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Epidermal growth factor potentiates in vitro metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel.

Uysal-Onganer P, Djamgoz MB - Mol. Cancer (2007)

Bottom Line: The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated.EGF increased VGSC Nav1.7 (predominant isoform in PCa) mRNA and protein expressions.Co-application of the highly specific VGSC blocker tetrodotoxin (TTX) suppressed the effect of EGF on all three metastatic cell behaviours studied. 1) EGF has a major involvement in the upregulation of functional VGSC expression in human PCa PC-3M cells. (2) VGSC activity has a significant intermediary role in potentiating effect of EGF in human PCa.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neuroscience Solutions to Cancer Research Group, Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, UK. p.onganer@imperial.ac.uk

ABSTRACT

Background: Although a high level of functional voltage-gated sodium channel (VGSC) expression has been found in strongly metastatic human and rat prostate cancer (PCa) cells, the mechanism(s) responsible for the upregulation is unknown. The concentration of epidermal growth factor (EGF), a modulator of ion channels, in the body is highest in prostatic fluid. Thus, EGF could be involved in the VGSC upregulation in PCa. The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated. A quantitative approach, from gene level to cell behaviour, was used. mRNA levels were determined by real-time PCR. Protein expression was studied by Western blots and immunocytochemistry and digital image analysis. Functional assays involved measurements of transverse migration, endocytic membrane activity and Matrigel invasion.

Results: Exogenous EGF enhanced the cells' in vitro metastatic behaviours (migration, endocytosis and invasion). Endogenous EGF had a similar involvement. EGF increased VGSC Nav1.7 (predominant isoform in PCa) mRNA and protein expressions. Co-application of the highly specific VGSC blocker tetrodotoxin (TTX) suppressed the effect of EGF on all three metastatic cell behaviours studied.

Conclusion: 1) EGF has a major involvement in the upregulation of functional VGSC expression in human PCa PC-3M cells. (2) VGSC activity has a significant intermediary role in potentiating effect of EGF in human PCa.

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Effects of TTX (500 nM) and TTX+EGF on respective relative levels of total and plasma membrane (PM) VGSC protein expression, presented as a percentages of respective controls (Cont). Two concentrations of EGF were used: 50 ng/ml (EGF1) and 100 ng/ml (EGF2). Relative levels of total VGSC were deduced from Western blots (as in Fig. 2). Relative levels of PM expression were obtained from immunocytochemistry/digital analysis(as in Fig. 4). Each histobar denotes mean ± standard error (n = 3–6). Light bars, total VGSC protein. Shaded bars, VGSC protein expressed in plasma membrane (PM).
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Figure 6: Effects of TTX (500 nM) and TTX+EGF on respective relative levels of total and plasma membrane (PM) VGSC protein expression, presented as a percentages of respective controls (Cont). Two concentrations of EGF were used: 50 ng/ml (EGF1) and 100 ng/ml (EGF2). Relative levels of total VGSC were deduced from Western blots (as in Fig. 2). Relative levels of PM expression were obtained from immunocytochemistry/digital analysis(as in Fig. 4). Each histobar denotes mean ± standard error (n = 3–6). Light bars, total VGSC protein. Shaded bars, VGSC protein expressed in plasma membrane (PM).

Mentions: Taking together the data obtained here (using the strongly metastatic human PCa PC-3M cell line) and the data obtained previously from analogous rat PCa Mat-LyLu cells [10] (both with Nav1.7 dominant), VGSC expression would appear to be under auto-regulation. Accordingly, long-term (24+ h) treatment with TTX led to down-regulation of functional VGSC expression. Thus, application of TTX (500 nM) alone reduced total and plasma membrane VGSC protein expressions by 12 ± 1.5 % and 10 ± 1.4 %, respectively (p < 0.01 for both; n = 5–6; Fig. 6). Interestingly, even when EGF (50 and 100 ng/ml) was added in the presence of TTX, protein expressions remained below control levels. Thus, the auto-regulation by positive feed-back appeared to dominate the normally potentiating effect of EGF.


Epidermal growth factor potentiates in vitro metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel.

Uysal-Onganer P, Djamgoz MB - Mol. Cancer (2007)

Effects of TTX (500 nM) and TTX+EGF on respective relative levels of total and plasma membrane (PM) VGSC protein expression, presented as a percentages of respective controls (Cont). Two concentrations of EGF were used: 50 ng/ml (EGF1) and 100 ng/ml (EGF2). Relative levels of total VGSC were deduced from Western blots (as in Fig. 2). Relative levels of PM expression were obtained from immunocytochemistry/digital analysis(as in Fig. 4). Each histobar denotes mean ± standard error (n = 3–6). Light bars, total VGSC protein. Shaded bars, VGSC protein expressed in plasma membrane (PM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211503&req=5

Figure 6: Effects of TTX (500 nM) and TTX+EGF on respective relative levels of total and plasma membrane (PM) VGSC protein expression, presented as a percentages of respective controls (Cont). Two concentrations of EGF were used: 50 ng/ml (EGF1) and 100 ng/ml (EGF2). Relative levels of total VGSC were deduced from Western blots (as in Fig. 2). Relative levels of PM expression were obtained from immunocytochemistry/digital analysis(as in Fig. 4). Each histobar denotes mean ± standard error (n = 3–6). Light bars, total VGSC protein. Shaded bars, VGSC protein expressed in plasma membrane (PM).
Mentions: Taking together the data obtained here (using the strongly metastatic human PCa PC-3M cell line) and the data obtained previously from analogous rat PCa Mat-LyLu cells [10] (both with Nav1.7 dominant), VGSC expression would appear to be under auto-regulation. Accordingly, long-term (24+ h) treatment with TTX led to down-regulation of functional VGSC expression. Thus, application of TTX (500 nM) alone reduced total and plasma membrane VGSC protein expressions by 12 ± 1.5 % and 10 ± 1.4 %, respectively (p < 0.01 for both; n = 5–6; Fig. 6). Interestingly, even when EGF (50 and 100 ng/ml) was added in the presence of TTX, protein expressions remained below control levels. Thus, the auto-regulation by positive feed-back appeared to dominate the normally potentiating effect of EGF.

Bottom Line: The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated.EGF increased VGSC Nav1.7 (predominant isoform in PCa) mRNA and protein expressions.Co-application of the highly specific VGSC blocker tetrodotoxin (TTX) suppressed the effect of EGF on all three metastatic cell behaviours studied. 1) EGF has a major involvement in the upregulation of functional VGSC expression in human PCa PC-3M cells. (2) VGSC activity has a significant intermediary role in potentiating effect of EGF in human PCa.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neuroscience Solutions to Cancer Research Group, Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, UK. p.onganer@imperial.ac.uk

ABSTRACT

Background: Although a high level of functional voltage-gated sodium channel (VGSC) expression has been found in strongly metastatic human and rat prostate cancer (PCa) cells, the mechanism(s) responsible for the upregulation is unknown. The concentration of epidermal growth factor (EGF), a modulator of ion channels, in the body is highest in prostatic fluid. Thus, EGF could be involved in the VGSC upregulation in PCa. The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated. A quantitative approach, from gene level to cell behaviour, was used. mRNA levels were determined by real-time PCR. Protein expression was studied by Western blots and immunocytochemistry and digital image analysis. Functional assays involved measurements of transverse migration, endocytic membrane activity and Matrigel invasion.

Results: Exogenous EGF enhanced the cells' in vitro metastatic behaviours (migration, endocytosis and invasion). Endogenous EGF had a similar involvement. EGF increased VGSC Nav1.7 (predominant isoform in PCa) mRNA and protein expressions. Co-application of the highly specific VGSC blocker tetrodotoxin (TTX) suppressed the effect of EGF on all three metastatic cell behaviours studied.

Conclusion: 1) EGF has a major involvement in the upregulation of functional VGSC expression in human PCa PC-3M cells. (2) VGSC activity has a significant intermediary role in potentiating effect of EGF in human PCa.

Show MeSH
Related in: MedlinePlus