Functional expression of voltage-gated Na+ channels (VGSCs) occurs in human carcinomas and promotes invasiveness in vitro and metastasis in vivo. Both neonatal and adult forms of Nav1.5 (nNav1.5 and aNav1.5, respectively) have been reported to be expressed at mRNA level in human colorectal cancer (CRCa) cells. The main aims of this PhD on human CRC were (1) to characterise the functional role of the 'neonatal' splice variant of Navl.5 (nNavl.5) in cellular invasiveness under normoxia and hypoxia, and (2) to elucidate possible mechanisms underlying the pro-invasive role of nNav1.5.
In this study, nNav1.5 mRNA and protein expression was detected in all CRCa cell lines (HT29, HCT116 and SW620) tested using RT-PCR, western blots and immunocytochemistry techniques. Comparison with the effect of the 20µM tetrodotoxin (TTX, a specific blocker of VGSCs) suggested that matrigel invasion was driven pre-dominantly by nNav1.5. SW620 cells was the most consistent in terms of nNav1.5 expression levels (mRNA and protein) and VGSC (mainly nNav1.5)-mediated invasiveness. Under hypoxia, SW620 cells exhibited increased invasiveness which was then inhibited by 5µM ranolazine, a blocker of the persistent VGSC current (INaP). The latter suggested nNav1.5-mediated INaP played a significant role in the hypoxia-induced increase in invasiveness. This effect of ranolazine was dose-dependent but was lost in cells pre-treated with siRNA-silenced nNav1.5. There was no effect of ranolazine under normoxia. Similar to hypoxia, aconitine (a VGSC opener) increased the invasiveness of SW620 cells, which was reduced by ranolazine and TTX (positive control).
The possible role of a functional ‘axis’ involving “VGSC - protein kinase A (PKA) - Na+/H+ exchanger 1 (NHE1)” in controlling CRCa invasion was investigated. Aconitine increased PKA phosphorylation in SW620 cells. Conversely, the phosphorylated-PKA level was then reduced by TTX or siRNA targeting nNav1.5. Aconitine and forskolin (a PKA-activator) separately increased, whilst KT5720 (a PKA inhibitor) reduced the invasiveness. Co-treatment of aconitine / KT5720 or forskolin / TTX significantly reduced the invasiveness. Also, cariporide (a specific Na+/H+ exchanger 1 blocker) reduced the invasiveness. Interestingly, both aconitine- and forskolin-stimulated invasiveness was significantly reduced upon cariporide co-treatment. VGSC-NHE1 relationship was found to be dominant over VGSC-PKA or PKA-NHE1 in controlling invasion.
VGSC activity-mediated glucose transporter 1 (GLUT1) expression and its effect on ‘growth’ of SW620 cells were investigated. VGSC inhibition reduced the expression of GLUT1 (mRNA and protein) under hypoxia only. Under high-glucose condition, cellular proliferation was increased and this effect was inhibited by TTX treatment
Overall, the main conclusions were as follows: 1) VGSC-dependent invasiveness was driven predominantly by nNav1.5 under both normoxic and hypoxic conditions; (2) the hypoxia-induced increase in invasiveness was mediated by the persistent current component of nNav1.5; (3) VGSC activity-induced increase in invasiveness involved PKA and NHE1 as intermediaries; and (4) a novel ‘hypoxic’ pathway involving VGSC activity and GLUT1 expression controlled SW620 cell proliferation under high-glucose conditions