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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Exogenous EGF enhanced the cells' <it>in vitro </it>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.</p> <p>Conclusion</p> <p>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.</p

Overview of the oncogenic signaling pathways in colorectal cancer: Mechanistic insights.

Colorectal cancer is a multifaceted disease which is therapeutically challenging. Based on insights gleaned from almost a quarter century of research, it is obvious that deregulation of spatio-temporally controlled signaling pathways play instrumental role in development and progression of colorectal cancer. High-throughput technologies have helped to develop a sharper and broader understanding of the wide ranging signal transduction cascades which also contribute to development of drug resistance, loss of apoptosis and, ultimately, of metastasis. In this review, we have set the spotlight on role of JAK/STAT, TGF/SMAD, Notch, WNT/β-Catenin, SHH/GLI and p53 pathways in the development and progression of colorectal cancer. We have also highlighted recent reports on TRAIL-mediated pathways and molecularly distinct voltage-gated sodium channels in colorectal cancer

Confocal microscopy and densitometric analysis of VGSC protein expression in PC-3M cells

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor potentiates metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel"</p><p>http://www.molecular-cancer.com/content/6/1/76</p><p>Molecular Cancer 2007;6():76-76.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2211503.</p><p></p> (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)

Functional evidence for EGF-induced enhancement of metastatic cell behaviours via VGSC expression/activity in PC-3M cells

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor potentiates metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel"</p><p>http://www.molecular-cancer.com/content/6/1/76</p><p>Molecular Cancer 2007;6():76-76.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2211503.</p><p></p> (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)

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)

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor potentiates metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel"</p><p>http://www.molecular-cancer.com/content/6/1/76</p><p>Molecular Cancer 2007;6():76-76.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2211503.</p><p></p> 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)

Tetracaine downregulates matrix metalloproteinase activity and inhibits invasiveness of strongly metastatic MDA-MB-231 human breast cancer cells

Tetracaine, a long-acting amino ester-type local anesthetic, prevents the initiation and propagation of action potentials by reversibly blocking voltage-gated sodium channels (VGSCs). These channels, which are highly expressed in several carcinomas (e.g. breast, prostate, colon and lung cancers) have been implicated in promoting metastatic behaviours. Recent evidence suggests that local anesthetics can suppress cancer progression. In this paper, we aimed to explore whether tetracaine would reduce the invasive characteristics of breast cancer cells. In a comparative approach, we used two cell lines of contracting metastatic potential: MDA-MB-231 (strongly metastatic) and MCF-7 (weakly metastatic). Tetracaine (50 μM and 75 μM) did not affect the proliferation of both MDA-MB-231 and MCF-7 cells. Importantly, tetracaine suppressed the migratory, invasive, and adhesive capacities of MDA-MB-231 cells; there was no effect on the motility of MCF-7 cells. Tetracaine treatment also significantly decreased the expression and activity levels of MMP-2 and MMP-9, whilst increasing TIMP-2 expression in MDA-MB-231 cells. On the other hand, VGSC α/Nav1.5 and VGSC-β1 mRNA and protein expression levels were not affected. We conclude that tetracaine has anti-invasive effects on breast cancer cells and may be exploited clinically, for example, in surgery and/or in combination therapies