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Withacnistin inhibits recruitment of STAT3 and STAT5 to growth factor and cytokine receptors and induces regression of breast tumours.

Zhang X, Blaskovich MA, Forinash KD, Sebti SM - Br. J. Cancer (2014)

Bottom Line: The inhibition of P-STAT3 and P-STAT5 occurred rapidly, within minutes of Wit treatment and growth factor stimulation.Wit also inhibits STAT3 nuclear translocation, DNA binding, promoter transcriptional activation, and it suppresses the expression levels of STAT3 target genes such as Bcl-xL and Mcl-1.These data warrant further development of Wit as a novel anticancer drug for targeting tumours that harbour hyperactivated STAT3 and STAT5.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Department, Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.

ABSTRACT

Background: The binding of STAT3 and STAT5 to growth factor and cytokine receptors such as EGFR and IL-6 receptor gp130 is critical to their activation and ability to contribute to malignant transformation. Therefore, interfering with these biochemical processes could lead to the discovery of novel anticancer agents.

Methods: Co-immunoprecipitation, western blotting, microscopy, DNA binding, invasion, and soft agar assays as well as a mouse model were used to investigate the mechanism by which the natural product Withacnistin (Wit) inhibits STAT 3/5 tyrosine phosphoryaltion and activation.

Results: Wit blocks EGF- and IL-6-stimulated binding of STAT3 and STAT5 to EGFR and gp130. Wit inhibits EGF-, PDGF-, IL-6-, IFNβ-, and GM-CSF-stimulation of tyrosine phosphorylation of STAT3 and STAT5 but not of EGFR or PDGFR. The inhibition of P-STAT3 and P-STAT5 occurred rapidly, within minutes of Wit treatment and growth factor stimulation. Wit also inhibits STAT3 nuclear translocation, DNA binding, promoter transcriptional activation, and it suppresses the expression levels of STAT3 target genes such as Bcl-xL and Mcl-1. Finally, Wit induces apoptosis, inhibits anchorage-dependent and -independent growth and invasion, and causes breast tumour regression in an ErbB2-driven transgenic mouse model.

Conclusions: These data warrant further development of Wit as a novel anticancer drug for targeting tumours that harbour hyperactivated STAT3 and STAT5.

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Related in: MedlinePlus

Withacnistin inhibits EGF-, PDGF-, IFN-β-, and GM-CSF-stimulated tyrosine phosphorylation of STAT3 and STAT5. (A) Cells were serum starved for 48 h and pretreated with Wit at increasing concentrations for 2 h, and then stimulated for 10 min with EGF at 100 ng ml−1 for 10 min (MDA-MB-468 cells), IL-6 at 10 ng ml−1 (U266 cells), GM-CSF at 2 ng ml−1 (TF-1 cells), IFN-β at 1000 U ml−1 (U266 cells), and PDGF-BB at 10 ng ml−1 (NIH-3T3 cells). The cells were then harvested and processed for western blotting with the indicated antibodies as described under Materials and Methods. (B) Cells were serum starved and pretreated with Wit as above, and then stimulated with EGF at 100 ng ml−1 for 10 min (MDA-MB-468 cells), IL-6 at 10 ng ml−1 (U266 cells), GM-CSF at 2 ng ml−1 (TF-1 cells), and PDGF-BB at 10 ng ml−1 (NIH-3T3 cells). The cells were then harvested and processed for western blotting with the indicated antibodies as described under Materials and Methods. The data in A and B are representative of at least three independent experiments for each panel.
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fig2: Withacnistin inhibits EGF-, PDGF-, IFN-β-, and GM-CSF-stimulated tyrosine phosphorylation of STAT3 and STAT5. (A) Cells were serum starved for 48 h and pretreated with Wit at increasing concentrations for 2 h, and then stimulated for 10 min with EGF at 100 ng ml−1 for 10 min (MDA-MB-468 cells), IL-6 at 10 ng ml−1 (U266 cells), GM-CSF at 2 ng ml−1 (TF-1 cells), IFN-β at 1000 U ml−1 (U266 cells), and PDGF-BB at 10 ng ml−1 (NIH-3T3 cells). The cells were then harvested and processed for western blotting with the indicated antibodies as described under Materials and Methods. (B) Cells were serum starved and pretreated with Wit as above, and then stimulated with EGF at 100 ng ml−1 for 10 min (MDA-MB-468 cells), IL-6 at 10 ng ml−1 (U266 cells), GM-CSF at 2 ng ml−1 (TF-1 cells), and PDGF-BB at 10 ng ml−1 (NIH-3T3 cells). The cells were then harvested and processed for western blotting with the indicated antibodies as described under Materials and Methods. The data in A and B are representative of at least three independent experiments for each panel.

Mentions: We next sought to determine whether the effects of Wit were limited to EGF and IL-6 stimulation of STAT3 and/or STAT5 tyrosine phosphorylation. To this end, we first chose human and murine cell lines in which growth factors or cytokines are known to stimulate STAT3 phosphorylation. Starting with MDA-MB-468 cells, a human breast cancer cell line where EGF is known to induce STAT3 phosphorylation, we demonstrated as shown in Figure 2A, that Wit inhibited EGF-stimulated tyrosine phosphorylation of STAT3, with an IC50 of about 1–3 μM. Wit, however, did not affect the ability of EGF to stimulate EGFR autophosphorylation. Furthermore, Figure 2A also shows that pretreatment with Wit of starved NIH-3T3 cells before stimulation with PDGF-BB inhibited PDGF stimulation of STAT3 tyrosine phosphorylation. Importantly, Wit did not inhibit PDGF-stimulated PDGFR tyrosine autophosphorylation. We next used the U266 human multiple myeloma cell line, where IL-6 and IFN-β stimulate STAT3 phosphorylation. Figure 2A shows that IL-6- and IFN-β-stimulated STAT3 tyrosine phosphorylation was also inhibited by Wit in U266 cells. The effect of Wit was more potent after IL-6 stimulation than after IFN-β stimulation. Finally, Figure 2A shows that Wit also inhibited potently the ability of GM-CSF to stimulate STAT3 tyrosine phosphorylation in TF-1 cells.


Withacnistin inhibits recruitment of STAT3 and STAT5 to growth factor and cytokine receptors and induces regression of breast tumours.

Zhang X, Blaskovich MA, Forinash KD, Sebti SM - Br. J. Cancer (2014)

Withacnistin inhibits EGF-, PDGF-, IFN-β-, and GM-CSF-stimulated tyrosine phosphorylation of STAT3 and STAT5. (A) Cells were serum starved for 48 h and pretreated with Wit at increasing concentrations for 2 h, and then stimulated for 10 min with EGF at 100 ng ml−1 for 10 min (MDA-MB-468 cells), IL-6 at 10 ng ml−1 (U266 cells), GM-CSF at 2 ng ml−1 (TF-1 cells), IFN-β at 1000 U ml−1 (U266 cells), and PDGF-BB at 10 ng ml−1 (NIH-3T3 cells). The cells were then harvested and processed for western blotting with the indicated antibodies as described under Materials and Methods. (B) Cells were serum starved and pretreated with Wit as above, and then stimulated with EGF at 100 ng ml−1 for 10 min (MDA-MB-468 cells), IL-6 at 10 ng ml−1 (U266 cells), GM-CSF at 2 ng ml−1 (TF-1 cells), and PDGF-BB at 10 ng ml−1 (NIH-3T3 cells). The cells were then harvested and processed for western blotting with the indicated antibodies as described under Materials and Methods. The data in A and B are representative of at least three independent experiments for each panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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fig2: Withacnistin inhibits EGF-, PDGF-, IFN-β-, and GM-CSF-stimulated tyrosine phosphorylation of STAT3 and STAT5. (A) Cells were serum starved for 48 h and pretreated with Wit at increasing concentrations for 2 h, and then stimulated for 10 min with EGF at 100 ng ml−1 for 10 min (MDA-MB-468 cells), IL-6 at 10 ng ml−1 (U266 cells), GM-CSF at 2 ng ml−1 (TF-1 cells), IFN-β at 1000 U ml−1 (U266 cells), and PDGF-BB at 10 ng ml−1 (NIH-3T3 cells). The cells were then harvested and processed for western blotting with the indicated antibodies as described under Materials and Methods. (B) Cells were serum starved and pretreated with Wit as above, and then stimulated with EGF at 100 ng ml−1 for 10 min (MDA-MB-468 cells), IL-6 at 10 ng ml−1 (U266 cells), GM-CSF at 2 ng ml−1 (TF-1 cells), and PDGF-BB at 10 ng ml−1 (NIH-3T3 cells). The cells were then harvested and processed for western blotting with the indicated antibodies as described under Materials and Methods. The data in A and B are representative of at least three independent experiments for each panel.
Mentions: We next sought to determine whether the effects of Wit were limited to EGF and IL-6 stimulation of STAT3 and/or STAT5 tyrosine phosphorylation. To this end, we first chose human and murine cell lines in which growth factors or cytokines are known to stimulate STAT3 phosphorylation. Starting with MDA-MB-468 cells, a human breast cancer cell line where EGF is known to induce STAT3 phosphorylation, we demonstrated as shown in Figure 2A, that Wit inhibited EGF-stimulated tyrosine phosphorylation of STAT3, with an IC50 of about 1–3 μM. Wit, however, did not affect the ability of EGF to stimulate EGFR autophosphorylation. Furthermore, Figure 2A also shows that pretreatment with Wit of starved NIH-3T3 cells before stimulation with PDGF-BB inhibited PDGF stimulation of STAT3 tyrosine phosphorylation. Importantly, Wit did not inhibit PDGF-stimulated PDGFR tyrosine autophosphorylation. We next used the U266 human multiple myeloma cell line, where IL-6 and IFN-β stimulate STAT3 phosphorylation. Figure 2A shows that IL-6- and IFN-β-stimulated STAT3 tyrosine phosphorylation was also inhibited by Wit in U266 cells. The effect of Wit was more potent after IL-6 stimulation than after IFN-β stimulation. Finally, Figure 2A shows that Wit also inhibited potently the ability of GM-CSF to stimulate STAT3 tyrosine phosphorylation in TF-1 cells.

Bottom Line: The inhibition of P-STAT3 and P-STAT5 occurred rapidly, within minutes of Wit treatment and growth factor stimulation.Wit also inhibits STAT3 nuclear translocation, DNA binding, promoter transcriptional activation, and it suppresses the expression levels of STAT3 target genes such as Bcl-xL and Mcl-1.These data warrant further development of Wit as a novel anticancer drug for targeting tumours that harbour hyperactivated STAT3 and STAT5.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Department, Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.

ABSTRACT

Background: The binding of STAT3 and STAT5 to growth factor and cytokine receptors such as EGFR and IL-6 receptor gp130 is critical to their activation and ability to contribute to malignant transformation. Therefore, interfering with these biochemical processes could lead to the discovery of novel anticancer agents.

Methods: Co-immunoprecipitation, western blotting, microscopy, DNA binding, invasion, and soft agar assays as well as a mouse model were used to investigate the mechanism by which the natural product Withacnistin (Wit) inhibits STAT 3/5 tyrosine phosphoryaltion and activation.

Results: Wit blocks EGF- and IL-6-stimulated binding of STAT3 and STAT5 to EGFR and gp130. Wit inhibits EGF-, PDGF-, IL-6-, IFNβ-, and GM-CSF-stimulation of tyrosine phosphorylation of STAT3 and STAT5 but not of EGFR or PDGFR. The inhibition of P-STAT3 and P-STAT5 occurred rapidly, within minutes of Wit treatment and growth factor stimulation. Wit also inhibits STAT3 nuclear translocation, DNA binding, promoter transcriptional activation, and it suppresses the expression levels of STAT3 target genes such as Bcl-xL and Mcl-1. Finally, Wit induces apoptosis, inhibits anchorage-dependent and -independent growth and invasion, and causes breast tumour regression in an ErbB2-driven transgenic mouse model.

Conclusions: These data warrant further development of Wit as a novel anticancer drug for targeting tumours that harbour hyperactivated STAT3 and STAT5.

Show MeSH
Related in: MedlinePlus