after (Q + Fe) the treatment with quercetin. D: SK-Br3 cells were treated with quercetin (200 μM), DFX (200 μM), or both for 4 h. After Her-2/neu protein was immunoprecipitated, ubiquitination level and interacting amount of Hsp90 was compared by Western blot analysis.

Fig. 5

Effect of Hsp90 inhibition on Her-2/neu protein level and ubiquitination during treatment with quercetin. A: SK-Br3 cells were pretreated with 200 μM of quercetin for 30 min, and then indicated amount of specific Hsp90 inhibitor, GA, was added for additional 4 h. Cellular levels of Her-2/neu protein were compared from whole cell lysate. B: GA was applied in combination with 200 μM of quercetin for 1.5 h. After Her-2/neu proteins were immunoprecipitated, ubiquitination and Hsp90 level was compared by Western blot analysis. In order to inhibit Hsp90 activity, intracellular ATP level was reduced by glucose deprivation for 4 h (C) or by addition of non-metabolizable glucose analog, 2-deoxyglucose (5 mM), in the culture medium for an h in advance (D). After treatment of quercetin (200 μM) for additional 4 h, Her-2/neu protein was immunoprecipitated and then ubiquitination and Hsp90 level was compared by Western blot analysis.

Fig. 5

Effect of Hsp90 inhibition on Her-2/neu protein level and ubiquitination during treatment with quercetin. A: SK-Br3 cells were pretreated with 200 μM of quercetin for 30 min, and then indicated amount of specific Hsp90 inhibitor, GA, was added for additional 4 h. Cellular levels of Her-2/neu protein were compared from whole cell lysate. B: GA was applied in combination with 200 μM of quercetin for 1.5 h. After Her-2/neu proteins were immunoprecipitated, ubiquitination and Hsp90 level was compared by Western blot analysis. In order to inhibit Hsp90 activity, intracellular ATP level was reduced by glucose deprivation for 4 h (C) or by addition of non-metabolizable glucose analog, 2-deoxyglucose (5 mM), in the culture medium for an h in advance (D). After treatment of quercetin (200 μM) for additional 4 h, Her-2/neu protein was immunoprecipitated and then ubiquitination and Hsp90 level was compared by Western blot analysis.

Fig. 6

CHIP is involved in quercetin-induced ubiquitination of Her-2/neu. A: COS7 cells were cotransfected with pCMV-Her-2/neu and pcDNA-HIS-CHIP. After 24 h, cells were either untreated or treated with quercetin for 4 h. Her-2/neu protein was immunoprecipitated and then ubiquitination of Her-2/neu and the level of interaction between Her-2/neu and CHIP were compared. B: COS7 cells were cotransfected with pCMV-Her-2/neu and CHIP siRNA. After 24 h, cells were either untreated or treated with quercetin for 4 h. Her-2/neu protein was immunoprecipitated and then ubiquitination level was compared.

Fig. 7

Quercetin inhibited Her-2/neu tyrosine kinase activity. A: SK-Br3 cells were treated with 200 μM of quercetin for the indicated time. Her-2/neu protein was immunoprecipitated and then tyrosine phosphorylation was detected with anti-phospho-tyrosine monoclonal antibody. B: Her-2/neu protein was isolated by immunoprecipitation and then autophosphorylation activity was compared by incubating with [γ-³²p]ATP in the presence of different amounts of quercetin. C: Her-2/neu protein was isolated by immunoprecipitation and then tyrosine kinase activity was measured in the presence of DMSO or 200 μM of quercetin using a synthetic peptide as a substrate.

Fig. 7

Quercetin inhibited Her-2/neu tyrosine kinase activity. A: SK-Br3 cells were treated with 200 μM of quercetin for the indicated time. Her-2/neu protein was immunoprecipitated and then tyrosine phosphorylation was detected with anti-phospho-tyrosine monoclonal antibody. B: Her-2/neu protein was isolated by immunoprecipitation and then autophosphorylation activity was compared by incubating with [γ-³²p]ATP in the presence of different amounts of quercetin. C: Her-2/neu protein was isolated by immunoprecipitation and then tyrosine kinase activity was measured in the presence of DMSO or 200 μM of quercetin using a synthetic peptide as a substrate.

Fig. 8

Quercetin enhanced adriamycin-induced cell death. SK-Br3 cells were treated with 100 μM of quercetin in combination with indicated amount of adriamycin for 2 days. Cell viability was determined by the trypan blue exclusion assay. Error bars represent standard error from the mean (SEM) for three separate experiments.