Limits...
Insulin-like growth factor-binding protein-2 promotes prostate cancer cell growth via IGF-dependent or -independent mechanisms and reduces the efficacy of docetaxel.

Uzoh CC, Holly JM, Biernacka KM, Persad RA, Bahl A, Gillatt D, Perks CM - Br. J. Cancer (2011)

Bottom Line: Understanding the resistance to therapy could aid the development of more effective treatments.The IGFBP-2 promoted cell growth in both cell lines but with PC3 cells this was in an IGF-dependent manner, whereas with DU145 cells the effect was independent of IGF receptor activation.This IGF-independent effect of IGFBP-2 was mediated by interaction with β-1-containing integrins and a consequent increase in PTEN phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: IGFs and Metabolic Endocrinology Group, School of Clinical Sciences, Learning and Research Building, Southmead Hospital, Bristol, UK.

ABSTRACT

Background: The development of androgen independence, chemo-, and radioresistance are critical markers of prostate cancer progression and the predominant reasons for its high mortality. Understanding the resistance to therapy could aid the development of more effective treatments.

Aim: The aim of this study is to investigate the effects of insulin-like growth factor-binding protein-2 (IGFBP-2) on prostate cancer cell proliferation and its effects on the response to docetaxel.

Methods: DU145 and PC3 cells were treated with IGFBP-2, insulin-like growth factor I (IGF-I) alone or in combination with blockade of the IGF-I receptor or integrin receptors. Cells were also treated with IGFBP-2 short interfering ribonucleic acid with or without a PTEN (phosphatase and tensin homologue deleted on chromosome 10) inhibitor or docetaxel. Tritiated thymidine incorporation was used to measure cell proliferation and Trypan blue cell counting for cell death. Levels of IGFBP-2 mRNA were measured using RT-PCR. Abundance and phosphorylation of proteins were assessed using western immunoblotting.

Results: The IGFBP-2 promoted cell growth in both cell lines but with PC3 cells this was in an IGF-dependent manner, whereas with DU145 cells the effect was independent of IGF receptor activation. This IGF-independent effect of IGFBP-2 was mediated by interaction with β-1-containing integrins and a consequent increase in PTEN phosphorylation. We also determined that silencing IGFBP-2 in both cell lines increased the sensitivity of the cells to docetaxel.

Conclusion: The IGFBP-2 has a key role in the growth of prostate cancer cells, and silencing IGFBP-2 expression reduced the resistance of these cells to docetaxel. Targeting IGFBP-2 may increase the efficacy of docetaxel.

Show MeSH

Related in: MedlinePlus

Involvement of integrin receptors in the intrinsic effects of IGFBP-2 on DU145 cells. (A) Graph shows total cell number of DU145 cells following treatment with RGD peptide (0–40 μg ml−1) for 48 h after initial plating (as in Figures 1A and B) in GM for 24 h and serum starving for a further 24 h. Insert shows a representative PCR blot (of IGFBP-2 m RNA) and WIBs (of IGFBP-2 protein from cell lysates and conditioned cell supernatants). A WIB for tubulin is also shown as a loading control for IGFBP-2 in the cell lysates. (B) Graph shows total cell number of DU145 cells pre-dosed for 1 h with RGD peptide (40 μg ml−1) or SFM and then re-dosed with SFM or IGFBP-2 (250 ng ml−1) for 48 h after initial plating (as in Figures 1C and D) in GM for 24 h and serum starving for a further 24 h. (C) Graph shows total cell number of DU145 cells pre-dosed for 1 h with β1-integrin receptor-blocking antibody (200 ng ml−1) or control mouse IgG1 antibody (200 ng ml−1) or SFM before they were then spiked with IGFBP-2 (250 ng ml−1) for 48 h after initial plating (as in Figures 1C and D) in GM for 24 h and serum starving for a further 24 h. All graphs show the mean±s.e.m. of at least three independent experiments each repeated in triplicate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3101905&req=5

fig2: Involvement of integrin receptors in the intrinsic effects of IGFBP-2 on DU145 cells. (A) Graph shows total cell number of DU145 cells following treatment with RGD peptide (0–40 μg ml−1) for 48 h after initial plating (as in Figures 1A and B) in GM for 24 h and serum starving for a further 24 h. Insert shows a representative PCR blot (of IGFBP-2 m RNA) and WIBs (of IGFBP-2 protein from cell lysates and conditioned cell supernatants). A WIB for tubulin is also shown as a loading control for IGFBP-2 in the cell lysates. (B) Graph shows total cell number of DU145 cells pre-dosed for 1 h with RGD peptide (40 μg ml−1) or SFM and then re-dosed with SFM or IGFBP-2 (250 ng ml−1) for 48 h after initial plating (as in Figures 1C and D) in GM for 24 h and serum starving for a further 24 h. (C) Graph shows total cell number of DU145 cells pre-dosed for 1 h with β1-integrin receptor-blocking antibody (200 ng ml−1) or control mouse IgG1 antibody (200 ng ml−1) or SFM before they were then spiked with IGFBP-2 (250 ng ml−1) for 48 h after initial plating (as in Figures 1C and D) in GM for 24 h and serum starving for a further 24 h. All graphs show the mean±s.e.m. of at least three independent experiments each repeated in triplicate.

Mentions: We tested the effect of a short disintegrin, RGD containing, peptide, which represents an amino acid sequence common to several ECM components that bind to integrins. We observed a dose-dependent decrease in total cell number with increasing RGD peptide concentrations (0–40 μg ml−1; Figure 2A). We found that there was no increase in IGFBP-2 mRNA. However, conditioned media from this experiment showed a progressive increase in levels of IGFBP-2 with increasing RGD peptide concentration (Figure 2A insert), suggesting displacement of IGFBP-2 from the cell surface as opposed to an increase in its synthesis. We anticipated that the increase in IGFBP-2 in the cell supernatants would be accompanied by a decrease in the cell lysates. However, levels of IGFBP-2 in the cell lysate remained the same suggesting that the association of the RGD peptide with the cell surface was in some way inhibiting degradation of IGFBP-2. We went on to show that the proliferative effect of exogenous IGFBP-2 was markedly inhibited in the presence of a sub-apoptotic dose of RGD peptide (Figure 2B). This indicated that IGFBP-2 was promoting growth via interaction with an integrin receptor. We then showed that IGFBP-2 acted specifically via integrin receptors containing the β1 subunit, as a β1-integrin receptor blocking antibody similarly inhibited cell proliferation stimulated by exogenous IGFBP-2, whereas a control mouse IgG1 antibody was without effect (Figure 2C).


Insulin-like growth factor-binding protein-2 promotes prostate cancer cell growth via IGF-dependent or -independent mechanisms and reduces the efficacy of docetaxel.

Uzoh CC, Holly JM, Biernacka KM, Persad RA, Bahl A, Gillatt D, Perks CM - Br. J. Cancer (2011)

Involvement of integrin receptors in the intrinsic effects of IGFBP-2 on DU145 cells. (A) Graph shows total cell number of DU145 cells following treatment with RGD peptide (0–40 μg ml−1) for 48 h after initial plating (as in Figures 1A and B) in GM for 24 h and serum starving for a further 24 h. Insert shows a representative PCR blot (of IGFBP-2 m RNA) and WIBs (of IGFBP-2 protein from cell lysates and conditioned cell supernatants). A WIB for tubulin is also shown as a loading control for IGFBP-2 in the cell lysates. (B) Graph shows total cell number of DU145 cells pre-dosed for 1 h with RGD peptide (40 μg ml−1) or SFM and then re-dosed with SFM or IGFBP-2 (250 ng ml−1) for 48 h after initial plating (as in Figures 1C and D) in GM for 24 h and serum starving for a further 24 h. (C) Graph shows total cell number of DU145 cells pre-dosed for 1 h with β1-integrin receptor-blocking antibody (200 ng ml−1) or control mouse IgG1 antibody (200 ng ml−1) or SFM before they were then spiked with IGFBP-2 (250 ng ml−1) for 48 h after initial plating (as in Figures 1C and D) in GM for 24 h and serum starving for a further 24 h. All graphs show the mean±s.e.m. of at least three independent experiments each repeated in triplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101905&req=5

fig2: Involvement of integrin receptors in the intrinsic effects of IGFBP-2 on DU145 cells. (A) Graph shows total cell number of DU145 cells following treatment with RGD peptide (0–40 μg ml−1) for 48 h after initial plating (as in Figures 1A and B) in GM for 24 h and serum starving for a further 24 h. Insert shows a representative PCR blot (of IGFBP-2 m RNA) and WIBs (of IGFBP-2 protein from cell lysates and conditioned cell supernatants). A WIB for tubulin is also shown as a loading control for IGFBP-2 in the cell lysates. (B) Graph shows total cell number of DU145 cells pre-dosed for 1 h with RGD peptide (40 μg ml−1) or SFM and then re-dosed with SFM or IGFBP-2 (250 ng ml−1) for 48 h after initial plating (as in Figures 1C and D) in GM for 24 h and serum starving for a further 24 h. (C) Graph shows total cell number of DU145 cells pre-dosed for 1 h with β1-integrin receptor-blocking antibody (200 ng ml−1) or control mouse IgG1 antibody (200 ng ml−1) or SFM before they were then spiked with IGFBP-2 (250 ng ml−1) for 48 h after initial plating (as in Figures 1C and D) in GM for 24 h and serum starving for a further 24 h. All graphs show the mean±s.e.m. of at least three independent experiments each repeated in triplicate.
Mentions: We tested the effect of a short disintegrin, RGD containing, peptide, which represents an amino acid sequence common to several ECM components that bind to integrins. We observed a dose-dependent decrease in total cell number with increasing RGD peptide concentrations (0–40 μg ml−1; Figure 2A). We found that there was no increase in IGFBP-2 mRNA. However, conditioned media from this experiment showed a progressive increase in levels of IGFBP-2 with increasing RGD peptide concentration (Figure 2A insert), suggesting displacement of IGFBP-2 from the cell surface as opposed to an increase in its synthesis. We anticipated that the increase in IGFBP-2 in the cell supernatants would be accompanied by a decrease in the cell lysates. However, levels of IGFBP-2 in the cell lysate remained the same suggesting that the association of the RGD peptide with the cell surface was in some way inhibiting degradation of IGFBP-2. We went on to show that the proliferative effect of exogenous IGFBP-2 was markedly inhibited in the presence of a sub-apoptotic dose of RGD peptide (Figure 2B). This indicated that IGFBP-2 was promoting growth via interaction with an integrin receptor. We then showed that IGFBP-2 acted specifically via integrin receptors containing the β1 subunit, as a β1-integrin receptor blocking antibody similarly inhibited cell proliferation stimulated by exogenous IGFBP-2, whereas a control mouse IgG1 antibody was without effect (Figure 2C).

Bottom Line: Understanding the resistance to therapy could aid the development of more effective treatments.The IGFBP-2 promoted cell growth in both cell lines but with PC3 cells this was in an IGF-dependent manner, whereas with DU145 cells the effect was independent of IGF receptor activation.This IGF-independent effect of IGFBP-2 was mediated by interaction with β-1-containing integrins and a consequent increase in PTEN phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: IGFs and Metabolic Endocrinology Group, School of Clinical Sciences, Learning and Research Building, Southmead Hospital, Bristol, UK.

ABSTRACT

Background: The development of androgen independence, chemo-, and radioresistance are critical markers of prostate cancer progression and the predominant reasons for its high mortality. Understanding the resistance to therapy could aid the development of more effective treatments.

Aim: The aim of this study is to investigate the effects of insulin-like growth factor-binding protein-2 (IGFBP-2) on prostate cancer cell proliferation and its effects on the response to docetaxel.

Methods: DU145 and PC3 cells were treated with IGFBP-2, insulin-like growth factor I (IGF-I) alone or in combination with blockade of the IGF-I receptor or integrin receptors. Cells were also treated with IGFBP-2 short interfering ribonucleic acid with or without a PTEN (phosphatase and tensin homologue deleted on chromosome 10) inhibitor or docetaxel. Tritiated thymidine incorporation was used to measure cell proliferation and Trypan blue cell counting for cell death. Levels of IGFBP-2 mRNA were measured using RT-PCR. Abundance and phosphorylation of proteins were assessed using western immunoblotting.

Results: The IGFBP-2 promoted cell growth in both cell lines but with PC3 cells this was in an IGF-dependent manner, whereas with DU145 cells the effect was independent of IGF receptor activation. This IGF-independent effect of IGFBP-2 was mediated by interaction with β-1-containing integrins and a consequent increase in PTEN phosphorylation. We also determined that silencing IGFBP-2 in both cell lines increased the sensitivity of the cells to docetaxel.

Conclusion: The IGFBP-2 has a key role in the growth of prostate cancer cells, and silencing IGFBP-2 expression reduced the resistance of these cells to docetaxel. Targeting IGFBP-2 may increase the efficacy of docetaxel.

Show MeSH
Related in: MedlinePlus