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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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Confocal microscopy and densitometric analysis of VGSC protein expression in PC-3M cells. (A). Typical confocal images. (i) Control. (ii) EGF (100 ng/ml). (iii) AG1478 (100 nM). Each treatment was for 24 h. Scale bar, 20 μm (applicable to all panels). (B) Signal density, ie optical density of plasma membrane (PM) VGSC immunocytochemistry, corresponding to images such as (A). Each histobar denotes mean ± standard error (n = 50 cells/3 separate experiments). (C) Effects of EGF (similar treatment as in A) on total and PM VGSC expression. The PM fraction was immunoprecipitated by biotin labelling. Key: 1) Control total VGSC protein. (2) EGF-treated total VGSC protein. (3) Control PM VGSC protein. (4) EGF-treated PM VGSC protein. Note the change in the molecular size of the biotinylated fractions (lanes 3 & 4).
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Figure 4: Confocal microscopy and densitometric analysis of VGSC protein expression in PC-3M cells. (A). Typical confocal images. (i) Control. (ii) EGF (100 ng/ml). (iii) AG1478 (100 nM). Each treatment was for 24 h. Scale bar, 20 μm (applicable to all panels). (B) Signal density, ie optical density of plasma membrane (PM) VGSC immunocytochemistry, corresponding to images such as (A). Each histobar denotes mean ± standard error (n = 50 cells/3 separate experiments). (C) Effects of EGF (similar treatment as in A) on total and PM VGSC expression. The PM fraction was immunoprecipitated by biotin labelling. Key: 1) Control total VGSC protein. (2) EGF-treated total VGSC protein. (3) Control PM VGSC protein. (4) EGF-treated PM VGSC protein. Note the change in the molecular size of the biotinylated fractions (lanes 3 & 4).

Mentions: The effect of EGF (exogenous and endogenous) in increasing VGSC protein expression was also apparent in plasma membrane (PM). Thus, exogenous EGF (100 ng/ml) and AG1478 (100 nM) altered PM expression by +20 ± 2.0 and -15 ± 2.6 %, respectively (for both: p < 0.01; n = 5; Figs. 4Ai–iii &3B). In a preliminary experiment (n = 2), an alternative method, involving cell surface biotinylation similarly showed that EGF increased plasma membrane VGSC protein expression (Fig. 4C, lane 4 vs 3).


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)

Confocal microscopy and densitometric analysis of VGSC protein expression in PC-3M cells. (A). Typical confocal images. (i) Control. (ii) EGF (100 ng/ml). (iii) AG1478 (100 nM). Each treatment was for 24 h. Scale bar, 20 μm (applicable to all panels). (B) Signal density, ie optical density of plasma membrane (PM) VGSC immunocytochemistry, corresponding to images such as (A). Each histobar denotes mean ± standard error (n = 50 cells/3 separate experiments). (C) Effects of EGF (similar treatment as in A) on total and PM VGSC expression. The PM fraction was immunoprecipitated by biotin labelling. Key: 1) Control total VGSC protein. (2) EGF-treated total VGSC protein. (3) Control PM VGSC protein. (4) EGF-treated PM VGSC protein. Note the change in the molecular size of the biotinylated fractions (lanes 3 & 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Confocal microscopy and densitometric analysis of VGSC protein expression in PC-3M cells. (A). Typical confocal images. (i) Control. (ii) EGF (100 ng/ml). (iii) AG1478 (100 nM). Each treatment was for 24 h. Scale bar, 20 μm (applicable to all panels). (B) Signal density, ie optical density of plasma membrane (PM) VGSC immunocytochemistry, corresponding to images such as (A). Each histobar denotes mean ± standard error (n = 50 cells/3 separate experiments). (C) Effects of EGF (similar treatment as in A) on total and PM VGSC expression. The PM fraction was immunoprecipitated by biotin labelling. Key: 1) Control total VGSC protein. (2) EGF-treated total VGSC protein. (3) Control PM VGSC protein. (4) EGF-treated PM VGSC protein. Note the change in the molecular size of the biotinylated fractions (lanes 3 & 4).
Mentions: The effect of EGF (exogenous and endogenous) in increasing VGSC protein expression was also apparent in plasma membrane (PM). Thus, exogenous EGF (100 ng/ml) and AG1478 (100 nM) altered PM expression by +20 ± 2.0 and -15 ± 2.6 %, respectively (for both: p < 0.01; n = 5; Figs. 4Ai–iii &3B). In a preliminary experiment (n = 2), an alternative method, involving cell surface biotinylation similarly showed that EGF increased plasma membrane VGSC protein expression (Fig. 4C, lane 4 vs 3).

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