Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC312

Poster Communications

Epidermal growth factor-driven expression of a TTX-sensitive voltage-gated sodium channel potentiates human non-small cell lung cancer invasion via elevation of intracellular sodium concentration

T. Campbell1, L. Gorham1, M. Main2, E. Fitzgerald1

1. Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom. 2. Cancer Bioscience, AstraZeneca, Alderley Park, United Kingdom.

Expression of NaV channels has been reported in a range of cancer cells and appears to be specifically linked to cell invasion. However, the cellular mechanisms underlying how NaV channels promote invasion remain unclear. We have shown previously that EGF receptor stimulation enhances H460 NSCLC invasion via expression of TTX-sensitive NaV1.7. We are currently attempting to define the interplay between intracellular sodium and metastatic potential in invasive NSCLC cell lines. The presence of NaV channel currents in strongly metastatic (H460) and weakly metastatic (A549) NSCLC cell lines was determined using patch clamp electrophysiology with physiological saline solutions. ICC was carried out on formaldehyde-fixed cells permeabilised with saponin. Gene expression was assessed by qPCR using cDNA obtained from 25ng total cell RNA, with all results normalised to 18S rRNA expression. Protein levels were determined with western blots using 20μg total cell protein. Intracellular Na+ levels were measured using the ratiometric dye SBFI. Cell invasion was analysed using a Boyden chamber setup with Matrigel™. Values are means ± SEM, compared by unpaired Student’s t-test or ANOVA. EGF-regulated, TTX-sensitive NaV1.7 currents are present in H460 cells but absent in A549 cells. Moreover, the current in the H460 cells is a sustained one at the resting membrane potential of the cells (-27±4mV, n=6). H460 cells have an internal Na+ concentration more than double that of the A549 cells (H460: 22.3±4.3mM, n=4, A549: 9.7±3.2mM, n=4, p<0.01) and when NaV1.7 channels are blocked in H460 cells with 1μM TTX, or downregulated with 5nM siRNA directed against NaV1.7, cell invasion is significantly reduced (CTL: relative invasion = 1.00±0.05, n=9, TTX: 0.60±0.07, n=9, siRNA: 0.50±0.02, n=9, p<0.01 and p<0.001 for TTX and siRNA vs. CTL respectively). Additionally, when the sodium/hydrogen exchanger protein NHE1, whose expression is also regulated by EGF signalling, is blocked in H460 cells with EIPA, cell invasion is also significantly reduced and there is no additive effect following co-application of EIPA and TTX (CTL: relative invasion = 1.00±0.03, n=9, EIPA: 0.60±0.02, n=9, EIPA+TTX: 0.62±0.04, n=9, p<0.001 vs. CTL for both treatments). These data therefore suggest that elevated intracellular Na+ is driving H460 cell invasion. Preliminary work reveals that treatment of H460 cells with 10μM monensin (Na+ ionophore) significantly enhances cell invasion (CTL: relative invasion = 1.00±0.03, n=6, monensin: 1.80±0.19, n=8, p<0.001), thus supporting the idea that elevated intracellular Na+ alone can drive cancer cell invasion. Work is in progress to determine the precise mechanism downstream of Na+ that may be driving cell invasion, with specific focus on Ca2+ signalling and pH.

Where applicable, experiments conform with Society ethical requirements