Limits...
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.

Show MeSH

Related in: MedlinePlus

Functional evidence for EGF-induced enhancement of metastatic cell behaviours via VGSC expression/activity in PC-3M cells. (A) Migration index (MiI), expressed relative to the control level (Cont), fixed as 100 %. Effects of EGF (50 ng/ml), TTX (500 nM) and EGF+TTX are shown. (B) Dose dependence of the effect of EGF on MiI. ΔMiI denotes the percentage change (increase) in MiI induced by increasing concentrations of EGF, expressed relative to the maximum (fixed as 100 %) seen for 50 ng/ml. (C) Endocytosis index (EI), expressed as percentage of the control level (Cont). Effects of EGF (20 ng/ml), TTX (500 nM) and EGF+TTX are shown. (D) Dose dependence of the effect of EGF on EI. ΔEI denotes the change (increase) in EI induced by given concentrations of EGF. (E) Boyden chamber invasion assay data. Effects of EGF (100 ng/ml), TTX (500 nM), EGF+TTX and AG1478 (100 nM) are shown. Invasion index (InvI) denotes the percentage of cells crossing the membrane in Transwell assays. Each data point or histobar denotes mean ± standard error (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2211503&req=5

Figure 2: Functional evidence for EGF-induced enhancement of metastatic cell behaviours via VGSC expression/activity in PC-3M cells. (A) Migration index (MiI), expressed relative to the control level (Cont), fixed as 100 %. Effects of EGF (50 ng/ml), TTX (500 nM) and EGF+TTX are shown. (B) Dose dependence of the effect of EGF on MiI. ΔMiI denotes the percentage change (increase) in MiI induced by increasing concentrations of EGF, expressed relative to the maximum (fixed as 100 %) seen for 50 ng/ml. (C) Endocytosis index (EI), expressed as percentage of the control level (Cont). Effects of EGF (20 ng/ml), TTX (500 nM) and EGF+TTX are shown. (D) Dose dependence of the effect of EGF on EI. ΔEI denotes the change (increase) in EI induced by given concentrations of EGF. (E) Boyden chamber invasion assay data. Effects of EGF (100 ng/ml), TTX (500 nM), EGF+TTX and AG1478 (100 nM) are shown. Invasion index (InvI) denotes the percentage of cells crossing the membrane in Transwell assays. Each data point or histobar denotes mean ± standard error (n = 4).

Mentions: Exogenous EGF (1–100 ng/ml) significantly increased transverse migration of PC-3M cells in a dose dependent manner (p < 0.05 for all concentrations; n = 9; Fig. 2A and 2B). The greatest effect was seen for 50 ng/ml EGF, which increased migration by 39 ± 1.2 % (Fig. 2A &2B). In most of the experiments that followed, working concentrations of EGF around this peak (i.e., 20, 50 or 100 ng/ml) were used. In endocytosis assays, treatment with EGF (20 ng/ml) enhanced HRP uptake by 23 ± 5.4 % (p = 0.01; n = 9; Fig. 2C). This effect was also concentration dependent (Fig. 2D). Interestingly, in both assays, increasing the EGF concentration ultimately produced reduced effects (Fig. 2B &2D). In Boyden chamber invasion assays, EGF (100 ng/ml) increased the cells' invasiveness by 20 ± 5.2 % (p < 0.03 cf. control; n = 4; Fig. 2E). Application of 100 nM AG1478, an inhibitor of EGF receptor, alone had the opposite effect, reducing cell invasion by 19 ± 5.4 % (p = 0.02, n = 4; Fig. 2E). This result suggested that the potentiating effect of EGF on MCBs also occurred endogenously.


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)

Functional evidence for EGF-induced enhancement of metastatic cell behaviours via VGSC expression/activity in PC-3M cells. (A) Migration index (MiI), expressed relative to the control level (Cont), fixed as 100 %. Effects of EGF (50 ng/ml), TTX (500 nM) and EGF+TTX are shown. (B) Dose dependence of the effect of EGF on MiI. ΔMiI denotes the percentage change (increase) in MiI induced by increasing concentrations of EGF, expressed relative to the maximum (fixed as 100 %) seen for 50 ng/ml. (C) Endocytosis index (EI), expressed as percentage of the control level (Cont). Effects of EGF (20 ng/ml), TTX (500 nM) and EGF+TTX are shown. (D) Dose dependence of the effect of EGF on EI. ΔEI denotes the change (increase) in EI induced by given concentrations of EGF. (E) Boyden chamber invasion assay data. Effects of EGF (100 ng/ml), TTX (500 nM), EGF+TTX and AG1478 (100 nM) are shown. Invasion index (InvI) denotes the percentage of cells crossing the membrane in Transwell assays. Each data point or histobar denotes mean ± standard error (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Functional evidence for EGF-induced enhancement of metastatic cell behaviours via VGSC expression/activity in PC-3M cells. (A) Migration index (MiI), expressed relative to the control level (Cont), fixed as 100 %. Effects of EGF (50 ng/ml), TTX (500 nM) and EGF+TTX are shown. (B) Dose dependence of the effect of EGF on MiI. ΔMiI denotes the percentage change (increase) in MiI induced by increasing concentrations of EGF, expressed relative to the maximum (fixed as 100 %) seen for 50 ng/ml. (C) Endocytosis index (EI), expressed as percentage of the control level (Cont). Effects of EGF (20 ng/ml), TTX (500 nM) and EGF+TTX are shown. (D) Dose dependence of the effect of EGF on EI. ΔEI denotes the change (increase) in EI induced by given concentrations of EGF. (E) Boyden chamber invasion assay data. Effects of EGF (100 ng/ml), TTX (500 nM), EGF+TTX and AG1478 (100 nM) are shown. Invasion index (InvI) denotes the percentage of cells crossing the membrane in Transwell assays. Each data point or histobar denotes mean ± standard error (n = 4).
Mentions: Exogenous EGF (1–100 ng/ml) significantly increased transverse migration of PC-3M cells in a dose dependent manner (p < 0.05 for all concentrations; n = 9; Fig. 2A and 2B). The greatest effect was seen for 50 ng/ml EGF, which increased migration by 39 ± 1.2 % (Fig. 2A &2B). In most of the experiments that followed, working concentrations of EGF around this peak (i.e., 20, 50 or 100 ng/ml) were used. In endocytosis assays, treatment with EGF (20 ng/ml) enhanced HRP uptake by 23 ± 5.4 % (p = 0.01; n = 9; Fig. 2C). This effect was also concentration dependent (Fig. 2D). Interestingly, in both assays, increasing the EGF concentration ultimately produced reduced effects (Fig. 2B &2D). In Boyden chamber invasion assays, EGF (100 ng/ml) increased the cells' invasiveness by 20 ± 5.2 % (p < 0.03 cf. control; n = 4; Fig. 2E). Application of 100 nM AG1478, an inhibitor of EGF receptor, alone had the opposite effect, reducing cell invasion by 19 ± 5.4 % (p = 0.02, n = 4; Fig. 2E). This result suggested that the potentiating effect of EGF on MCBs also occurred endogenously.

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