Limits...
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.

Show MeSH

Related in: MedlinePlus

Withacnistin induces cancer cell apoptosis, inhibits cell invasion and anchorage-independent growth, and causes breast tumour regression in ErbB2 transgenic mice. (A) MDA-MB-468 cells were treated with Wit at the indicated concentration as described in Materials and Methods. The cells were fixed and the TUNEL assay was performed to determine apoptosis as described under Materials and Methods. The fold apoptosis induction was calculated by dividing the % of TUNEL-positive cells in the Wit-treated samples by those in the vehicle-treated samples. The % of TUNEL-positive cells in the vehicle-treated samples was 2.6±1.1%. (B) MDA-MB-468 cells were seeded over Matrigel in the top chamber of invasion well inserts. Cells were treated for 48 h with vehicle or Wit, and the invaded cells were fixed with methanol, stained with crystal violet, and photographed as described under Materials and Methods. MDA-MB-468 (C) and A549 (D) cells were seeded into 12-well plates in regular growth media containing 0.3% agar; Wit was added the following day, and colonies were allowed to grow for 3–4 weeks as described in Materials and Methods. The data in A–D are representative of three independent experiments each. (E) The sizes of the breast tumours from the ErbB2 transgenic mice were followed by orthogonal measurements 2–3 times per week, and the tumour volume calculated using the formula: V=(a2b)/2, where ‘a' and ‘b' are defined as the smaller and larger measurements, respectively. Drug treatment was initiated when the tumours grew to about 1200–2400 mm3 and treatment lasted up to 14 days. Wit (20 mpk per day) was administered i.p. into tumour-bearing mice. The percent change in tumour volume was calculated based on the tumour volume on the day of measurement relative to that on the day of initiation of treatment (T0—see Supplementary Table S1) for each mouse tumour.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150266&req=5

fig5: Withacnistin induces cancer cell apoptosis, inhibits cell invasion and anchorage-independent growth, and causes breast tumour regression in ErbB2 transgenic mice. (A) MDA-MB-468 cells were treated with Wit at the indicated concentration as described in Materials and Methods. The cells were fixed and the TUNEL assay was performed to determine apoptosis as described under Materials and Methods. The fold apoptosis induction was calculated by dividing the % of TUNEL-positive cells in the Wit-treated samples by those in the vehicle-treated samples. The % of TUNEL-positive cells in the vehicle-treated samples was 2.6±1.1%. (B) MDA-MB-468 cells were seeded over Matrigel in the top chamber of invasion well inserts. Cells were treated for 48 h with vehicle or Wit, and the invaded cells were fixed with methanol, stained with crystal violet, and photographed as described under Materials and Methods. MDA-MB-468 (C) and A549 (D) cells were seeded into 12-well plates in regular growth media containing 0.3% agar; Wit was added the following day, and colonies were allowed to grow for 3–4 weeks as described in Materials and Methods. The data in A–D are representative of three independent experiments each. (E) The sizes of the breast tumours from the ErbB2 transgenic mice were followed by orthogonal measurements 2–3 times per week, and the tumour volume calculated using the formula: V=(a2b)/2, where ‘a' and ‘b' are defined as the smaller and larger measurements, respectively. Drug treatment was initiated when the tumours grew to about 1200–2400 mm3 and treatment lasted up to 14 days. Wit (20 mpk per day) was administered i.p. into tumour-bearing mice. The percent change in tumour volume was calculated based on the tumour volume on the day of measurement relative to that on the day of initiation of treatment (T0—see Supplementary Table S1) for each mouse tumour.

Mentions: Having determined that Wit prevents the activation of STAT3 by blocking its ability to associate with the EGF receptor in MDA-MB-468 cells, we next sought to determine whether the inhibition of STAT3 signalling with Wit leads to inhibition of malignant transformation. To this end, we determined the effects of Wit on apoptosis, anchorage-dependent and -independent growth, invasion, and tumour growth in vivo. Figure 5A shows that in MDA-MB-468 cells, Wit induced apoptosis (TUNEL-positive cells) in a concentration-dependent manner. We next determined the effect of Wit on anchorage-dependent and -independent growth by MTT and soft agar assays, respectively. Cell invasion assays were performed in the BD BioCoat Matrigel Invasion Chamber as described under Materials and Methods. Figure 5B shows that Wit inhibited the ability of MDA-MB-468 cells to invade in a dose-dependent manner with an IC50 value of 3 μM. Similarly, Wit also inhibited the ability of MDA-MB-468 cells to grow in soft agar, but the effects of Wit on soft agar growth were more potent than those on invasion (Figure 4C). This potent effect of Wit was also seen in another cell line, the lung cancer cell line A549 (Figure 5D). Wit also inhibited the ability of MDA-MB-468 cells to grow in an anchorage-dependent manner in plastic (Supplementary Figure S1).


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 induces cancer cell apoptosis, inhibits cell invasion and anchorage-independent growth, and causes breast tumour regression in ErbB2 transgenic mice. (A) MDA-MB-468 cells were treated with Wit at the indicated concentration as described in Materials and Methods. The cells were fixed and the TUNEL assay was performed to determine apoptosis as described under Materials and Methods. The fold apoptosis induction was calculated by dividing the % of TUNEL-positive cells in the Wit-treated samples by those in the vehicle-treated samples. The % of TUNEL-positive cells in the vehicle-treated samples was 2.6±1.1%. (B) MDA-MB-468 cells were seeded over Matrigel in the top chamber of invasion well inserts. Cells were treated for 48 h with vehicle or Wit, and the invaded cells were fixed with methanol, stained with crystal violet, and photographed as described under Materials and Methods. MDA-MB-468 (C) and A549 (D) cells were seeded into 12-well plates in regular growth media containing 0.3% agar; Wit was added the following day, and colonies were allowed to grow for 3–4 weeks as described in Materials and Methods. The data in A–D are representative of three independent experiments each. (E) The sizes of the breast tumours from the ErbB2 transgenic mice were followed by orthogonal measurements 2–3 times per week, and the tumour volume calculated using the formula: V=(a2b)/2, where ‘a' and ‘b' are defined as the smaller and larger measurements, respectively. Drug treatment was initiated when the tumours grew to about 1200–2400 mm3 and treatment lasted up to 14 days. Wit (20 mpk per day) was administered i.p. into tumour-bearing mice. The percent change in tumour volume was calculated based on the tumour volume on the day of measurement relative to that on the day of initiation of treatment (T0—see Supplementary Table S1) for each mouse tumour.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150266&req=5

fig5: Withacnistin induces cancer cell apoptosis, inhibits cell invasion and anchorage-independent growth, and causes breast tumour regression in ErbB2 transgenic mice. (A) MDA-MB-468 cells were treated with Wit at the indicated concentration as described in Materials and Methods. The cells were fixed and the TUNEL assay was performed to determine apoptosis as described under Materials and Methods. The fold apoptosis induction was calculated by dividing the % of TUNEL-positive cells in the Wit-treated samples by those in the vehicle-treated samples. The % of TUNEL-positive cells in the vehicle-treated samples was 2.6±1.1%. (B) MDA-MB-468 cells were seeded over Matrigel in the top chamber of invasion well inserts. Cells were treated for 48 h with vehicle or Wit, and the invaded cells were fixed with methanol, stained with crystal violet, and photographed as described under Materials and Methods. MDA-MB-468 (C) and A549 (D) cells were seeded into 12-well plates in regular growth media containing 0.3% agar; Wit was added the following day, and colonies were allowed to grow for 3–4 weeks as described in Materials and Methods. The data in A–D are representative of three independent experiments each. (E) The sizes of the breast tumours from the ErbB2 transgenic mice were followed by orthogonal measurements 2–3 times per week, and the tumour volume calculated using the formula: V=(a2b)/2, where ‘a' and ‘b' are defined as the smaller and larger measurements, respectively. Drug treatment was initiated when the tumours grew to about 1200–2400 mm3 and treatment lasted up to 14 days. Wit (20 mpk per day) was administered i.p. into tumour-bearing mice. The percent change in tumour volume was calculated based on the tumour volume on the day of measurement relative to that on the day of initiation of treatment (T0—see Supplementary Table S1) for each mouse tumour.
Mentions: Having determined that Wit prevents the activation of STAT3 by blocking its ability to associate with the EGF receptor in MDA-MB-468 cells, we next sought to determine whether the inhibition of STAT3 signalling with Wit leads to inhibition of malignant transformation. To this end, we determined the effects of Wit on apoptosis, anchorage-dependent and -independent growth, invasion, and tumour growth in vivo. Figure 5A shows that in MDA-MB-468 cells, Wit induced apoptosis (TUNEL-positive cells) in a concentration-dependent manner. We next determined the effect of Wit on anchorage-dependent and -independent growth by MTT and soft agar assays, respectively. Cell invasion assays were performed in the BD BioCoat Matrigel Invasion Chamber as described under Materials and Methods. Figure 5B shows that Wit inhibited the ability of MDA-MB-468 cells to invade in a dose-dependent manner with an IC50 value of 3 μM. Similarly, Wit also inhibited the ability of MDA-MB-468 cells to grow in soft agar, but the effects of Wit on soft agar growth were more potent than those on invasion (Figure 4C). This potent effect of Wit was also seen in another cell line, the lung cancer cell line A549 (Figure 5D). Wit also inhibited the ability of MDA-MB-468 cells to grow in an anchorage-dependent manner in plastic (Supplementary Figure S1).

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