Prof. and Chief of Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology,
Wuhan, 430022 China
Ion channels are pore-forming proteins and their function is to facilitate the diffusion of ion across cell membranes by flow of ions down the electrochemical gradient. Voltage-gated ion channels are a class of transmembrane ion channels and are found throughout the body which allowed a rapid and coordinated depolarization in response to voltage change in excitable cells (Catterall, 2010). Recent studies have demonstrated that Voltage-gated ion channels had been found in non-excitable cells. Among these channels, Voltage-gated sodium channels (VGSC) are the new topic and increasing evidences have suggested that which participated in the oncogenic process (Fiske et al., 2006; Mechaly et al., 2005).
VGSC is a large transmembrane glycoprotein complexes, composed of a highly processed α subunits (VGSCαs) with 24 transmembrane domains and one or more regulatory β subunits (VGSCβs), which mediate the all-or-none action potential initiation and propagation in excitable cells and tissues. When VGSC is activated, influx of sodium ions through channel depolarizes the cell membrane and initiates the rising phase of the action potential (Catterall, 2000).
VGSCαs are approximately 260 kDa, which have been identified ten encoding genes up to now. According to the phylogeny difference, Nav1.1 to Nav1.9 of nine isoforms constitute the functional subunits of VGSCαs and name Nav1 family, there are greater than 50% identical in amino acid sequence in these structure. As for another isoform Nax, its structure and function are different with Nav1 family and which seem to be regulated by sodium concentration but not by voltage. In case of Nav1 family, each isform is made up of a single polypeptide with 4 homologous domains (D1-D4) and every domain has 6 transmembrane segments (S1-S6). There are several functional parts about Nav1 structure. Among them, the S4 serves as a voltage sensor, which responds to the levels of the membrane potential, the S5/S6 pore-forming regions determine its ion selectivity, which is quite selective for sodium ions (Goldin et al., 2000; Goldin, 2001). Tetrodotoxin (TTX) is a potent neurotoxin, which blocks action potentials by binding to the VGSC in cell membranes. The binding site of TTX is located at the pore opening of VGSC (Narahashi, 2008). According to the sensitivity to TTX, Nav1 family was described as TTX-sensitive (TTX-S; Nav1.1-1.4, Nav1.6-1.7) and TTX-resistant (TTX-R; Nav1.5, Nav1.8, Nav1.9).
VGSCβs are approximately 30-40 kDa, which have been found that modified the channel and current density, kinetics and voltage-dependence of gating. To date, β1 to β4 of four isoforms encoded by SCN1B to SCN4B have been defined and they were the transmembrane proteins which contain a similar structure, consisting of one N-terminal extracellular immunoglobulin domain, one transmembrane segment, and a small intracellular domain (Brackenbury and Isom, 2008).
With the development of patch clamp technique and molecular biology, the functional expressions of VGSCαs have been reported in cancer cells (Palmer et al., 2008). The group of Mustafa Djamgoz in England first found the sodium current in highly metastatic rat prostate cancer cell line Mat-Ly-Lu using patch clamp technique, but not in the weakly metastatic counterparts of AT-2 cells (Grimes et al., 1995). Over the past few years, researchers have described that inhibiting the activity of VGSCαs could significantly reduce the invasion of the highly metastatic cancer cells but without obvious effect on weakly metastatic cancer cells (Roger et al., 2007; Gao et al., 2010; Diaz et al., 2007). At present, the group of Mustafa Djamgoz in England and the group of Le Guennec in France have made the best understood about VGSCαs and cancer (Roger et al., 2006). In this section, we would summarize the functional expression of VGSC Nav1.5 and Nav1.7 in cancer cells.
Individual VGSCαs subtypes can generate unique physiological signatures in different cell types and generate multiple expressions of individual VGSCαs subtypes in single cells (Diss et al., 2004). During recent years, increasing evidence has been accumulated in supporting that Nav1.5 can cause a variety of pathophysiological phenotypes in cancer cells (Schroeter et al., 2010). As we have known, Nav1.5 is belonging to the TTX-R isoform and encoded by the SCN5A gene, which is normally associated with human cardiac tissue and located on chromosome 3p21-24. It plays important roles in the excitability of atrial and ventricular cardiomyocytes and in rapid impulse propagation (Schroeter et al., 2010).
In 2003, the group of Le Guennec in France reported that VGSCαs were expressed in a highly metastatic breast cancer cell MDA-MB-231 but was not detected in the weakly metastatic MCF-7 cells (Roger et al., 2003). Later on, the group of Mustafa Djamgoz in England further found that the mainly isoform expressed in breast cancer highly metastatic cells was the Nav1.5, and inhibition of Nav1.5 with TTX could markedly reduced the invasion of MDA-MB-231 cells (Fraser et al., 2005). Interestingly, our research also demonstrated that the abnormal expression of Nav1.5 could be an integral component of the metastatic process in human ovarian cancer and inhibiting the activity of Nav1.5 could significant reduce the invasion of cancer cells (Gao et al., 2010). In addition, many similar discovery have also been reported a predominant expression of Nav1.5 in colon cancer and T-lymphocyte Jurkat cells (House et al., 2010; Fraser et al., 2004).
Alternative splicing are sophisticated and ubiquitous nuclear process, they are important in normal development by creating protein diversity in complex organisms and also are natural source of cancer-causing errors in gene expression (Venables, 2004). Up to now, six Nav1.5 splice variants have been reported in Gene Bank (NM_000335.4; NM_001099404.1; NM_001099405.1; NM_001160160.1; NM_001160161.1; NM_198056.2). To date, Diss et al. and Schroeter et al. subsequently summarized the currently known functional splice variants of Nav1.5, they both mentioned the naturally occurring Nav1.5 splice variants (Diss et al., 2004; Schroeter et al., 2010). Among these splice variants, the D1:S3 3' splice variant and D1:S3 5' splice variant are the predominant, which can lead to multiple 5'- and 3'-noncoding regions, resulting in the mutations of SCN5A. Because D1:S3 5' splice variant differs from D1:S3 3' splice variant at 31 nucleotides and result in 7 amino acid substitutions (Fraser et al., 2005), Brackenbury et al. designed the siRNA and a polyclonal antibody of targeting D1:S3 5' splice variant, which could rapidly reduce the activity of Nav1.5 and the invasion of MDA-MB-231 cells (Brackenbury et al., 2007). Further research has also shown that the D1:S3 5' splice variant were expressed in cancer cells, the expression levels of different Nav1.5 splice variants were diversity at different stage of development, the D1:S3 3' splice variant gradually increased during the developing of organism, while D1:S3 5' splice variant was decreased significantly in the mature cells even though which is abundant at embryonic stage (Zimmer et al., 2002). However, House et al. recently found that the functional isoforms in colon cancer was the D1:S3 3' splice of Nav1.5 but not the D1:S3 5' correspondence (House et al., 2010). In this study, researchers presumed that the recruitment of Nav1.5 expression might facilitate the regulation of a colon cancer invasion network involving downstream genes which encompass the Wnt signaling way.
Until now, the exact mechanisms by which different Nav1.5 splice variants functional expressed in different cancer cells remain unknown. Pan et al. reported that biochemical constitution of extracellular medium was critical in control of MDA-MB-231 cell motility (Pan and Djamgoz, 2009). Nav1.5 involving in the metastasis might have an indirect effect through the regulation of intracellular sodium homeostasis, the influx of Na+ could alter the release or uptake of Ca2+ from intracellular stores by deregulation of intracellular H+ concentration. Gillet et al. propose that Nav1.5 enhance the invasiveness of MDA-MB-231 cells by favoring the pH-dependent activity of cysteine cathepsins (Gillet et al., 2009). Subsequently, Brisson et al. demonstrated that Nav1.5 could enhance MDA-MB-231 cells invasiveness by increasing Na+/H+ exchanger type 1-dependent H+ efflux (Brisson et al., 2011). Furthermore, the rise influx of Na+ could also activate protein kinase A (PKA), which could lead to phosphorylation of cytoskeletal components. Chioni et al. also founded that PKA plays an important role in functional expression of Nav1.5 in MDA-MB-231 cells by mediating activity-dependent positive feedback, and which enhances the metastatic of MDA-MB-231 cells in turn (Chioni et al., 2010). Therefore, this general mechanism could lead to the identification of new targets allowing the therapeutic prevention of metastases.
Nav1.4 was first found functionally expressed in rat and human prostate cancer cell lines other than skeletal muscle in 1995. In this paper, the author reported that Nav1.4 is not only present in the highly metastatic MAT-LyLu and PC-3 cells, but also was detected in the weakly metastatic AT-2 and LNCaP cells (Diss et al., 1998). Interestingly, Bennett et al. reported that when Nav1.4 was transiently expressed in non-metastatic LNCaP cell, its invasion was sharply increased and the increased invasion could be completely reversed by treatment with TTX (Bennett et al., 2004). However, further research revealed that main isoform functionally expressed in prostate cancer was NaV1.7 but not Nav1.4, and researcher concluded that the NaV1.4 expressed in weakly metastatic cells might be at a sub-threshold density (Roger et al., 2006; Bennett et al., 2004).
As we have known, the TTX-S isoform Nav1.4 is encoded by SCN4A gene and located on chromosome 2, it is responsible for the generation and propagation of action potentials in neurons and muscle and broadly expressed in skeletal muscle (Jurkat-Rott et al., 2010). While the TTX-S isoform Nav1.7 is encoded by SCN9A gene and located on chromosome 2q23-24, it is necessary for pain sensation and broadly expressed in neurons (Wood et al., 2004). Recently research have shown that loss of function of Nav1.7 could cause a congenital inability to experience pain in humans, while gain of function of Nav1.7 could enhance the invasion of rat and human prostate cancer cells (Fischer and Waxman, 2010).
In 2001, Diss et al. reported that the main mRNA isoform expressed in Mat-Ly-Lu and PC3 cells was NaV1.7 (Diss et al., 2001). Later on, the same research group reported that the functional expression of NaV1.7 was related with the development of prostate cancer, NaV1.7 might be a potential novel marker for human prostate cancer (Diss et al., 2005). Over the past few years, NaV1.7 also been reported that participated in the invasion in human non-small-cell lung cancer cells, cervical cancer cells and so on (Roger et al., 2007; Diaz et al., 2007; Roger et al., 2006).
However, the mechanisms responsible for the functional expression of NaV1.7 in metastatic cancer cells also remain unknown. Growth factors have been shown to play the important roles in regulation of Nav1.7 transcription in prostate cancer strongly metastatic cells (Uysal-Onganer and Djamgoz, 2007; Ding et al., 2008). The expressions of Nav1.7 were under auto-regulation by activity-dependent positive feed-back which dominated the effect of different growth factors and which might be made effect on by PKA activation. In addition, over expressions of Nav1.7 also induced Ca2+ influx led to protein kinase C α (PKCα) phosphorylation and glycogen synthase kinase-3β (GSK-3β) phosphorylation by activating activated ERK1/ERK2 and p38 pathway (Kanai et al., 2009).
Due to VGSCβs subunits are multi-functional molecules, they are homologous to the immunoglobulin superfamily of cell adhesion molecules (CAMs) and play important roles in regulating cellular excitability, adhesion and metastatic activity in cancer (Isom, 2001). So far, the study of VGSCβs and cancer was mainly in breast cancer and prostate cancer cells. The group of Mustafa Djamgoz in London first found VGSCβs were expressed in prostate cancer cells and reducing the expression of β1 or disturbing its association with Nav1.7 could significantly reducing metastatic cell behaviour (Diss et al., 2008). In 2008, the same group further shown that the expression levels of β1 were significantly higher in weakly metastatic MCF-7 cells than strongly metastatic MDA-MB-231 cells, and silencing SCN1B using siRNA could increase the migration of MCF-7 cells, which suggested that β1 might as a novel cell adhesion molecule control the expression levels of Nav1.5 and cellular migration in breast cancer cells (Chioni et al., 2009). Therefore, it is inferred that VGSCβs might be involved in VGSCαs intracellular trafficking and functional expression by protein-protein interactions may, and the co-expression of VGSCβs and VGSCαs might increase the current density and the efficiency with which the mature channel protein was targeted to plasma membrane (Johnson and Bennett, 2006).
In conclusion, VGSC expressions were functionally up-regulated in many cancer cells by involving in transcriptional, pre-translational, translational, post-translational regulation (Shao et al., 2009). In addition, recent studies have reported that using of local anesthetics (the blockers of VGSC) during surgical resection of cancers was associated with a reduced risk of clinical cancer reoccurrence and metastasis (Wuethrich et al., 2010). Therefore, it should be noted that in the future studies that how VGSC activity participate in the progress of cancer, and VGSC functional expression in cancer cells might represent a novel mechanism for potentiating cellular metastasis and promising a new therapeutic strategy against cancers.